Tandem antigen capture assays for measuring QSOX1 isoforms

Abstract

Provided are antibodies specific for the long isoform of QSOX1 and methods of using these antibodies to detect and measure the levels of the presence of the long and short isoforms of QSOX1 in a sample.

Description

TANDEM ANTIGEN CAPTURE ASSAYS FOR MEASURING QSOX1 ISOFORMS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 578,467 filed on August 24, 2023, the content of which is incorporated by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (112624.01469.xml; Size: 24,576 bytes; and Date of Creation: August 26, 2024) is herein incorporated by reference in its entirety. BACKGROUND

[0003] Quiescin sulfhydryl oxidase 1 (QSOX1) is a potential biomarker for multiple cancer types as well as acute decompensated heart failure. Differentiation of the two major isoforms of QSOX1, QSOX1-L and QSOX1-S, may also be useful in distinguishing various disease states, and developing better treatment strategies. Therefore, mechanisms for detecting and distinguishing QSOX1 and its isoforms are needed. SUMMARY

[0004] Provided herein is QSOX1-L antibody comprising: a heavy chain variable domain (VH) complementary determining region (CDR) CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 21, a VL CDR2 comprising SEQ ID NO: 22, and a VL CDR3 comprising SEQ ID NO: 23. The VH may comprise SEQ ID NO: 24 or a sequence having at least 90% identity thereto, and the VL may comprise SEQ ID NO: 25 or a sequence having at least 90% identity thereto. The antibody may bind to a peptide comprising SEQ ID NO: 17.

[0005] Also provided herein is a method for detecting a long isoform of QSOX1 (QSOX1-L) in a sample, the method comprising: (a) contacting a first portion of the sample with a capture antibody under conditions that promote binding of the capture antibody with QSOX1-L and a short isoformof QSOX1 (QSOX1-S); (b) removing unbound sample; (c) contacting bound QSOX1-L and QSOX1-S with a QSOX1-L antibody under conditions that promote binding of the QSOX1-L antibody with QSOX1-L, wherein the QSOX1-L antibody does not bind to QSOX1-S; (d) removing unbound QSOX1-L antibody; and (e) detecting bound QSOX1-L antibody; wherein the QSOX1-L antibody comprises: a VH CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a VL CDR1 comprising SEQ ID NO: 21, a VL CDR2 comprising SEQ ID NO: 22, and a VL CDR3 comprising SEQ ID NO: 23; wherein the capture antibody comprises: a VH CDR1 comprising SEQ ID NO: 1 or 7, a VH CDR2 comprising SEQ ID NO: 2 or 8, a VH CDR3 comprising SEQ ID NO: 3 or 9, a VL CDR1 comprising SEQ ID NO: 4 or 10, a VL CDR2 comprising SEQ ID NO: 5 or 11, and a VL CDR3 comprising SEQ ID NO: 6 or 12. The VH of the QSOX1-L antibody may comprise SEQ ID NO: 24 or a sequence having at least 90% identity thereto, and the VL of the QSOX1-L antibody may comprise SEQ ID NO: 25 or a sequence having at least 90% identity thereto. The VH of the capture antibody may comprise SEQ ID NO: 13 or 15, and the VL of the capture antibody may comprise SEQ ID NO: 14 or 16. The QSOX1-L antibody may be linked to a detectable label.

[0006] The method may further comprise detecting QSOX1-S in the sample, wherein detecting QSOX1-S comprises: (f) contacting a second portion of the sample with the capture antibody under conditions that promote binding of the capture antibody with QSOX1-L and QSOX1-S; (g) removing unbound sample; (h) contacting bound QSOX1-L and QSOX1-S with a total QSOX1 antibody under conditions that promote binding of the total QSOX1 antibody with QSOX1-L and QSOX1-S; (i) removing unbound total QSOX1 antibody; (j) detecting bound total QSOX1 antibody; (k) measuring the amount of bound QSOX1-L antibody and bound total QSOX1 antibody, wherein a difference between the amount of bound QSOX1-L antibody and bound total QSOX1 antibody detects the presence of QSOX1-S; wherein the capture antibody comprises: the VH CDR1 comprising SEQ ID NO: 1, the VH CDR2 comprising SEQ ID NO: 2, the VH CDR3 comprising SEQ ID NO: 3, the VL CDR1 comprising SEQ ID NO: 4, the VL CDR2 comprising SEQ ID NO: 5, and the VL CDR3 comprising SEQ ID NO: 6; and wherein the total QSOX1 antibody comprises: the VH CDR1 comprising SEQ ID NO: 7, the VH CDR2 comprising SEQ ID NO: 8, the VH CDR3 comprising SEQ ID NO: 9, the VL CDR1 comprising SEQ ID NO: 10, the VL CDR2 comprising SEQ ID NO: 11, and the VL CDR3 comprising SEQ ID NO: 12. The capture antibody may comprise the VH comprising SEQ ID NO: 13 or a sequence having at least90% identity thereto, and the VL comprising SEQ ID NO: 14 or a sequence having at least 90% identity thereto; and the total QSOX1 antibody may comprise the VH comprising SEQ ID NO: 15 or a sequence having at least 90% identity thereto, and the VL comprising SEQ ID NO: 16 or a sequence having at least 90% identity thereto.

[0007] The total QSOX1 antibody may be linked to the detectable label. The capture antibody may be attached to a solid support. The sample may comprise a serum or a plasma. The sample may be a human sample.

[0008] Also provided herein is a method for detecting a long isoform of QSOX1 (QSOX1-L) in a sample, the method comprising: (a) contacting the sample with a QSOX1-L antibody under conditions that promote binding of the QSOX1-L antibody with QSOX1-L; (b) detecting bound QSOX1-L in the sample; wherein the QSOX1-L antibody comprises: a VH CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a VL CDR1 comprising SEQ ID NO: 21, a VL CDR2 comprising SEQ ID NO: 22, and a VL CDR3 comprising SEQ ID NO: 23. The VH may comprise SEQ ID NO: 24 or a sequence having at least 90% identity thereto, and the VL may comprise SEQ ID NO: 25 or a sequence having at least 90% identity thereto.

[0009] The QSOX1-L antibody may be attached to a solid support. The QSOX1-L antibody may be linked to a detectable label. The sample may comprise serum or plasma. The sample may be a human sample.

[0010] Also provided herein is a kit comprising a QSOX1-L antibody, a total QSOX1 antibody, and a capture antibody; wherein the QSOX1-L antibody comprises:a VH CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a VL CDR1 comprising SEQ ID NO: 21, a VL CDR2 comprising SEQ ID NO: 22, and a VL CDR3 comprising SEQ ID NO: 23; wherein the total QSOX1 antibody comprises: a VH CDR1 comprising SEQ ID NO: 7, a VH CDR2 comprising SEQ ID NO: 8, a VH CDR3 comprising SEQ ID NO: 9, a VL CDR1 comprising SEQ ID NO: 10, a VL CDR2 comprising SEQ ID NO: 11, and a VL CDR3 comprising SEQ ID NO: 12; and wherein the capture antibody comprises: a VH CDR1 comprising SEQ ID NO: 1, a VH CDR2 comprising SEQ ID NO: 2, a VH CDR3 comprising SEQ ID NO: 3, a VL CDR1 comprising SEQ ID NO: 4, a VL CDR2 comprising SEQ ID NO: 5, and a VL CDR3 comprising SEQ ID NO: 6.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGS.1A-1C. Characterization of anti-QSOX1 mAbs. A) Western blot of rQSOX1-S and rQSOX1-L probed with mAbs 2F1, 3A10, and 56-3 following electrophoresis of 50 ng of protein by SDS-PAGE. Location of molecular weight markers (kDa) are noted. B) Epitope mapping of mAb 2F1 by western blot probing rQSOX1-S mutants at enzymatically active CxxC motifs following electrophoresis of 50 ng of protein by SDS-PAGE. Location of molecular weight markers (kDa) are noted. C) Diagram of known epitopes of anti-QSOX1 mAbs. Different colors represent the domains of QSOX1 with the requisite amino acid residues. “NEQ” indicates the location of the NEQEQPLGQWHLS (SEQ ID NO: 17) peptide which was used as an immunogen to generate mAb 56-3. Diagram created with Biorender.com.

[0012] FIGS.2A-2B. LC-MS / MS analysis of rQSOX1-S and rQSOX1-L. Coverage maps from Proteome Discoverer of A) rQSOX1-S (59.71% coverage) and B) rQSOX1-L (57.83% coverage) against the reference sequence for QSOX1 (Uniprot O00391, SEQ ID NO: 26). Underlines indicate high-confidence peptides. Carbamidomethylation and oxidation modifications are annotated as “C” and “O,” respectively. O-linked glycosylation annotated in Uniprot are labelled as “P.”

[0013] FIGS. 3A-3D. Development of Tandem anti-QSOX1 Antigen Capture ELISAs. Schematics of optimized antigen capture ELISAs to detect A) Total QSOX1 with Mab 2F1 and Mab 3A10 and B) QSOX1-L with Mab 2F1 and Mab 56-3. Schematics created with Biorender.com. C) Specificity of Total QSOX1 and QSOX1-L ELISAs to rQSOX1 isoforms. OD450 values reported were blanked against a PBS only control. Bars represent the mean OD450 for each group. D) Standard curves showing limit of detection of tandem ELISAs detecting Total QSOX1 and QSOX1-L. Data points show the mean OD450 with a 4-parameter logarithmic fit line and dotted error bars representing the 95% confidence interval from three replicates. The standard curve for the Total QSOX1 ELISA starts at 5 nM rQSOX1-L while the standard curve for the QSOX1-L ELISA starts at 625 pM. The different x-axis values should be noted.

[0014] FIGS. 4A-4C. Measuring QSOX1 isoform levels in healthy donor sera and plasma. A) Comparison of levels of QSOX1 and isoforms between serum and plasma, n=76 paired sera and plasma. The mean concentrations of QSOX1 were as follows: total QSOX1 was at 9.40 nM in serum and 14.7 nM in plasma, QSOX1-L was at 3.21 nM in serum and 5.63 nM in plasma, QSOX1-S was at 6.55 nM in serum and 9.19 nM in plasma. B) Comparison of QSOX1 and isoforms within serum and plasma by age, <40 years old n=15, ≥40 years old n=15. C) Comparisonof QSOX1 and isoforms within serum and plasma by sex, female n=36, male n=40. Concentrations of QSOX1 were determined using a standard curve of QSOX1-L. Black bars display median with error bars representing upper and lower quartiles. Statistical significance determined by two-way ANOVA with Geisser-Greenhouse correction followed by Šídák’s multiple comparisons test (* = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001). Significance brackets are colored by the sample type in accordance with the plot legend in which different isoforms are being compared, with brackets comparing a single isoform between sample types colored black. A lack of a significance bracket indicates the difference was not significant. DETAILED DESCRIPTION

[0015] The present disclosure provides antibodies and methods for detecting and measuring the long and short isoforms of QSOX1.

[0016] Antibodies

[0017] In a first aspect, provided herein is a QSOX1-L antibody produced by hybridoma 56-3. The QSOX1-L antibody may comprise a heavy chain variable domain (VH) complementary determining region (CDR) CDR1 comprising or consisting of SEQ ID NO: 18 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, a VH CDR2 comprising or consisting of SEQ ID NO: 19 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, a VH CDR3 comprising or consisting of SEQ ID NO: 20 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, a variable light chain region (VL) CDR1 comprising or consisting of SEQ ID NO: 21 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, a VL CDR2 comprising or consisting of SEQ ID NO: 22 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and a VL CDR3 comprising or consisting of SEQ ID NO: 23 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. The VH of the QSOX1-L antibody may comprise or consist of SEQ ID NO: 24, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL of the QSOX1-L antibody may comprise or consist of SEQ ID NO: 25, or a sequence having at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0018] Quiescin sulfhydryl oxidase 1 (QSOX1) is a sulfhydryl oxidase expressed by multiple histological tumor types. It is expressed as fibroblasts begin to exit the proliferative cycle and enter quiescence. There are two major isoforms of QSOX1: a long form isoform (QSOX1-L) and a short form isoform (QSOX1-S). The two isoforms have different tissue distribution. The long isoform has a transmembrane helix in the carboxy terminal, and the short isoform is a secreted soluble protein. Differentiating the levels of the QSOX1 long and short isoforms in patient samples may be used to distinguish disease states. The QSOX1-L antibody is specific to the long isoform of QSOX1. The QSOX1-L antibody may bind a peptide at the C-terminal end of QSOX. The QSOX1-L antibody bind a peptide comprising or consisting of NEQEQPLGQWHLS (SEQ ID NO: 17).

[0019] The terms “antibody” and “antibody molecule” are used herein interchangeably and refer to immunoglobulin molecules or other molecules which comprise an antigen binding domain. Antibodies include whole antibodies (e.g., IgG, IgA, IgE, IgM, or IgD), monoclonal antibodies, chimeric antibodies, humanized antibodies, and antibody fragments, including single chain variable fragments (ScFv), single domain antibodies, and antigen-binding fragments, genetically engineered antibodies, among others, as long as the characteristic properties (e.g. ability to bind to QSOX1) are retained.

[0020] The term antibody includes “antibody fragments” or “antibody-derived fragments” and “antigen binding fragments” which comprise an antigen binding domain and displays antigen binding function, for example, Fab, Fab', F(ab')2, scFv, Fv, dsFv, ds-scFv, Fd, mini bodies, monobodies, and multimers thereof and bispecific antibody fragments. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), (see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al. , PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context.

[0021] As mentioned above, fragments may comprise a heavy chain variable region (VHdomain) and light chain variable region (VL). Fragments may comprise one or more of the heavy chaincomplementarity determining regions (CDRHs) of the antibodies or of the VH domains, and one or more of the light chain complementarity determining regions (CDRLs), or VL domains to form the antigen binding site.

[0022] The terms “complementarity determining region” and “CDR” refer to part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B-cells and T-cells respectively, where these molecules bind to their specific antigen. As the most variable parts of the molecules, CDRs are crucial to the diversity of antigen specificities generated by lymphocytes. There are three CDRs (CDR1, CDR2 and CDR3), arranged non-consecutively, on the amino acid sequence of a variable domain of an antigen binding site. Since the antigen binding sites are typically composed of two variable domains (on two different polypeptide chains, heavy and light chain), there are six CDRs for each antigen binding site that can collectively come into contact with the antigen. A single whole antibody molecule has two antigen binding sites and therefore contains twelve CDRs. For further example, sixty CDRs can be found on a pentameric IgM molecule.

[0023] Within the variable domain, CDR1 and CDR2 may be found in the variable (V) region of a polypeptide chain, and CDR3 includes some of V, and all of diversity (D, heavy chains only) and joining (J) regions. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Among these, CDR3 shows the greatest variability as it is encoded by a recombination of VJ in the case of a light chain region and VDJ in the case of heavy chain regions. The tertiary structure of an antibody is important to analyze and design new antibodies.

[0024] The human VHcomplex is composed of approximately 100 gene segments per haploid genome, including at least 51 functional genes, as judged by successful rearrangement in cloned cDNA. On the basis of nucleic acid sequence homology, the VH genes have been grouped into 6– 7 families (VH 1-7). Among the seven families, the VH3 family is the largest.

[0025] Antibodies can be genetically engineered from the CDRs, VH, VL, and monoclonal antibody sequences described herein into antibodies and antibody fragments by using conventional techniques such as, for example, synthesis by recombinant techniques or chemical synthesis. Techniques for producing antibody fragments are well known and described in the art.

[0026] One may wish to engraft one or more CDRs from the monoclonal antibodies described herein into alternate scaffolds. For example, standard molecular biological techniques can be usedto transfer the DNA sequences encoding the antibody's CDR(s) to (1) full IgG scaffold of human or other species; (2) a scFv scaffold of human or other species, or (3) other specialty vectors. If the CDR(s) have been transferred to a new scaffold all of the previous modifications described can also be performed. For example, one could consult Biotechnol Genet Eng Rev, 2013, 29:175-86 for a review of useful methods.

[0027] The antibodies or antibody fragments can be wholly or partially synthetically produced. Thus, the antibody may be from any appropriate source, for example recombinant sources and / or produced in transgenic animals or transgenic plants. Thus, the antibody molecules can be produced in vitro or in vivo. The antibody or antibody fragment can be made that comprises all or a portion of a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region.

[0028] Furthermore, the antibody or antibody fragment can further comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region. All or part of such constant regions may be produced wholly or partially synthetic. Appropriate sequences for such constant regions are well known and documented in the art.

[0029] The terms “monoclonal antibody” and “monoclonal antibody composition” refer to a preparation of antibody molecules of a single amino acid composition that specifically binds to a single epitope of an antigen.

[0030] The term “chimeric antibody” refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Other forms of “chimeric antibodies” are those in which the class or subclass has been modified or changed from that of the original antibody. Such “chimeric” antibodies are also referred to as “class-switched antibodies.” Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. The antibodies described herein may be chimeric antibodies including heavy chain constant domains from non-human mammals (e.g., mouse, rat, rabbit, or non-human primate). The antibodies described herein may be chimeric antibodies including constant regions from rabbit heavy chain immunoglobulin sequences. Suitable heavy chain constant region sequences from non-human mammals, including mouse, rat, rabbit, and non-human primate are known in the art.

[0031] Antibodies can be made by well-known methods, such as described in Harlow and Lane, Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988). Monoclonal antibodies can be produced by immunizing inbred mice with a peptide antigen. The QSOX1-L antibody producing hybridoma cells disclosed herein may be produced by immunizing mice with the peptide NEQEQPLGQWHLS (SEQ ID NO: 17). The mice may be immunized by the IP or SC route in an amount and at intervals sufficient to elicit an immune response. The mice may receive an initial immunization on day 0 and be rested for about 3 to about 30 weeks. Immunized mice may be given one or more booster immunizations of by the intravenous (IV) or subcutaneous (SC) route. Lymphocytes, from antibody positive mice may be obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells may be produced by mixing the splenic lymphocytes with an appropriate fusion partner under conditions which will allow the formation of stable hybridomas. The antibody producing cells and fusion partner cells may be fused in polyethylene glycol at concentrations from about 30% to about 50%. Fused hybridoma cells may be selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids may be collected from growth-positive wells and screened for antibody production by an immunoassay such as an Enzyme Linked Immunosorbent Assay. Hybridoma cells from antibody positive wells may be cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques or by limiting dilution in which hybridomas are diluted in suitable growth media and re-plated such that ~1 hybridoma cell is pipetted into a single well of a 96-well plate where it grows over the course of 10 days as a single clone, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.

[0032] The QSOX1 antibodies described herein may comprise a polypeptide or protein comprising the antigen binding regions of the antibodies described herein, e.g., the CDRs (1-3) of the heavy and light chain that form the antigen binding region. The peptide may further comprise a detectable tag or other molecules.

[0033] As used herein, the terms “proteins” and “polypeptides” are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein” and “polypeptide” refer to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated,glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The antibodies described herein are polypeptides, as well the antigen- binding fragments and fragments thereof.

[0034] The sequences provided are only for the variable domains of the antibody. Those skilled in the art will appreciate that these regions determine the specificity of the antibody but that the effector function of the antibody is generally dependent on the constant regions (and the specific isotype) of the antibody. Those skilled in the art can engineer antibodies for specific purposes based on the variable domains provided herein. For example, any of the antibodies described herein may comprise an Fc portion of a human or humanized antibody. The Fc regions may be engineered for a suitable effector function(s).

[0035] The term “identity”, as recognized by those skilled in the art, represents a comparison between two or more amino acid sequences performed using published methods and software known in the art. For example, the compared amino acid sequences are optimally aligned, and the number of amino acid differences are counted and converted to a percentage. For example, if a first amino acid sequence of 50 amino acids is optimally aligned with a second amino acid sequence of 50 amino acids, and 5 out of 50 amino acids differ from the second amino acid sequence, then the first amino acid sequence is said to have 10% identity with the second amino acid sequence.

[0036] Methods

[0037] In a second aspect, provided herein is a method for detecting the long isoform of QSOX1 (QSOX1-L) in a sample, the method comprising contacting the sample with a capture antibody under conditions that promote binding of the capture antibody with QSOX1-L and with the short isoform of QSOX1 (QSOX1-S); removing unbound sample; contacting the bound QSOX1-L and QSOX1-S with a QSOX1-L antibody under conditions that promote binding of the QSOX1-L antibody with QSOX1-L, wherein the QSOX1-L antibody does not bind to QSOX1-S; removing unbound QSOX1-L antibody; and detecting bound QSOX1-L antibody.

[0038] The QSOX1-L antibody may be the antibody produced from hybridoma 56-3 described herein.

[0039] The capture antibody is capable of binding QSOX1-L and QSOX1-S. In preferred embodiments, the capture antibody is the 2F1 antibody described in International Publication No. WO2022 / 132859, which is incorporated herein by reference in its entirety. The 2F1 antibody VH CDR1 comprises or consists of SEQ ID NO: 1 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, the VH CDR2 comprises or consists of SEQ ID NO: 2 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the VH CDR3 comprises or consists of SEQ ID NO: 3 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the VL CDR1 comprises or consists of SEQ ID NO: 4 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the VL CDR2 comprises or consists of SEQ ID NO: 5 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and the VL CDR3 comprises or consists of SEQ ID NO: 6 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. The 2F1 antibody VH comprises or consists of SEQ ID NO: 13, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of SEQ ID NO: 14 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0040] In embodiments, the capture antibody is the 3A10. The 3A10 antibody is described in International Publication No. WO2022 / 132859, which is incorporated herein by reference in its entirety. The 3A10 antibody VH CDR1 comprises or consists of SEQ ID NO: 7 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, the VH CDR2 comprises or consists of SEQ ID NO: 8 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, the VH CDR3 comprises or consists of SEQ ID NO: 9 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, the VL CDR1 comprises or consists of SEQ ID NO: 10 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity thereto, the VL CDR2 comprises or consists of SEQ ID NO: 11 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, the VL CDR 3 comprises or consists of SEQ ID NO: 12 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. The 3A10 antibody VH comprises or consists of SEQ ID NO: 15, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of SEQ ID NO: 16 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0041] The method may further comprise detecting both the QSOX1-L isoform and the QSOX1- S isoform. The method comprises detecting the QSOX1-L isoform in a first portion of the sample. In a second portion of the sample, the method comprises contacting the second portion of the sample with the capture antibody under conditions that promote binding of the capture antibody with QSOX1-L and QSOX1-S; removing unbound sample; contacting bound QSOX1-L and QSOX1-S with a total QSOX1 antibody under conditions that promote binding of the total QSOX1 antibody with QSOX1-L and QSOX1-S; removing unbound total QSOX1 antibody; detecting bound total QSOX1 antibody; and measuring the amount of bound QSOX1-L antibody and bound total QSOX1 antibody, wherein a difference between the amount of bound QSOX1-L antibody and bound total QSOX1 antibody detects the presence of QSOX1-S.

[0042] In preferred embodiments, the capture antibody comprises the 2F1 antibody described above, and the total QSOX1 antibody comprises the 3A10 antibody described above.

[0043] Any of the antibodies provided herein may comprise a detectable label. Non-limiting examples of detectable labels include fluorophores (e.g. GFPs and fluoresceins), horseradish peroxidases, alkaline phosphatases, luminescent agents, luciferase, radioisotopes, biochemical tags (e.g. biotin or avidin), colorimetric reagents, and combinations thereof. In exemplary embodiments, the QSOX1-L antibody and the total QSOX1 antibody are linked to biotin. In these embodiments, the detection step may comprise adding HRP-conjugated streptavidin (SA-HRP), and detecting the HRP. In exemplary embodiments, the detection methods are performed with an ELISA assay.

[0044] In a third aspect, provided herein is a method for detecting a long isoform of QSOX1 (QSOX1-L) in a sample, the method comprising contacting the sample with a QSOX1-L antibodyunder conditions that promote binding of the QSOX1-L antibody with QSOX1-L; and detecting bound QSOX1-L in the sample. The QSOX1-L antibody may be linked to a detectable label. The QSOX1-L antibody maybe linked to a solid support.

[0045] In the methods described herein, the capture antibody may be attached to a solid support. Non-limiting examples of a solid support include substrates, polymers, beads, microarrays, microtiter plates, slides, tissue culture plates, and tissue culture wells. When the capture antibody is attached to a solid support, the steps of removing unbound sample and removing unbound detection antibodies may be performed by washing the captured antibodies and proteins.

[0046] The methods of detecting the QSOX1 isoforms may further comprise measuring the levels and / or concentrations of the antibody-QSOX1 complexes. The methods may comprise comparing the levels and / or concentrations of the QSOX1-L and QSOX1-S isoforms, where the level / concentration of the QSOX1-S isoform is determined by subtracting the QSOX1 levels / concentrated measured in a sample contacted with the QSOX1-L antibody from the QSOX1 levels / concentrations measured in a sample contacted with the total QSOX1 antibody. The level of QSOX1 in a sample and / or the level of QSOX1-L compared to QSOX1-S may be correlated with a disease or condition in the individual from which the sample was derived. Thus, a determined level of the QSOX1 isoforms from a sample can provide information, e.g., diagnostic, prognostic and / or therapeutic response information, about a subject, including, such as, whether the subject would benefit from a particular therapeutic modality, survival prospects, and / or whether the subject would benefit from additional screening for a particular cancer or additional diagnostic method.

[0047] The sample may be a plasma sample, a serum sample, a blood sample, or any other bodily substance that expresses QSOX1 at sufficient levels for detection and measurement. The sample may be taken from a subject having or suspected of having a disease or disorder related to expression of the QSOX1-L and / or QSOX1-S isoform. For example, the disease or condition may be a cancer or acute decompensated heart failure. The terms “subject” and “individual” are used interchangeably herein to refer to a mammal, preferably a human.

[0048] The subject may be in need of treatment with either an antibody that binds human QSOX1, or a combination of such antibody with an immune checkpoint inhibitor. Therefore, the method may further comprise treating the subject with a humanized form of any of the QSOX1 antibodies described herein.

[0049] A method of diagnosis cancer in an individual may comprise obtaining a sample from the subject and determining the level of QSOX1 in the sample using at least one of the antibodies provided herein, where an increase in QSOX1 in the sample as compared to a control is indicative of cancer in the subject. In some embodiments, a level of QSOX1 of greater than 500 ng / mL, 750 ng / mL, 1,000 ng / mL, 1,250 ng / mL, 1,500 ng / mL, 3,000 ng / mL, 4,000 ng / mL, 5,000 ng / mL, 7,500 ng / mL, 10,000 ng / mL, and / or 15,000 ng / mL is indicative of the presence of a cancer in the subject.

[0050] The term “cancer” is used herein to refer to all types of cancer, neoplasm or malignant tumors found in mammals (e.g., humans), including leukemias, lymphomas, carcinomas and sarcomas. Exemplary cancers that may be treated with a composition, combination or method provided herein include solid, non-lymphoid tumors, B cell leukemias, Non-Hodgkin's Lymphoma, and multiple myeloma.

[0051] The term “solid, non-lymphoid tumor” is used herein, for purposes of the specification and claims, to mean any primary tumor of epithelial cell origin, including tumors originating in an organ or gland such as liver, lung, brain, adrenal gland, breast, colon, bladder, pancreas, stomach, prostate, gastrointestinal tract, or reproductive tract (cervix, ovaries, endometrium etc.), or metastases thereof. For the purposes of the present invention, “solid, non-lymphoid tumor” also includes melanoma.

[0052] The phrase “therapeutically effective amount” means an amount of a composition or combination that results in a therapeutic effect following administration to an individual in need of such composition or combination. In immunotherapy, the therapeutic effect may be represented by activation of a T cell response that is suppressed prior to treatment with a method described herein. Such activation may be measured by an increase in one or more T cell subpopulations (e.g., CD4+T cells, CD8+T cells) using methods known in the art (e.g., labeling with detectable markers followed by flow cytometry analyses) or of the induced or increased expression of activation markers for such T cell subpopulations (e.g., increased CD44 or decreased CD62L expression). Alternatively, activation may also be measured by a decrease in the number or function of regulatory T cells (e.g., CD25+FoxP3+, CD4+cells). Thus, therapeutic efficacy may be assessed by clinical outcome; an increase in the number of anti-tumor T cells or activated T cells as compared with the number prior to treatment or in absence of treatment with a combination of an antibody that binds human QSOX1 and an immune checkpoint inhibitor.

[0053] The terms “treat”, “treating”, or “treatment” as used herein, embrace one or more of preventative (prophylactically) or therapeutically (palliative). As used herein, the terms “treat”, “treating”, or “treatment” include but are not limited to initiating, enhancing, or prolonging an anti-cancer response. In treatment of cancer, a therapeutic effect may include but is not limited to, one or more of (a) an immune-related response, as known to those skilled in the art as an immune- related complete response or an immune-related partial response relative to total tumor burden (e.g., an anti-tumor immune response); and (b) traditional overall objective response rate using the appropriate response assessment criteria known to those skilled in the art and depending on the type of cancer treated (e.g., for lymphoma, see Cheson et al., 2014, J. Clin. Oncology 32 (27): 3059-3067; for solid nonlymphoid tumors, Response Evaluation Criteria In Solid Tumors (RECIST)) (e.g., an antitumor response).

[0054] Methods for treating cancer or initiating, enhancing, or prolonging an anti-tumor or anti- cancer response in a subject may include administering an antibody provided herein to the subject. In any of the methods of treatment provided herein, the dosage of an antibody will depend on such factors as the mode of administration, the formulation for administration, type of cancer, stage of cancer, the size and health of the individual to receive such a composition, and other factors which can be taken into consideration by a medical practitioner whom is skilled in the art of determining appropriate dosages for treatment. For example, for methods of treatment provided herein, an antibody may be administered in a dosage range (per body weight of the individual) that is between about 0.1 mg / kg to about 50 mg / kg, about 0.5 mg / kg to about 20 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 5 mg / kg, or 0.5 mg / kg to about 1 mg / kg. One skilled in the art can apply known principles and models of drug delivery and pharmacokinetics to ascertain a likely range of dosages to be tested in preclinical and clinical studies for determining a therapeutically effective amount of a composition or combination used in the methods of treatment provided herein. A composition or combination, useful in a method of treatment provided herein, may further comprise a pharmaceutically acceptable carrier to facilitate one or more of storage, stability, administration, and delivery. The carrier may be particulate, so that the composition or combination may be in, for example, powder or solid form. The carrier may be in a semi-solid, gel, or liquid formula, so that the composition or combination may be injected, applied, or otherwise administered. The mode of administration of a composition or combination, useful in a method of treatment provided herein, to an individual (such as a human) in need of thereof maybe any mode known in the art to be suitable for delivering a pharmaceutical composition, and particularly suitable for treating cancer. A mode of administration may include but is not limited to, intravenously, intraperitoneally, subcutaneously, intramuscularly, by perfusion, and by peristaltic techniques. A composition or combination, useful in a method of treatment provided herein, may also be combined with other cancer treatments known to those skilled in the art, including but not limited to chemotherapeutic treatment and radiation therapies.

[0055] In a method of treatment provided herein, an antibody may be administered to an individual at a suitable frequency to be therapeutically effective. For example, an antibody may be administered once, more than once or over the course of multiple days or even weeks.

[0056] Kits

[0057] In a fourth aspect, provided herein is a kit comprising reagents that may be used to detect the presence of the QSOX1 isoforms. The kit may comprise the QSOX1-L antibody, the total QSOX1 antibody, and the capture antibody described herein. The kit may further comprise any buffers and reagents needed to perform an ELISA assay. The kit may comprise one or more control samples. Negative control samples may include samples lacking QSOX1. Positive control samples may include samples comprising various concentrations of QSOX1. The concentrations may be provided in a serial dilution.

[0058] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0059] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0060] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted tobe partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of” and “consisting of” those certain elements.

[0061] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0062] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0063] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and theapplicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0064] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0065] The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

[0066] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0067] EXAMPLES

[0068] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0069] Example 1

[0070] Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme which catalyzes disulfide bond formation in proteins in the secretory pathway [1–3] by utilizing a thioredoxin (Trx) domain, an ERV / ALR domain, and a FAD-binding domain [4,5]. Two CxxC motifs (C70xxC73, C449xxC452) shuttle electrons to FAD, where O2 is reduced to H2O2 [6–9]. This conversion of O2to H2O2 allows for measurement of QSOX1 activity using an oxygen electrode

[0010] or a fluorescent-based H2O2 detection assay using homovanillic acid (HVA)

[0011] , in addition to measuring a decrease in thiols with DTNB

[0012] . Some of the enzymology of QSOX1 has been performed with dithiothreitol (DTT), a surrogate substrate [1], but it has been suggested that the cellular substrates of QSOX1 include secreted and extracellular matrix (ECM) proteins, e.g. laminin α4 [13,14] and matrix metalloproteinases

[0015] . This suggests that QSOX1 may play a significant role in modulating the ECM and cellular microenvironment.

[0071] There are two major isoforms of QSOX1 [2,16]; a short isoform (QSOX1-S) that is 604 amino acids and a long isoform (QSOX1-L) that is 747 amino acids. Residues 1-602 are identical between the two isoforms, with residues 603-604 being a leucine and isoleucine in the short form and alanine and serine in the long form. The long form contains an additional 143 amino acids which includes a putative transmembrane domain at the C-terminal end. QSOX1 contains 12 exons, and these isoforms are the result of alternative splicing within exon 12, which is subdivided into exons 12a, 12b, and 12c. The entirety of exon 12 is included in QSOX1-L transcripts while exon 12b is spliced out to generate QSOX1-S transcripts [1]. Soloviev et al. identified additional splice variants of QSOX1, but they did not find evidence that these splice variants are translated into protein

[0017] .

[0072] QSOX1-S is thought to be predominantly secreted by cells into the ECM [18,19] while QSOX1-L is thought to be localized to the endoplasmic reticulum (ER) and Golgi apparatus [3,18,20] in addition to being secreted, though this secretion was shown only in a laboratory setting with cell lines overexpressing QSOX1 and has yet to be shown in serum or plasma. Secretion of QSOX1-L involves a proteolytic cleavage between residues 644-694 which releases QSOX1-L from the membrane

[0020] , but QSOX1-S lacks these residues and a transmembrane domain and does not undergo cleavage.

[0073] Studies have uncovered potential clinical importance for QSOX1. Our group identified a peptide (NEQEQPLGQWHLS) (SEQ ID NO: 17) specific to QSOX1-L in plasma from pancreatic cancer patients but not in healthy donors

[0021] . Additional studies show QSOX1 to be a viable biomarker for breast cancer

[0022] , neuroblastoma

[0023] , mesothelioma

[0024] , and colorectal cancer

[0025] . Further investigation from our group has revealed that QSOX1 promotes tumor cell invasion in pancreatic

[0026] , breast

[0015] , and renal cancer [27,28], while other groups have linked QSOX1 to tumor invasiveness and progression in a number of other cancer types including prostate

[0029] ,lung

[0030] , and glioblastoma

[0031] . QSOX1 has also been identified as a possible biomarker for acute decompensated heart failure (ADHF)

[0032] . Further, the role of QSOX1 in acute heart failure (AHF) was described as helping to resolve acute stress by folding proteins in the ER

[0033] . Taken together, there appears to be a clinical need to detect and measure QSOX1 in patient serum or plasma samples.

[0074] To address the need to detect QSOX1 in patient samples, we developed tandem antigen capture ELISAs: one which measures both isoforms of QSOX1 (Total QSOX1 ELISA) and one which specifically measures QSOX1-L (QSOX1-L ELISA), allowing for extrapolation of QSOX1-S levels. While QSOX1 ELISA kits are commercially available, they claim to measure total QSOX1 only, and our identification of a QSOX1-L-specific peptide in pancreatic cancer patient plasma

[0021] suggests that differentiating isoforms could be important in distinguishing disease states. In addition, a novel version of the previously mentioned fluorescence-based H2O2 detection assay was used to quantify QSOX1 levels in human serum by measuring sulfhydryl oxidase activity

[0034] ; however, as noted by the authors, this innovative, rapid assay has the potential to be influenced by residual H2O2 or free thiols (e.g. free cysteine or glutathione) present in the sample aside from the DTT added. The antigen capture ELISAs reported here are unaffected by H2O2or free thiols in serum or plasma.

[0075] Our ELISAs show for the first time that intact QSOX1-L is present in human serum and plasma and that QSOX1-L levels are slightly higher in plasma than serum. We also demonstrate that individuals ≥40 years of age are more likely to have lower levels of QSOX1-L and higher levels of QSOX1-S in serum than individuals <40 years old. Our results suggest that these quantitative assays could be used for QSOX1 biomarker studies.

[0076] METHODS

[0077] Human Subjects

[0078] Serum and plasma samples were obtained from peripheral blood in serum and EDTA plasma collection tubes from individuals with no knowledge of disease. All donors were consented to protocols approved by ASU Institutional Review Board (IRB) under protocol #0601000548 and Mayo Clinic IRB approval #13-002439. Regarding these samples, reference to “sex” indicates sex assigned at birth.

[0079] Recombinant protein production

[0080] The genes for the short and long isoforms of QSOX1 were amplified from MDA-MB-231 cDNA, obtained with a Cells-to-cDNA kit (Ambion), using Phusion Flash Master Mix (Thermo) and cloned into pcDNA3.1 V5 / His A (Thermo) using Cold Fusion cloning (System Biosciences). Mutants of rQSOX1-S were generated using splicing by overlap extension with primers containing point mutations which caused the cysteine to serine change. All genes were sequenced by Sanger sequencing (Arizona State University Genomics Core) and verified against the NCBI database. Plasmids were prepared using the Nucleobond Xtra Maxi Plus kit (Macherey-Nagel) and transfected into FreeStyle™ 293-F Cells (Invitrogen) cells at a 1:2 DNA:lipid ratio using the FreeStyle™ MAX Reagent (Invitrogen). Supernatants were harvested 6-7 days post transfection and filtered using a 0.45 um PES filtration unit. rQSOX1 was purified by immobilized metal affinity chromatography using Ni+ Agarose beads (Cube Biotech) followed by ion exchange chromatography using SP-Sepharose beads (Cytiva). Proteins were buffer-exchanged into phosphate-buffered saline (PBS) and concentrated using a 30 kDa Amicon centrifuge filter (Millipore), quantified using the BCA Protein Assay Kit (Thermo), and stored at -80°C.

[0081] In-gel protein digestion and mass spectrometry

[0082] SDS-PAGE was performed by running 1 μg rQSOX1-S and 1 μg rQSOX1-L on a 10% acrylamide gel under reducing conditions at 150V for one hour. Gels were stained with “blue silver” stain

[0035] for one hour and destained in LC-MS-grade H2O (Honeywell) for 30 minutes. Protein bands corresponding to the molecular weights of rQSOX1-S and rQSOX1-L were excised, cut into 2 mm cubes, and processed in low protein binding tubes for LC-MS / MS as previously reported

[0036] . In short, gels cubes were destained in 100 mM ammonium bicarbonate and 50-100% acetonitrile (CAN), reduced in 10 mM DTT, alkylated with 55 mM iodoacetamide (IAA), and digested with 20 ng / μL MS-grade trypsin (Thermo) overnight at 37°C. Peptides were extracted with 5% formic acid and ACN and dried in a CentriVap (Labconco) at 30°C. Prepared samples were submitted to the Mass Spectrometry Core at Arizona State University where LC-MS / MS was run on an Orbitrap Fusion Lumos Tribrid (Thermo). Spectra were analyzed in Proteome Discoverer against a human proteome FASTA database (Proteome ID: UP000005640).

[0083] Generation of hybridomas and purification of monoclonal antibodies

[0084] Murine monoclonal antibodies (mAbs) were generated as previously reported

[0037] . Briefly, BALB / c mice were immunized with 50 μg of rQSOX1-S in Magic Mouse Adjuvant (Creative Diagnostics). Mice were boosted twice and sacrificed after serum titers exceeded 1:10,000 byELISA. Spleens were excised and processed to obtain a single cell suspension of splenocytes which were fused with P3X63Ag8.653 myeloma cells and cultured in hypoxanthine-aminopterin- thymidine (HAT) selective medium (Sigma Aldrich) in a 96-well plate for ten days at 37°C with 5% CO2. Supernatants from each well were screened for anti-rQSOX1-S by indirect ELISA and positive wells were subcloned by limiting dilution and screened again after ten days of incubation. Positive subclones were cultured in DMEM (Corning) supplemented with 20% fetal bovine serum (FBS), NCTC-109 (Invitrogen), OPI (Sigma), HEPES (Sigma), Glutamax I (LTI), and Penicillin / Streptomycin (Invitrogen), termed “HY media.” A peptide previously identified from patients with pancreatic ductal adenocarcinoma (NEQEQPLGQWHLS) (SEQ ID NO: 17)

[0021] was synthesized and sent to Spring Bioscience Corp (now part of Roche Molecular Systems Inc) for generation of rabbit hybridomas secreting mAbs specific for the peptide. A hybridoma producing rabbit mAb 56-3 was generated. The hybridoma was cultured in HY media (see above). MAbs were purified from both mouse and rabbit hybridoma supernatants on Mabselect Protein A resin (Cytiva), then buffer exchanged, concentrated, and quantified the same as rQSOX1. MAbs were stored at -20°C.

[0085] Western blot

[0086] SDS-PAGE was performed by running 50 ng rQSOX1 on 10% acrylamide SDS-PAGE gels under reducing conditions at 150 V for one hour. Proteins were transferred onto a PVDF membrane (BioRad) at 90 V for one and a half hours. Membranes were blocked in 1% bovine serum albumin (BSA) in PBS overnight at 4°C then probed with anti-QSOX1 mAbs 2F1, 3A10, and 56-3 at 1 ug / mL in 1% BSA in PBS with 0.1% Tween-20 (PBST) for one hour followed by four washes with PBST. Goat anti-mouse IgG and goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase (HRP) (Jackson ImmunoResearch) were added respectively at a 1:50,000 dilution in 1% BSA in PBST for one hour followed by four washes with PBST and two washes with PBS. Membranes were developed using SuperSignal West Pico PLUS chemiluminescent substrate (Thermo) and imaged on an Azure 600 (Azure Biosystems). Western blot figures were formatted and generated in Sciugo.

[0087] Antigen capture ELISA

[0088] MAb 2F1 was coated onto 96-well high-binding flat bottom plates (Corning) at 1 ug / mL in carbonate coating buffer (pH 9.2) overnight at 4°C. Plates were washed four times with PBST and blocked with 1% BSA in PBS for one hour. Blocking solution was discarded, followed by theaddition of rQSOX1 as a standard and / or sera or plasma samples. Two-fold serial dilutions of rQSOX1-L were added starting at 5 nM for the Total QSOX1 ELISA and 625 pM for the QSOX1- L ELISA. Sera and plasma were initially diluted 1:10 in 1% BSA in PBST then 5-fold to ensure an OD450 which fell within the standard curve. Standards and samples were incubated for one hour followed by four washes with PBST. Biotinylated mAb 3A10 and biotinylated mAb 56-3 were added as detection antibodies at 1 ug / mL for one hour. Plates were then washed four times as before with PBST followed by the addition of HRP-conjugated streptavidin (SA-HRP) at a pre- determined dilution of 1:5000 for one hour. A final round of four washes was done, and plates were developed with 3,3’,5,5’ tetramethyl benzidine (TMB) for ten minutes then stopped with 2N H2SO4. Plates were read on a Spectramax i3X (Molecular Devices) at a wavelength of 450 nm. Concentrations of QSOX1 in serum and plasma were calculated in SoftMax Pro 7.1 by fitting a 4- parameter logistic curve to the standard dilutions and averaging calculated concentrations from OD450 values which fell within the linear portion of the standard curve. QSOX1-S values were calculated by subtracting the QSOX1-L concentration from the total QSOX1 concentration. For cases where the calculated concentration of QSOX1-S was negative (six serum samples and six plasma samples), these values were set to zero for analysis and graphing.

[0089] Assay limits

[0090] The limit of blank (LoB) and limit of detection (LoD) of the Total QSOX1 and QSOX1-L ELISAs were calculated based on equations from Armbruster and Pry

[0038] . rQSOX1-L was added to the Total QSOX1 ELISA at 150 pM and to the QSOX1-L ELISA at 10 pM as low concentrations of analyte along with PBS only (blank) in 24 replicates each. The mean OD450 of blank, standard deviation of blank, and standard deviation of low concentration of QSOX1 were used to determine the limit of blank and limit of detection according to Armbruster and Pry’s equations. The limit of blank and limit of detection from these calculations were given as an OD450, which was converted to a concentration using the standard curves in FIG. 3D and the Four Parameter Logistic (4PL) Curve Calculator from AAT Bioquest.

[0091] Statistical analysis

[0092] The statistical significance of differences in the QSOX1 levels between sample types and isoforms was determined by two-way ANOVA (with matched data points) with Geisser- Greenhouse correction (for lack of assumption of sphericity) followed by Šídák’s multiple comparisons test. Statistical analyses and graphing were performed in GraphPad Prism 9.

[0093] RESULTS

[0094] Characterization of anti-QSOX1 monoclonal antibodies

[0095] MAbs purified from hybridoma clones which were reactive in ELISA screenings were tested by western blotting. Western blots run with both isoforms of rQSOX1 show that mAb 56-3 specifically binds to rQSOX1-L while mAbs 2F1 and 3A10 bind both isoforms (FIG. 1A). The binding of mAb 2F1 was further characterized by probing mutated rQSOX1-S in which C70, C73, C449, and C452 were independently mutated to a serine. MAb 2F1 did not bind to the C70S or C73S mutants (FIG.1B), suggesting the epitope of mAb 2F1 includes the first CxxC motif. Since mAb 56-3 was generated against the NEQEQPLGQWHLS (SEQ ID NO: 17) peptide from QSOX1-L residues 631-643, the epitope is known and displayed in FIG.1C. We have not mapped to epitope of mAb 3A10 at this time.

[0096] To ensure mAb specificity, protein bands corresponding to mAb 2F1, 3A10, and 56-3 reactivity on western blotting were cut from an SDS-PAGE and subjected to LC-MS / MS. As shown in FIG. 2 and consistent with our western blots, peptide coverage maps for the bands correspond to rQSOX1-S and rQSOX1-L with 59.71% coverage for rQSOX1-S and 57.83% coverage for rQSOX1-L. Importantly, the rQSOX1-L sample contained peptides specific for QSOX1-L while the rQSOX1-S did not contain any peptides specific for QSOX1-L.

[0097] Development of tandem antigen capture ELISAs

[0098] Multiple pairs and orientations of antigen capture ELISAs were iteratively tested using the three monoclonal antibodies as either capture or biotinylated detection mAbs. The optimal pairings and orientations used mAb 2F1 as the capturing mAb for both Total QSOX1 and QSOX1-L assays and used biotinylated mAb 3A10 to detect total QSOX1 and biotinylated mAb 56-3 to detect QSOX1-L (FIGS. 3A and 3B). The specificity of each assay for isoforms of QSOX1 was tested by individually adding equimolar amounts of rQSOX1-S and rQSOX1-L to both assays. The Total QSOX1 ELISA detected both isoforms of QSOX1, although it produced higher signal with rQSOX1-S than rQSOX1-L, and the QSOX1-L ELISA discriminated rQSOX1-L from rQSOX1- S (FIG.3C). Standard curves were generated for each ELISA, which show a quantifiable range of 5 nM rQSOX1-L diluted 2-fold to 0.039 nM for the Total QSOX1 ELISA and 625 pM rQSOX1- L diluted 2-fold to 4.88 pM for the QSOX1-L ELISA (FIG.3D). LoB and LoD for each ELISA are found in Table 1.

[0099] Measuring QSOX1 isoform levels in healthy donor sera and plasma

[0100] With parameters for the ELISAs established, we tested 76 paired serum and plasma samples from healthy donors and quantified the concentration of total QSOX1 and QSOX1-L, followed by calculation of the concentration of QSOX1-S by subtracting QSOX1-L from total QSOX1. The range of QSOX1 concentrations measured by each assay in these samples is displayed in Table 1.

[0101] Table 1. Limit of blank, limit of detection, and range of tandem QSOX1 ELISAs.

[0102] Limit of blank and limit of detection were calculated with 24 replicates each of a low concentration of rQSOX1-L and PBS based on equations determined by Armbruster and Pry

[0038] . Range was determined by the lowest and highest concentrations of QSOX1 measured. QSOX1 concentrations were analyzed by sample type (serum vs. plasma), age (<40 n=15, ≥40 n=61), and sex (female n=36, male n=40). The mean concentrations of QSOX1 in serum and plasma were as follows: total QSOX1 was measured at 9.40 nM in serum and 14.7 nM in plasma, QSOX1-L was measured at 3.21 nM in serum and 5.63 nM in plasma, QSOX1-S was measured at 6.55 nM in serum and 9.19 nM in plasma (FIG. 4A). The difference in QSOX1-L concentration between serum and plasma was statistically significant (**p=0.006), while the differences for total QSOX1 and QSOX1-S were not (FIG. 4A). Sera from individuals ≥40 years old had less than half the concentration of QSOX1-L than samples from individuals <40 years old, 2.57 nM and 5.82 nM, respectively (***p=0.0005; FIG.4B). There were slight differences in QSOX1-L between females and males in sera and plasma, though they were not very statistically significant (serum p=0.3672, plasma *p=0.0295; FIG.4C).

[0103] DISCUSSION

[0104] QSOX1 has been identified as a possible biomarker for multiple cancer types [21–25] and ADHF

[0032] , suggesting that quantitative assays to measure QSOX1 may be useful. Because there were no assays to detect QSOX1-L and QSOX1-S splice variants independently, we developed tandem ELISAs to meet the potential clinical need for measuring both isoforms of this potential biomarker in serum and plasma. By pairing mAb 56-3, which binds to a C-terminal peptide from QSOX1-L, with mAb 2F1 which binds to C70xxC73 near the N-terminus of the protein, we detected only QSOX1-L. Total QSOX1 was detected using mAb 2F1 paired with mAb 3A10which binds both QSOX1-S and L. Since the 604 amino acid sequence of QSOX1-S is contained entirely within QSOX1-L (except for the last two amino acids at the end of QSOX1-S), subtracting levels of QSOX1-L from total QSOX1 protein provides levels of QSOX1-S. LC-MS / MS of protein bands corresponding to those which mAbs 2F1, 3A10, and 56-3 bind to confirmed that mAbs 2F1 and 3A10 bind to QSOX1-S and QSOX1-S (FIGS.1 and 2A), which suggests that our mAbs can specifically detect total QSOX1 and that mAb 56-3 discriminates QSOX1-L from QSOX1-S. Additionally, the novel detection of QSOX1-L with mAbs 2F1 and 56-3 suggests that QSOX1-L exists in plasma and sera in an intact state because mAb 2F1 binds at C70xxC73 and mAb 56-3 binds at residues 631-643.

[0105] In healthy donor serum, our ELISAs detected QSOX1 at a wide range of concentrations using neat serum and plasma (Table 1). The average concentration of total QSOX1 in serum determined by our ELISA was 9.4 nM. Israel et al. reported a concentration of 25 nM QSOX1 using their enzymatic assay to measure sulfhydryl oxidase activity in serum and compare that to the activity of rQSOX1 at known concentrations

[0034] . The disparity between our findings and Israel et al. could be attributed to the previously mentioned possibility of H2O2 or thiol sources being present in the serum natively which would inflate the apparent sulfhydryl oxidase activity and thusly report a higher concentration of QSOX1 than is actually present. Our ELISAs are unaffected by H2O2 or free thiols in serum and / or plasma. Another reason could be that Israel et al. measured QSOX1 levels in pooled serum. As we show in our ELISA, individual donors may have very different levels of QSOX1 such that a few samples with elevated levels of QSOX1 could skew an “average level” of QSOX1 in pooled serum.

[0106] Of the several statistically significant differences across sample designations and isoforms, the most notable are the higher QSOX1-L concentration in plasma compared to serum (FIG.4A) and the decrease in QSOX1-L concentrations with increased age in serum (FIG.4C). A potential reason for the higher concentration of QSOX1-L in plasma could be that QSOX1-L is consumed by or involved in the clotting cascade as this is a major difference between plasma and serum. The role of thiol oxidoreductases, including QSOX1, have been noted for their role in blood coagulation

[0039] . This function has been better characterized for protein disulfide isomerase (PDI) specifically

[0040] , but QSOX1 shares a PDI-like Trx domain and may play a similar role. Regarding the decrease in QSOX1-L with increasing age in serum, this difference may be an artifact of the smaller sample size (n=15) of the <40 years old samples compared to the ≥40 years old samples(n=61). The younger samples here could all have lower levels of QSOX1-L by happenstance, and collecting and quantifying more samples from younger individuals could reduce the difference between the two groups.

[0107] The differing limits of blank and detection between the two ELISAs (Table 1) are likely due to differences in the binding characteristics of the detecting antibodies for each assay, mAbs 3A10 and 56-3. MAb 56-3 may have a higher binding affinity than mAb 3A10, allowing for detection of lower concentrations of QSOX1-L; however, this requires further characterization of the mAbs.

[0108] The tandem antigen capture ELISAs described here might be useful as clinically relevant quantitative assays in detecting cancer or ADHF (where appropriate) with their high sensitivity and ability to distinguish and quantify QSOX1 isoforms. While samples from patients with cancer or ADHF need to be tested, these assays could prove to be a valuable diagnostic tool for physicians in oncology and cardiology.

[0109] REFERENCES [1] C. Thorpe, K.L. Hoober, S. Raje, N.M. Glynn, J. Burnside, G.K. Turi, D.L. Coppock, Sulfhydryl oxidases: Emerging catalysts of protein disulfide bond formation in eukaryotes, Arch. Biochem. Biophys.405 (2002) 1–12. https: / / doi.org / 10.1016 / S0003-9861(02)00337-5. [2] V.K. Kodali, C. Thorpe, Oxidative protein folding and the quiescin-sulfhydryl oxidase family of flavoproteins, Antioxidants Redox Signal. 13 (2010) 1217–1230. https: / / doi.org / 10.1089 / ars.2010.3098. [3] S. Chakravarthi, C.E. Jessop, M. Willer, C.J. Stirling, N.J. Bulleid, Intracellular catalysis of disulfide bond formation by the human sulfhydryl oxidase, QSOX1, Biochem. J.404 (2007) 403– 411. https: / / doi.org / 10.1042 / BJ20061510. [4] D.L. Coppock, D. Cina-Poppe, S. Gilleran, The Quiescin Q6 gene (QSCN6) is a fusion of two ancient gene families: Thioredoxin and ERV1, Genomics. 54 (1998) 460–468. https: / / doi.org / 10.1006 / geno.1998.5605. [5] K. Limor-Waisberg, S. Ben-Dor, D. Fass, Diversification of Quiescin sulfhydryl oxidase in a preserved framework for redox relay, BMC Evol. Biol.13 (2013). https: / / doi.org / 10.1186 / 1471- 2148-13-70.[6] E.J. Heckler, A. Alon, D. Fass, C. Thorpe, Human quiescin-sulfhydryl oxidase, QSOX1: Probing internal redox steps by mutagenesis, Biochemistry. 47 (2008) 4955–4963. https: / / doi.org / 10.1021 / bi702522q. [7] W. Zheng, Y. Chu, Q. Yin, L. Xu, C. Yang, W. Zhang, Y. Tang, Y. Yang, Crucial effect of the first CXXC motif of human QSOX 1b on the activity to different substrates, J. Biochem. 149 (2011) 293–300. https: / / doi.org / 10.1093 / jb / mvq143. [8] W. Zheng, W. Zhang, W. Hu, C. Zhang, Y. Yang, Exploring the smallest active fragment of HsQSOX1b and finding a highly efficient oxidative engine, PLoS One. 7 (2012) e40935. https: / / doi.org / 10.1371 / journal.pone.0040935. [9] Y. Gat, A. Vardi-Kilshtain, I. Grossman, D.T. Major, D. Fass, Enzyme structure captures four cysteines aligned for disulfide relay, Protein Sci. 23 (2014) 1102–1112. https: / / doi.org / 10.1002 / pro.2496.

[0010] K.L. Hoober, B. Joneja, H.B. White, C. Thorpe, A sulfhydryl oxidase from chicken egg white, J. Biol. Chem.271 (1996) 30510–30516. https: / / doi.org / 10.1074 / jbc.271.48.30510.

[0011] S. Raje, N.M. Glynn, C. Thorpe, A continuous fluorescence assay for sulfhydryl oxidase, Anal. Biochem.307 (2002) 266–272. https: / / doi.org / 10.1016 / S0003-2697(02)00050-7.

[0012] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82 (1959) 70–77. https: / / doi.org / 10.1016 / 0003-9861(59)90090-6.

[0013] T. Ilani, A. Alon, I. Grossman, B. Horowitz, E. Kartvelishvily, S.R. Cohen, D. Fass, A secreted disulfide catalyst controls extracellular matrix composition and function, Science (80-. ). 341 (2013) 74–76. https: / / doi.org / 10.1126 / science.1238279.

[0014] T. Feldman, I. Grossman-Haham, Y. Elkis, P. Vilela, N. Moskovits, I. Barshack, T.M. Salame, D. Fass, T. Ilani, Inhibition of fibroblast secreted QSOX1 perturbs extracellular matrix in the tumor microenvironment and decreases tumor growth and metastasis in murine cancer models, Oncotarget.11 (2020) 386–398. https: / / doi.org / 10.18632 / oncotarget.27438.

[0015] B.A. Katchman, I.T. Ocal, H.E. Cunliffe, Y.H. Chang, G. Hostetter, A. Watanabe, J. LoBello, D.F. Lake, Expression of quiescin sulfhydryl oxidase 1 is associated with a highly invasive phenotype and correlates with a poor prognosis in Luminal B breast cancer, Breast Cancer Res.15 (2013) 28. https: / / doi.org / 10.1186 / bcr3407.

[0016] J. Radom, D. Colin, F. Thiebault, M. Dognin-Bergeret, G. Mairet-Coello, A. Esnard-Feve, D. Fellmann, M. Jouvenot, Identification and expression of a new splicing variant of FAD-sulfhydryloxidase in adult rat brain, Biochim. Biophys. Acta - Gene Struct. Expr. 1759 (2006) 225–233. https: / / doi.org / 10.1016 / j.bbaexp.2006.04.008.

[0017] M. Soloviev, M.P. Esteves, F. Amiri, M.R. Crompton, C.C. Rider, Elevated Transcription of the Gene QSOX1 Encoding Quiescin Q6 Sulfhydryl Oxidase 1 in Breast Cancer, PLoS One. 8 (2013). https: / / doi.org / 10.1371 / journal.pone.0057327.

[0018] G. Mairet-Coello, A. Tury, A. Esnard-Feve, D. Fellmann, P.Y. Risold, B. Griffond, FAD- Linked Sulfhydryl Oxidase QSOX: Topographic, Cellular, and Subcellular Immunolocalization in Adult Rat Central Nervous System, J. Comp. Neurol. 473 (2004) 334–363. https: / / doi.org / 10.1002 / cne.20126.

[0019] C. Amiot, J.F. Musard, M. Hadjiyiassemis, M. Jouvenot, D. Fellmann, P.Y. Risold, P. Adami, Expression of the secreted FAD-dependent sulfydryl oxidase (QSOX) in the guinea pig central nervous system, Mol. Brain Res. 125 (2004) 13–21. https: / / doi.org / 10.1016 / j.molbrainres.2004.02.024.

[0020] J. Rudolf, M.A. Pringle, N.J. Bulleid, Proteolytic processing of QSOX1A ensures efficient secretion of a potent disulfide catalyst, Biochem. J. 454 (2013) 181–190. https: / / doi.org / 10.1042 / BJ20130360.

[0021] K. Antwi, G. Hostetter, M.J. Demeure, B.A. Katchman, G.A. Decker, Y. Ruiz, T.D. Sielaff, L.J. Koep, D.F. Lake, Analysis of the plasma peptidome from pancreas cancer patients connects a peptide in plasma to overexpression of the parent protein in tumors, J. Proteome Res. 8 (2009) 4722–4731. https: / / doi.org / 10.1021 / pr900414f.

[0022] V. Kulasingam, E.P. Diamandis, Proteomics analysis of conditioned media from three breast cancer cell lines: A mine for biomarkers and therapeutic targets, Mol. Cell. Proteomics.6 (2007) 1997–2011. https: / / doi.org / 10.1074 / mcp.M600465-MCP200.

[0023] D.G.B. Araújo, L. Nakao, P. Gozzo, C.D.A. Souza, V. Balderrama, E.S. Gugelmin, A.P. Kuczynski, M. Olandoski, L. De Noronha, Expression level of quiescin sulfhydryl oxidase 1 (QSOX1) in neuroblastomas, Eur. J. Histochem. 58 (2014) 52–56. https: / / doi.org / 10.4081 / ejh.2014.2228.

[0024] S. Lacerenza, F. Ciregia, L. Giusti, A. Bonotti, V. Greco, G. Giannaccini, V. D’Antongiovanni, P. Fallahi, L. Pieroni, A. Cristaudo, A. Lucacchini, M.R. Mazzoni, R. Foddis, Putative biomarkers for malignant pleural mesothelioma suggested by proteomic analysis of cellsecretome, Cancer Genomics and Proteomics. 17 (2020) 225–236. https: / / doi.org / 10.21873 / cgp.20183.

[0025] N. Ganig, F. Baenke, M.L. Thepkaysone, K. Lin, V.S. Rao, F.C. Wong, H. Polster, M. Schneider, D. Helm, M. Pecqueux, A.M. Seifert, L. Seifert, J. Weitz, N.N. Rahbari, C. Kahlert, Proteomic Analyses of Fibroblast- and Serum-Derived Exosomes Identify QSOX1 as a Marker for Non-invasive Detection of Colorectal Cancer, Cancers (Basel). 13 (2021) 1–22. https: / / doi.org / 10.3390 / CANCERS13061351.

[0026] B.A. Katchman, K. Antwi, G. Hostetter, M.J. Demeure, A. Watanabe, G.A. Decker, L.J. Miller, D.D. Von Hoff, D.F. Lake, Quiescin sulfhydryl oxidase 1 promotes invasion of pancreatic tumor cells mediated by matrix metalloproteinases, Mol. Cancer Res. 9 (2011) 1621–1631. https: / / doi.org / 10.1158 / 1541-7786.MCR-11-0018.

[0027] P.D. Hanavan, C.R. Borges, B.A. Katchman, D.O. Faigel, T.H. Ho, C.T. Ma, E.A. Sergienko, N. Meurice, J.L. Petit, D.F. Lake, Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines, Oncotarget. 6 (2015) 18418–18428. https: / / doi.org / 10.18632 / oncotarget.4099.

[0028] A.L. Fifield, P.D. Hanavan, D.O. Faigel, E. Sergienko, A. Bobkov, N. Meurice, J.L. Petit, A. Polito, T.R. Caulfield, E.P. Castle, J.A. Copland, D. Mukhopadhyay, K. Pal, S.K. Dutta, H. Luo, T.H. Ho, D.F. Lake, Molecular inhibitor of QSOX1 suppresses tumor growth in vivo, Mol. Cancer Ther.19 (2020) 112–122. https: / / doi.org / 10.1158 / 1535-7163.MCT-19-0233.

[0029] J.A. Baek, P.H. Song, Y.H. Ko, M.J. Gu, High expression of QSOX1 is associated with tumor invasiveness and high grades groups in prostate cancer, Pathol. Res. Pract.214 (2018) 964–967. https: / / doi.org / 10.1016 / j.prp.2018.05.019.

[0030] H.J. Sung, J.M. Ahn, Y.H. Yoon, S.S. Na, Y.J. Choi, Y.I. Kim, S.Y. Lee, E.B. Lee, S. Cho, J.Y. Cho, Quiescin sulfhydryl oxidase 1 (QSOX1) secreted by lung cancer cells promotes cancer metastasis, Int. J. Mol. Sci.19 (2018). https: / / doi.org / 10.3390 / ijms19103213.

[0031] Y. Geng, C. Xu, Y. Wang, L. Zhang, Quiescin sulfhydryl oxidase 1 regulates the proliferation, migration and invasion of human glioblastoma cells via PI3K / Akt pathway, Onco. Targets. Ther. 13 (2020) 5721–5729. https: / / doi.org / 10.2147 / OTT.S255941.

[0032] A. Mebazaa, G. Vanpoucke, G. Thomas, K. Verleysen, A. Cohen-Solal, M. Vanderheyden, J. Bartunek, C. Mueller, J.M. Launay, N. Van Landuyt, F. D’Hondt, E. Verschuere, C. Vanhaute,R. Tuytten, L. Vanneste, K. De Cremer, J. Wuyts, H. Davies, P. Moerman, D. Logeart, C. Collet, B. Lortat-Jacob, M. Tavares, W. Laroy, J.L. Januzzi, J.L. Samuel, K. Kas, Unbiased plasma proteomics for novel diagnostic biomarkers in cardiovascular disease: Identification of quiescin Q6 as a candidate biomarker of acutely decompensated heart failure, Eur. Heart J.33 (2012) 2317– 2324. https: / / doi.org / 10.1093 / eurheartj / ehs162.

[0033] A. Caillard, M. Sadoune, A. Cescau, M. Meddour, M. Gandon, E. Polidano, C. Delcayre, K. Da Silva, P. Manivet, A.M. Gomez, A. Cohen-Solal, N. Vodovar, Z. Li, A. Mebazaa, J.L. Samuel, QSOX1, a novel actor of cardiac protection upon acute stress in mice, J. Mol. Cell. Cardiol.119 (2018) 75–86. https: / / doi.org / 10.1016 / j.yjmcc.2018.04.014.

[0034] B.A. Israel, L. Jiang, S.A. Gannon, C. Thorpe, Disulfide bond generation in mammalian blood serum: Detection and purification of quiescin-sulfhydryl oxidase, Free Radic. Biol. Med.69 (2014) 129–135. https: / / doi.org / 10.1016 / j.freeradbiomed.2014.01.020.

[0035] G. Candiano, M. Bruschi, L. Musante, L. Santucci, G.M. Ghiggeri, B. Carnemolla, P. Orecchia, L. Zardi, P.G. Righetti, Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis, Electrophoresis. 25 (2004) 1327–1333. https: / / doi.org / 10.1002 / ELPS.200305844.

[0036] N.M. Mitchell, A.L. Sherrard, S. Dasari, D.M. Magee, T.E. Grys, D.F. Lake, Proteogenomic Re-Annotation of Coccidioides posadasii Strain Silveira, Proteomics. 18 (2018) 1700173. https: / / doi.org / 10.1002 / pmic.201700173.

[0037] F.J. Grill, T.E. Grys, M.F. Grill, A. Roeder, J.E. Blair, D.F. Lake, Development of a Quantitative Antigen Assay to Detect CoccidioidalChitinase-1 (CTS1), Open Forum Infect. Dis. 8 (2021). https: / / doi.org / 10.1093 / OFID / OFAB344.

[0038] D.A. Armbruster, T. Pry, Limit of Blank, Limit of Detection and Limit of Quantitation, Clin. Biochem. Rev.29 (2008) S49. / pmc / articles / PMC2556583 / (accessed March 27, 2023).

[0039] L.Y. Tanaka, P.V.S. Oliveira, F.R.M. Laurindo, Peri / Epicellular Thiol Oxidoreductases as Mediators of Extracellular Redox Signaling, Https: / / Home.Liebertpub.Com / Ars.33 (2020) 280– 307. https: / / doi.org / 10.1089 / ARS.2019.8012.

[0040] R.H. Bekendam, R. Flaumenhaft, Inhibition of Protein Disulfide Isomerase in Thrombosis, Basic Clin. Pharmacol. Toxicol.119 (2016) 42–48. https: / / doi.org / 10.1111 / bcpt.12573.

[0110] SEQUENCES

[0111] Table 2. SequencesSEQ ID Sequence IMGT NO: CDR region 1 GFSLTDYG 2F1; CDR1 VH 2 IWGDGST 2F1; CDR2 VH 3 AGALYDGYYGY 2F1; CDR3 VH 4 QDVSTA 2F1; CDR1 VL 5 SAS 2F1; CDR2 VL 6 QQHYSTPLT 2F1; CDR3 VL 7 GLTFSDAW 3A10; CDR1 VH 8 IRSKAHNHAT 3A10; CDR2 VH 9 TRYGNPFVY 3A10; CDR3 VH 10 SPSLLYSSNQKNY 3A10; CDR1 VL 11 WAS 3A10; CDR2 VL 12 QQYYSYPWT 3A10; CDR3 VL 13 EVQLEESGPGLVAPSQSLSITCTVSGF 2F1; VH SLTDYGVTWIRQPPGKGLEWLGVIW GDGSTYYNSALKSRLSISKDDSKSQV FLKMNSLQTDDTAMYYCAGALYDG YYGYWGQGTTLTVSS 14 DIVLTQTHKFMSTSVGDRVSITCKAS 2F1; CDR3 VL QDVSTAVAWYQQKPGQSPKLLIYSA SYRYSGVPDRFTGSGSGTDFTFTISSV QAEDLAVYYCQQHYSTPLTFGAGTK LELK 15 EVQLQQSGGGLVQPGGSMKLSCAAS 3A10 VH GLTFSDAWMDWVRQSPEKGLEWVA EIRSKAHNHATYYAESVKGRFTISRD DSKSSVYLQMNSLRAEDTGIYYCTR YGNPFVYWGQGTLVTVSADIVMTQSPSSLAVSVGEKVTMSCKSS 3A10 VL PSLLYSSNQKNYLAWYQQKPGQSPK LLIYWASTRESGVPDRFTGSGSGTDF TLTISSVKAEDLAVYYCQQYYSYPW TFGGGTKLEIK NEQEQPLGQWHLS mAB 56-3 targeting peptide GFSLSNYA 56-3; CDR1 VH IASSGFT 56-3; CDR2 VH ARRGPSVGTGTFYFSL 56-3; CDR3 VH QSVYNNKN 56-3; CDR1 VL EAS 56-3; CDR2 VL QGEFACSNTDCIL 56-3; CDR3 VL QSVEESGGRLVMPGTPLTLTCTVSGF 56-3 VH SLSNYAVGWVRQAPGEGLEWIGGIA SSGFTYYASWAKGRFTISKTSSTTVD LKVTSLTAADTATYFCARRGPSVGT GTFYFSLWGPGTLVTISS ALVMTQTPSSVSAAVGGTVTINCQA 56-3 VL SQSVYNNKNLAWYQQKPGQPPKLLI YEASKLASGVPSRFKGSGSGTQFTLTI SSVQCDDAATYYCQGEFACSNTDCIL FGGGTEVVVK

Claims

CLAIMS What is claimed:

1. A QSOX1-L antibody comprising: a heavy chain variable domain (VH) complementary determining region (CDR) CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a light chain variable domain (VL) CDR1 comprising SEQ ID NO: 21, a VL CDR2 comprising SEQ ID NO: 22, and a VL CDR3 comprising SEQ ID NO:

23.

2. The QSOX1-L antibody of claim 2, wherein the VH comprises SEQ ID NO: 24 or a sequence having at least 90% identity thereto, and the VL comprises SEQ ID NO: 25 or a sequence having at least 90% identity thereto.

3. The antibody of claim 1 or 2, wherein the antibody binds to a peptide comprising SEQ ID NO:

17.

4. A method for detecting a long isoform of QSOX1 (QSOX1-L) in a sample, the method comprising: (a) contacting a first portion of the sample with a capture antibody under conditions that promote binding of the capture antibody with QSOX1-L and a short isoform of QSOX1 (QSOX1-S); (b) removing unbound sample; (c) contacting bound QSOX1-L and QSOX1-S with a QSOX1-L antibody under conditions that promote binding of the QSOX1-L antibody with QSOX1-L, wherein the QSOX1-L antibody does not bind to QSOX1-S; (d) removing unbound QSOX1-L antibody; and (e) detecting bound QSOX1-L antibody; wherein the QSOX1-L antibody comprises: a VH CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a VL CDR1 comprising SEQ ID NO: 21,a VL CDR2 comprising SEQ ID NO: 22, and a VL CDR3 comprising SEQ ID NO: 23; wherein the capture antibody comprises: a VH CDR1 comprising SEQ ID NO: 1 or 7, a VH CDR2 comprising SEQ ID NO: 2 or 8, a VH CDR3 comprising SEQ ID NO: 3 or 9, a VL CDR1 comprising SEQ ID NO: 4 or 10, a VL CDR2 comprising SEQ ID NO: 5 or 11, and a VL CDR3 comprising SEQ ID NO: 6 or 12.

5. The method of claim 4, wherein the VH of the QSOX1-L antibody comprises SEQ ID NO: 24 or a sequence having at least 90% identity thereto, and the VL of the QSOX1-L antibody comprises SEQ ID NO: 25 or a sequence having at least 90% identity thereto.

6. The method of claim 4 or 5, wherein the VH of the capture antibody comprises SEQ ID NO: 13 or 15, and the VL of the capture antibody comprises SEQ ID NO: 14 or 16.

7. The method of any one of claims 4-6, wherein the QSOX1-L antibody is linked to a detectable label.

8. The method of any one of claims 4-7, further comprising detecting QSOX1-S in the sample, wherein detecting QSOX1-S comprises: (f) contacting a second portion of the sample with the capture antibody under conditions that promote binding of the capture antibody with QSOX1-L and QSOX1-S; (g) removing unbound sample; (h) contacting bound QSOX1-L and QSOX1-S with a total QSOX1 antibody under conditions that promote binding of the total QSOX1 antibody with QSOX1-L and QSOX1- S; (i) removing unbound total QSOX1 antibody; (j) detecting bound total QSOX1 antibody; (k) measuring the amount of bound QSOX1-L antibody and bound total QSOX1 antibody, wherein a difference between the amount of bound QSOX1-L antibody and bound total QSOX1 antibody detects the presence of QSOX1-S; wherein the capture antibody comprises: the VH CDR1 comprising SEQ ID NO: 1, the VH CDR2 comprising SEQ ID NO: 2, the VH CDR3 comprising SEQ ID NO: 3, the VLCDR1 comprising SEQ ID NO: 4, the VL CDR2 comprising SEQ ID NO: 5, and the VL CDR3 comprising SEQ ID NO: 6; and wherein the total QSOX1 antibody comprises: the VH CDR1 comprising SEQ ID NO: 7, the VH CDR2 comprising SEQ ID NO: 8, the VH CDR3 comprising SEQ ID NO: 9, the VL CDR1 comprising SEQ ID NO: 10, the VL CDR2 comprising SEQ ID NO: 11, and the VL CDR3 comprising SEQ ID NO:

12.

9. The method of claim 8, wherein the capture antibody comprises the VH comprising SEQ ID NO: 13 or a sequence having at least 90% identity thereto, and the VL comprising SEQ ID NO: 14 or a sequence having at least 90% identity thereto; and wherein the total QSOX1 antibody comprises the VH comprising SEQ ID NO: 15 or a sequence having at least 90% identity thereto, and the VL comprising SEQ ID NO: 16 or a sequence having at least 90% identity thereto.

10. The method of any one of claims 4-9, wherein the total QSOX1 antibody is linked to the detectable label.

11. The method of any one of claims 4-10, wherein the capture antibody is attached to a solid support.

12. The method of any one of claims 4-11, wherein the sample comprises a serum or a plasma.

13. The method of any one of claims 4-12, wherein the sample is a human sample.

14. A method for detecting a long isoform of QSOX1 (QSOX1-L) in a sample, the method comprising: (a) contacting the sample with a QSOX1-L antibody under conditions that promote binding of the QSOX1-L antibody with QSOX1-L; (b) detecting bound QSOX1-L in the sample; wherein the QSOX1-L antibody comprises a VH CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a VL CDR1 comprising SEQ ID NO: 21, a VL CDR2 comprising SEQ ID NO: 22, and a VL CDR3 comprising SEQ ID NO: 23.

15. The method of claim 14, wherein the VH comprises SEQ ID NO: 24 or a sequence having at least 90% identity thereto, and the VL comprises SEQ ID NO: 25 or a sequence having at least 90% identity thereto.

16. The method of claim 14 or 15, wherein the QSOX1-L antibody is attached to a solid support.

17. The method of any one of claims 14-16, wherein the QSOX1-L antibody is linked to a detectable label.

18. The method of any one of claims 14-17, wherein the sample comprises serum or plasma.

19. The method of any one of claims 14-18, wherein the sample is a human sample.

20. A kit comprising a QSOX1-L antibody, a total QSOX1 antibody, and a capture antibody; wherein the QSOX1-L antibody comprises: a VH CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a VL CDR1 comprising SEQ ID NO: 21, a VL CDR2 comprising SEQ ID NO: 22, and a VL CDR3 comprising SEQ ID NO: 23; wherein the total QSOX1 antibody comprises: a VH CDR1 comprising SEQ ID NO: 7, a VH CDR2 comprising SEQ ID NO: 8, a VH CDR3 comprising SEQ ID NO: 9, a VL CDR1 comprising SEQ ID NO: 10, a VL CDR2 comprising SEQ ID NO: 11, and a VL CDR3 comprising SEQ ID NO: 12; and wherein the capture antibody comprises: a VH CDR1 comprising SEQ ID NO: 1, a VH CDR2 comprising SEQ ID NO: 2, a VH CDR3 comprising SEQ ID NO: 3, a VL CDR1 comprising SEQ ID NO: 4, a VL CDR2 comprising SEQ ID NO: 5, and a VL CDR3 comprising SEQ ID NO: 6.

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