Ultra-sensitive assay for detecting ORF1p in biological fluids

Abstract

Described herein are methods and compositions for the accurate detection of cancer using ultra-sensitive immunoassays, such as digital ELISA, to detect the open reading frame 1 protein (ORF1p) encoded by LINE-1 reverse transcriptase in a biological fluid.

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 423,696, filed on November 8, 2022. The entire contents of the foregoing are hereby incorporated by reference. Technical Field

[0003] Described herein are methods and compositions for accurately detecting cancer using ultrasensitive immunoassays, such as digital ELISAs, to detect open reading frame 1 protein (ORF1p) encoded by LINE-1 retrotransposon in biological fluids. Background Art

[0004] Early detection of cancer is crucial to improving prognosis. For example, ovarian cancer is the fifth leading cause of cancer-related death among women in the United States because the disease is primarily diagnosed at an advanced stage, with high-grade serous ovarian cancer (HGSOC) accounting for 70-80% of ovarian cancer deaths. 1 However, for cases diagnosed at stage I, the 5-year survival rate is >90%. 2 Although circulating tumor DNA (ctDNA) has shown great potential in cancer detection, the level of ctDNA is extremely low and usually undetectable in the early stages of the disease, which remains a major challenge for ovarian cancer. 3 and other cancers. MicroRNAs (miRNAs) have also shown great potential, but further clinical validation of miRNA signatures is needed for early cancer detection. Proteins are a promising class of biomarkers because they are direct functional players in biological processes and are present in higher abundance in the blood compared to ctDNA. However, protein-based liquid biopsies are still limited by serious gaps in biomarker specificity and protein measurement technology. For example, in the case of ovarian cancer, although the FDA has approved the blood-based biomarkers carbohydrate antigen 125 (Ca125) and human epididymis protein 4 (HE4), they have poor sensitivity and specificity for early detection, limiting their usefulness in screening. 4 . Summary of the Invention

[0005] The methods provided herein include obtaining a sample comprising blood from a subject (e.g., a subject suspected of having or being at risk of developing cancer), and determining the level of ORF1p in the sample using an ultrasensitive protein assay (i.e., an assay with a limit of detection of less than 1 picomolar (0.1 femtomolar in 100 μl)). In some embodiments, the method further includes comparing the level of ORF1p in the sample to a disease reference, wherein an ORF1p level higher than the reference indicates that the subject has or is at risk of developing cancer.

[0006] In some embodiments, the cancer is a carcinoma, such as ovarian cancer, breast cancer, liver cancer, colon / colorectal cancer, lung cancer, esophageal cancer, prostate cancer, stomach cancer, head and neck cancer, soft tissue cancer, kidney cancer, gallbladder cancer, bile duct cancer (cholangiocarcinoma), bladder cancer, uterine cancer or pancreatic cancer; in some embodiments, the cancer is a non-brain cancer. In some embodiments, the cancer is ovarian cancer, such as high-grade serous ovarian cancer (HGSOC). In some embodiments, the cancer is not breast cancer. In some embodiments, the cancer is ovarian cancer, breast cancer, liver cancer, colon / colorectal cancer, lung cancer, esophageal cancer, prostate cancer, stomach cancer, head and neck cancer, brain cancer (optionally glioblastoma), soft tissue cancer, kidney cancer, gallbladder cancer, bile duct cancer (cholangiocarcinoma), bladder cancer, uterine cancer or pancreatic cancer, blood cancer or bone marrow cancer (optionally lymphoma, leukemia or myeloma) or skin cancer (optionally melanoma).

[0007] The methods provided herein include obtaining a sample comprising blood from a subject and determining the level of ORF1p in the sample using an ultrasensitive protein assay. In some embodiments, the method further includes comparing the level of ORF1p to a disease reference, wherein an ORF1p level higher than the reference indicates that the subject has cancer or is at risk of developing cancer. In some embodiments, the sample is a biological fluid that is whole blood, plasma, or serum; or, in some embodiments, the sample is or includes feces, cervical fluid (such as a Pap smear), uterine lavage fluid, urine, or sputum. The sample can also be a tissue sample, such as from a biopsy (e.g., a puncture, needle, or scrape biopsy, or a surgical biopsy), such as a tissue lysate.

[0008] In some embodiments, cancer is carcinoma. In some embodiments, cancer is ovarian cancer, breast cancer, liver cancer, colon / colorectal cancer, lung cancer, esophageal cancer, prostate cancer, stomach cancer, head and neck cancer, brain cancer, soft tissue cancer, kidney cancer, gallbladder cancer, bile duct cancer (cholangiocarcinoma), bladder cancer, uterine cancer or pancreatic cancer. In some embodiments, cancer is not brain cancer. In some embodiments, ovarian cancer is high-grade serous ovarian cancer (HGSOC). In some embodiments, cancer is not breast cancer. In some embodiments, cancer originates from blood, bone marrow, brain, skin or soft tissue, especially lymphoma, leukemia, myeloma, glioblastoma or melanoma.

[0009] In some embodiments, the ultrasensitive assay is a single-molecule array (SIMOA); Molecular On-bead Signal Amplification for Individual Counting (MOSAIC); Meso Scale Discovery (MSD); single molecule counting (SMC); nucleic acid-linked immunosorbent assay (NULISA); LUMINEX; SOMAscan assay; mass spectrometry (e.g., MALDI-MS) and / or mass cytometry (e.g., CyTOF).

[0010] In some embodiments, determining the level of ORF1p comprises contacting the sample with a capture or detection reagent comprising a Nanobody selected from Nb2, Nb5, Nb9, Nb10 or NB21 or an ORF1p binding derivative comprising its CDRs (as shown in Table B) or a concatemer thereof, optionally MT1032, MT1033, MT1034, MT1035, MT1036, MT1037, MT1038, MT1039 or MT1040 and / or selected from 62H12, 64C6 , 33A8, 61A11, 36D12, 34H7, 50E9, 34C5, 55A6, 42D10, 4H1, Ab6 (ab246317, Abeam), Ab54 (ab246320, Abeam), or an antigen-binding fragment thereof, optionally wherein the capture / detection reagent is 34H7 / Ab6, 62H12 / Ab6, 34H7 / Nb5-5LL, 62H12 / Nb5-5LL, 4H1 / Nb5-5 or 4H1 / Nb5-5LL.

[0011] In some embodiments, the Nanobody comprises a sequence that is at least 80%, 85%, 90% or 95% identical to a sequence in Table B or a multimer thereof, preferably wherein the CDRs of the Nanobody are identical to the CDRs of a sequence in Table B or a multimer thereof. Exemplary Nanobody concatemers include MT1032, MT1033, MT1034, MT1035, MT1036, MT1037, MT1038, MT1039 and MT1040 (see Figure 22A).

[0012] In some embodiments, the method further comprises recommending or sending the subject for additional evaluation, such as by imaging and / or biopsy. In some embodiments, the method further comprises administering a cancer treatment to a subject identified as having or at risk of developing cancer. In some embodiments, the treatment comprises chemotherapy, hormone therapy, immunotherapy, radiation, or surgical resection.

[0013] In some embodiments, the method further comprises measuring the level of ORF1p in the subject after administering the treatment, and comparing the level of ORF1p before the treatment with the level of ORF1p during and / or after the treatment, wherein a decrease in the level of ORF1p indicates that the treatment is effective in treating the cancer. The method can thus be used to monitor the efficacy of the treatment. If the treatment is effective, the method can include continuing the treatment. If the treatment is ineffective (e.g., the level of ORF1p does not decrease or increase), the method can include selecting and optionally administering a different treatment.

[0014] Also provided herein are single domain antibodies or antigen-binding fragments thereof that bind to human ORF1p, as described herein, e.g., comprising a sequence that is at least 90%, 95%, 97% or 99% identical to a Nanobody sequence as shown in Table B, or CDR1, CDR2 and CDR3 thereof, or comprising CDR1, CDR2 and CDR3 as shown in Table B, and multimers thereof.

[0015] Additionally provided herein are fusion constructs comprising at least two (e.g., three, four or five) single domain antibodies or antigen-binding fragments thereof as described herein, optionally with a linker therebetween, optionally as shown in Table C. Exemplary Nanobody concatemers include MT1032, MT1033, MT1034, MT1035, MT1036, MT1037, MT1038, MT1039 and MT1040 (see Figure 22A).

[0016] Additionally, provided herein are antibodies or antigen-binding portions thereof that specifically bind to human ORF1p, as described herein, e.g., wherein the antibody or antigen-binding portion thereof comprises at least one of: a heavy chain variable region (VH) comprising a heavy chain variable region (VH) that is identical to that in Table D or Figure 20A - A VH sequence that is at least 95% identical to the sequence shown in Table D or J, or its CDR1, CDR2 and CDR3, or consists thereof; and / or a light chain variable region (VL) comprising a VH sequence that is at least 95% identical to the sequence shown in Table D or J, or its CDR1, CDR2 and CDR3, or consists thereof; Figure 20A - or consists of a VL sequence or CDR1, CDR2 and CDR3 thereof that is at least 95% identical to the sequence shown in J, preferably wherein the VH and VL or CDRs are from the same antibody.

[0017] In some embodiments, the antibody comprises a constant region, optionally as shown in Table A.

[0018] In some embodiments, a single domain antibody or antigen binding fragment thereof, a fusion protein, or an antibody or antigen binding portion thereof is fused to a tag (e.g., an oligonucleotide, peptide, chemiluminescent, fluorescent, radioactive, or colorimetric label). In some embodiments, the radiolabel is 125 I.

[0019] Also provided herein are nucleic acid molecules encoding the single domain antibodies, or antigen-binding fragments thereof, fusion constructs, or antibodies or antigen-binding portions thereof as described herein, as well as vectors comprising the nucleic acid molecules and, optionally, a promoter, and host cells comprising the nucleic acid molecules and optionally expressing the single domain antibodies, or antigen-binding fragments thereof, fusion constructs, or antibodies or antigen-binding portions thereof as described herein.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0021] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Mobilization of LINE-1 retrotransposons: (i) RNA pol II-mediated transcription of LINE-1 RNA; (ii) translation of open reading frame 1 protein (ORF1p) and ORF2p, and ribonucleoprotein assembly; (iii) insertion of LINE-1 cDNA into the genome via ORF2p-mediated target-triggered reverse transcription (TPRT) of LINE-1 RNA.

[0023] Figure 2 The signal-to-noise ratios of the indicated capture antibody pairs (x-axis) and detection antibody pairs (y-axis) were evaluated in samples spiked with recombinant protein (top) or breast cancer cell lysates (bottom). Darker colors indicate higher signal-to-noise ratios.

[0024] Figure 3 Representative calibration curves for several affinity reagent pairs (capture / detection).

[0025] Figure 4 . Antibody pair screening in breast cancer serum samples (8-fold dilution).

[0026] Figure 5 .Antibody pair screening in BioIVT serum samples (4-fold dilution).

[0027] Figure 6The Simoa assay measures plasma ORF1p levels in patients with high-grade serous ovarian cancer (HGSOC) compared to healthy controls. Two different capture and detection antibody combinations (Ab54 / Ab6 and C5 / Ab6) were used as mentioned above.

[0028] Figure 7A A, B, Plasma ORF1p levels in a preliminary pan-cancer pilot study measured by SIMOA; triplicate measurements were performed on each 25 μL volume using the Nb5 (clone 5) nanobody (capture) / Ab6 (detection). The pie chart above shows the percentage of detectable levels. Of the 400 "healthy" patients assayed, four tested positive; one was found to have prostate cancer, and information on the other patients was limited. Specificity is >99%. B, Pilot study in patients with high-grade serous ovarian cancer (HGSOC) and healthy patients (Penn cohort).

[0029] Figure 8 Schematic diagram of an exemplary ultrasensitive Simoa assay for detecting ORF1p in biological fluids.

[0030] Figure 9A-D. Digital ELISA based on femtoliter-size well arrays (11). (a, b) Single protein molecules are captured and labeled on beads using standard ELISA reagents (a), and the beads are then loaded into the femtoliter-size well array (b). (c) SEM image of a cross-section of the femtoliter-size well array after bead loading. (d) Fluorescence image of a cross-section of the femtoliter-size well array after a single enzyme generates a signal. Only a small fraction of the beads have enzymatic activity, indicating the presence of a single bound protein molecule.

[0031] Figure 10A-E. Improved detection of ORF1p using a second-generation assay. (A) Schematic diagram of the affinity reagents used. 34H7 and 62H2 are custom mAbs; Nb5-5LL is an engineered homodimeric nanobody. (B) We performed triplicate measurements of 25 μL of plasma from ovarian cancer patients (Penn cohort) using first- and second-generation assays; the affinity reagents used are labeled [Capture::Detection]. The second-generation assay includes a novel capture reagent (mAb 34H7 or 62H12) and a detection reagent (engineered dimeric nanobody Nb5-5LL). ORF1p was detected in four of five stage I patients in the cohort; assay #3 appears to have increased sensitivity but may have reduced specificity. (C) Triplicate second-generation assays were performed using 25 μL of plasma from the MGH cohort of advanced gastroesophageal and ovarian cancers. The ovarian cohort included 102 HGSOC and 30 other ovarian malignancies (mucinous, clear cell, and low-grade serous). 80-90% of ovarian cancers were detectable, with increased sensitivity for both cancer types. (D) Measurement results of the 34H7::Nb5-5LL second-generation assay in a multi-cancer cohort. (E) ROC curves for the single marker ORF1p in all healthy and ovarian cancer patients (top, n=128-132 cancer, 447-455 healthy), and a multivariate model for ovarian cancer (bottom, n=51-53 cancer, 50 healthy).

[0032] Figure 11A-C. ORF1p is an early predictor of response in 19 patients with gastroesophageal (GE) disease undergoing chemotherapy / chemoradiotherapy and is prognostic for GE and colorectal cancer (CRC). Responders and non-responders were retrospectively characterized by medical oncologists blinded to the results of post-treatment, pre-operative imaging. (A) Plasma ORF1p measured by all three second-generation Simoa assays before and during / after treatment; Left: Pre-treatment ORF1p was higher in non-responders than in responders (p = 0.02, t-test); Right: Pre-treatment and during / after treatment ORF1p categorized as responders and non-responders; p < 0.0001, Fisher's exact test. (B) Representative CT and PET-CT images of patients from this cohort. A representative non-responder had the second highest plasma ORF1p before treatment (25.8 pg / ml), which increased to 43.0 pg / ml on day 28 of FOLFOX treatment (47 days after diagnosis), with an increase in the size and number of liver metastases observed on CT on day 61. A representative responder had the fourth highest plasma ORF1p value in the responder cohort (0.83 pg / ml), which decreased to undetectable on day 26 of CROSS treatment (48 days after diagnosis); the PET-CT shown was 59 days after the start of treatment, 31 days after the second ORF1p measurement. (C) Kaplan-Meier survival analysis categorizing patients into plasma ORF1p high and ORF1p low based on the median plasma ORF1p assay showed significantly longer survival in patients with ORF1p low for GE (stages III-IV, p = 0.0017, log-rank test) and CRC (all stage IV, p = 0.011, log-rank test). The shaded area represents the 95% confidence interval.

[0033] Figure 12 A pilot large-volume second-generation assay was performed using the flow cytometry-based digital ELISA platform MOSAIC. A pilot cohort consisting of 10 healthy patients and 10 gastroesophageal (GE) cancer patients whose ORF1p levels were undetectable in a 25 μL assay volume (left panel) was assayed using 20-fold more plasma and read by flow cytometry, with results isolated from 9 of the 10 healthy subjects (right panel).

[0034] Figure 13 Simoa's novel rabbit monoclonal α-ORF1p antibody demonstrates a 5-fold improvement in signal-to-noise ratio (SNR) compared to our current best antibody, Abcam Ab6 (left). Two capture beads with different epitopes, nanobody C5 (Nb-5) and 4H1, were used.

[0035] Figure 14Nanobody / antibody pair screening was performed in plasma samples from healthy and cancer (colorectal and gastroesophageal) patients. The capture / detector pair is indicated in each panel. For comparison, measurements from a first-generation assay (Nb5 / Ab6) are depicted. All assays were performed using the three-step Simoa assay.

[0036] Figure 15 A newly developed monoclonal antibody (GenScript) and a commercially available monoclonal antibody were screened using dimeric nanobodies for detection of ORF1p using Simoa. Signal-to-noise comparison of affinity reagents used as capture / detector pairs on Simoa using recombinant ORF1p protein. All labeled affinity reagents were monoclonal antibodies, except for Nb5-5(LL), which represents the homodimeric form of the nanobody Nb5.

[0037] Figure 16 .Plasma screening round #3: Newly developed monoclonal antibody and dimeric nanobody reagent pairs were screened in the plasma of eight healthy patients and eight patients with cancer (colorectal or gastroesophageal).

[0038] Figure 17 Plasma screening round #4: Newly developed monoclonal antibody and dimeric nanobody reagent pairs were screened in the plasma of eight healthy patients and eight patients with cancer (colorectal or gastroesophageal).

[0039] Figure 18 Plasma screening round #5: Screening of newly developed monoclonal antibody and dimeric nanobody reagent pairs in patient plasma. Affinity reagent pairs selected from previous rounds of screening ( Figure 16-17 Each assay is represented by a capture / detection reagent pair. For comparison, the measurement results of the first generation assay (Nb5 / Ab6) are depicted.

[0040] Figure 19A -C, second generation assay 1, 34H7 / Nb5-5LL; B, second generation assay 2, 62H12 / Nb5-5LL; C, second generation assay 3, 62H12 / Ab6.

[0041] Figure 20A -J. Monoclonal antibody sequences shown in Table D.

[0042] Figure 21A-D. Improved detection of ORF1p using the third-generation Simoa assay and the MOSAIC assay. (A) Comparison of second- and third-generation Simoa assays (25 μL) in 25 patients with barely detectable gastroesophageal (GE) cancer and healthy controls. (B) Schematic diagram of the MOSAIC assay. The captured single-molecule "immunosandwich" is formed similarly to the Simoa assay. DNA-conjugated streptavidin allows rolling circle amplification, generating a strong localized fluorescent signal on the bead surface. "On" and "off" beads are then quantified by flow cytometry, allowing for efficient sampling of a large number of captured beads. This improves sensitivity and multiplexing capabilities. (C) 37H7::Nb5-5LL MOSAIC and Simoa assays in 10 patients with previously undetectable GE cancer and healthy controls. The dashed line in the left panel and the bottom dashed line in the right panel represent the analytical limit of detection (LoD) of recombinant ORF1p in buffer. The top dashed line in the right panel represents the plasma-specific background in the bulk MOSAIC assay and is used to determine positivity in the pie chart. (D) Similar results were observed in the breast cancer cohort.

[0043] Figure 22A-B Engineered nanobody constructs. (A) Schematic design of engineered dimeric and trimeric nanobody constructs with flexible (GGGGS×4) and rigid helical (EAAAK×3 or DAAAR×3) linkers. The 5xCys tag sequence is CGSGRCGSGRCGSGRCGSGRC. (B) Representative preparation of engineered nanobody constructs, stained with Coomassie Brilliant Blue.

[0044] Figure 23 Calibration curve for the "third generation" Simoa assay. Two "second generation" assays are compared, grey boxes. Dashed lines indicate assay detection limits.

[0045] Figure 24 .Second round of screening of newly developed "third generation" assays using dimeric nanobody detectors. Affinity reagent pairs selected from the first round of screening in plasma samples were screened in plasma samples from 25 healthy patients and 25 patients with GE cancer. Each assay is represented by a capture agent / detector reagent pair. A second generation assay was performed for comparison (upper left and lower left panels). The dotted line represents the assay detection limit, taking into account a four-fold dilution. The middle row of the chart represents the three third generation assays that were finally selected. MT1032 and MT1035 are Nb5-5 homodimers with different linkers. MT1036 is a Nb2-Nb9 heterodimer, MT1037 is a Nb5-Nb9 heterodimer, and MT1038 is a Nb9-Nb9 homodimer.

[0046] Figure 25 . Figure 5 Calibration curve for the large volume (500 μL) MOSAIC assay used in . The dashed line indicates the assay detection limit. DETAILED DESCRIPTION

[0047] Liquid biopsies are very popular because they are minimally invasive and can facilitate widespread screening. Many liquid biopsies currently available can detect circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), or microRNAs (miRNAs). However, key challenges remain: (1) Very low levels of ctDNA and CTCs in the early stages of the disease are often undetectable and require large amounts of blood. 3、 12. 13 (2) DNA mutations unrelated to malignant tumors will reduce the specificity of ctDNA testing 14、15 (3) Although miRNA has shown promise in detecting cancers such as ovarian cancer, further clinical validation is needed to determine whether miRNA signatures can be used for early detection. 4 .

[0048] Overexpression of LINE-1 retrotransposons is a hallmark of various human cancers.

[0049] Transposable elements constitute nearly half of the human genome and are receiving increasing attention due to their aberrant activity in human cancers. 7 In particular, expression of the retrotransposon long interspersed element-1 (LINE-1) has become a hallmark of various human malignancies and is associated with specific mutational signatures in cancer genomes. 17-20 LINE-1 is the only protein-coding transposable element still active in humans: its 6-kilobase sequence encodes two proteins essential for retrotransposition, open reading frame 1 protein (ORF1p) and ORF2p ( Figure 1 Although LINE-1 expression in somatic cells is usually suppressed by promoter methylation and histone modifications, LINE-1 promoter hypomethylation has been observed in many human malignancies. 7、17、21 Consistent with this observation, ORF1p is expressed in many tumors, especially in ovarian cancer. 8、10、22 and esophageal cancer 18、 43. 44 Importantly, ORF1p is a stable homotrimer and is highly expressed once it is derepressed. Taking ovarian cancer as an example, ORF1p expression is observed in more than 90% of high-grade serous ovarian cancer (HGSOC) cases, which is the most aggressive and lethal subtype of ovarian cancer. 8ORF1p is a particularly promising "binary" biomarker for ovarian cancer: while ORF1p is not expressed in normal fallopian tube epithelium, its expression is activated in serous tubal intraepithelial carcinoma (STIC) lesions, the early precursors of ovarian cancer. 9、10 , suggesting its potential use in early ovarian cancer detection ( Figure 2 A). However, the presence of ORF1p in blood remains a completely unexplored opportunity for liquid biopsy. Importantly, the absence of ORF1p expression in non-malignant tumor cells suggests that it could potentially serve as a "binary" blood-based biomarker with greater specificity than existing protein cancer biomarkers, which often have varying individual baseline levels and are expressed in normal tissues. 23-25 .

[0050] Although ORF1p expression is elevated in tumor tissue, ORF1p shed from tumors is diluted to very low levels in the bloodstream, far below the detection limit of conventional methods including mass spectrometry, thus requiring ultrasensitive detection. Ultrasensitive single molecule detection technology, single molecule array (SIMOA, Figure 2 D), and the recently reported detection of ORF1p in blood 26、48 .

[0051] Diagnostic methods

[0052] Included herein are methods for diagnosing cancer. The methods rely on detecting ORF1p in a biological fluid (e.g., whole blood, plasma or serum, feces, cervical fluid such as a Pap smear, uterine lavage fluid, urine or sputum) or a tissue sample (e.g., from a biopsy (e.g., a puncture, needle or scrape biopsy, or a surgical biopsy of suspected cancerous tissue), such as a tissue lysate), as described herein. In some embodiments, the disclosed methods provide a blood test for cancer detection and diagnosis using circulating ORF1p. In some embodiments, the cancer is a carcinoma, such as ovarian cancer, breast cancer, liver cancer, colon / colorectal cancer, lung cancer, esophageal cancer, prostate cancer, gastric cancer, head and neck cancer, brain cancer, soft tissue cancer, kidney cancer, gallbladder cancer, bile duct cancer (cholangiocarcinoma), bladder cancer, uterine cancer or pancreatic cancer. 8、22 In some embodiments, the cancer originates in the blood, bone marrow, brain, skin, or soft tissue, particularly lymphoma, leukemia, myeloma, glioblastoma, or melanoma. In some embodiments, the biological fluid is whole blood, plasma, or serum.

[0053] As used herein, the terms "cancer," "hyperproliferation," and "neoplasm" refer to cells that have the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states can be classified as pathological, i.e., characterizing or constituting a disease state, or as non-pathological, i.e., a deviation from normal but not associated with a disease state. The terms are intended to include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or stage of invasiveness. "Pathologically hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include cell proliferation associated with wound repair.

[0054] The term "cancer" or "tumor" includes malignancies of various organ systems, such as those affecting the lung, breast, thyroid, lymphoma, brain, soft tissue, gastrointestinal and genitourinary tracts, as well as adenocarcinomas, which include malignancies such as most colorectal cancers, kidney or renal cell carcinoma, gallbladder cancer, bile duct cancer (cholangiocarcinoma), bladder cancer, uterine cancer, prostate and / or testicular tumors, non-small cell lung cancer, small intestine cancer, and esophageal cancer.

[0055] The term "cancer" is recognized in the art and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory cancer, gastrointestinal cancer, genitourinary cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. In some embodiments, the disease is renal cancer or melanoma. Exemplary cancers include cancers formed by tissues of cervical cancer, lung cancer, prostate cancer, breast cancer, head and neck cancer, kidney cancer, gallbladder cancer, bile duct cancer (cholangiocarcinoma), bladder cancer, uterine cancer, colon cancer / colorectal cancer, and ovarian cancer. The term also includes carcinosarcoma, for example, which includes a malignant tumor composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to a cancer that is derived from glandular tissue or tumor cells forming a recognizable glandular structure.

[0056] In some embodiments, the cancer is a non-brain cancer.

[0057] The term "sarcoma" is recognized and refers to a malignant tumor of mesenchymal origin. In some embodiments, the cancer is not a sarcoma.

[0058] In some embodiments, the cancer is ovarian cancer, such as high-grade serous ovarian cancer. In some embodiments, the cancer is not breast cancer.

[0059] In some embodiments, the cancer originates in the blood, bone marrow, brain, skin, or soft tissue, particularly lymphoma, leukemia, myeloma, glioblastoma, or melanoma.

[0060] An exemplary sequence of human ORF1p is:

[0061] MGKKQNRKTGNSKTQSASPPPKERSSSPATEQSWMENDFDELREEGFRRSNYSELREDIQTKGKEVENFEKNLEECITRITNTEKCLKELMELKTKARELREECRSLRSRCDQLEERVSAMEDEMNEMKREGKFREKRIKRNEQSLQEIWDYVKRPNLRLIGVPESDVENGTKLENTLQDIIQENFPNLARQANVQIQEIQRTP QRYSSRRATPRHIIVRFTKVEMKEKMLRAAREKGRVTLKGKPIRLTADLSAETLQARREWGPIFNILKEKNFQPRISYPAKLSFISEGEIKYFIDKQMLRDFVTTRPALKELLKEALNMERNNRYQPLQNHAKM* (derived from the L1 insertion in the X-linked retinitis pigmentosa locus of Homo sapiens retrotransposon, complete sequence, GenBank: AF148856.1).

[0062] The method comprises obtaining a sample from a subject and assessing the presence and / or level of ORF1p in the sample. In some embodiments, the subject is a mammal, such as a human or a non-human veterinary subject, such as a cat, dog, cow, horse, goat, or non-human primate. In some embodiments, the subject is suspected of having cancer or being at risk of developing cancer, such as having one or more clinical symptoms associated with cancer, or having a family or personal history of cancer, a genetic or environmental risk factor for cancer, or an increased risk of developing cancer compared to a reference cohort of subjects.

[0063] As used herein, the term "sample," when referring to a material to be tested for the presence of ORF1p using the methods described herein, specifically includes biological fluids, such as whole blood, plasma, or serum. In some embodiments, the sample is or includes stool, cervical fluid (such as a Pap smear), uterine lavage fluid, urine, or sputum. The sample may also be a tissue sample, such as from a biopsy (e.g., a puncture, needle, or scrape biopsy, or a surgical biopsy); for example, a tissue lysate may be used. If desired, ORF1p protein may be identified and / or isolated and / or purified from the sample using various methods known in the art. An "isolated" or "purified" biomarker (such as ORF1p) is substantially free of cellular material or other contaminants from the cell or tissue from which the biomarker is derived, i.e., is partially or completely altered or removed from its natural state by human intervention. For example, the protein contained in the sample may be isolated according to standard methods, such as using a digestive enzyme, a chemical solution, or by separation using a protein-binding resin according to the manufacturer's instructions.

[0064] The method can include incubating a sample (e.g., 7.5 μl, 25 μl, 50 μl, 100 μl, 250 μl, 500 μl, 750 μl, 1 ml, 2 ml, 2.5 ml, or 10 ml of sample) with a capture reagent (e.g., an antibody, nanobody, or antigen-binding fragment thereof as described herein). In some embodiments, the sample is diluted, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:10, or 1:20, and any range therebetween, for example, 1:1 to 1:20, or 1:1 or 1:10, with the foregoing as endpoints. In some embodiments, the sample is diluted with a buffer; exemplary sample dilution buffers are described herein and can include a detergent, for example, Triton-X 100, Tween 20, NP-40, Brij35, Brij58, or C12E8, for example, in an amount of about 0.05%-2% of the sample. As used herein, "about" means plus or minus 10%.

[0065] In some embodiments, the sample is contacted with the capture reagent for a time sufficient to allow ORF1p present in the sample to bind to the capture reagent, for example, at least 5, 10, 15, 20, 30, or 45 minutes, or at least 1, 2, 3, 4, 5, or 6 hours, and up to 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours. In a preferred embodiment, the capture reagent is on beads, such as paramagnetic beads. The capture reagent bound to ORF1p is then incubated in the presence of a detection antibody (e.g., an antibody, nanobody, or antigen-binding fragment thereof as described herein), and the presence and / or amount of bound antibody is determined.

[0066] The presence and / or level of ORF1p protein can be assessed using methods known in the art. In preferred embodiments, the method comprises the use of highly sensitive or ultrasensitive and preferably multiplexed detection methods, including mesoscale discovery (MSD); single molecule array (SIMOA); droplet digital ELISA (ddELISA); 26 Molecular Signal Amplification on Beads Individual Counting (MOSAIC), 30 Single molecule counting (SMC); nucleic acid-linked immunosorbent assay (NULISA); Spear Bio's NAB-SURE (a cell-free assay that quantifies neutralizing antibodies (NAb) using a real-time PCR system); LUMINEX (an immunoassay that accurately measures multiple analytes in one sample); SOMAscan assay; mass spectrometry (e.g., MALDI-MS) and mass cytometry (e.g., CyTOF) (see, e.g., Cohen and Walt, Chem. Rev. 2019, 119, 293-321).

[0067] In some embodiments, the SIMOA or MOSAIC assay is used to measure ORF1p protein in blood for cancer detection (11, 30, 39). The SIMOA assay has several advantages over conventional ELISA, the current gold standard for protein detection in blood. First, SIMOA is 1000 times more sensitive than ELISA and allows for quantification of low concentrations of analyte (11). SIMOA can detect as low as 10 -19 M protein concentration, while conventional ELISA can only detect 10 -12 Second, because of the high sensitivity of SIMOA, serum samples can be diluted more dilutely, thereby reducing nonspecific binding caused by matrix effects (40, 41). Third, SIMOA has a wide dynamic range, spanning four orders of magnitude in concentration, so that both low-abundance and high-abundance markers can be detected using a single assay (42). In some embodiments, SIMOA technology achieves this high sensitivity by labeling each immune complex and physically isolating it into femtoliter-sized wells, digitally counting the number of molecules in the sample ( Figure 8 , 9A-D). These advantages offer the potential for detecting and quantifying blood biomarkers such as ORF1p, allowing for the development of robust assays.

[0068] In preferred embodiments, an ELISA method is used, such as SIMOA, MOSAIC, or other ultrasensitive methods; in preferred embodiments, the capture antibody is Ab54 (ab246320, AbCam) or Nb5, which are further described herein. In some embodiments, the detection antibody is Ab6 (ab246317, AbCam). In some embodiments, the capture / detection pair is a pair described herein, such as Table 2 or 34H7 / Ab6, 62H12 / Ab6, 34H7 / Nb5-5LL, 62H12 / Nb5-5LL, 4H1 / Nb5-5, or 4H1 / Nb5-5LL.

[0069] In some embodiments, mass spectrometry, particularly matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) and surface-enhanced laser desorption / ionization mass spectrometry (SELDI-MS), is used to detect biomarkers (see U.S. Patent Nos. 5,118,937; 5,045,694; 5,719,060; 6,225,047).

[0070] In some embodiments, other methods may be used, such as standard electrophoresis and quantitative immunoassay methods for ORF1p protein, including but not limited to Western blot; enzyme-linked immunosorbent assay (ELISA); enzyme-linked immunospot (ELISPOT); biotin / avidin-type assays; protein array detection, such as protein microarrays; radioimmunoassay; immunohistochemistry (IHC); immunoprecipitation assays; flow cytometry / FACS (fluorescence-activated cell sorting); proximity ligation assay (PLA); lateral flow assay; surface plasmon resonance (SPR); optical imaging; Spear Bio's NAB-SURE (a cell-free assay that quantifies neutralizing antibodies (NAbs) using a real-time PCR system); and mass spectrometry (Kim (2010) Am J Clin Pathol 134:157-162; Yasun (2012) Anal Chem 84(14):6008-6015; Brody (2010) Expert Rev Mol Diagn 10(8):1013-1022;Philips(2014)PLOS One9(3):e90226;Pfaffe(2011)ClinChem 57(5):675-687;Cohen and Walt,Chem.Rev.2019,119,293-321). The method generally includes revealing a label that directly or indirectly provides a signal, such as fluorescence, chemiluminescence, radioactivity and enzymatic or dye molecules, or oligonucleotide labels that can be used for example MOSAIC or other digital ELISA platforms, immuno-PCR. As used herein, the term "label" refers to coupling (i.e., physically linking) a detectable substance (such as a radioactive agent or fluorophore (e.g., phycoerythrin (PE) or indocyanine (Cy5))) to an antibody or probe, as well as indirectly labeling a probe or antibody (e.g., horseradish peroxidase, HRP) by reactivity with a detectable substance.

[0071] The method can also include comparing the presence and / or level to one or more references, such as a control reference representing normal levels of ORF1p (e.g., levels in unaffected subjects) and / or a disease reference representing levels of the protein associated with cancer (e.g., levels in subjects with cancer). Suitable reference values ​​can include undetectable levels of ORF1p or levels below, for example, 0.01, 0.005, or 0.001 pg / mL for subjects without cancer.

[0072] In some embodiments, if the presence and / or level of ORF1p is comparable to the presence and / or level of ORF1p in a disease reference, and the subject has one or more symptoms associated with cancer, then the subject has cancer. In some embodiments, if the subject has no obvious signs or symptoms of cancer, but the presence and / or level of one or more proteins being assessed is comparable to the presence and / or level of proteins in a disease reference, then the subject has cancer or is at increased risk of having cancer. In some embodiments, once a person is determined to have cancer or to be at increased risk of having cancer, the subject can be selected or identified for further evaluation, such as using other blood-based diagnostics (e.g., a biomarker panel), imaging, or biopsy to identify a tumor or cancer, and / or a treatment, such as known in the art or as described herein, can be selected and / or administered.

[0073] Suitable reference values ​​can be determined using methods known in the art, such as using standard clinical trial methods and statistical analysis. The reference value can have any relevant form. In some cases, the reference value includes a predetermined value of a meaningful ORF1p level, such as a control reference level representing a normal ORF1p level, such as the level of an unaffected subject or a subject not at risk of developing a disease described herein, and / or a disease reference level representing an ORF1p level associated with cancer, such as the level of a subject suffering from cancer.

[0074] The predetermined level can be a single cutoff value (threshold value), such as median or mean value, or the level of the boundary above or below other sections of definition quartiles, tertiles or clinical trial colony, and the section is determined to be statistically different from other sections. It can be a series of cutoff values ​​(or threshold values), such as confidence intervals. It can be determined according to a comparison group, such as, in a limited group, the correlation of the risk of illness or disease existence is higher or lower several times (for example, about 2 times, 4 times, 8 times, 16 times or more) than the correlation of the risk of illness or disease existence in another limited group. It can be a scope, for example, a group of subjects (for example, control subjects) are equally (or unequally) divided into groups, such as low-risk group, medium-risk group and high-risk group, or divided into quartiles, the lowest quartile is the subject with the lowest risk, and the highest quartile is the subject with the highest risk, or divided into n quartiles (that is, n regularly spaced intervals), the lowest quartile in the n quartiles is the subject with the lowest risk, and the highest quartile in the n quartiles is the subject with the highest risk.

[0075] In some embodiments, the predetermined level is the level or occurrence in the same subject, eg, at a different time point, eg, an earlier time point.

[0076] A subject associated with a predetermined value is often referred to as a reference subject. For example, in some embodiments, a control reference subject does not have cancer, is not at risk of developing cancer, or will not later develop cancer.

[0077] A disease reference subject is a subject having cancer (or having an increased risk of developing cancer). Increased risk is defined as being higher than the risk for subjects in the general population.

[0078] In some embodiments, a subject's ORF1p level greater than or equal to an ORF1p reference level indicates the presence of cancer or a risk of developing cancer, while a subject's ORF1p level less than or equal to the ORF1p reference level indicates the absence of disease or a normal risk of disease.

[0079] Thus, in some embodiments, to assess whether a subject clinically has cancer, the method can include first performing a logarithmic transformation on the ORF1p value and then assigning a predicted probability, such as using a logistic regression model, to generate a probability score. If the predicted probability score for the subject is above a selected threshold (e.g., at least 50%), the subject is predicted to have cancer (e.g., assigned to a certain cancer category). If the predicted probability score is below a selected threshold (e.g., 50%), the subject is predicted to be healthy (e.g., assigned to a healthy category).

[0080] In some embodiments, the level of ORF1p is used to calculate a score, for example, together with one or more additional variables (e.g., age). The score can be calculated using an algorithm such as summing or weighted summing the (normalized) levels of the variables. Specific algorithms can be identified using known statistical methods, including PCA, linear regression, SVM (support vector machine), decision tree, KNN (K nearest neighbor), K means, gradient boosting, or random forest methods.

[0081] For example, in some embodiments, the exemplary model uses logistic regression analysis, where each variable (X) is assigned a weight (B). In the exemplary equation below, a weight (B) is calculated for each marker, and each biomarker can have a unique B value.

[0082]

[0083] In the clinic, the probability score of a patient having or will have cancer can be obtained by substituting the measured ORF1 p value (X value) into the equation and then calculating the probability value (P). In some embodiments, the clinical procedure for obtaining the probability of an individual having cancer is as follows:

[0084] First, a blood draw is performed on the person being screened. Second, the ORF1p protein concentration in the person's blood is measured, for example, using Simoa. Third, the predicted probability of the person developing cancer is calculated based on a logistic regression formula, where the dependent variable is the natural logarithm of [(probability of developing cancer) / (probability of not developing cancer)] and the independent variables are age and ORF1p. The predicted probability can inform discussions between the person being screened and their physician to determine how best to proceed, such as deciding whether further follow-up or confirmatory radiological imaging is necessary.

[0085] In some embodiments, the subject's level (or score) is lower than the reference level (or score) by an amount sufficient to distinguish the subject from a control subject, and is optionally statistically significantly lower than the control subject's level (or score). When a subject's level (or score) of a biomarker is equal to a reference level (or score) of that biomarker, "equal" means approximately equal (e.g., not statistically different).

[0086] The predetermined value can depend on the selected specific subject (e.g., human subject) population. For example, a seemingly healthy population may have a different "normal" level range of a biomarker than a population that suffers from, may suffer from, or has a higher risk of suffering from a disease described herein. Therefore, the selected predetermined value can take into account the category (e.g., sex, age, health status, risk, the presence of other diseases) to which the subject (e.g., human subject) belongs. Those of ordinary skill in the art can select appropriate ranges and categories only through routine experiments.

[0087] In characterizing likelihood or risk, a number of predetermined values ​​may be established.

[0088] In some embodiments, multiple assays are performed using different combinations of antibodies as described herein, eg, to increase sensitivity and / or specificity.

[0089] Nanobodies

[0090] Nanobodies (also known as VHH antibodies) and their antigen binding domains are described herein. In one aspect, the antibodies provided herein comprise an antigen binding site in a single polypeptide. Therefore, the antibodies are referred to herein as "single domain antibodies". Single domain antibodies are also referred to as nanobodies. However, in certain embodiments, the single antibodies disclosed herein can be bispecific or multispecific single domain antibodies as described elsewhere herein, in which two single domain antibodies are coupled.

[0091] Single-domain antibodies are antibody fragments composed of a single monomeric variable antibody domain. Like whole antibodies, they are able to selectively bind to specific antigens. The molecular weight of single-domain antibodies is typically in the range of 12-15 kDa, which is much lower than that of ordinary antibodies, which typically have a molecular weight in the range of 150 to 160 kDa. Single-domain antibodies are also smaller than the Fab fragment (approximately 50 kDa) of heterotetrameric antibodies, which are composed of one light chain and half a heavy chain.

[0092] In some embodiments, the antibodies used in the methods of the invention are single domain antibodies, preferably derived from camelid antibodies, preferably camel antibodies, including functional homologues, fragments thereof and fusion macromolecules comprising a VHH domain covalently linked to a glycan, nucleic acid, protein or non-macromolecular chemical group.

[0093] The single domain VHH antibodies described herein preferably comprise one or more CDRs in Table B, such as SEQ ID NO: 1. Specifically, CDRs can identify the specificity of the antibody, and therefore preferably the antigen binding site comprises one or more CDRs, preferably at least 1, more preferably at least 2, and even more preferably 3 or more CDRs. In one embodiment, the single domain antibody comprises 1 CDR. In one embodiment, the single domain antibody comprises 2 CDRs. In a preferred embodiment, the single domain antibody comprises 3 CDRs and four framework regions. Methods for CDR exchange in VHH antibodies are known, see, for example, Zupancic et al., Cell Chem Biol. 2021 Sep 16; 28(9): 1379-1388.e7; Saerens et al., J Mol Biol. 2005 Sep 23; 352(3): 597-607; Muyldermans et al., FEBS J. 2021 Apr; 288(7): 2084–2102.

[0094] In some embodiments, the Nanobody comprises the VHH sequence QVQLVESGGDLVQAGGSLRLSCAVSGGTSSNYGMGWFRQAPGK EREFVSSISWSGSRTLYSDSVKGRFTISRDNAKNTVDLQMNSLKPEDTAVYYCTAVREYRDYPQRDNFDYWGQGTQVTVS (SEQ ID NO: 1). The bold sequences represent CDR1 (GGTSSNYG, SEQ ID NO: 2), CDR2 (ISWSGSRT, SEQ ID NO: 3) and CDR3 (TAVREYRDYPQRDNFDY, SEQ ID NO: 4) (identified based on IMGT numbering).

[0095] In preferred embodiments, the Nanobody comprises a sequence that is at least 90%, 95%, 97%, 99% or 100% identical to SEQ ID NO: 1. In some embodiments, any mutations or substitutions are located in the framework regions (and not in the CDRs) and do not significantly affect binding to the target antigen (ORF1p).

[0096] In some embodiments, concatemers of nanobody sequences are used, for example, wherein 2, 3, 4, 5 or more C5 nanobody fusions are present, optionally with intervening linkers. Exemplary nanobody concatemers include MT1032, MT1033, MT1034, MT1035, MT1036, MT1037, MT1038, MT1039 and MT1040 (see Figure 22A). A variety of suitable linkers are known to those skilled in the art and are not limited to any particular sequence disclosed herein. In some embodiments, the polypeptide linker is composed of naturally occurring or non-naturally occurring amino acids. In some embodiments, the linker comprises an amino acid that allows flexibility. In some embodiments, the linker comprises an amino acid that allows suitable solubility. In some embodiments, the linker comprises a glycine amino acid. In some embodiments, the linker comprises a glycine and a serine amino acid. In certain embodiments, the linker comprises one or more glycine / serine repeat sequence sets. In some embodiments, the polypeptide linker is selected from the group consisting of (GGGGS)n (where n=1-4) (SEQ ID NO:5), GGGGS (SEQ ID NO:6), GGGGSGGGGS (SEQ ID NO:7), GGGGSGGGGS GGGGS (SEQ ID NO:8), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:9, and (GGGGA)n (where n=1-4) (SEQ ID NO:20), or a rigid helical linker, such as (EAAAK)n or (DAAAR)n, where n=1-4, preferably n=3, SEQ ID NO:11 and SEQ ID NO:12, respectively). In some embodiments, the linker comprises GGGGSGGGGSGGGGS (SEQ ID NO:8). In some embodiments, the linker is preferably 5-100, 5-80, or 10-80 amino acids in length and comprises GGGG SGGGGSGGGGSGGGGS (SEQ ID NO: 9) or GGGGSGGGGSGGGG SGGGGSEAAAKEAAAKEAAAKSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 13).

[0097] Anti-ORF1p monoclonal antibody

[0098] Described herein are monoclonal antibodies 62H12, 64C6, 33A8, 61A11, 36D12, 34H7, 50E9, 34C5, 55A6, and 42D10, and derivatives and antigen-binding fragments thereof. The term "antibody" refers to an immunoglobulin molecule or an immunologically active portion thereof, i.e., an antigen-binding portion (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence). In preferred embodiments, the antibody comprises a sequence that is at least 90%, 95%, 97%, 99%, or 100% identical to the sequence set forth herein. In some embodiments, any mutation or substitution is in the framework region (rather than in the CDRs) and does not significantly affect binding to the target antigen (ORF1p).

[0099] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in trace amounts. The antibody may be a monoclonal antibody. The antibody may be a human antibody or a humanized antibody. The term "monoclonal antibody" encompasses complete and full-length monoclonal antibodies and antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies (e.g., scFv), fusion proteins comprising antibody fragments, and any other modified immunoglobulin molecules comprising at least one antigen binding site. In addition, "monoclonal antibody" refers to antibodies made by a variety of techniques including, but not limited to, hybridoma production, phage library display, recombinant expression, and transgenic animals.

[0100] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a first source or species, while the remaining heavy and / or light chain is derived from a different source or species.

[0101] As used herein, the term "humanized antibody" refers to an antibody comprising a human heavy chain variable region and a light chain variable region, wherein the native CDR residues are replaced by residues of the corresponding CDRs from a non-human antibody (e.g., mouse, rat, rabbit, or non-human primate), wherein the non-human antibody has the desired specificity, affinity, and / or activity. In some embodiments, one or more framework region residues of a human heavy chain or light chain variable region are replaced by corresponding residues from a non-human antibody. In addition, the humanized antibody may be included in residues not seen in human antibodies or in non-human antibodies. In some embodiments, these modifications are in order to further improve and / or optimize antibody properties. In some embodiments, the humanized antibody comprises at least a portion of an immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc), typically at least a portion of an immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc) of a human immunoglobulin.

[0102] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human and / or an antibody prepared using any technique known to those skilled in the art for preparing human antibodies. Such techniques include, but are not limited to, phage display libraries, yeast display libraries, transgenic animals, recombinant protein production, and B cell hybridoma technology.

[0103] "Antibody fragments" may include a portion of an intact antibody, preferably the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, enabling the scFv to form the structure required for antigen binding.

[0104] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to the portion of an antigen or target that can be recognized and bound by a specific antibody. When the antigen or target is a polypeptide, the epitope can be formed by continuous amino acids and non-continuous amino acids juxtaposed by the tertiary folding of the protein. The epitope formed by continuous amino acids (also referred to as a linear epitope) is typically retained after protein denaturation, while the epitope formed by the tertiary folding (also referred to as a conformational epitope) is typically lost after protein denaturation. An epitope typically comprises at least 3 amino acids, more typically at least 5, 6, 7 or 8-10 amino acids, in a unique spatial conformation. Epitopes can be predicted using any of the large amount of software bioinformatics tools available on the internet. X-ray crystallography can be used to characterize epitopes on a target protein by analyzing the interactions of the amino acid residues of the antigen / antibody complex.

[0105] "Fv" encompasses the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. It is this interaction of the three CDRs from each variable domain that defines the antigen-binding site on the surface of the VH-VL dimer. Together, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the complete binding site. Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of several residues to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab' fragments in which one or more cysteine ​​residues of the constant domains have free thiol groups are referred to herein as Fab'-SH. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them.Other chemical conjugates of antibody fragments are also known.

[0106] Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant domain. There are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgB1, IgG2, IgG3, IgG4, IgA, and IgA2. "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the required structure for antigen binding.

[0107] In various embodiments, the antibody or its antigen-binding fragment comprises people or humanized antibodies.The humanized form of non-human (for example, mouse) antibody is chimeric immunoglobulin, immunoglobulin chain or its fragment (such as Fv, Fab, Fab ', F (ab ') 2 or other antigen-binding subsequences of antibody), which contains the minimum sequence derived from non-human immunoglobulin.Humanized antibody comprises such human immunoglobulin (acceptor antibody), wherein the residue from the complementary determining region (CDR) of the acceptor is replaced by the residue from the CDR of the non-human species (donor antibody) such as mouse, rat or rabbit with required specificity, affinity and capacity.In some cases, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.Humanized antibody can also be included in the residue that does not exist in the CDR or framework sequence of acceptor antibody or import. Typically, the humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Methods for humanizing non-human antibodies are well known in the art.

[0108] The ORF1p antibodies described herein can be affinity matured, for example, using selection and / or mutagenesis methods known in the art. Typically, an "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions that result in an increase in the affinity of the antibody for the antigen compared to a parent antibody that does not have the one or more alterations. In one embodiment, the affinity matured antibody has a nanomolar or even picomolar affinity for the target antigen. Preferred affinity matured antibodies have an affinity that is 5 times, more preferably 10 times, even more preferably 20 or 30 times higher than the starting antibody (typically murine, humanized, or human) from which the mature antibody was prepared.

[0109] An antibody that "binds to," "specifically binds to," or "is specific for" a particular polypeptide or an epitope on a particular polypeptide is an antibody that binds to a particular polypeptide or an epitope on a particular polypeptide and does not substantially bind to any other polypeptide or polypeptide epitope. As used herein, the term "specifically binds" refers to an interaction of an ORF1p agent (e.g., an anti-ORF1p antibody) with a particular antigen, epitope, protein, or target molecule that is more frequent, more rapid, longer lasting, greater in affinity, or some combination thereof than with an alternative substance. In some cases, an ORF1p antibody may or may not cross-react with an ORF1p-related protein (e.g., having the highest affinity for one ORF1p-related protein, such as human ORF1p, and a lower affinity for other ORF1p-related proteins, such as ORF2p).

[0110] In some embodiments, the antibody VH and VL domains described herein are fused to constant regions, e.g., as shown in Table A.

[0111] Table A. Sequences of exemplary constant regions

[0112]

[0113]

[0114] Identity

[0115] The term "identical" or "identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acid residues when comparing and aligning (if necessary, introducing spaces) to obtain maximum correspondence, without considering any conservative amino acid substitutions as part of sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain the alignment of amino acid or nucleotide sequences are well known in the art. These include but are not limited to BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package and their modifications. In some embodiments, two nucleic acids of the present disclosure or polypeptides are substantially identical, which means that when comparing and aligning to obtain maximum correspondence, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid identity, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a region of sequence that is at least about 10, at least about 20, at least about 20-40, at least about 40-60, at least about 60-80 nucleotides or amino acids in length, or any integer value therebetween. In some embodiments, the identity exists over a region longer than 60-80 nucleotides or amino acids, e.g., at least about 80-100 nucleotides or amino acids, and in some embodiments, the sequences are substantially identical over the entire length of the compared sequences (e.g., (i) the coding region of a nucleotide sequence or (ii) an amino acid sequence).

[0116] As used herein, the phrase "conservative amino acid substitution" refers to a substitution in which one amino acid residue is replaced by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, replacing valine with alanine is considered to be a conservative substitution. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate binding are well known in the art.

[0117] In some embodiments, a nanobody or antibody can be conjugated to another protein or peptide, for example to form a multifunctional protein / peptide. Exemplary conjugates can include an Fc fragment. See, for example, Bao et al., EJNMM I Res. 2021; 11: 6; Hoey et al., Exp Biol Med (Maywood). 2019 Dec; 244(17): 1568–1576; Bever et al., Anal Bioanal Chem. 2016 Sep; 408(22): 5985–6002.

[0118] In some embodiments, the nanobody or antibody may be conjugated to or comprise a tag, such as a label (detectable moiety) or a purification moiety, such as FLAG, hexahistidine (6-HIS) or hemagglutinin (HA). Examples of detectable substances used as labels include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive materials include 125 I. 131 I. 35 S or 3 H.

[0119] Also provided herein are kits and compositions comprising Nanobodies and / or antibodies as described herein, and nucleic acids encoding said Nanobodies and / or antibodies, vectors comprising the nucleic acids (e.g. viral vectors or plasmids, preferably comprising regulatory sequences such as promoters to drive expression of the Nanobodies and / or antibodies), and host cells (e.g. bacterial cells, yeast cells, insect cells or mammalian cells) comprising the nucleic acids and optionally expressing the Nanobodies and / or antibodies.

[0120] Methods for treating, screening, and monitoring treatment efficacy

[0121] As shown herein, plasma ORF1p levels determined at diagnosis can predict overall survival rates for cancers (including colorectal and gastroesophageal cancers) and can be used to monitor treatment responses over time. This application can stratify patients into high-risk and low-risk groups to receive additional treatments, such as chemotherapy, more aggressive chemotherapy, or additional surgery, particularly in colorectal, breast, or prostate cancers for which there are multiple treatments. Thus, the methods described herein include methods for treating cancer. In general, the methods comprise selecting and optionally administering a therapeutically effective amount of a cancer treatment to a subject who has been determined to be in need of such treatment by the methods described herein. Treatment of cancer depends on the type of cancer and may include radiation, surgical resection, chemotherapy, hormone / endocrine therapy, and / or immunotherapy. In some embodiments, the cancer is a carcinoma, such as ovarian cancer, breast cancer, liver cancer, colon / colorectal cancer, lung cancer, esophageal cancer, prostate cancer, gastric cancer, head and neck cancer, brain cancer, soft tissue cancer, kidney cancer, gallbladder cancer, bile duct cancer (cholangiocarcinoma), bladder cancer, uterine cancer, or pancreatic cancer. In some embodiments, the cancer originates in the blood, bone marrow, brain, skin, or soft tissue, particularly lymphoma, leukemia, myeloma, glioblastoma, or melanoma.

[0122] In some embodiments, when a subject is identified as possibly having ovarian cancer, the subject undergoes surgical resection and optionally chemotherapy and / or immunotherapy. Chemotherapy may include, for example, paclitaxel and carboplatin, docetaxel and carboplatin, or carboplatin and pegylated liposomal doxorubicin, gemcitabine, topotecan, etoposide, and / or bevacizumab; a PARP inhibitor, such as olaparib; or hormonal therapy, such as tamoxifen or letrozole.

[0123] The method can also include sending the subject for additional screening, such as referral for additional testing, e.g., transvaginal ultrasound, uterine lavage, or falloposcopy, based on the updated posterior probability of having ovarian cancer (optionally combining the ORF1p result with other clinical characteristics and potentially other biomarkers (e.g., CA125 and / or HE4 for ovarian cancer)).

[0124] The methods can also be used to monitor response to treatment, such as radiation, surgical resection, chemotherapy, hormone / endocrine therapy, and / or immunotherapy. The methods can include determining a baseline level of ORF1p in a subject using the methods described herein; administering treatment, such as one or more doses of treatment, and determining subsequent levels of ORF1p in the subject, such as during treatment (obtained while treatment is ongoing) and / or after treatment (obtained after treatment is completed). A decrease in the subject's ORF1p level from baseline to a subsequent level indicates that the subject is responding to or has responded to the treatment. The methods can also be used to monitor subjects in remission to determine whether the subject is still in remission (e.g., ORF1p level is equal to or below a threshold, such as a detected level in a sample, or a level in a subject who does not have cancer). If the treatment is effective, the methods can include continuing the treatment. If the treatment is ineffective (e.g., ORF1p level does not decrease or increase), the methods can include selecting and optionally administering a different treatment.

[0125] Kits and assay reagents

[0126] Also provided herein are kits and assay reagents comprising nanobodies and antibodies as described herein (including antigen-binding fragments thereof). In some embodiments, the kit or reagent comprises a solid surface to which the nanobody or antibody is coupled. In some embodiments, the surface is a bead, such as a paramagnetic bead or a polymer bead.

[0127] Example

[0128] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0129] Materials and methods

[0130] The following materials and methods were used in the following examples.

[0131] Materials. All affinity reagents used in this study are listed in Table E. Conjugation reagents, paramagnetic beads, and assay buffer were obtained from Quanterix Corporation. DNA oligonucleotides used in the MOSAIC assay were obtained from Integrated DNA Technologies. The antibodies used in the final Simoa and MOSAIC assays (monoclonal Ab6, Ab54, 62H12, 34H7) were also validated by Western blotting.

[0132] Table E. Affinity reagents used in all screening experiments.

[0133]

[0134]

[0135] Preparation of capture and detector reagents. All capture antibodies and nanobodies were obtained in or dialyzed into phosphate-buffered saline (PBS). For the first-generation Simoa assay, 7 × 10 beads were first washed with 400 μL of bead wash buffer (Quanterix Corp.). 8 Each carboxylated paramagnetic 2.7-μm bead (Homebrew Singleplex Beads, Quanterix Corp.) was washed three times and then washed twice with 400 μL of cold bead conjugation buffer (Quanterix Corp.) before being resuspended in 390 μL of cold bead conjugation buffer. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Thermo Fisher Scientific) in a 1 mg vial was then dissolved in cold bead conjugation buffer to 10 mg / mL, and 10 μL was added to the beads. The beads were shaken at 4°C for 30 minutes to activate the carboxyl groups on the beads, then washed once with 400 μL of cold bead conjugation buffer and resuspended in the capture nanobody solution (a total of 10 μg nanobody) and diluted to a final volume of 400 μL in bead conjugation buffer. The beads were shaken at 4°C for two hours, washed twice with 400 μL of bead wash buffer, and resuspended in 400 μL of bead blocking buffer (Quanterix Corp.), then shaken at room temperature for 30 minutes to block the beads. After washing once with 400 μL of bead wash buffer and bead diluent (Quanterix Corp.), the beads were resuspended in bead diluent and stored at 4°C. Before use in the assay, the beads were counted using a BeckmanCounter Z series particle counter. For the second-generation Simoa assay, the following bead coupling conditions were used: 4.2 × 10 8 Starting beads, 300 μL wash volume, 6 μL EDC and 40 μg antibody.

[0136] For the biotinylation of the detection agent antibody or nanobody, the Sulfo-NHS-LC-LC-biotin in the 1 mg vial was dissolved in 150 μL of water and added to the 1 mg / mL antibody or nanobody solution with an 80-fold molar excess. The reaction mixture was incubated at room temperature for 30 minutes and then purified with an Amicon Ultra-0.5 mL centrifugal filter (the cutoff values ​​for antibodies and dimeric nanobodies were 50K and 10K, respectively). Five 14,000 x g centrifugation cycles were performed for five minutes each, with 450 μL of PBS added to each cycle. The purified biotinylated detection agent reagent was recovered by inverting the filter into a new tube and centrifuging at 1000 x g for two minutes. The concentration was quantified using a NanoDrop spectrophotometer.

[0137] Recombinant ORF1p protein production. ORF1p was prepared as described (25); briefly, codon-optimized human ORF1p with an N-terminal His6-TEV corresponding to L1RP (L1 insertion in the X-linked retinitis pigmentosa locus, GenBank AF148856.1) was expressed in Escherichia coli (E. coli), purified by Ni-NTA affinity, eluted, the tag cleaved in the presence of RNase A, and refined by size exclusion in a buffer containing 50 mM HEPES pH 7.8, 500 mM NaCl, 10 mM MgCl2, and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP) to obtain monodisperse trimeric ORF1p carrying an N-terminal glycine scar.

[0138] Production and screening of nanobodies. Nanobodies were produced essentially as described in (49, 52) using mass spectrometry / lymphocyte cDNA sequencing to identify antigen-specific nanobody candidates. Briefly, camels were immunized with monodisperse ORF1p, and serum and bone marrow were separated. Only the heavy chain IgG fraction (VHH) was isolated from the serum and bound to an immobilized ORF1p column. The bound protein was eluted in SDS and sequenced by mass spectrometry using a library derived from sequencing of VHH fragments amplified by PCR from bone marrow-derived plasma cells. The candidate sequence was cloned into an E. coli expression vector with a C-terminal His6 tag and expressed overnight in 50 ml culture in E. coli Arctic Express RP (Agilent) at 12°C with 0.2 mM IPTG induction. Periplasmic extracts were generated by resuspending the pellet in 10 ml / L culture TES buffer (200 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, and 500 mM sucrose), adding 20 ml / L hypotonic lysis buffer (TES buffer diluted 1:4 with ddH2O), supplemented with 1 mM PMSF and 3 μg / ml pepstatin A, incubating at 4°C for 45 min, and centrifuging at 25,000 x g for 30 min. The supernatant (periplasmic extract) was bound to ORF1p-conjugated sepharose, washed three times, eluted with SDS at 70°C for 10 min, and the periplasmic extract and eluate were analyzed by SDS-PAGE to determine expression and yield. ORF1p binding candidates were purified as follows and analyzed by ELISA.

[0139] Nanobody and multimeric nanobody purification. C-terminal His6-tagged nanobody constructs were expressed and purified essentially as described in (49). Briefly, proteins were expressed overnight in E. coli ArcticExpress RP (Agilent) at 12°C using 0.2 mM IPTG induction. Periplasmic extracts (generated as above) were supplemented with 5 mM MgCl2, 500 mM NaCl, and 20 mM imidazole, purified by Ni-NTA chromatography, dialyzed into 150 mM NaCl, 10 mM HEPES, pH 7.4, and concentrated to 1-3 mg / ml by ultrafiltration. The "5xCys tail" construct was purified by resuspension, washing, elution, and dialysis buffer with the addition of 5 mM TCEP-HCl.

[0140] Surface plasmon resonance (SPR) assays. The binding kinetics (k) of antibodies and nanobody constructs to ORF1p were obtained on a Biacore 8K instrument (Cytiva). a 、k d and K D ). Recombinant ORF1p was immobilized on an S-series CM5 sensor chip at 1.5 μg / ml using EDC / NHS coupling chemistry following the manufacturer's instructions. Nanobodies and antibodies were prepared as analytes and run in a buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl, and 0.05% Tween-20. In single-cycle kinetic experiments, analytes were injected at 30 μl / min at concentrations of 0.1, 0.3, 1, 3.3, and 10 nM, with association times of 120-180 seconds and dissociation times of 1200-7200 seconds, depending on the observed dissociation rate. Residual bound protein was removed between experiments using 10 mM glycine-HCl pH 3.0. Data were analyzed using Biacore software, and a Langmuir 1:1 binding model was fitted to the sensorgrams to calculate kinetic parameters.

[0141] For epitope binning, antibody pairs were sequentially flowed over immobilized ORF1p using Biacore tandem double injection according to the manufacturer's instructions. Antibodies were injected at a concentration of 200 nM at a flow rate of 10 μl / min. The primary antibody contact time was 120 seconds, followed by a 150-second contact time for the secondary antibody, followed by a 30-second dissociation period. The secondary antibody response signal was measured within a 10-second window at the onset of dissociation. The chip was regenerated with glycine pH 3.0 between experiments as described above. Data were analyzed using the Biacore software Epitope Binning module.

[0142] ORF1p Simoa assay. The Simoa assay was performed on an HD-X analyzer (Quanterix Corp.), and all assay reagents and consumables were loaded into the instrument according to the manufacturer's instructions. Each Simoa assay used 250,000 capture beads and 250,000 auxiliary (non-conjugated) beads. Both first- and second-generation assays used a three-step assay configuration, including a 15-minute target capture step (incubating the capture beads with 100 μL of sample), a 5-minute incubation with the detector reagent (0.3 μg / mL for both first- and second-generation assays), and a 5-minute incubation with streptavidin-β-galactosidase (150 pM for the first-generation assay; 300 pM for the second-generation assay). After each assay step, the beads were washed with system wash buffer 1 (Quanterix Corp.). In the final wash cycle, beads were loaded into a 216,000 microwell array along with the fluorescent enzyme substrate resorufin β-D-galactopyranoside and subsequently sealed with oil. The instrument automatically imaged and counted the "open" and "closed" wells and calculated the average enzyme per bead (AEB). 2 The 4PL weighting factors were fitted to the calibration curve, and the limit of detection (LOD) was determined as three standard deviations above the blank.

[0143] All plasma and serum samples were diluted fourfold in Homebrew sample diluent (Quanterix Corp.) with 1x Halt protease inhibitor cocktail (ThermoFisher), and 1% Triton-X 100 was added in the second-generation assay. All recombinant ORF1p calibrators were run in triplicate, the blank calibrator was run in quadruplicate, and all plasma and serum samples were run in duplicate. The average LOD for all sample runs for each assay was determined and plotted in each graph.

[0144] Plasma and serum samples from healthy individuals were obtained from the Mass General Brigham Biobank, with samples from the Penn Ovarian Cancer Research Center and Tomas Mustelin (University of Washington).

[0145] ORF1p large volume MOSAIC assay. The MOSAIC assay was performed as described previously, using a 2 ml microcentrifuge tube for the initial capture step. For each sample, 500 μL of plasma was diluted fourfold with protease inhibitors and 1% Triton-X 100 in Homebrew sample diluent to a total volume of 2 mL. In short, 100,000 capture beads were incubated with the sample and mixed for two hours at room temperature, then magnetically separated and resuspended in 250 μL system wash buffer 1, and then transferred to a 96-well plate. The beads were then washed with system wash buffer 1 using a Biotek 405TS microplate washing machine, followed by addition of 100 μL of nanobody detector reagent (0.3 μg / mL, diluted in Homebrew sample diluent) and the plate was shaken at room temperature for 10 minutes. After washing with a microplate washer, the beads were incubated with 100 μL of streptavidin-DNA (100 pM, diluted in Homebrew sample diluent containing 5 mM EDTA and 0.02 mg / mL heparin) at room temperature under shaking for 10 minutes, followed by another wash step. The beads were transferred to a new 96-well plate, manually washed with 180 μL of system wash buffer 1, and then resuspended in 50 μL of reaction mixture for rolling circle amplification (RCA). The RCA reaction mixture consisted of 0.33 U / uL phi29 polymerase, 1 nM ATTO647N-labeled DNA probe, 0.5 mM deoxyribonucleotide mixture, 0.2 mg / mL bovine serum albumin, and 50 mM Tris-HCl (pH 7.5), 10 mM (NH4)2SO4, and 10 mM MgCl2 containing 0.1% Tween-20. The beads were shaken at 37°C for one hour, followed by addition of 160 μL of PBS containing 5 mM EDTA and 0.1% Tween-20. After washing the beads once with 200 μL of the same buffer, the beads were resuspended in 140 μL of buffer containing 0.2% BSA. All samples were analyzed using a NovoCyte flow cytometer (Agilent) equipped with three lasers. As previously described, analysis of the average number of molecules per bead (AMB) values ​​was performed using FlowJo software (BD Biosciences) and Python. All code used for MOSAIC data analysis can be downloaded as part of the waltlabtools.mosaic Python module, which can be obtained at github.com / tylerdougan / waltlabtools.

[0146] Targeted proteomic analysis of immunoprecipitated ORF1p. Isotope-labeled standard peptides (AQUAQuantProHeavy peptides with 13 C 15The protein levels of LINE-1 ORF1p (UniProt ID: Q9UN81) were determined by targeted proteomics using a 5-mer 5-well plate (N-labeled C-terminal lysine or arginine, Thermo Fisher) for accurate quantification. Assays were developed for two quantotypic peptides of ORF1p, LSFISEGEIK and cysteine-alkylated NLEECITR (the approach was similar to assay development previously described for other proteins (53)). Briefly, 3–6 mL of patient plasma were diluted with an equal volume of 2× dilution buffer (PBS containing 2% Triton X-100, 10 mM EDTA, and 1 Pierce protease inhibitor tablet (2× concentration, Thermo) per 25 mL) to a final concentration of 1% Triton X-100, 5 mM EDTA, and 1× protease inhibitor and bound to 7 million 62H12-conjugated magnetic beads for 1 hour at room temperature. The beads were washed three times with 5x PBS containing 0.1% tween 20 and 1x protease inhibitors, then washed once with the same buffer without tween 20, and eluted in 50 μl of buffer containing 2% SDS and 50 mM Tris pH 8.5 by heating at 95°C for 5 minutes with stirring. The separated eluate was reduced with 10 mmol / L dithiothreitol and protein alkylated with 55 mmol / L iodoacetamide, followed by in-gel digestion with trypsin (150 ng sequencing grade modified trypsin V5111; Promega), followed by LC-MS analysis of the target peptides (53).

[0147] Classification Models. Classification models were trained on: (1) all healthy and all ovarian cancer patients measured by second-generation assays; and (2) a subset of 51 ovarian cancer patients and 50 age-matched healthy female patients obtained from Ronny Drapkin (University of Pennsylvania). Each dataset contained no missing values, and the measurements in the dataset were log-transformed and normalized beforehand for classification analysis of healthy subjects and ovarian cancer subjects. The univariate classifier used logistic regression, while the multivariate classifier used the best-performing k-nearest neighbor (KNN) and lightweight gradient boosting machine (LightGBM) classifiers and was implemented in Python 3.7.15 using the scikit-learn version 1.0.2 package. Each classifier underwent inter-class weight optimization to handle the data imbalance between healthy and cancer subjects, and grid search was used for hyperparameter tuning.

[0148] The performance of each biomarker in distinguishing ovarian cancer patients from healthy controls was evaluated using five-fold cross-validation by calculating accuracy, precision, recall, f1-score, sensitivity, specificity, and the area under the receiver operating characteristic (ROC) curve (AUC). The stratified five-fold cross-validation strategy randomly divided the positive and negative samples into five equal-sized subsets. Each positive and negative subset was selected as the test dataset, while the remaining samples were used to train the classification model.

[0149] In multivariate analysis, variance inflation factors (VIFs) were calculated for the biomarkers, and any biomarker with a very high correlation with VIF (greater than 10) was excluded a priori from the classification model.

[0150] Cases of Barrett's esophagus. We assembled a cohort of 75 patients with esophageal biopsies and varying degrees of dysplasia. Negative cases without a previous history of dysplasia were screened. The mean age of the cohort was 67 years, with a male predominance (M:F ratio = 3.7:1). All samples were reanalyzed for histologic features of dysplasia by three experienced gastrointestinal pathologists (LRZ, VD, OHY), who were blinded to the original diagnosis. Consensus was reached in 72 cases, and the consensus diagnosis was used as the gold standard. Interpathologist agreement was moderate (κ 0.43-0.51).

[0151] Colon cancer tissue microarray. Between 2011 and 2013, 178 consecutive CRC cases resected by unit surgeons were pooled on 3-mm core tissue microarrays. All cases were independently scored by two pathologists. The mean age of the cohort was 65 years, and 49.8% were male. The mean follow-up was 25 months. At the time of resection, 23% were stage I, 33% were stage II, 44% were stage III, and 1% were stage IV.

[0152] Ovarian cancer samples. Age-matched plasma samples from patients with ovarian cancer (n=53) and healthy controls (n=50) were obtained from the Ovarian Cancer Research Center, University of Pennsylvania, OCRC Tumor BioTrust Collection, Research Resource Identifier (RRID): SCR_02287.

[0153] Gastroesophageal cancer treatment cohort. Nineteen patients received systemic therapy, three of whom also underwent surgical resection. Patients were treated with concurrent chemotherapy (carboplatin / paclitaxel) and radiotherapy (N=3), fluorouracil / leucovorin / oxaliplatin / docetaxel (FLOT, N=2), fluorouracil / leucovorin / irinotecan / oxaliplatin (FOLFIRINOX, N=2), fluorouracil / leucovorin / oxaliplatin (FOLFOX, N=9), FOLFOX plus trastuzumab (N=1), pembrolizumab (N=1), or FOLFOX followed by chemoradiotherapy (1). The mean age of the cohort was 76 years. All patients were male (100%). At initial diagnosis, 58% had locally advanced disease (stage II-III) and 42% had advanced disease (stage IV). After reviewing restaging imaging (CT and / or PET-CT) blinded to the assay results, researchers considered 68% (N=13) responders to treatment and 32% (N=6) non-responders to standard of care. Note that on-treatment / post-treatment blood draws for Simoa measurements were typically performed prior to these imaging studies.

[0154] Patient consent. All plasma samples were obtained with written informed consent at Mass General Brigham (MGB), the University of Pennsylvania, and the University of Washington under IRB-approved protocols. All experiments using patient samples were performed with IRB approval and in accordance with the ethical guidelines outlined in the Belmont Report. Tissue samples were obtained with consent or, where appropriate, with waiver of consent under MGB-approved protocols.

[0155] Histochemistry: ORF1p immunohistochemistry was performed essentially as described using anti-ORF1 4H1 (Millipore) (8) at a dilution of 1:3000 and reoptimized on a Leica Bond system (17). Cases were scored by three experienced gastrointestinal pathologists (MST, VD, OHY) at two institutions. LINE-1 in situ hybridization was performed as described using RNAscope catalog 565098 (Advanced Cell Diagnostics) on a Leica Bond system (17). This probe is complementary to the 5′ end of L1RP (L1 insertion in the X-linked retinitis pigmentosa locus). Cases were scored by three experienced gastrointestinal pathologists (MST, VD, OHY).

[0156] Survival analysis: Kaplan-Meier (KM) curves (54) were calculated to investigate the association between overall survival and plasma ORF1p concentrations in ovarian, colorectal, and esophageal cancers. To investigate the association with survival, we categorized ORF1p concentrations in two different ways. First, if the signal was above the limit of detection (LoD) in at least two of the three assays, at least one of the three assays was classified as positive (majority voting). Second, we assessed whether ORF1p concentrations measured by only the most sensitive assay (62H12::Ab6) were associated with survival and categorized patients as ORF1p high and low according to the cohort-specific median. The time variable was defined as the number of days after diagnosis (GE and CRC) or treatment initiation (ovarian). Surviving patients were censored at the last assessment date. Because age at diagnosis was significantly associated with poor prognosis in CRC and male sex was significantly associated with poor prognosis in GE cancer, we employed a Cox proportional hazards regression model (55); ORF1p was found to be independently prognostic. Survival objects and KM curves were calculated using the survival, ggpubr, and survminer packages in R. All tests were performed using R version 4.3.1 (the R project for statistical computing, R-project.org / ). The proportional hazards hypothesis was tested by drawing Schoenfeld residuals and applying the Grambsch-Therneau test using the ggcoxdiagnostics function in R. The effect of the influential observations was assessed by drawing the deviance residuals using the ggcoxdiagnostics function in R. The raw data of survival are provided in the file "Supplementary Raw Survival Data."

[0157] Example 1. Ultrasensitive detection of circulating ORF1p.

[0158] Although ORF1p expression is elevated in tumor tissue from ovarian and other cancers, the levels of ORF1p and many other biomarkers shed from tumors are diluted into the bloodstream, reaching very low levels. These circulating concentrations can be well below the detection limits of conventional enzyme-linked immunosorbent assays (ELISAs) and mass spectrometry methods, necessitating ultrasensitive detection methods. Single molecule array (Simoa) is a digital ELISA technology that offers 1000-fold greater analytical sensitivity than conventional ELISAs ( Figure 2 B) 11 Using Simoa technology, we recently reported an ultrasensitive digital assay for detecting ORF1p in blood. 26 .

[0159] The assay was optimized and expanded to detect circulating ORF1p concentrations as low as sub-pg / mL (low femtomolar) in patients with ovarian and other cancers, demonstrating high specificity for cancer detection ( Figure 2 C). Briefly, affinity reagents were combinatorially screened as capture and detector pairs on the Simoa platform. The capture affinity reagent was conjugated to 2.7-μm paramagnetic beads via EDC chemistry, while the detector affinity reagent was biotinylated using SulfoNHS-LC-LC-biotin reagent. For each pair, several concentrations of recombinant human ORF1p protein and a buffer blank were measured to determine the signal-to-noise ratio. Selected pairs were used to measure ORF1p levels in breast cancer cell lysates and subsequently in serum samples from breast cancer and healthy patients.

[0160] The reagents tested included 4H1 (a mouse monoclonal antibody targeting amino acids 35 to 44 of human ORF1p, MABC1152, Sigma-Aldrich), JH73 (a rabbit monoclonal antibody raised against the C-terminus of human ORF1p, Taylor et al., Cell. 2013 Nov 21; 155(5): 1034-48) and JH74 (a rabbit monoclonal antibody raised against the C-terminus of human ORF1p, Doucet-O'Hare et al., Proc Natl Acad Sci US A. 2015 Sep 1; 112(35): E4894-900), see, e.g., Mita et al., eLife. 2018; 7: e30058; commercial antibodies Ab6 (ab246317, AbCam), Ab54 (ab246320, AbCam), D3W9O (#88701, Cell Signaling Technology); and 168006 (NovoPro Labs); Nanobody 5 (Nb5) (a Nanobody described herein), and monoclonal antibodies 62H12, 64C6, 33A8, 61A11, 36D12, 34H7, 50E9, 34C5, 55A6 and 42D10 as described herein.

[0161] The signal-to-noise ratios of various pairs on the Simoa platform are as follows: Figure 2 These values ​​were determined using recombinant human ORF1p protein and diluted breast cancer cell lysates, and affinity reagent pairs with the highest signal-to-noise ratios were selected for further screening in a pilot cohort of healthy and breast cancer patient serum samples. Figure 3 Representative calibration curves for selected affinity reagent pairs are shown, where the measured signal is the average enzyme amount per bead (AEB). Figure 4-5 Shown are ORF1p levels measured in healthy and breast cancer patient serum samples using selected affinity reagent pairs on the Simoa platform. Figure 6Shown are ORF1p levels measured in high-grade serous ovarian cancer and healthy patient plasma samples using the Nb5 Nanobody / Ab6 and Ab54 / Ab6 capture / detection pairs.

[0162] The Nanobody / Antibody pair of Nb5 Nanobody / Ab6 was ultimately chosen due to its high specificity in healthy patients (undetectable at baseline levels) and good sensitivity in cancer patients.

[0163] like Figure 6 and 7A As shown in Figure 7, the results show that ORF1p levels are elevated in multiple cancer types, with approximately 63% of HGSOC patients (36 / 57) having detectable levels in two independent pilot cohorts. Importantly, the data show that circulating ORF1p is highly specific: in over 400 healthy individuals in the independent cohorts, almost all individuals had undetectable ORF1p levels (4x LOD (accounting for dilution factor) of 0.28 pg / mL) (Figure 7); four of them tested positive; one of them was found to have prostate cancer, and information on the other patients was limited, resulting in a specificity of >99%.

[0164] Thus, in the vast majority of healthy individuals, ORF1p baseline levels were undetectable (less than 0.28 pg / mL (considering a 4-fold dilution factor, using 25 uL of plasma or serum per replicate, diluted fourfold to a total volume of 100 uL), which helps to distinguish healthy people from cancer patients through a robust threshold, while many existing ovarian cancer protein biomarkers may have different baseline levels in different individuals. These results provide strong support for the feasibility and translational potential of ORF1p as a blood-based biomarker for multiple types of cancer, including ovarian, pancreatic, liver, colorectal, lung, and head and neck cancers.

[0165] Example 2. Improved Assay - Second Generation

[0166] Improved “second-generation assays” were developed using custom reagents that increased analytical sensitivity by approximately 2-5 fold and now detect ORF1p with a sensitivity of approximately 50 attomoles, equivalent to 1000 ORF1 homotrimers among approximately 1019 molecules in 25 μL of plasma. These assays employ optimized buffers and rabbit mAbs for capture and either mAbs or engineered bivalent nanobody “Nb5-5LL” detection agents, which consist of two linked Nb5 molecules. We applied these assays, along with our “first-generation assay,” to a pilot cohort of 53 plasma samples from patients with ovarian cancer and 50 healthy patients. These results ( Figure 10A) showed that the first-generation assay detected approximately 50% of patients, including 3 of 5 stage I patients assayed; a "second-generation assay" used three of the best-performing capture:detection pairs, rabbit monoclonal antibodies 34H7 and 62H12 as capture reagents and either Ab6 or the homodimeric form of Nb5 (Nb5-5LL) as detectors, increasing the detection rate to 70-80% (4 of 5 stage I patients) while maintaining high specificity ( Figure 10A -D). Addition of detergent further improved performance, presumably by limiting bead aggregation and improving bead loading into the microwells. These second-generation assays achieved detection limits of 0.016–0.029 pg / mL (130–240 aM trimeric ORF1p), and the four different reagents had primarily non-overlapping epitopes in binning experiments (with partial overlap for 34H7 and 62H12).

[0167] Somewhat unexpectedly, the analytical sensitivity of the assay (for detection of recombinant ORF1p in buffer) did not fully correspond to the clinical sensitivity (for detection of ORF1p in plasma from cancer patients). Although the second-generation assay improved analytical sensitivity by less than an order of magnitude over the first-generation assay, the detectability of circulating ORF1p relative to background in buffer was significantly improved in re-measured samples from a large cohort of healthy and cancer patients. This discrepancy may be due to different accessibility of circulating ORF1p epitopes or different nonspecific binding patterns in plasma.

[0168] Next, we applied these assays to a larger cohort of patients with stage III and IV MGH ovarian and gastroesophageal cancers ( Figure 10B In ovarian cancer, we found similar or higher detection rates with the improved assay in a larger cohort, including detection of mucinous ovarian cancer subtypes, where CA-125 levels tend not to be elevated. In esophagus, we demonstrated improved detection rates. Together, these results demonstrate the utility of earlier detection of ovarian cancer, including 80% of patients with stage I ovarian cancer at clinical diagnosis, as well as detection of esophageal cancer.

[0169] Undetectable or very low ORF1p levels in healthy individuals could be easily distinguished from ORF1p levels measured in ovarian cancer patients, resulting in a single-marker model with strong discriminatory power (area under the receiver operating characteristic curve, AUC, 0.93 to 0.948, sensitivity 41% to 81%, specificity 98%, Figure 10D This large cohort included pre-treatment plasma samples from a subcohort of ovarian cancer patients (mostly high-grade serous ovarian cancer, the "Penn cohort"), as well as age-matched controls (n = 51-53 women, Figure 10C); Similarly, the second-generation assay showed higher sensitivity while maintaining high specificity, notably detecting five of six stage I / II patients with a specificity >98%. In addition, a multivariate model combining ORF1p (34H7::Nb5-5LL assay) with the ovarian cancer biomarkers CA125 and HE4 achieved improved diagnostic performance compared to these existing markers (CA125 and HE4 alone, AUC = 0.94, 59% sensitivity and 98% specificity; ORF1p, CA125 and HE4, AUC = 0.98, 91% sensitivity and 98% specificity); Figure 10D Although it is unclear whether the low ORF1p levels detected in several healthy individuals are due to nonspecific binding, true background levels of ORF1p, or an unrecognized premalignant state, several positive healthy controls were positive in only one of the three second-generation assays (n = 4 were positive only by 62H12::Nb5-5LL, and n = 75 were positive only by 62H12::Ab6), suggesting that nonspecific binding is present in at least some of these cases and that specificity can be improved by combining data from multiple assays. Our results demonstrate that by developing improved affinity reagents, we have achieved higher clinical sensitivity for detecting circulating ORF1p in cancer patients, with a sensitivity of 83% and a specificity of >98% for early detection of ovarian cancer.

[0170] The breast cancer cohort included 30 patients with metastatic disease and 30 patients with localized disease for whom receptor subtype was available. Triple-negative cases tended to have a higher positivity rate across all assays, but the most sensitive second-generation assay (62H12::Ab6) detected 96% of triple-negative cases and 91% of the remaining cases, with a sensitivity of 93% for both localized and metastatic disease. Overall, metastatic disease was detected more frequently than localized disease (43% vs. 6.7% for first-generation assays and 67-93% vs. 23-93% for second-generation assays, depending on the assay), and all three second-generation assays had higher sensitivity than first-generation assays.

[0171] To test whether ORF1p can be used to monitor treatment response, we identified 19 patients with gastroesophageal cancer who had detectable plasma ORF1p at diagnosis and for whom follow-up samples were available during or after treatment (mean 80 days after treatment initiation, range 26-179 days). The primary tumors were all adenocarcinomas located in the esophagus (n=7), gastroesophageal junction (n=7), and stomach (n=5). All patients received systemic therapy, either chemotherapy or chemoradiotherapy: CROSS (n=3), FLOT (n=3), FOLFIRINOX (n=2), FOLFOX (n=9), FOLFOX plus trastuzumab (n=1), or FOLFOX followed by CROSS (n=1). A small number of patients also received radiation and / or surgery. Clinical responses ("responders" and "non-responders") were determined by review of restaging CT and PET-CT imaging by a clinician blinded to the assay results. Over a median of 465 days (range, 98-1098), 12 patients died, six were still alive at the time of last follow-up (all “responders”), and one patient was lost to follow-up. Non-responders had higher pre-treatment plasma ORF1p ( Figure 11A , left panel, p = 0.02). All six patients had detectable ORF1p (defined as positive above background in two of three assays) at follow-up sampling and were non-responders by imaging ( Figure 11A , right, p<0.0001, Fisher's exact test) and lower survival (overall survival log-rank test p=0.001). In contrast, in all 13 responders, circulating ORF1p decreased to undetectable levels in subsequent sampling. Plasma ORF1p was measured in four responders and two non-responders at early time points of 26-33 days. There was no difference in sampling time between the groups (average 93 days for non-responders and 74 days for responders, p=0.5). Pre-treatment blood was drawn an average of 20 days after diagnosis (range -8-48, average 22 days for non-responders and average 19 days for responders, p=0.6). Representative PET and PET-CT images are shown ( Figure 11B ), both images were taken approximately two months after the start of treatment and one month after the plasma ORF1p results were available. Thus, a decrease in circulating ORF1p paralleled treatment response and survival, while persistent circulating ORF1p corresponded to patients with refractory disease, demonstrating the prognostic potential of this marker.

[0172] Because these results suggest that pretreatment plasma ORF1p levels may have prognostic value, we evaluated the prognostic value of the second-generation ORF1p Simoa assay in cohorts of patients with GE, CRC, and ovarian cancer. We stratified patients based on median ORF1p values ​​or ORF1p detectability (Methods) and found that higher pretreatment plasma ORF1p levels were significantly associated with worse survival in patients with GE and CRC ( Figure 11C , p = 0.0017 and 0.011, respectively, by log-rank test), but not in patients with ovarian cancer. ORF1p still had significant prognostic value in multivariate analysis of GE and CRC.

[0173] Example 3. Improving analytical sensitivity

[0174] Assuming that the assay described in Example 1 is limited by the number of ORF1p molecules in a 25 μL sample, using a larger volume of patient plasma can increase the number of target molecules present. For example, a teaspoon (5 ml) contains 200 times more than what is used in a 25 μL assay sample. Furthermore, a recently developed flow-based detection platform, Molecular Signal Amplification on Beads Individual Counting (MOSAIC), can improve sensitivity. 30 Cohorts using 20-fold greater volumes were evaluated. 0.5 ml of plasma (500 μl) was diluted 1:4 in diluent binding buffer containing additional 1% Triton X-100 detergent and bound to 34H7-conjugated beads for 90 minutes, washed, detected by binding to biotinylated Nb5-5LL (also known as MT997), and signal development on the beads was measured using MOSAIC and read on a flow cytometer. Figure 12 As shown, 9 out of 10 previously undetected patients showed signals higher than healthy controls.

[0175] We collected large volumes (3-10 ml) from 20 HGSOC patients, 20 benign disease controls, 20 EAC, and 20 Barrett's esophagus patients (with matched tissue) to provide a "ground truth" of ORF1p expression, along with bulk healthy plasma and bulk gastroesophageal plasma. The MOSAIC assay was performed using the reagents described herein, with variables including reagent selection, buffer additives (such as bead number and loading, salts and detergents), binding time, and wash conditions varied to optimize sensitivity, specificity, and reproducibility.

[0176] Example 3. Development of Nanobodies

[0177] Using Fridy et al., 2014 49 Nanobodies that bind to ORF1p were generated as described in

[15] . Briefly, the method was followed by Carter et al., 2020 35The method described in [1] produces highly purified ORF1p from Escherichia coli. Camels are immunized, bone marrow is extracted, and candidate DNA regions are amplified and sequenced to generate a library. Serum is separated and then bound to immobilized purified ORF1p. Bound heavy chain antibodies are eluted, and VHH fragments are extracted and sequenced by mass spectrometry using a library derived from the bone marrow library. Candidate sequences are then assembled, synthesized, cloned, and screened for their ability to bind to ORF1p. The identified sequences are shown in Table B.

[0178] Table B Nanobodies (CDRs in bold)

[0179]

[0180]

[0181] NB5 (also referred to herein as clone 5) was selected for further development. An exemplary nucleic acid encoding Nb5 is as follows:

[0182]

[0183] Since ORF1p is a homotrimer, it is possible to achieve a variety of high-affinity binding reagent combinations. After two rounds of affinity mass spectrometry and cloning, further screening was performed and 21 high-affinity clones were obtained. 49 , wherein preferably have medium picomolar affinity (Table 1), and identified clones with non-overlapping epitopes. As shown in Table 1, engineered nano antibody reagents have low picomolar affinity, similar to or exceeding existing antibody reagents (Ab6, 4H1 and Ab54). In the first round, dimer and trimer reagents were engineered and the linker length (a "long linker" of 56 residues containing flexible (GGGGS) (SEQ ID NO: 6) and rigid (EAAAK) (SEQ ID NO: 11) sections was optimized, while a linker comprising 20 flexible (GGGGS) (SEQ ID NO: 6) residues also had an advantage) was optimized, and affinity was improved, with both reagents outperforming the best mAb before, Ab6. Table C provides the sequences of some developed concatemer constructs.

[0184] Table C. Exemplary concatemer constructs

[0185]

[0186]

[0187]

[0188]

[0189]

[0190] Through SPR binning experiment 49 Reagents with non-overlapping epitopes were identified and combinations were empirically tested using calibration curves of recombinant ORF1p. The best performing reagents were then tested against a pilot cohort of cancer and healthy patient plasma. Combinations of these affinity reagents were tested using Simoa on spiked healthy plasma and demonstrated a 7-fold improvement in the limit of detection (Table 2). Figure 14 As shown, this figure plots ORF1p measurements for selected reagent pairs in a small sample of plasma from healthy and cancer patients. The reagent with the highest affinity is not always the best performer in plasma, so a combination of rational and empirical screening is needed.

[0191] Table 1. Surface plasmon resonance (SPR) affinity measurements of anti-ORF1 affinity reagents

[0192]

[0193]

[0194] Table 2. Combinations of SIMOA measurements

[0195] Capture agent Detection Agent ug / mL detection reagent LOD (fg / mL) 4H1 Nb 5-5 0.3 7 Ab6 Ab6 0.6 19 4H1 Ab6 0.3 30 Ab6 Nb 5-5 long 0.3 44 Nb 5-5 Ab6 0.3 50 Nb5 Ab6 0.3 53 4H1 Nb 5-5 long 0.3 56 Ab6 Nb 5-5 0.15 59 Nb 5-5 4H1 0.3 72 Nb 5-5 long Ab6 0.3 75 4H1 Nb 5-5 0.15 91 Ab6 Nb 5-1 0.3 101 Ab6 Ab6 0.3 102 Ab6 Nb 5-5 long 0.15 111

[0196] Simoa optimization using novel and existing reagents resulted in a candidate assay with a seven-fold lower limit of detection (LOD) of 7 fg / ml (approximately 50 aM) for ORF1p. Because the target is trimeric, some combinations could potentially yield very high affinities.

[0197] Example 4. Development of monoclonal antibodies

[0198] MonoRab by GenScript TM The immunization and B cell cloning platform was used with purified recombinant human ORF1p trimer. The 50 best performing clones, identified by ELISA screening of multiple dilutions of conditioned cell culture medium, were selected and used in a modified Simoa assay using two different detector beads (Nb-5, the C5 nanobody clone mentioned above, or 4H1 (Sigma-Aldrich)). The ten best clones (62H12, 64C6, 33A8, 61A11, 36D12, 34H7, 50E9, 34C5, 55A6, and 42D10) all showed a signal-to-noise ratio that was 5-fold higher than our best available reagent, Ab6 ( Figure 13 A). Table D and Figure 20A -J provides the variable region heavy chain and light chain sequences of each of these ten antibodies.

[0199] These antibodies were initially screened against recombinant ORF1p protein using SIMOA in buffer (sample diluent from Quanterix Corp. supplemented with Triton-X 100) to identify antibody pairs that provided improved sensitivity and specificity. Table 3 shows representative limits of detection (LODs) for selected antibody pairs in dilution buffer. The LODs for the original Nb5 / Ab6 pair averaged approximately 0.05 pg / ml.

[0200] Table 3 Representative LODs for selected capture / detector antibody pairs

[0201]

[0202] Simoa also measured several signal / background pairs using recombinant ORF1p protein in dilution buffer; the results are shown in Figure 2. Figure 15 As shown. When rabbit monoclonal antibodies were used as both capture and detector, background was high. The best combinations were rabbit monoclonal antibody capture / Ab6 detector; Nb5-5 capture / rabbit monoclonal antibody detector; and 4H1 capture / rabbit monoclonal antibody detector.

[0203] Selected pairs (including the original Nb5 / Ab6 for comparison) were used to test ORF1p levels in plasma samples diluted four-fold in sample diluent containing Triton-X 100 (Quanterix Corp.). Figure 16 and 17 In these assays, the original Nb5 / Ab6 sample was diluted in sample diluent buffer, while the new assay used samples diluted in sample diluent + 1% Triton-X. Sample diluent consisted of 20 g BSA (Millipore #820451); 100 mL 10X PBS (Sigma #P5493-1L); 10 mL 10% Tween-20 (Sigma #P9416-50ML); 500 uL Proclin 300 (Sigma 48912-U); 10 mL 0.5 M EDTA (Sigma #E7889-100ML), and was made up to 1 L with MilliQ water.

[0204] All assays were performed as three-step assays (separate capture agent, detector labeling, and streptavidin β-galactosidase labeling steps) unless otherwise stated as two-step assays (combined capture agent and detector labeling steps). The results show that sensitivity varies with the choice of capture agent / detector. Figure 18 Shown are the results of assays performed in a cohort of 25 healthy subjects and 25 patients with breast, colorectal, or esophageal cancer.

[0205] The selected pairs were then evaluated in a panel of cancers. Figure 19A - shown in C.

[0206] Table D. Variable region sequences of rabbit anti-ORF1p monoclonal antibodies

[0207]

[0208]

[0209] Example 5. Development of a third generation assay

[0210] Building on improvements achieved through nanobody engineering in our second-generation assay, we developed an expanded set of nanobody concatemers, including homodimeric, heterodimeric, and heterotrimeric anti-ORF1p nanobodies, and screened them in combination with the 34H7 and 62H12 capture antibodies, generating a “third-generation” assay ( Figure 22A-B 、 23 , 24). We noticed that reagents containing Nb2 performed very well in SPR but poorly in the Simoa assay, and we hypothesized that this was because Nb2 contains a lysine in the CDR that would be biotinylated during the procedure, reducing affinity. Therefore, we designed new reagents with C-terminal biotinylation at a cysteine ​​residue and different linker sequences. Five of these assays utilized constructs containing Nb2 and Nb9 and outperformed our second-generation assay in a cohort of 25 GE cancer patients with previously mostly undetectable ORF1p measurements while maintaining high specificity compared to healthy individuals ( Figure 21A 、 Figure 24 ).

[0211] To detect ORF1p using a more sensitive assay, we next investigated the expression of ORF1p on our recently developed molecular bead signal amplification individual counting platform (MOSAIC, Figure 21B ) were tested on an ORF1p affinity reagent from one of the second-generation Simoa assays. Unlike the microwell array format in Simoa, MOSAIC can generate localized on-bead signals from single captured molecules and improve analytical sensitivity by an order of magnitude by increasing the number of beads counted (26). Furthermore, since the Simoa assay was developed using only 25 μL of plasma, we hypothesized that using larger volumes of plasma would enhance the detectability of ORF1p by increasing the number of analyte molecules present. By using a 20-fold higher sample volume (500 μL of plasma) and the MOSAIC platform, we achieved a tenfold higher analytical sensitivity with a limit of detection of 0.002 pg / ml ORF1p (17 aM trimer, Figure 25Indeed, in a pilot cohort of gastroesophageal cancer and healthy patients, nine out of ten patients with previously undetectable cancer had ORF1p levels that were easily distinguishable from healthy individuals ( Figure 21C Similar results were observed in a breast cancer cohort ( Figure 21D Therefore, in addition to improved affinity reagents, using larger sample volumes and more sensitive analytical techniques could further enhance the sensitivity and discrimination of circulating ORF1p levels between healthy controls and cancer patients. The relative contributions of increased volume and improved assay platforms to the improved sensitivity remain to be explored; assay background, seen in patient plasma but not in buffer, could also be further optimized.

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[0268] Other implementation plans

[0269] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims

1. A method comprising: A sample from a subject is provided, preferably a sample comprising a biological fluid or tissue, preferably wherein the biological fluid is blood, serum or plasma, or the tissue is a tissue lysate, and the level of ORF1p in the sample is determined using an ultrasensitive protein assay. 2 . The method of claim 1 , further comprising comparing the ORF1p level to a disease reference, wherein an ORF1p level higher than the reference indicates that the subject has cancer or is at risk of developing cancer.

3. The method of claim 1 or 2, wherein the cancer is carcinoma.

4. The method of claim 3, wherein the cancer is ovarian cancer, breast cancer, liver cancer, colon / colorectal cancer, lung cancer, esophageal cancer, prostate cancer, stomach cancer, head and neck cancer, brain cancer (optionally glioblastoma), soft tissue cancer, kidney cancer, gallbladder cancer, bile duct cancer (cholangiocarcinoma), bladder cancer, uterine cancer or pancreatic cancer, blood cancer or bone marrow cancer (optionally lymphoma, leukemia or myeloma) or skin cancer (optionally melanoma).

5. The method of claim 4, wherein the ovarian cancer is high-grade serous ovarian cancer (HGSOC).

6. The method of claims 1 to 5, wherein the ultrasensitive assay is single molecule array (SIMOA); molecular on-bead signal amplification individual counting (MOSAIC); mesoscale discovery (MSD); single molecule counting (SMC); nucleic acid-linked immunosorbent assay (NULISA); LUMINEX; SOMAscan assay; NAB-SURE; mass spectrometry (optionally MALDI-MS) and / or mass cytometry (optionally CyTOF).

7. The method of claims 1 to 6, wherein determining the level of ORF1p comprises contacting the sample with a capture or detection reagent comprising a Nanobody selected from Nb2, Nb5, Nb9, Nb10 or NB21 or an ORF1p binding derivative comprising its CDRs, or concatemers thereof, optionally MT1032, MT1033, MT1034, MT1035, MT1036, MT1037, MT1038, MT1039 or MT1040 and / or a Nanobody selected from 62H12, 64C6, 3 or an antigen-binding fragment thereof, wherein the capture / detection reagent is 62H12 / MT1036, 62H12 / MT1037, 62H12 / MT1038, 62H12 / Ab6, 34H7 / Nb5-5LL, 34H7 / Ab6, 62H12 / Nb5-5LL, 4H1 / Nb5-5, or 4H1 / Nb5-5LL.

8. The method of claim 7, wherein the Nanobody comprises a sequence that is at least 80%, 85%, 90% or 95% identical to a sequence in Table B, preferably wherein the CDRs of the Nanobody are identical to the CDRs of a sequence in Table B.

9. The method of claims 1 to 8, further comprising recommending or sending the subject for additional evaluation, optionally by imaging and / or biopsy.

10. The method of claims 1 to 9, further comprising administering a cancer therapy to a subject identified as having cancer or being at risk of developing cancer.

11. The method of claim 8, wherein the treatment comprises chemotherapy, hormone therapy, immunotherapy, radiation, or surgical resection.

12. The method of claim 1, further comprising determining the level of ORF1p in the subject after administering the treatment, and comparing the level of ORF1p before treatment with the level of ORF1p during and / or after treatment, wherein a decrease in the level of ORF1p indicates that the treatment is effective in treating cancer.

13. A single domain antibody or antigen binding fragment thereof that binds to human ORF1p, comprising a sequence that is at least 90% identical to a Nanobody sequence as shown in Table B or to its CDR1 , CDR2 and CDR3.

14. A single domain antibody or antigen-binding fragment thereof comprising CDR1, CDR2 and CDR3 as shown in Table B.

15. A fusion construct comprising at least two, optionally three, four or five single domain antibodies or antigen binding fragments thereof according to claim 13 or 14, optionally with a linker therebetween, optionally as shown in Table C, optionally MT1032, MT1033, MT1034, MT1035, MT1036, MT1037, MT1038, MT1039 and MT1040.

16. An antibody or antigen-binding portion thereof that specifically binds to human ORF1p, wherein the antibody or antigen-binding portion thereof comprises at least one of the following: a heavy chain variable region (VH) comprising or consisting of a VH sequence at least 95% identical to a sequence shown in Table D or Figures 20A-J, or CDR1, CDR2, and CDR3 thereof; and / or A light chain variable region (VL) comprising or consisting of a VL sequence at least 95% identical to a sequence shown in Table D or Figures 20A-J, or CDR1, CDR2 and CDR3 thereof, preferably wherein the VH and VL or CDRs are from the same antibody.

17. The antibody, or antigen binding portion thereof, of claim 16, wherein the antibody comprises a constant region, optionally as shown in Table A.

18. The single domain antibody or antigen binding fragment thereof of claims 13-14, the fusion construct of claim 15, or the antibody or antigen binding portion thereof of claim 16 or 17, fused to a tag.

19. The single domain antibody or antigen binding fragment thereof, fusion construct or antibody or antigen binding portion thereof according to claim 18, wherein the tag is an oligonucleotide, peptide, chemiluminescent, fluorescent, radioactive or colorimetric label.

20. The single domain antibody or antigen binding fragment thereof, fusion construct or antibody or antigen binding portion thereof of claim 19, wherein the radiolabel is 125 I.

21. A nucleic acid molecule encoding the single domain antibody or antigen binding fragment thereof, fusion construct or antibody or antigen binding portion thereof according to claims 13-20.

22. A vector comprising the nucleic acid molecule of claim 21 and optionally a promoter.

23. A host cell comprising the nucleic acid molecule of claim 21 and optionally expressing the single domain antibody or antigen binding fragment thereof, fusion construct or antibody or antigen binding portion thereof of claims 13-19.

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