RO52 targeting antibody or antigen binding fragments thereof for use in treating cancer

Abstract

Provided herein are methods of treating a cancer in a subject, wherein the cancer expresses a Ro52 antigen, comprising administering an anti-Ro52 binding agent to the subject, thereby treating the cancer in the subject. In some embodiments, the Ro52 antigen is expressed on a cancer cell surface. In some embodiments, the anti-Ro52 binding agent comprises a Fab, Fab', F(ab')2, Fahl-SH, Fv, diabody, linear antibody or single-chain variable fragment (scFv). In some embodiments, the anti-Ro52 binding agent is a humanized or chimeric antibody. In some embodiments, the anti-Ro52 binding agent is conjugated to a therapeutic agent.

Description

[0001] RO52 TARGETING ANTIBODY OR ANTIGEN BINDING FRAGMENTS THEREOF

[0002] FOR USE IN TREATING CANCER

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Patent Application No. 63 / 543,136, filed on October 9, 2023, which is incorporated herein by reference in its entirety.

[0005] SEQUENCE LISTING

[0006] This application contains a Sequence Listing that has been submitted electronically as an XML file named 44807-0463WOl_ST26_SL.XML.” The XML file, created on October 8, 2024, is 43,319 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0007] TECHNICAL FIELD

[0008] Described herein are methods of treating and identifying cancers that employ Ro52 targeting antibodies or antigen binding fragments thereof, wherein a Ro52 antigen is expressed by a cancer cell.

[0009] BACKGROUND

[0010] Antibody-based immunotherapy is considered to be one of the main components of cancer therapy, wherein antibodies can directly target cancer cells while simultaneously inducing anti-tumor immune responses. Antibody-drug conjugates (ADCs) are a promising cancer treatment that includes delivering therapeutic drugs to specific tumor cells that exhibit specific antigens connected to malignancy. In some embodiments, ADCs are expected to provide therapeutic modalities against various cancers by combining the selectivity of monoclonal antibodies (mAbs) and the efficacy of various chemotherapeutics. Antibodies or antigen binding fragments thereof, together with a therapeutic payload can provide a highly effective anti-tumor agent directly and selectively providing therapeutic anti-cancer drugs to cancer cells, directed by the antibodies with exceptional specificity and affinity. SUMMARY

[0011] Provided herein are methods of treating a cancer in a subject, wherein the cancer expresses a Ro52 antigen, the method comprising administering an anti-Ro52 binding agent to the subject, thereby treating the cancer in the subject. In some embodiments, the Ro52 antigen is expressed on a cancer cell surface.

[0012] In some embodiments, the anti-Ro52 binding agent comprises a Fab, Fab’, F(ab’)2, Fabl-SH, Fv, diabody, linear antibody or single-chain variable fragment (scFv). In some embodiments, the anti-Ro52 binding agent is a humanized or chimeric antibody. In some embodiments, the anti-Ro52 binding agent is conjugated to a therapeutic agent. In some embodiments, the therapeutic agent comprises a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent comprises DM1, MMAE, MMAF, mertansine, calicheamicin, SN-38, or exatecan. In some embodiments, the chemotherapeutic agent mediates cytotoxicity of the cancer cell.

[0013] In some embodiments, the cancer is a metastatic cancer. In some embodiments, the metastatic cancer is a breast cancer, colon cancer, ovarian cancer, gastric cancer, or pancreatic cancer.

[0014] In some embodiments, prior to administration, the Ro52 antigen is present on the cancer cell surface. In some embodiments, the cancer cell is from a primary tumor sample or a metastatic tumor sample. In some embodiments, the primary tumor sample or the metastatic tumor sample is from a fresh biopsy sample. In some embodiments, the primary tumor sample or the metastatic tumor sample is from a fixed biopsy sample. In some embodiments, the cancer cell comprises circulating tumor cells.

[0015] Also provided herein are methods for identifying a tumor in a subject, the method comprising (a) administering an anti-Ro52 binding agent to the subject; and (b) detecting the anti-Ro52 binding agent on a cell of the tumor in the subject, thereby identifying the tumor in the subject. In some embodiments, the anti-Ro52 binding agent is bound to a cell surface of the cell.

[0016] In some embodiments, the anti-Ro52 binding agent is conjugated to a label. In some embodiments, the label is a detectable label. In some embodiments, the label is a fluorophore. In some embodiments, the detecting step (b) comprises fluorescence imaging, in some embodiments, the anti-Ro52 binding agent is conjugated to an imaging agent, in some embodiments, the detecting step (b) comprises magnetic resonance imaging (MRI) or positron emission tomography (PET) imaging. In some embodiments, the administering step (a) comprises intravenous, intrathecal, intra-thoracic, or intraperitoneal administration.

[0017] 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 this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0018] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0019] BRIEF DESCRIPTION OF DRAWINGS

[0020] FIGs. 1A-1B show autoantibody containing sera from rheumatic disease patients selectively binds breast cancer organoids from unrelated patients without known rheumatic disease. (FIG. 1A) Whole IgG containing sera was isolated from both rheumatic disease patients and healthy donors and used to stain breast cancer organoids generated from tumors in unrelated patients. IgG staining was visualized using fluorescent labeled anti-human secondary antibodies, which can recognize the Fc region of autoantibodies. (FIG. IB) Number of sera isolated from rheumatic disease patients that was able to positively recognize and stain breast cancer organoids.

[0021] FIGs. 2A-2C show patient derived autoantibodies mediate cytotoxic killing of cancer cells through the innate immune system. (FIG. 2A) PDX cell clusters were grown in either monoculture or co-culture with human derived NK cells and treated with whole sera isolated from autoimmune patients. Limited cytotoxicity was observed when PDX cancer cells were treated with sera alone. In the presence of NK cells, sera was able to stimulate a cytotoxic effect against the cancer cells, reducing their ability to form metastatic colonies. (FIG. 2B) Purified SSA / Ro52 autoantibodies isolated from patients stimulate human PBMCs to attack PDX organoids, resulting in a reduction in organoid viability, size, and growth. (FIG. 2C) Purified SSA / Ro52 autoantibodies added to macrophage-MCF7 cocultures stimulate macrophage mediated phagocytosis of the cancer cells. Isotype control antibodies did not cause this effect, which can be observed through the overlay of red and green fluorescence, indicating that the cancer cells were engulfed by the macrophage. FIGs. 3A-3C show the SSA / Ro52 antigen can be recognized on the surface of live cancer cells. (FIG. 3A) Monoclonal antibodies raised against various autoantigens are able to recognize their predicted target in chemically fixed MCF7 cancer cells. (FIG. 3B) Monoclonal antibodies targeting SSA / Ro52 can bind to the surface of living cells, in a pattern distinct from their binding to fixed cells. Antibodies targeting select common autoimmune antigens were not able to bind to the surface of live cells. (FIG. 3C) Antibodies targeting the SSA / Ro52 antigen do not bind to the surface of live MCF10A cells.

[0022] FIGs. 4A-4C show patient derived SSA / Ro52 autoantibodies are internalized into living cells MCF7 and PDX cancer cells over time. (FIG. 4A) Scheme detailing how autoantibody internalization can be detected in live cells. Two fluorescent tagged autoantibodies are introduced to growing cells, one which fluoresces continuously at 594 nm, and second which fluoresces at 647 nm only when endocytosed into acidic cell compartments. (FIG. 4B) SSA / Ro52 patient derived autoantibodies bind to living MCF7 cells after 4 hours and are internalized into cells after 24 hours. Antibody internalization is indicated by the overlapping signal between total and acidic compartment localized antibody. (FIG. 4C) Autoimmune patient derived SSA / Ro52 antibodies are internalized into living PDX cell clusters grown in 3D extracellular matrices. FIG. 5 shows anti-Ro52 antibodies bind to the surface of PDX organoids. Anti-Ro52 antibodies isolated from rheumatology patients bind to the surface of living PDX cancer organoids. PDX organoids were derived from three different patient tumors, and anti-Ro52 antibodies were able to positively bind each.

[0023] FIGs. 6A-6D show interferon treatment induces Ro52 (TRIM21) mRNA and protein expression and type I interferons induce surface expression of the antigen. (FIG. 6A) PDX organoids were treated with high and low doses of interferons for 24 hours, triggering a robust upregulation of TRIM21 mRNA and protein. (FIG. 6B) Interferon treatment of normal breast and breast cancer cell lines resulted in a strong upregulation of TRIM21 mRNA. (FIG. 6C) Between the different models used in this study, type I and II interferon expression was variable. Results show expression of interferon producing genes relative to a housekeeping gene (RPLPO) in each model. (FIG. 6D) Type I interferons induced surface translocation of the Ro52 antigen in MDA-MB-231 cells. Cells were treated with interferons for 72 hours, after which the Ro52 antigen was detected on the cell surface of live cells using monoclonal antibodies. Untreated cells showed little surface expression of Ro52, which selectively increased after type I interferon treatment.

[0024] FIGs. 7A-7B show anti-Ro52 antibodies from multiple sources bind to the surface of multiple breast cancer models. (FIG. 7A) Mouse anti-Ro52 monoclonal antibodies were injected into the tail-vein of a mouse containing spontaneous breast cancer metastases to the lung. After 1 hour, the mouse was euthanized and selective labeling of metastatic cancer cells in the lung was observed by the anti-Ro52 antibody. PyMT oncogene staining confirmed the presence of metastasis. (FIG. 7B) Human derived anti-Ro52 antibodies label primary organoids isolated from breast cancer patients at the time of tumor resection. Anti-Ro52 antibodies are selectively enriched in the invasive cells of the organoid. (Key) PyMT- Polyomavirus Middle T Antigen oncogene.

[0025] FIGs. 8A-8C show anti-Ro52 antibody drug conjugates exhibit potent cytotoxicity against breast cancer cell lines. (FIG. 8A) Anti-Ro52 antibodies bind to the surface of live breast cancer cells in amounts similar to FDA-approved Cetuximab and Herceptin. Antibodies were bound to live MCF7 cells for 1 hour then visualized with immunofluorescence. (FIG. 8B) Antibody drug conjugates are created by conjugating DM1 and VcMMAE microtubule targeting drugs to the Fc region of the antibody. Antibody-drug loading was confirmed by SDS- PAGE, where antibody conjugated to drug runs at a higher molecular weight than antibody heavy chain alone. (FIG. 8C) MDAMB-

[0026] 231 and MCF7 cell lines were treated with ADCs for 72 hours and cell viability7was assessed using a luminescence-based assay. A strong cytotoxic effect was observed when cells were treated with Ro52 ADCs.

[0027] FIGs. 9A-9D show autoimmune patient antibodies recognize and bind breast cancer cells from unrelated patients without known rheumatic disease. (FIG. 9A) Scheme to identify rheumatology patient sera containing IgGs capable of binding MCF7 breast cancer cells. Patient sera was allowed to bind either fixed or live cells for 1 hour, after which staining was visualized using anti -human secondary antibodies. (FIG. 9B) Number of autoimmune patient sera able to bind to live MCF7 cells in each of three distinct patterns. (FIG. 9C) Representative images of sera from autoimmune patients binding to both live and fixed MCF7 cells. Results are compared to sera from a healthy control patient, without known rheumatic disease. SSc- Systemic Sclerosis; Myo- Myositis; SLE- Systemic Lupus Erythematosus. Scale bars = 25 pm. (FIG. 9D) Flow cytometry results of live MCF7 cells incubated with autoimmune and healthy control patient sera. FIGs. 10A-10C show rheumatology patient sera contain antibodies that bind to the surface of live breast cancer cells. (FIG. 10A) Large field of view immunofluorescence of rheumatology patient sera bound to live MCF7 cells. Patient sera was bound to cells for 1 hour, after which the cells were fixed and IgG on the cell surface was visualized with anti-human secondary antibodies. Orthogonal projections detail antibody binding to the cell surface Live cell binding pattern is indicated, along with the autoimmune disease subtype. Scale bars = 25 pm. (FIG. 10B) Flow cytometry gating strategy to analyze live cell binding of antibodies in rheumatology patient sera to MCF7 cells. (FIG. IOC) Flow cytometry results of rheumatology patient sera IgG binding to MCF7 cells.

[0028] FIGs. 11A-11F show rheumatology patient sera mediate cytotoxic clearance of breast cancer cells through multiple immune pathways. (FIG. 11A) Exemplary schematic of complement pathway overview. Positive complement protein C3c staining marks activation. (FIG. 11B) Representative images of the ability of patient sera to activate complement against MCF7 breast cancer cells. Cells were incubated with sera and complement proteins for 1 hour, after which they were fixed and complement activation was assessed. (FIG. 11C) TNBC PDX Colony formation assay to measure the effect of rheumatology' patient sera either alone (in monoculture) or in the presence of NK cells (in co-culture) to measure ADCC. (FIG. 11D) Representative images showing the effect of rheumatology patient sera on mono- and coculture colony formation. Images are a maximum intensity projection of the entire extracellular matrix. Scale bars = 100 pm. (FIG. HE) Percent TNBC PDX colony formation in the presence of rheumatology patient sera. Results are normalized to the healthy control patient condition. (FIG. HF) Percent TNBC PDX colony formation in the presence of rheumatology patient sera and NK cells. Results are normalized to the healthy control patient condition where NK cells were also present.

[0029] FIGs. 12A-12F show anti-Ro52 antibodies bind to the surface of live human breast cancer cells and live mouse metastatic breast cancer cells. (FIG. 12A) Exemplary scheme to identify autoimmune antigens on the surface of MCF7 breast cancer cells. Monoclonal antibodies were incubated with live cells for 1 hour, after which cells were fixed and antibody binding was visualized by immunofluorescence. (FIG. 12B) Antigens screened for MCF7 cell surface expression, as well as the observed fixed and live cell binding patterns. (FIG. 12C) Representative live cell immunofluorescence of anti-Ro52 and matched isotype control antibodies. Orthogonal projections are shown to visualize antibody binding to the cell surface. Scale bars = 25 pm. (FIG. 12D) Scheme to label metastatic breast cancer cells in live MMTV- PyMT mice. Antibodies were injected into the tail-vein of mice, which were euthanized after an hour and labeled metastases were identified in resulting lung sections. (FIG. 12E) Fluorescence intensity of metastatic cancer cells in mice injected with anti-Ro52 and isotype control antibodies. Metastatic cells were identified by expression of the PyMT oncogene, and antibody fluorescence was determined using Nikon NIS elements software. (FIG. 12F) Representative images of single- and multi-cellular metastases labeled live with anti-Ro52 and isotype control antibodies. Scale bars = 25 pm.

[0030] FIGs. 13A-13C show selective antigens, including Ro52, can be recognized on the surface of live MCF7 breast cancer cells. (FIG. 13A) MCF7 cell immunofluorescence using isotype control, Cetuximab, and Herceptin antibodies. Cells were treated live with antibodies for 1 hour, then fixed, counterstained, and imaged. Scale bars = 25 pm. (FIG. 13B) Live MCF7 cell immunofluorescence using monoclonal antibodies targeting various autoantigens. Scale bars = 25 pm. (FIG. 13C) Fixed MCF7 cell immunofluorescence using monoclonal antibodies targeting various autoantigens. Scale bars = 25 pm.

[0031] FIGs. 14A-14E show type 1 and 2 interferons induce autoantigen expression and Ro52 presentation on the surface of breast cancer cells. (FIG. 14A) Representative live cell immunofluorescence of monoclonal anti-Ro52 antibodies binding to MCF7, MDA-MB-231, and MCF10A cells. Antibodies were bound to live cells for 1 hour. Scale bars = 25 pm. (FIG. 14B) Scheme to study changes in autoantigen gene, protein, and cell surface expression in response to interferons and other stimuli. (FIG. 14C) qRT-PCR of autoantigen producing genes in response to low (10 ng / mL) and high (100 ng / mL) interferon treatments. MCF7, MDA-MB-231, and MCF10A cells were treated for 24 hours and resulting log2 fold change values were calculated by normalizing results to non-treated control cells. (FIG. 14D) Ro52 protein expression in MCF7, MDA-MB-231, and MCF10A cells following treatment with interferons for 24 hours, fl-actin is used as the loading control. (FIG. 14E) Anti-Ro52 live cell immunofluorescence of cell lines following treatment with interferons for 48 hours. Results are shown in comparison to the untreated control. Scale bars = 25 pm.

[0032] FIGs. 15A-15E show breast cancer models heterogeneously express interferons and TNBC PDXs show variable responses to interferon treatment. (FIG. ISA) Fixed cell Ro52 staining of MDA-MB-231 and MCF10A cells. Scale bars = 25 pm. (FIG. 15B) Expression oi TRIM21 mRNA in MCF7, MDA-MB-231, and MCF10A cell lines. To show relative gene expression per cell line, the ACt to a housekeeping gene (RPLPO) is calculated. Higher relative gene expression per cell lines results in a ACt approaching zero. (FIG. 15C) Expression of interferon receptor mRNAs in MCF7, MDA-MB-231, and MCF10A cell lines. (FIG. 15D) Expression of interferon molecule mRNAs in MCF7, MDA-MB-231, and MCF10A cell lines. (FIG. 15E) Quantification of change in protein expression in MCF7, MDA-MB-231, and MCF10A cells in response to interferon treatment. Results are normalized to the control condition, where no interferons were added. Results are the quantification of FIG. 15D.

[0033] FIGs. 16A-16G show anti-Ro52 antibodies that bind to live cancer cells recognize an epitope derived from the fourth exon of the Ro52 gene. (FIG. 16A) Ro52 purified protein constructs lacking different exons and regions of the Ro52 protein. (FIG. 16B) Western blots to identify the protein binding patterns of monoclonal anti-Ro52 antibodies. A single purified protein was run in each lane of a gel, transferred to a PVDF membrane, and blotted with anti-Ro52 antibodies. (FIG. 16C) Western blots of Ro52 epitope binding of rheumatology patient sera known to have anti-Ro52 antibodies and live cell surface binding to MCF7 cells. (FIG. 16D) Western blot assay to determine the ability of Ro52 exon knock-out constructs to compete the anti-Ro52 binding ability of anti-Ro52 antibodies. The monoclonal antibody was incubated with Ro52 proteins for 1 hour prior to use as the primary antibody for the western blots. (FIG. 16E) Peptide competition assay scheme to determine the specificity of anti-Ro52 antibodies capable of binding to the surface of live cells. Antibodies / sera were pre-incubated with Ro52 peptides for 1 hour and then used in live cell immunofluorescence experiments. (FIG. 16F) Representative live cell immunofluorescence images of MCF7 cells incubated with an exon 4 targeting anti-Ro52 antibody alone or antibody blocked with a Ro52 or Ro52Ex3-4 protein. Anti-Ro52 fluorescence intensity was determined for the cells in each condition and results were normalized to those from the exon 4 antibody alone. Scale bars = 25 pm. (FIG. 16G) Live cell immunofluorescence images of MCF7 cells incubated with patient sera alone or patient sera pre-incubated with Ro52Ex3-4 protein. The anti-Ro52 antibody status of each sera is identified, as well as whether or not live cell binding was blocked using the Ro52Ex3-4 peptide. Anti-fgG fluorescence intensity was determined for the cells in each condition, and results were normalized to the healthy patient control sera condition. Scale bars = 25 pm.

[0034] FIGs. 17A-17F show Ro52 antibodies are internalized into cells and can be used as potent ADCs to selectively target breast cancer cells. (FIG. 17A) Exemplary graphic depicting monoclonal antibody labeling scheme, where antibodies are tagged with a pH sensitive dye that fluoresces once internalized into the cell. (FIG. 17B) Representative images of isotype control and anti-Ro52 antibodies internalized into MCF7 breast cancer cells over 24 hours. (FIG. 17C) Quantification of antibody internalization into MCF7 cells over 24 hours. Images were taken every 5 minutes and total fluorescence intensity in the pHrodo-antibody channel was quantified, background subtracted, and normalized to timepoint 0. (FIG. 17D) Representative images of isotype control and anti-Ro52 antibodies internalized into normal MCF10A breast cells over 24 hours. (FIG. 17E) Quantification of antibody internalization into MCF10A cells over 24 hours. Images were taken every 5 minutes and total fluorescence intensity in the pHrodo-antibody channel was quantified, background subtracted, and normalized to timepoint 0. (FIG. 17F) Cell viability assays using VcMMAE and DM1 conjugated isotype control and anti-Ro52 antibody -drug conjugates. Results are normalized to no treatment control wells.

[0035] FIGs. 18A-18B show anti-Ro52 antibodies recognize their target antigen on the surface of diverse cancer types and in freshly isolated human breast cancer organoids. (FIG. 18A) Representative images of various carcinoma and normal cell lines stained live with isotype control and anti-Ro52 antibodies. Cells were incubated live with primary antibodies for 1 hour prior to being fixed, counterstained, and imaged. Scale bars = 25 pm. (FIG. 18B) Primary human breast cancer organoids stained live with anti-Ro52 antibodies. Organoids were generated from human tumors the day of resection, grown in collagen for 5 days, then stained with anti-Ro52 antibodies overnight. Merged images are counterstained with DAPI and phalloidin, and scale bars = 25 pm.

[0036] FIG. 19 shows various cancer and normal cell lines express the Ro52 protein under homeostatic conditions. Representative images of various cell lines stained with monoclonal anti-Ro52 and matched isotype control antibodies following fixation and permeabilization. The fixed and live cell staining patterns are indicated for each cell line. Scale bars = 25 pm.

[0037] DETAILED DESCRIPTION

[0038] The type and severity of rheumatic disease correlates closely with the targets of the corresponding autoantibodies, with specific autoantigens serving as powerful diagnostic and prognostic markers. In some embodiments, paraneoplastic rheumatic diseases can be characterized by the close temporal succession of diagnosis of cancer and autoimmune disease and outcomes for these patients correlate with their autoantibody repertoire. For example, in some patients, there are recognizable correlations between mutated genes and autoantibodies targeting the corresponding protein. These clinical data have focused interest on the mechanisms underlying autoantigen generation and the role that specific autoantibodies play in generating the distinct phenotypes of the various rheumatic diseases as well as the altered cancer risks observed in these patient populations. Both rheumatic disease and cancer arise in the context of a dysregulated immune system, and both involve the generation of novel autoantigens. Since these processes occur simultaneously in paraneoplastic rheumatic disease, there are long standing hypotheses that autoantibodies are a mechanistic link between autoimmune rheumatic diseases and cancer. In some embodiments, it has been shown that patients with rheumatic disease are producing an immune response that has both anti-cancer and autoimmune effects and that these diseases represent an important model of natural cancer immunoediting in humans. In some cases, studies have shown a differential incidence of cancer associated with the onset of multiple autoimmune rheumatic diseases, including dermatomyositis (DM), scleroderma (SSc), and systemic lupus erythematosus (SLE). In some embodiments, these altered risks can be mapped to the presence of antibodies against specific autoantigens. For example, autoantibodies recognizing TIFl-y (TRIM33) can be found in the subgroup of dermatomyositis patients most likely to manifest an early cancer. Within this group, additional autoantibodies (e.g. anti-CCARl) are associated with a significantly diminished frequency of cancer - and when cancer does emerge - it is diagnosed later after autoimmune disease presentation, is smaller, and is less likely to be metastatic. In some embodiments, patient-derived autoantibodies may have favorable anti-cancer properties, potentially through binding to the cancer cell surface. In some embodiments, autoantibodies from DM, SSc, and SLE patients can bind to live cancer cells and activate multiple immune effector pathways.

[0039] A relatively small number of autoantigens are recurrently targeted in rheumatic disease, suggesting that some of these antigens could represent conserved features of cancer cells that can be therapeutically targeted. A prominent autoimmune antigen is Ro52, a cytoplasmic FC receptor that is not normally expressed on the surface of healthy cells. In some embodiments, monoclonal antibodies targeting Ro52 recapitulate the live cancer cell surface binding observed with rheumatology patient sera and they can also recognize metastatic cancer cells in vivo. In some embodiments, competitive binding assays with recombinant Ro52 proteins revealed that patient autoantibodies recurrently target the fourth exon, and that the fourth exon is selectively localized to the cancer cell surface. In some embodiments, Ro52 can be targeted by antibodydrug conjugates (ADCs) and an Ro52 antigen can be accessible on the surface of breast, colorectal, ovarian, gastric, and pancreatic cancer cells. In some embodiments, Ro52 can be a representative of a class of antigens that are conditionally localized to the surface of cells experiencing stresses, such as cancer and metastasis. In some embodiments, immune strategies targeting these conserved surface antigens could be broadly effective anti-cancer therapeutics.

[0040] Provided herein are methods of treating a cancer in a subject, wherein the cancer expresses a Ro52 antigen (e.g., wherein the cancer expresses a Ro52 antigen on a cancer cell surface), the method including administering an anti-Ro52 binding agent to the subject. Also provided herein are methods for identifying a tumor in a subject that include detecting the presence of Ro52 on a cell (e.g., the surface of the cell). In some embodiments, methods for identifying a tumor in a subject include (a) administering an anti-Ro52 binding agent to the subject; and (b) detecting the anti-Ro52 binding agent on a cell of the tumor in the subject. In some embodiments, the anti-Ro52 binding agent is bound to a cell surface of the cell.

[0041] Various non-limiting aspects of these methods are described herein and can be used in any combination without limitation.

[0042] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0043] As used herein, the term “about”, when used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0044] As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be intravenous, intra-arterial, intratumor, intraperitoneal, intrathecal, intra-thoracic, or intraventricular. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0045] As used herein, the terms “antibody” or “antigen-binding fragment” refer to an immunoglobulin molecule that includes one or more antigen-binding domains that specifically bind to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibodies include, but are not limited to monoclonal antibodies, polyclonal antibodies, and fragments thereof. In some embodiments, an antibody may include one or more sequence elements are humanized, primatized, chimeric, etc., as is known in the art. In many embodiments, the term “antibody” is used to refer to one or more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, an antibody utilized in accordance with the present invention can be in a format selected from, but not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety , a catalytic moiety, etc.], or other pendant group [e.g., poly-ethylene glycol, etc.]. In many embodiments, an antibody is or comprises a polypeptide whose ammo acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 ammo acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an ammo acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.

[0046] As used herein, the term “antigen” refers to a molecule or molecular structure that binds to a specific antibody, B-cell receptor, or T-cell receptor. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid sequence (e.g., DNA or RNA), a peptide, a polypeptide, a protein, a carbohydrate, a glycoprotein, a lipid or phospholipid, a lipoprotein, a polymer (including biologic polymers [e.g., nucleic acid and / or amino acid polymers] and polymers other than biologic polymers [e.g., other than a nucleic acid or amino acid polymer]), etc. In some embodiments, an antigen is or comprises a peptide. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a protein. Those of ordinary skill in the art will appreciate that, in general, an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source). In some certain embodiments, an antigen is present in a cellular context (e.g., an antigen is expressed on the surface of a cell or expressed in a cell). In some embodiments, an antigen is present on the surface of a cell in a complex with HLA. In some embodiments, an antigen is a recombinant antigen.

[0047] As used herein, the term “autoantibody” refers to an antibody produced by the immune system that is directed against one or more of a host’s own antigens, such as an epitope of a protein, a peptide, or a non-protein epitope. Such autoantibodies can be associated with autoimmune disease. In some embodiments, an autoimmune antibody is obtained from a subject (or the therapeutic antibody used (e.g., affinity matured antibody) is derived from an autoantibody from a subject) with an autoimmune disease. As used herein, the term “autoimmune disease” means a disease resulting from an immune response against a self-tissue or tissue component, including both self-antibody responses and cell-mediated responses. In some embodiments, an autoimmune disease encompasses organ-specific autoimmune diseases, in which an autoimmune response is directed against a single tissue, such as type I diabetes mellitus (T1D), Crohn’s disease, ulcerative colitis, myasthenia gravis, vitiligo, Graves’ disease, Hashimoto’s disease, Addison’s disease and autoimmune gastritis and autoimmune hepatitis. In some embodiments, an autoimmune disease also encompasses non-organ specific autoimmune diseases, in which an autoimmune response is directed against a component present in several or many organs throughout the body. For example, such autoimmune diseases can include, but are not limited to, rheumatoid diseases, systemic lupus erythematosus, progressive systemic sclerosis and variants, polymyositis and dermatomyositis. Additional autoimmune diseases include pernicious anemia including some of autoimmune gastritis, primary biliary cirrhosis, autoimmune thrombocytopenia, Sjogren’s syndrome, multiple sclerosis and psoriasis.

[0048] As used herein, the term “binding” typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can ty pically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell).

[0049] As used herein, the terms “cancer”, “malignancy”, “neoplasm”, “tumor”, and “carcinoma”, refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. Exemplary cancers that may be treated with a compound or method provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, Medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, cancer of the head, Hodgkin's Disease, andNon-Hodgkin's Lymphomas. Exemplary cancers that may be treated with a compound or method provided herein include cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus. Additional examples include, thyroid carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, skin cutaneous melanoma, colon adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and nonHodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like.

[0050] As used herein, a “cell” can refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source.

[0051] As used herein, a “patient” or “subject” refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, a patient is human.

[0052] Methods of Treatment of Cancer

[0053] Described herein are methods of treating a cancer in a subject, wherein the cancer expresses a Ro52 antigen on a cancer cell (e.g. , on a cancer cell surface), the methods including administering an anti-Ro52 binding agent to the subject, thereby treating the cancer in the subject.

[0054] Anti-Ro52 binding agent Ro52 is a cytoplasmic protein and autoantigen that is a common target of circulating autoantibodies in autoimmune disease. Ro52 can be placed within the family of tripartite motif proteins (TRIM) and has been shown to function as an Fc receptor and E3 ubiquitin ligase. Also, Ro52 is expressed in the immune system as a predominantly cytoplasmic protein that can be upregulated and translocate to the nucleus in a proinflammatory environment, wherein anti- Ro52 antibodies have been associated with autoimmune disorders, infection, and malignancies.

[0055] In some embodiments, Ro52 is present at the surface of cancer cells, wherein the Ro52 antigen localizes to the cell surface spontaneously and when the cancer cells are exposed to inflammatory signals (e.g., interferons). In some embodiments, a Ro52 antigen is expressed on a cancer cell surface. In some embodiments, an anti-Ro52 binding agent can specifically bind to a Ro52 antigen on a cancer cell surface.

[0056] As used herein, the term ”anti-Ro52 binding agent” refers to an agent that specifically binds to an Ro52 antigen. In some embodiments, an anti-Ro52 binding agent comprises an anti- Ro52 antibody or a Ro52 antigen binding fragment thereof. In some embodiments, an anti- Ro52 binding agent comprises a Fab, Fab’, F(ab’)2, Fabl-SH, Fv, diabody, linear antibody or single-chain variable fragment (scFv). In some embodiments, an anti-Ro52 binding agent is a humanized or chimeric antibody. In some embodiments, an anti-Ro52 binding agent can comprise chimeric antigen receptors (CARs), polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); camel oid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; or KALBITOR®s.

[0057] Therapeutic Agents

[0058] In some embodiments, an anti-Ro52 binding agent can be conjugated to a therapeutic agent. In some embodiments, a therapeutic agent comprises a chemotherapeutic agent. As used herein, the term “therapeutic agent” and / or “chemotherapeutic agent” can refer to one or more pro-apoptotic, cytostatic and / or cytotoxic agents, for example specifically including agents utilized and / or recommended for use in treating one or more diseases, disorders or conditions associated with undesirable cell proliferation. In some embodiments, chemotherapeutic agents are useful in the treatment of cancer. In some embodiments, a chemotherapeutic agent may be or comprise one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g. microtubule targeting agents such as taxanes, maytansine and analogs thereof, of), one or more epothilones, one or more histone deacetylase inhibitors HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of one or more of the following (i.e., that share a relevant anti -proliferative activity). In some embodiments, the chemotherapeutic agent comprises vincristine, prednisone, dexamethasone, busulfan, cisplatin, carboplatin, paclitaxel, docetaxel, nab-paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), liposomal doxorubicin, melphalan, pemetrexed, topotecan, vinorelbine, goserelin, leuprolide, tamoxifen, letrozole, anastrozole, exemestane, bevacizumab, olaparib, rucaparib, niraparib, nivolumab, pembrolizumab, durvalumab, atezolizumab, radioisotopes, monomethyl auristatin E (MMAE; e.g., vedotin), calicheamicins, deruxtecan, DM1, and any combinations thereof. In some embodiments, the anti-cancer agent comprises DM1, MMAE, MMAF, mertansine, calicheamicin, SN-38, or exatecan.

[0059] In some embodiments, a chemotherapeutic agent may be utilized in the context of an antibody-drug conjugate. As used herein, an “antibody -drug conjugate” refers to an agent comprising an antibody chemically linked to a therapeutic agent (e.g., a chemotherapeutic drug). In some embodiments, the antibody binds to a specific protein or receptor found on certain types of cells (e.g., cancer cells, T-cells, B-cells). In some embodiments, the chemotherapeutic agent can be conjugated to the antibody or antigen-binding fragment thereof via a linker. In some embodiments, the linker is a cleavable linker.

[0060] Cell surface localization ofRo52

[0061] In some embodiments, an anti-Ro52 binding agent is administered to a subject that has a cancer, wherein the cancer expresses a Ro52 antigen on a cancer cell surface. In some embodiments, a cancer is a metastatic cancer. In some embodiments, a metastatic cancer is a breast cancer, colon cancer, pancreatic cancer, gastric cancer, or ovarian cancer.

[0062] As used herein, the term “cell surface localization” refers to the accumulation of a protein or antigen at the cell surface, wherein the protein is transported to and / or maintained at the cell surface. In some embodiments, an antigen can localize to a cell surface spontaneously. In some embodiments, surface-localization of an antigen when the cell is exposed to external factors (e.g., inflammatory signals). In some embodiments, surface-localization of an antigen when the cell is exposed to interferons. In some embodiments, surface-localization of an antigen when the cell is exposed to interferons that include interferon alpha 2A, interferon alpha 2B, interferon beta, or interferon gamma.

[0063] In some embodiments, a Ro52 antigen is present on a surface of a cancer cell. In some embodiments, a Ro52 antigen is detected on a cancer cell surface. In some embodiments, prior to administration of an anti-Ro52 binding agent to a subject, a Ro52 antigen is detected on a cancer cell surface.

[0064] In some embodiments, a cancer cell is from a primary tumor sample or a metastatic tumor sample from the subject. In some embodiments, a primary tumor sample or a metastatic tumor sample is from a fresh biopsy sample. In some embodiments, a cancer cell is a live cancer cell. In some embodiments, a primary tumor sample or a metastatic tumor sample is from a fixed biopsy sample. In some embodiments, a cancer cell is a fixed cancer cell. In some embodiments, a cancer cell comprises circulating tumor cells.

[0065] Treatment of cancer

[0066] In some embodiments, an anti-Ro52 binding agent can be administered to a subject, thereby treating a cancer in the subject. As used herein, the terms “treating”, or “treatment” can refer to any indicia of success in the therapy or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient’s physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and / or a psychiatric evaluation. The term "treating" and conjugations thereof, may include prevention of an injury, pathology, condition, or disease. In some embodiments, treating includes preventing.

[0067] “Treating” or “treatment” as used herein (and as well-understood in the art) can also broadly include any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. In some embodiments, "treatment" as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.

[0068] In some embodiments, a therapeutically effective amount of an anti-Ro52 antibody or antigen binding fragment thereof is administered to a subject, thereby treating a cancer in the subject. As used herein, an “effective amount” or a “therapeutically effective amount” is an amount sufficient for a compound to accomplish a stated purpose relative to the absence of the compound (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or reduce one or more symptoms of a disease or condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease. A “therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to ameliorate the disorder. For example, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2- fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

[0069] For any therapeutic agent (e.g., antibody-drug conjugate) described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art.

[0070] As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.

[0071] In some embodiments, the methods provided herein can be used to reduce or eliminate the number of cancer cells present within a subject having a cancer. For example, any one of the methods described herein can be used to reduce the number of cancer cells present within a subject having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, any one of the methods described herein can be used to reduce the size (e.g, volume) of one or more tumors present within a subject having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some embodiments, the number of cancer cells present within a subject being treated can be monitored. Any appropriate method can be used to determine whether or not the number of cancer cells present within a subject is reduced. For example, imaging techniques can be used to assess the number of cancer cells present within a subject.

[0072] In some embodiments, any one of the methods provided herein can be used to improve survival of a subject having a cancer. For example, any one of the methods described herein can be used to improve the survival of a subject having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, any one of the methods described herein can be used to improve the survival of a subject having cancer by, for example, at least 6 months (e.g, about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).

[0073] An effective amount (e.g., effective dose) of an anti-Ro52 binding agent can vary depending on the severity of the cancer, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and / or the judgment of the treating physician.

[0074] An effective amount of a composition (e.g., a pharmaceutical composition) containing an anti-Ro52 antibody or antigen binding fragment thereof can be any amount that can treat a subject having a cancer without producing significant toxicity to the subject. An effective amount of an anti-Ro52 binding agent can be any appropriate amount. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the subject’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g, a T cell cancer) may require an increase or decrease in the actual effective amount administered.

[0075] The frequency of administration of a therapeutic agent (e.g, a pharmaceutical composition) containing an anti-Ro52 binding agent can be any frequency that can treat a subject having a cancer without producing intolerable toxicity to the subject. For example, the frequency of administration can be once a day, once a week, once every 2 weeks, or once every 4 weeks. In some embodiments, an administration can include a continuous infusion of a composition containing an anti-Ro52 antibody or antigen binding fragment thereof. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing an anti-Ro52 binding agent can include rest periods. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., severity of cancer) may require an increase or decrease in administration frequency.

[0076] An effective duration for administering a composition (e.g., a pharmaceutical composition) containing an anti-Ro52 binding agent can be any duration that treat a subject having a cancer without producing significant toxicity to the subj ect. F or example, the effective duration can vary from several days to several weeks, months, or years. In some embodiments, the effective duration for the treatment of a subject can range in duration from about one month to about 10 years. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition (e.g., a cancer) being treated.

[0077] In some embodiments, an anti-Ro52 binding agent can be used as the sole active agent to treat a subject having a cancer. In some embodiments, one or more (e.g, one, two, three, four, five or more) additional treatments (e.g, therapeutic interventions) that are effective to treat cancers can be used. For example, a subject in need thereof (e.g., a subject having a cancer) can be administered an anti-Ro52 antibody or antigen binding fragment thereof in combination with one or more therapeutic interventions. Examples of therapeutic interventions that can be used as described herein to treat a cancer include, without limitation, cancer surgeries, radiation therapies, chemotherapies, and any combinations thereof. In some embodiments, the one or more additional treatments that are effective to treat cancers can be performed at the same time as the administration of an anti-Ro52 binding agent. In some embodiments, the one or more additional treatments that are effective to treat cancers can be performed before and / or after the administration of an anti-Ro52 antibody or antigen binding fragment thereof.

[0078] Methods for Identifying a Tumor in a Subject

[0079] Also provided herein are methods for identifying a tumor in a subject, the method including (a) administering an anti-Ro52 binding agent to the subject; and (b) detecting the anti-Ro52 binding agent on a cell surface of the tumor in the subject, thereby identifying the tumor in the subject.

[0080] In some embodiments, an anti-Ro52 binding agent is conjugated to a label. In some embodiments, a label is a detectable label. As used herein, the term “detectable label” can refer to a directly or indirectly detectable moiety that is associated with (e.g., conjugated to) a molecule to be detected (e.g., an antibody or antigen binding fragment thereof). A detectable label can be directly detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can be indirectly detectable, e.g., by catalyzing chemical alterations of a chemical substrate compound or composition, which chemical substrate compound or composition is directly detectable.

[0081] In some embodiments, a label is a fluorophore. In some embodiments, a label can include 7-AAD (7-Aminoactinomycin D), Acridine Orange (+DNA), Acridine Orange (+RNA), Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Allophycocyanin (APC), AMCA I AMCA-X, 7-Aminoactinomycin D (7-AAD), 7- Amino-4-methylcoumarin, 6-Aminoquinoline, Aniline Blue, ANS, APC-Cy7, ATTO-TAG™ CBQCA, ATTO-TAG™ FQ, Auramine O-Feulgen, BCECF (high pH), BFP (Blue Fluorescent Protein), BFP I GFP FRET, BOBO™-1 I BO-PRO™-!, BOBO™-3 I BO-PRO™-3, BODIPY® FL, BODIPY® TMR, BODIPY® TR-X, BODIPY® 530 / 550, BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 581 / 591, BODIPY® 630 / 650-X, BODIPY® 650-665-X, BTC, Calcein, Calcein Blue, Calcium Crimson™, Calcium Green-1™, Calcium Orange™, Calcofluor® White, 5-Carboxyfluoroscein (5-FAM), 5-Carboxynaphthofluoroscein, 6- Carboxyrhodamine 6G, 5-Carboxytetramethylrhodamine (5-TAMRA), Carboxy-X-rhodamine (5-ROX), Cascade Blue®, Cascade Yellow™, CCF2 (GeneBLAzer™), CFP (Cyan Fluorescent Protein), CFP I YFP FRET, Chromomycin A3, Cl-NERF (low pH), CPM, 6-CR 6G, CTC Formazan, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cychrome (PE-Cy5), Dansylamine, Dansyl cadaverine, Dansylchloride, DAPI, Dapoxyl, DCFH, DHR, DiA (4-Di- 16- ASP), DiD (DilCl 8(5)), DIDS, Dil (DilC 18(3)), DiO (DiOCl 8(3)), DiR (DilCl 8(7)), Di-4 ANEPPS, Di-8 ANEPPS, DM-NERF (4.5-6.5 pH), DsRed (Red Fluorescent Protein), EBFP, ECFP, EGFP, ELF® -97 alcohol, Eosin, Erythrosin, Ethidium bromide, Ethidium homodimer- 1 (EthD-1), Europium (III) Chloride, 5-FAM (5-Carboxyfluorescein), Fast Blue, Fluorescein- dT phosphoramidite, FITC, Fluo-3, Fluo-4, FluorX®, Fluoro-Gold™ (high pH), FluoroGold™ (low pH), Fluoro-Jade, FM® 1-43, Fura-2 (high calcium), Fura-2 / BCECF, Fura Red™ (high calcium), Fura Red™ / Fluo-3, GeneBLAzer™ (CCF2), GFP Red Shifted (rsGFP), GFP Wild Type, GFP / BFP FRET, GFP I DsRed FRET, Hoechst 33342 & 33258, 7- Hydroxy-4-methyl coumarin (pH 9), 1,5 IAEDANS, Indo-1 (high calcium), Indo-1 (low calcium), Indodi carbocyanine, Indotricarbocyanine, JC-1, 6-JOE, JOJO™-1 I JO-PRO™-1, LDS 751 (+DNA), LDS 751 (+RNA), LOLO™-1 I LO-PRO™-!, Lucifer Yellow, Ly soSensor™ Blue (pH 5), Ly soSensor™ Green (pH 5), Ly soSensor™ Yellow / Blue (pH 4.2), LysoTracker® Green, LysoTracker® Red, LysoTracker® Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium Green™, Marina Blue®, 4-Methylumbelliferone, Mithramycin, MitoTracker® Green, MitoTracker® Orange, MitoTracker® Red, NBD (amine), Nile Red, Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP (Peridinin chlorphyll protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (Propidium Iodide), PKH26, PKH67, POPO™-! I PO-PRO™-!, POPO™-3 / PO-PRO™-3, Propidium Iodide (PI), PyMPO, Pyrene, Pyronin Y, Quantam Red (PE-Cy5), Quinacrine Mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red) , Red Fluorescent Protein (DsRed), Resorufin, RH 414, Rhod-2, Rhodamine B, Rhodamine Green™, Rhodamine Red™, Rhodamine Phalloidin, Rhodamine 110, Rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS, SNAFL®-! (high pH), SNAFL®-2, SNARF®-! (high pH), SNARF®-1 (low pH), Sodium Green™, SpectrumAqua®, SpectrumGreen® #1, SpectrumGreen® #2, SpectrumOrange®, SpectrumRed®, SYTO® 11, SYTO® 13, SYTO® 17, SYTO® 45, SYTOX® Blue, SYTOX® Green, SYTOX® Orange, 5-TAMRA (5- Carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), Texas Red® I Texas Red®- X, Texas Red®-X (NHS Ester), Thiadicarbocyanine, Thiazole Orange, TOTO®-1 I TO- PRO®-1, TOTO®-3 / TO-PRO®-3, TO-PRO®-5, Tn-color (PE-Cy5), TRITC (Tetramethylrhodamine), TruRed (PerCP-Cy5.5), WW 781, X-Rhodamine (XRITC) , Y66F, Y66H, Y66W, YFP (Yellow Fluorescent Protein), YOYO®-1 I YO-PRO®-!, YOYO®-3 / YO-PRO®-3, 6-FAM (Fluorescein), 6-FAM (NHS Ester), 6-FAM (Azide), HEX, TAMRA (NHS Ester), Yakima Yellow, MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTO RholOl, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 5’ IRDye® 700, 5’ IRDye® 800, 5’ IRDye® 800CW (NHS Ester), WellRED D4 Dye, WellRED D3 Dye, WellRED D2 Dye, Lightcycler® 640 (NHS Ester), and Dy 750 (NHS Ester).

[0082] In some embodiments, detecting an anti-Ro52 binding agent on a cell surface of the tumor in the subject comprises fluorescence imaging. In some embodiments, an anti-Ro52 binding agent is conjugated to an imaging agent. In some embodiments, an imaging agent includes AdreView, air polymer type A, aminolevulinic acid oral, AMYViD, Axumin, Ceretec, Cerianna, Clariscan, copper CU 64 dotatate, Cystografin, Cysview, Cytalux, DaTscan, Definity, Definity RT, Detectnet, diatrizoate, diatrizoate meglumine / diatrizoate sodium, Dotarem, Drax Exametazime, Elucirem, Eovist, ethiodized oil, ExEm Foam, Feridex, ferumoxides, ferumoxsil, florbetaben F 18, florbetapir F 18, flortaucipir F 18, flotufolastat F 18, fluci cl ovine F 18, fluorine Fl 8 DCFPyL, fluorodeoxyphenylalanine 18F-DOPA, Fluorodopa Fl 8, fluoroestradiol F 18, flutemetamol F 18, Gadavist, gadobenate, gadobutrol, gadodiamide, gadopentetate, gadopiclenol, gadoterate meglumine, gadoteridol, gadoversetamide, gadoxetate, Gadoxetic Acid, gallium Ga 68 dotatate, gallium Ga 68 dotatoc, gallium Ga 68 gozetotide, gallium Ga 68 PSMA-11, Gastrografm, GastroMark, Gleolan, hexaminolevulinate, Illuccix, iobenguane l 123, iodixanol, ioflupane 1 123, iohexol, iopamidol, iopromide, ioversol, ioxilan, isosulfan blue, Isovue, Lexiscan, Lipiodol, Lumason, Lumisight, Lymphazurin, Lymphoseek, Magnevist, MD-Gastroview, MultiHance, Nephroscan, Netspot, Neuraceq, Omnipaque, Omniscan, OptiMARK, Optiray, Optison, Oraltag, Oxilan, pafolacianine, pegulicianine, perflutren, piflufolastat F 18, Posluma, Primovist, ProHance, Pulmotech MAA, PyL, Pylarify, regadenoson, Renocal 76, Renografin, Scanlux, sulfur hexafluoride, Tauvid, technetium Tc 99m albumin aggregated injection, technetium Tc 99m exametazime, technetium Tc 99m succimer, technetium Tc 99m tilmanocept, Ultravist, Visipaque, Vizamyl, Vueway, xenon Xe 129 hyperpolarized, or Xenoview. In some embodiments, the detecting of the anti-Ro52 antibody or antigen binding fragment thereof on a cell surface of the tumor in the subject comprises magnetic resonance imaging (MRI) or positron emission tomography (PET) imaging.

[0083] In some embodiments, an anti-Ro52 binding agent is conjugated to a pharmaceutical agent. In some embodiments, an anti-Ro52 binding agent is conjugated to a radioactive molecule. In some embodiments, an anti-Ro52 binding agent is conjugated to a radiosensitizer. In some embodiments, an anti-Ro52 binding agent is conjugated to a radionuclide.

[0084] EXAMPLES

[0085] Materials and Methods

[0086] Mice

[0087] Mouse husbandly' and procedures were performed in accordance with protocols approved by the Johns Hopkins Medical Institute Animal Care and Use Committee (ACUC; M023M200). FVB / N-Tg(MMTV-PyVT)634Mul / J (002374) and NOD scid gamma (005557) mice were obtained from the Jackson Laboratory. Patient Derived Xenograft (PDX) models of triple negative breast cancer were obtained from the Jackson Laboratory and passaged subcutaneously in NS G mice. PDX lines used in this study are as follows: PDX-A (TM00098), PDX-B (TM00096), and PDX-C (TM00089). Female MMTV-PyMT and PDX tumor bearing mice were euthanized when the cumulative maximum tumor diameters were 20 mm or less, consistent with ACUC approved protocol.

[0088] Cell Lines

[0089] MCF7 (HTB-22; ATCC), MDA-MB-231 (HTB-26; ATCC), PL45 (CRL-2558; ATCC), and MDST8 (99011801; Sigma Aldrich) cells were grown in DMEM (11885-84; Thermo Fisher Scientific) supplemented with 9.0% heat-inactivated FBS (F31016; SeraPrime), 1.0% penicillin-streptomycin (P0781; Sigma Aldrich), and 10 pg / mL insulin (19278; Sigma Aldrich). NCI-N87 (CRL-5822; ATCC) and SK-OV-3 (HTB-77; ATCC) cells were grown in RPMI (11875-093; Thermo Fisher Scientific) supplemented with 9.0% heat-inactivated FBS and 1.0% penicillin-streptomycin. LS 174T cells (CL-188; ATCC) were grown in EMEM (112-018-101; Quality Biological) containing 9.0% heat-inactivated FBS and 1.0% penicillin- streptomycin. MeT-5A cells (CRL-9444; ATCC) were grown in medium 199 (11150-059; Thermo Fisher Scientific) containing 9.0% heat-inactivated FBS and 1.0% penicillin- streptomycin. MCF10A cells (CRL-10317; ATCC) were grown in mammary epithelial growth media (CC-3150; Lonza) containing 0.1 pg / mL cholera toxin (C8052; Sigma Aldrich). All cell lines were grown in a 37 °C incubator with 5.0% CO2 and subcultured after reaching 80% confluency.

[0090] Patient Data and Serum Samples

[0091] Blood samples (10 ml) were collected into serum separator tubes from patients seen at the Johns Hopkins Hospital in the Rheumatology Centers of Excellence outpatient clinics. All were performed as part of studies approved by the Johns Hopkins Institutional Review Board. Patients provided signed, informed written consent. Once clotted, the de-identified samples were centrifuged (15 minutes at 1500 x g). The resulting serum was then aliquoted into appropriate volumes and stored in monitored -80°C freezers. Antibody profiles in each serum were assessed using a variety' of different assays, including line blot (Euroimmun platform), ELISA or immunoprecipitation with in vitro transcription / translated radiolabeled proteins as input. Tumor Organoid Isolation and Cluster Generation

[0092] Primary tumor organoids were isolated from PDX bearing mice through sequential digestion and enrichment steps. Briefly, harvested tumors were mechanically disrupted with a scalpel and shaken in a collagenase digestion solution for 1 hour at 37 °C. The digestion solution was made in DMEM / F-12 (10565-018; Thermo Fisher Scientific) with the following additives: 2 mg / mL collagenase (C2139; Sigma Aldrich), 2 mg / mL trypsin (27250-018; Sigma Aldrich), 5.0% FBS, 5 pg / mL insulin, and 50 pg / mL gentamicin (15750-060; Thermo Fisher Scientific). After digestion, the suspension was centrifuged for 10 minutes at 400 g then treated with 2 U / pL DNase (10104159001; Sigma Aldrich) for 5 minutes. A series of differential centrifugations were used to separate epithelial organoids from stromal cells, with resulting organoids between 100-250 cells in size. Cancer cell clusters (2-10 cells in size) were generated by digesting organoids for 10 minutes in IX TryPLE (12604-013; Thermo Fisher Scientific). The digestion reaction was neutralized through the addition of an equal volume of FBS, and resulting clusters were isolated through differential centrifugation. Organoids and cell clusters were embedded into either a collagen I (354236; Coming) or Matrigel (354230; Coming) extracellular matrix depending on the experiment. TNBC PDX organoids were cultured in OncoPro Tumoroid media (A5701201; Thermo Fisher Scientific) supplemented with 50 ng / mL EGF (E9644; Sigma Aldrich).

[0093] Human breast cancer samples were obtained from the Cooperative Human Tissue Network (CHTN). All human primary cancer organoids were generated within 24 hours of resection, as described above. Breast cancer organoids were cultured in OncoPro Tumoroid media supplemented with 10 ng / mL FGF-10 (PHG0372; Thermo Fisher Scientific).

[0094] Rheumatolog Patient Sera Screen

[0095] Cells were seeded into coverslip bottom imaging dishes at a density of 30,000 cells / cm2, and allowed to adhere and grow overnight. For live cell binding experiments, the cell culture media was replaced with fresh media containing either test autoimmune patient sera or sera from a healthy donor control (1 : 100). The media containing test sera was incubated with the live cells for 1 hour at 37 °C with 5.0% CO2. The cells were then washed 3 times with PBS (10010-023; Thermo Fisher Scientific) and fixed in 4.0% paraformaldehyde (15714-S; Electron Microscopy Sciences) for 10 minutes. For fixed cell binding experiments, cells were fixed in 4.0% paraformaldehyde for 10 minutes, permeabilized in 0.15% Triton X-100 (X100; Sigma Aldrich) for 10 minutes and blocked in 3.0% BSA (A1595; Sigma Aldrich) for 1 hour at room temperature. Patient and healthy control sera were diluted 1 : 100 in 1.0% BSA and cells were stained for 2 hours at room temperature then washed 3 times in PBS. For live and fixed cell experiments, cells were counterstained with DAPI (1 : 1000; 10236276001 ; Sigma Aldrich) and phalloidin (1:500; A12379; Thermo Fisher Scientific). Anti-human secondaries (1:200; A21090; Thermo Fisher Scientific) were used to visualize patient sera IgG binding. Resulting images were taken on a laser scanning confocal microscope equipped with a tunable GaAsp detector, 2k resonant scanner, and LUA-S6 laser unit (AXR; Nikon Instruments).

[0096] Flow Cytometry

[0097] Cells for flow cytometry were harvested using TryPLE and resuspended at a density of 1 x 106in flow cytometry buffer (00-4222-26; Thermo Fisher Scientific). The cells were then blocked using Fc block (564220; BD Biosciences) for 30 minutes, washed twice in PBS, and stained with human sera (1: 100) for 30 minutes on ice. After washing the cells twice in PBS, they were incubated with anti -human secondary antibodies, as well as SYTOX green (R37168; Thermo Fisher Scientific), for 30 minutes on ice. The cells were then washed twice in PBS and analyzed on an Attune flow cytometer equipped with blue (488 nm) and yellow (561 nm) excitation lasers (Thermo Fisher Scientific).

[0098] Complement Pathway Activation Assay

[0099] MCF7 cells were seeded into 96-well glass bottom imaging plates (655892; Grenier) at a density of 30,000 cells / cm2and allowed to adhere overnight. The media was then replaced with fresh media containing 1 : 100 autoimmune patient sera plus 5.0% normal human serum (NHS; CompTech) as a source of complement. Cobra venom factor activated normal human serum (CVF-NHS; CompTech) was used as negative control sera lacking complement activity. The live cells were then incubated for 1 hour at 37 °C with 5.0% CO2, after which they were washed three times in PBS and fixed in 4.0% paraformaldehyde. To visualize complement activity and autoantibody binding, cells were stained with anti-C3c Complement (1 :00; F0201; Dako) and anti-human secondary antibodies for 2 hours at room temperature. DAPI (1: 1000) and phalloidin (1 :500) were used to counterstain the cells. Representative fluorescence images were taken on a Nikon AXR using a 60X lens. To quantify complement activity. 16 images were taken in a tiled pattern using a 20X lens, cells were segmented by phalloidin staining, and anti-C3c signal fluorescence intensity was quantified using Nikon NIS -elements software. Fc-Gamma Receptor Activation Assay

[0100] Fc-Gamma receptor competition assays were purchased from Promega (CS3041A01; CS3041 A02; CS3041 A04) and used to determine the binding strength of IgG in human sera to purified receptors. The assays were scaled down to a 384 well format and conducted in white well luminescence compatible plates (HSP3805; Bio-Rad). Test sera were used in the assay at a starting concentration of 1 :100, with 11 sequential 4-fold dilutions conducted in duplicate. After combining assay components and incubating at room temperature for 1 hour, the detection reagent was added and results were read on a Cytation 5 Multimode Reader (BioTek) with an integration time of 0.5 seconds / well.

[0101] PDX ADCC Assays

[0102] PDX cancer cell clusters (2-10 cells in size) were generated as described above and embedded into a Matrigel ECM at a density of 200 clusters per pL either alone (monoculture) or in a 1 : 10 ratio with natural killer cells (co-culture). For the co-culture condition, human peripheral CD56+NK cells (70037; StemCell) were activated and expanded for 5 days prior to use in the assay. NK cells were grown in ImmunoCult T cell expansion media (10981; StemCell) supplemented with 500 lU / mL IL-2 (78036; StemCell) and activated using NKp46 and CD2 targeting antibodies (130-094-483; Miltenyi Biotec). After generating the mono- and co-culture conditions cells were resuspended in Matrigel and 15 pL domes were dispensed into each well of a 96-well glass bottom imaging plate and allowed to solidify for 1 hour prior to media addition. Human cancer organoid cultures were grown in OncoPro media as detailed above, supplemented with 500 lU / mL IL-2. Test rheumatology patient sera was added to each well at a final concentration of 5.0% and the cultures were incubated for 96 hours at 37 °C with 5.0% CO2. Following incubation, cultures were fixed in 1.0% paraformaldehyde containing 0.25% glutaraldehyde (16120; Electron Microscopy Sciences) and stained with phalloidin (1 :500). Resulting colonies were imaged on a Molecular Devices ImageXpress Micro using a 4X PlanApo objective and 15 pM z-steps to image the entire ECM. Maximum intensity projections were generated from the resulting Z-stacks, and colonies were counted using custom ImageJ and Python scripts. Percent colony formation was calculated as the number of colonies in each sera condition normalized to the healthy control sera. Results are from three independent biological replicates each containing technical triplicates.

[0103] MCF7 Cells and Macrophages Co-Culture CD 14+ monocytes were isolated from human PBMCs using magnetic positive selection (17858; StemCell) and grown in Immunocult Macrophage Media (10961; StemCell) supplemented with 50 ng / mL M-CSF (78057; StemCell). On day 3, 10 ng / mL LPS (1401 IS; Cell Signaling Technology) and 50 ng / mL IFN-y (78020; StemCell) were added to the culture to differentiate the monocytes to Ml macrophages, which were then grown for an additional 3 days. Ml macrophages were dissociated using Accutase (07920; StemCell) and labeled with CellTrace Far Red (C34553; Thermo Fisher Scientific) prior to co-culture with MCF7 cells. MCF7 cells were labeled with pHrodo green (P35373; Thermo Fisher Scientific) and then mixed at a 1: 1 ratio with Ml macrophages. The co-cultures were plated in coverslip bottom imaging dishes and grown in MCF7 media supplemented with 50 ng / mL M-CSF. Test antibodies were added at a concentration of 10 pg / mL and antibody dependent cellular phagocytosis (ADCP) was assessed after 24 hrs. To determine ADCP activity, cultures were imaged on a laser scanning confocal microscope (AXR; Nikon Instruments), with a red / green overlay indicating phagocytosis of target cells.

[0104] Live Antibody Binding to Cell Lines

[0105] Cells were seeded into coverslip bottom imaging dishes at a density of 30,000 cells / cm2, and allowed to adhere and grow overnight. The cell culture media was then replaced with fresh media containing either test autoimmune patient sera (1:100) or commercially available monoclonal antibody (5 pg / mL). The media containing test sera and antibodies was incubated with the live cells for 1 hr at 37 °C with 5.0% CO2. The cells were then washed 3 times with PBS (10010-023; Thermo Fisher Scientific) and fixed in 4.0 % paraformaldehyde for 10 minutes. Cells were counterstained with DAPI (1: 1000; 10236276001; Sigma Aldrich) and phalloidin (1 :500; A12379; Thermo Fisher Scientific). Antibody binding and localization was visualized using anti-Human (1 :200; A21090), anti-Mouse (1:200; A10037), and anti-Rabbit (1 :200; A11011) secondary antibodies from Thermo Fisher Scientific. Human IgG (12000C), Mouse IgG (31903), and Rabbit IgG (10500C) isotype controls were obtained from Thermo Fisher Scientific and raised in the same host species as the test antibodies. Images were taken on a laser scanning confocal microscope.

[0106] Antigen Screen with Commercial Antibodies

[0107] To determine if autoimmune antigens could be recognized on the surface of living cancer cells, cells were seeded into coverslip bottom imaging dishes at a density of 30,000 cells / cm2and grown overnight. Live and fixed cell binding experiments were conducted as described above. Commercial antibodies were added to the cell culture media at a concentration of 5 pg / mL, and those used in this study are detailed in Table 1. Mouse IgG (31903) and Rabbit IgG (10500C) isotype controls were obtained from Thermo Fisher Scientific and raised in the same host species as the test antibodies. Antibody binding and localization was visualized using anti-Mouse (1:200; A10037) and anti-Rabbit (1:200; Al 1011) secondary antibodies from Thermo Fisher Scientific. Representative images were taken on a laser scanning confocal microscope using a 40X lens. Live Antibody Binding to Organoids

[0108] Organoids were embedded into Matngel at a density of 1 organoid per microliter and grown at 37 °C with 5.0% CO2. After 5 days, the media was replaced with fresh media containing either test autoimmune patient sera (1 : 100) or commercially available monoclonal antibody (5 pg / mL). The media containing test sera and antibodies was incubated with the organoids for 12 hours at 37 °C with 5.0% CO2. Organoids in ECM were then washed 3 times with PBS and fixed in 1.0% paraformaldehyde with 0.25% glutaraldehyde for 10 minutes. After washing with PBS, organoids were counterstained with DAPI (1: 1000) and phalloidin (1 :500). Antibody binding and localization was visualized using anti-Human (1 :200) and antiMouse (1:200) secondary antibodies. Human and mouse IgG isotype controls were raised in the same host species as the test antibodies. Images were taken on a laser scanning confocal microscope.

[0109] Fixed Antibody Binding to Cell Lines

[0110] Adherent cells were seeded into coverslip bottom imaging dishes at a density of 30,000 cells / cm2 and grown overnight. Once the cells reached 80% confluency, they were fixed in 4.0% paraformaldehyde for 10 minutes. Cells were permeabilized in 0.15% Triton X-100 (X100; Sigma Aldrich) for 10 minutes and blocked in 3.0% BSA (A1595; Sigma Aldrich) for 1 hour at room temperature. Patient sera (1: 100) and test monoclonal antibodies (5 pg / mL) were diluted in 1.0% BSA and cells were stained for 4 hours at room temperature. Cells were washed 3 times in PBS then counterstained with DAPI (1: 1000) and phalloidin (1:500). Anti-human secondaries (1:200) were used to visualize patient sera IgG binding, and anti-mouse (1:200) and anti-rabbit (1:200) secondaries were used to visualize commercial monoclonal antibody binding. Images were taken on a laser scanning confocal microscope.

[0111] Antibody Internalization Assays

[0112] To study antibody internalization dynamics, antibodies were conjugated to Alexa Fluor 594 (A20185; Thermo Fisher Scientific) and pHrodo 647 (P35355; Thermo Fisher Scientific) fluorophores in separate reactions. Antibodies were added in tandem to live cells and organoids at a concentration of 5 pg / mL in media and incubated at 37 °C with 5.0% CO2 for either 4 or 24 hours. Cells and organoids were fixed in 4.0% paraformaldehyde for 10 min at room temperature, washed three times in PBS, and stained with DAPI (1: 1000). Antibody binding was visualized on a laser scanning confocal microscope, with total antibody signal in the 594 channel and internalized antibody signal in the 647 channel. Interferon Induction Western Blots in Organoids

[0113] Organoids were generated from PDX tumor bearing mice as detailed above and suspended in a Matrigel ECM at a concentration of 1 organoid per microliter. Organoids in ECM were plated, media was added, and they were incubated overnight. Interferons were added to the cultures, at both 10 ng / mL and 100 ng / mL, with untreated organoids used as the control. Interferon alpha 2A (78076), alpha 2B (78077), beta (78113), and gamma (78020) were obtained from StemCell. After a 24 hr treatment, the organoids were isolated by dissolving the Matrigel matrix in EDTA and proteins were isolated. For western blotting, 20 pg of protein was mixed with 7.5 pL of sample buffer (B0007; Thermo Fisher Scientific) in a 30 pL reaction. Samples were heated at 70 °C for 10 minutes, and then run in a 4-12% Bis-Tris gel (NW04120BOX; Thermo Fisher Scientific) at 140 volts for 1 hr. Proteins were transferred to a PVDF membrane (IB24001; Thermo Fisher Scientific) using an iBlot instrument (Thermo Fisher Scientific). Membranes were blocked for 1 hour at room temperature and primary antibodies were diluted in blocking buffer (927-60001 ; Li cor) at a concentration of 1 : 1000 and added to the shaking blots overnight at 4 °C. Primary antibodies used for western blotting were anti-P-Actin (A5441; Sigma Aldrich) and anti-TRIM21 (A1957; Abclonal). Membranes were washed 3 times for 10 minutes in tris-buffered saline with 0.1% Tween (TBST; 351-086-101; Quality Biological). IRDye secondary antibodies (Licor) were diluted 1 :7,000 in blocking buffer and added to the shaking membranes for 1 hour at room temperature. The membranes were then washed 3 times for 10 minutes in TBST, and imaged using an iBright instrument (Thermo Fisher Scientific).

[0114] Interferon Induction gPCR

[0115] Cells were seeded into tissue culture treated dishes at a density of 30,000 cells / cm2and grown overnight. Interferons were added to the cultures at low (10 ng / mL) and high (100 ng / mL) concentrations and cells were treated for 24 hours. RNA was extracted using TRIzol (15596026; Thermo Fisher Scientific) following the manufacturer’s protocol. cDNA was synthesized from 1.0 pg of total RNA using SuperScript IV Vilo Master Mix (11766050; Thermo Fisher Scientific). RT-qPCR was conducted in triplicate using SYBR Green (C14512; Thermo Fisher Scientific) with 4.5 ng of cDNA and 500 nM primers per reaction. Reactions were run on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Gene expression values were normalized to RPLP0. Live Labeling of MMTV-PyMT Metastases

[0116] Female MMTV-PyMT mice with a cumulative tumor burden of 20 mm were injected with 100 pL of test antibody (50 pg / mL dissolved in sterile PBS) into the tail-vein. Following injection, the mice were active for 1 hr, euthanized, and the lungs were perfused with 2 mM EDTA (46-034-CI; Coming). The lungs were fixed in 4.0% paraformaldehyde overnight, washed 3 times with PBS, and embedded into OCT (4583; Sakura). Frozen 20 pm lung sections were cut, permeabilized with 0.15% Triton X-100 for 10 minutes and blocked in 3.0% BSA for 1 hour. Slides were stained with an anti-PyMT (1 :250 in 1.0 % BSA; sc-53481 ; Santa Cruz) antibody overnight at 4 °C to label metastatic cancer cells. The sections were then counterstained with DAPI (1 : 1000) and secondary antibodies (1 : 200) in 1.0% BSA for 2 hours. After washing 3 times for 10 minutes in PBS, slides were mounted with fluoromount (F4680; Sigma Aldrich) and imaged on a laser scanning confocal microscope.

[0117] Antibody Drug Conjugate Creation and Cell Death Assays

[0118] Antibodies were conjugated to both VcMMAE (AT7001) and DM1 (AT7002) from AlphaThera following the manufacturer’s protocol. For ADC assays, 10,000 cells were seeded into each well of a tissue culture treated 96-well plate and allowed to adhere overnight. ADCs were added to cells the following morning and were dosed randomly into the plate using a D300e Digital Dispenser (Tecan). After incubating for 96 hours, cell viability was assessed using the CellTiter 96 Aqueous One Solution (G3580; Promega) and results were read on a Cytation 5 Multimode Reader (BioTek). Cell viability in response to ADC treatment was normalized to the non-treatment controls which were standardized to 100% viability.

[0119] In Vivo Metastasis Binding Assay

[0120] Female MMTV-PyMT mice with a cumulative tumor burden of 20 mm were injected with 100 pL of test antibody (50 pg / mL dissolved in sterile PBS) into the tail-vein. Following injection, the mice were active for 1 hr, euthanized, and the lungs were perfused with 2 mM EDTA (46-034-CI; Coming). The lungs were fixed in 4.0% paraformaldehyde overnight, washed 3 times with PBS, and embedded into OCT (4583; Sakura). Frozen 20 pm lung sections were cut, permeabilized with 0.15% Triton X-100 for 10 minutes, and blocked in 3.0% BSA for 1 hour. Slides were stained with an anti-PyMT (1 :250 in 1.0 % BSA; sc-53481 ; Santa Cruz) antibody overnight at 4 °C to label metastatic cancer cells. The sections were then counterstained with DAPI (1 : 1000) and secondary antibodies (1:200) in 1.0% BSA for 2 hours. After washing 3 times for 10 minutes in PBS, slides were mounted with fluoromount (F4680; Sigma Aldrich) and imaged on a laser scanning confocal microscope.

[0121] RNA Extraction and Quantitative PCR (qPCR)

[0122] RNA was extracted using TRIzol (15596026; Thermo Fisher Scientific) following the manufacturer’s protocol. cDNA was synthesized from 1.0 pg of total RNA using SuperScript IV Vilo Master Mix with an added genomic DNA digestion step (11766050; Thermo Fisher Scientific). RT-qPCR was conducted in triplicate using SYBR Green (C 14512; Thermo Fisher Scientific) with 4.5 ng of cDNA and 500 nM primers per reaction. Reactions were run on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Gene expression values were normalized to RPLP0 and fold change was calculated as 2'AACt. Primers used in this study are described in Table 2. For experiments where interferons were used to study changes in gene expression, cells and organoids were treated for 24 hours with low (10 ng / mL) and high (100 ng / mL) concentrations of the molecules. Interferon alpha 2A (78076), alpha 2B (78077), beta (78113), and gamma (78020) were obtained from StemCell.

[0123] Table 2. Primers used in this study.

[0124] Protein Extraction and Western Blots

[0125] Proteins were isolated from cell lines and organoids using RIPA lysis buffer (20188; Sigma Aldrich) containing proteases (67110300; Roche) and phosphatases (04906837001; Roche). For western blotting, 20 pg of protein was mixed with 7.5 pL of SDS containing sample buffer (B0007; Thermo Fisher Scientific) in a 30 pL reaction. Samples were heated at 70 °C for 10 minutes, and then run in a 4-12% Bis-Tris gel (NW04120BOX; Thermo Fisher Scientific) at 140 volts for 1 hour. Proteins were transferred to a PVDF membrane (IB24001; Thermo Fisher Scientific) using an iBlot instrument (Thermo Fisher Scientific). Membranes were blocked for 1 hour at room temperature in blocking buffer (927-60001; Licor) and primary antibodies were diluted in blocking buffer at a concentration of 1 : 1000 and added to the shaking blots overnight at 4 °C. Primary antibodies used are detailed in Table 1. Membranes were washed 3 times for 10 minutes in tris-buffered saline with 0.1% Tween (TBST; 351-086-101; Quality Biological). IRDye secondary antibodies (Licor) were diluted 1 : 7,000 in blocking buffer and added to the shaking membranes for 1 hour at room temperature. The membranes were then washed 3 times for 10 minutes in TBST, and imaged using an iBright instrument (Thermo Fisher Scientific).

[0126] Ro52 Surface Presentation Stimulation Immunofluorescence

[0127] MCF7, MDA-MB-231, and MCF10A cells were treated with 10 ng / mL type 1 and 2 interferons for 48 hours. Following incubation, 5 pg / mL of anti-Ro52 and matched isotype control antibodies were added to live cells and incubated for 1 hour at 37 °C with 5.0% CO2. Cells were then washed in PBS, fixed in 4.0% paraformaldehyde, and stained with DAPI (1 : 1000), phalloidin (1 :500), and anti-mouse secondary antibodies (1 :200) as described above. Imaging was completed on a Nikon AXR confocal microscope with 16 images taken per well in a tiled pattern using a 20X lens. Anti-mouse fluorescence intensity was determined by first identifying cells using phalloidin staining and then quantifying mean fluorescence intensity in the channel of interest.

[0128] Patient Sera Ro52 Domain Binding Mapping

[0129] Recombinant Ro52 isoforms were generated, using site directed mutagenesis to delete various exons and domains of the gene. To map the Ro52 binding patterns of patient sera IgG's and commercial anti-Ro52 antibodies, equal amounts of SDS reduced Ro52 proteins were ran in 4-12% Bis-Tris gel and transferred to aPVDF membrane as described above. After blocking the membranes for 1 hour at room temperature, the blots were incubated with patient sera or primary antibody, diluted in blocking buffer, overnight at 4 °C. Patient sera was diluted 1 :2,500 and commercial anti-Ro52 monoclonal antibodies were diluted 1:1000. For competition experiments, antibodies were pre-incubated with a 10 fold-molar excess of anti-Ro52 peptides for 1 hour at room temperature, then used as the primary antibody source for 1 hour. Patient sera IgG and commercial mAb binding were visualized using anti-human (926-32232), antimouse (926-32212), and anti-rabbit (926-68073) secondary antibodies (Licor) diluted 1:7,000 in blocking buffer. After incubating for an hour in secondary antibody solutions, blots were washed 3 times for 10 minutes in TBST and imaged using an iBright instrument.

[0130] Immunofluorescence Competition Assays

[0131] Rheumatology' patient sera, as well as monoclonal antibodies, were pre-incubated with a 10-fold molar excess of Ro52 peptides for 1 hour at room temperature in 10 pL of cell culture media. Control reactions lacked a competing peptide. Following incubation, 40 pL of media was added to each reaction, yielding a final antibody concentration of 5 pg / mL which was then added to live MCF7 cells for 1 hour at 37 °C. Following incubation, cells were fixed in 4.0% paraformaldehyde and stained with DAPI (1: 1000), phalloidin (1 :500), and either anti-human or anti-mouse secondary antibodies (1:200) as described above. High magnification representative images were taken on a Nikon AXR using a Plan Apo 60x oil lens. Cells were also imaged using a 20X lens with 16 images taken in a tiled pattern. Fluorescence intensity was measured as described above, and results were normalized to the no peptide competition control.

[0132] Antibody Internalization Assays

[0133] To study antibody internalization dynamics, antibodies were conjugated to the pHrodo 647 (P35355; Thermo Fisher Scientific) pH sensitive dye which fluoresces once an antibody has been internalized into an acidic cellular compartment. Live cells were labeled with NuclearMask blue stain (H10325; Thermo Fisher Scientific) for 30 minutes prior to live cell imaging experiments. For each experiment, 5 pg / mL of pHrodo labeled antibody in media was added to the respective well, and cells were imaged every 5 minutes for 24 hours using an AXR confocal microscope and Plan Apo 20x lens. Antibody internalization dynamics were tracked by quantifying the change in 647 fluorescence intensify over time minus the background value obtained from wells to which no antibody was added. Results were normalized to the zero timepoint and plotted as a function of time.

[0134] Antibody Drug Conjugate (ADC) Creation and Assays

[0135] Antibodies were conjugated to both VcMMAE (AT7001) and DM1 (AT7002) from AlphaThera following the manufacture’s protocol, and conjugation was validated by SDS- PAGE. For ADC assays, 10,000 cells were seeded into each well of a tissue culture treated 96- well plate (3596; Coming) and allowed to adhere overnight. ADCs were added to cells the following morning and were dosed randomly into the plate using a D300e Digital Dispenser (Tecan). After incubating for 72 hours, cell viability was assessed using the CellTiter 96 Aqueous One Solution (G3580; Promega) and results were read on a Cytation 5 Multimode Reader. Cell viability in response to ADC treatment was normalized to the non-treatment controls which were standardized to 100% viability.

[0136] Software and Statistical Analysis

[0137] Image processing and analysis were completed using the latest version of both Fiji and NIS-Elements (Nikon) software, and specific analysis methods are indicated where appropriate. Custom Python scripts were used to tabulate and analyze data generated after image processing. FlowJo software (BD Biosciences) was used for all flow cytometry analyses. GraphPad Prism (Dotmatics) was used to prepare figures and to conduct statistical analyses. Figure schematics were created using icons and illustrations from BioRender.com.

[0138] Example 1 - Autoantibodies from a rheumatic disease patient bind selectively to cancer cells

[0139] Autoantibodies from a rheumatic disease patient were shown to bind selectively to cancer cells from an unrelated patient that is not known to have a rheumatic disease. This recognition would be unlikely if the antibody was recognizing a ‘private’ neoantigen in the rheumatic disease patient and suggests that there are general features of cancer that are recognized recurrently by the immune system. Results show that total IgG fractions from patients with diverse rheumatic diseases bind to fresh primary breast tumor tissue removed at surgery, and to PDX tumors (FIGs. 1A-1B). Furthermore, affinity purified antibodies from patients with rheumatic disease were shown to bind to cancer cell lines and to PDX tumors (FIGs. 3A-3C, FIG. 5)

[0140] Autoantibody binding to live PDX cancer cells in monoculture induced varying effects on cancer cell viability and proliferation. Autoantibody binding to fresh primary human breast tumor tissue in culture induced widely varying effects on cancer cell viability and proliferation, with some replicates showing strong cancer cell killing, wherein it was hypothesized that varying quantities of co-purified immune cells from the surgical specimen could explain this variation.

[0141] Autoantibody treatment of live cancer cells in co-culture with total immune cells, macrophages, or natural killer cells result in strong enhancement of cancer cell killing, relative to monoculture controls or to healthy donor IgG co-culture controls. The magnitude of the cancer cell killing depended on the specific IgG fraction or purified autoantibody and also on the donor immune cells. Results were consistent between replicates for a specific autoantibody I donor immune cell combination, wherein ADCC was observed in cocultures containing natural killer cells and ADCP in cocultures containing macrophages (FIGs. 2A-2C).

[0142] Example 2 - Efficacious autoantibodies for preclinical development

[0143] It was reasoned that the most common mechanisms by which therapeutic antibodies recognize and eliminate cancer cells all benefit from cell surface localization of the antigen. Most autoantigens have a nuclear or cytoplasmic localization in normal cells under homeostatic conditions (e.g., TIF1 gamma and Ro52 respectively). Antibody binding to autoantigens could occur by shifting localization of the antigen to the cell surface or internalization of the antibody into the antigen’s subcellular compartment. The more useful scenario for therapeutic purposes is an autoantigen that selectively localizes to the cell surface during cancer progression, as this shift in location would enable the widest range of candidate antibodies to be tested and the widest range of antibody engineering approaches to be employed to optimize immune effector function.

[0144] Cancer cell lines were screened for the differential localization of the autoantigen when live cancer cells are treated with autoantibodies in the culture medium vs. when fixed dead cancer cells are treated with autoantibodies. Most autoantibodies exhibited modest if any binding to the surface of live cells and strong staining to fixed cells, in patterns consistent with the known localization of their antigens (FIGs. 3A-3B).

[0145] An autoantibody, anti-Ro52, showed strong binding to the surface of live cancer cells (MCF7). Its binding to fixed cells was predominantly intracellular (FIGs. 3A-3B). Anti-Ro52 antibodies showed negligible binding to the surface of live normal mammary epithelial cells (MCFlOa; FIG. 3C). When monitored over time in cancer cells, antibody-bound, surface- localized Ro52 was internalized to small vesicles via endocytosis (FIG. 4). This differential binding analysis required very small quantities of autoantibodies and cells and is amenable to large scale screening. It was anticipated that the differential binding to live vs. fixed cancer cells will provide an effective surrogate for antigen accessibility in tumors and that differential binding to live cancer cells vs. live normal cells would provide an effective surrogate for therapeutic index in patients.

[0146] Example 3 - Anti-Ro52 antibodies bind to the surface of cancer cells Anti-Ro52 antibodies were observed to bind to the surface of live cancer cells (PDX and MCF7) with both antigen-purified patient autoantibodies and with monoclonal antibodies raised against Ro52 peptides in mice (FIG. 5).

[0147] MDA-MB-231 breast cancer cells were treated with anti-Ro52 antibodies and minimal cell surface binding was observed. It was hypothesized that Ro52 expression and surface localization might be dependent on pro-inflammatory or ‘danger signals’ in the tumor microenvironment. Therefore, it was tested whether interferon proteins could induce expression or cell surface localization of Ro52.

[0148] It was shown that addition of any type 1 interferons (alpha 2A or 2B, interferon beta) increased mRNA levels (qRT-PCR), increased protein levels (Western blot), and increased surface localization of Ro52 (IF and FACS). Type 2 interferons (interferon gamma) were also shown to increase mRNA levels, increase protein levels, and modestly increase surface localization of Ro52 (FIGs. 6A-6D). The magnitude of increase induced by interferon proteins varied based on the cancer model, with some showing strong induction and others modest. Preliminarily, the degree to which Ro52 expression and localization was induced by exogenous interferons was inversely correlated to the level of endogenous interferon production by the cancer cells. It was hypothesized that cancer cells that already made large amounts of interferon did not change their Ro52 levels much in response to experimentally added interferons. Conversely, cancer cells that did not synthesize their own interferons typically displayed large increases in level and surface localization of Ro52 when interferons were experimentally introduced (FIGs. 6A-6C).

[0149] Taken together, these observations suggest that cancer cells typically localize Ro52 to the cell surface spontaneously and I or will do so when exposed to inflammatory signals, such as interferons. The fact that diverse cancer models responded to diverse interferons by increasing their level and surface localization of Ro52 suggests that this is a conserved response of cells to cancer and that it could therefore serve as the target of broadly effective therapies.

[0150] Both affinity-purified, rheumatic disease patient-derived Ro52 autoantibodies and monoclonal anti-Ro52 antibodies (raised in animals immunized with Ro52 peptides) bind to the surface of cancer cells, suggesting that the antigen is the Ro52 protein. Since Ro52 is surface localized in cancer cells, antibodies are only one example of a molecular system which could bind to and recognize Ro52 for diagnostic, prognostic, or therapeutic purposes.

[0151] In 3D culture-based invasion assays, anti-Ro52 antibodies were found to bind to PDX breast cancer cells, with selective enrichment to the most invasive cells (FIG. 5). In 3D culturebased invasion assays, anti-Ro52 antibodies were found to bind to fresh human breast tumor tissue, with selective enrichment to the invasive component (FIG. 7B). In vivo in mice, mouse anti-Ro52 monoclonal antibodies (delivered via the tail vein) bound to spontaneously disseminated murine breast cancer cells in distant organs, such as the lungs. Binding was observed to individual cancer cells, micrometastases, and macrometastases. Binding was selectively to the cancer cells and not the host cells, based on comparison of live cell binding of anti-Ro52 antibodies and fixed cell binding of antibodies that recognize the experimental oncogene (FIG. 7A). This selective binding suggests that systemically delivered anti-Ro52 antibodies, or other molecular recognition systems targeting Ro52, could effectively recognize metastatic cancer cells in distant organs and be used to diagnose cancer, monitor response to therapy, detect metastatic recurrence, and deliver therapy.

[0152] Example 4 - Conjugate either of two different ADC-warheads to anti-Ro52 antibodies, cetuximab, and Herceptin

[0153] Anti-Ro52 antibodies were also found to bind to the surface of breast cancer cells at similar or greater levels as FDA-approved therapeutic antibodies, such as cetuximab and Herceptin (FIG. 8A). Next, either of two different ADC-warheads, VcMMAE and DM1, were conjugated to anti-Ro52 antibodies and separately conjugated to both cetuximab and Herceptin. Ro52-ADC was highly effective at killing cancer cells in monoculture, compared to either unconjugated anti-Ro52 antibodies or to cetuximab-ADC or Herceptin-ADC (FIGs. 8B-8C).

[0154] Example 5 - Autoimmune patient antibodies recognize and bind live breast cancer cells

[0155] It was first sought to test the hypothesis that autoantibodies in the sera of patients with rheumatic disease would bind to the surface of cancers from unrelated patients. To test this hypothesis, a set of sera from 70 patients with diverse rheumatic diseases was used to stain breast cancer cells both live, to assay for surface accessible antigens, and fixed, to assay the intracellular distribution of these antigens (FIG. 9A). It was anticipated that some surface accessible antigens could be altered or extracted by fixation and permeabilization and so may not be evident in fixed staining. The MCF7 cell line was used as a model of luminal breast cancer. When cells were stained live with patient sera, three categories of positive staining was observed: ubiquitous binding to the plasma membrane, selective binding to sites of intercellular junctions, and cytoplasmic staining (FIG. 9B). Binding to the plasma membrane and to cellcell junctions was observed uniformly across all cells in the dish, while cytoplasmic staining was observed in a small fraction of cells (FIGs. 9C and 10A). Binding of healthy control sera to live or fixed MCF7 cells was not observed (FIG. 9C and 10A). Cell surface binding was observed in select sera from patients with DM, SSc, and SLE; other patient sera bound to the expected antigens in fixed cells but did not bind to live cells (Tables 3-4). To quantify the magnitude of binding, MCF7 cells in suspension were labeled and analyzed by flow cytometry, with select sera showing binding orders of magnitude higher than healthy control sera (FIGs.

[0156] 9D, 10B-10C).

[0157] Table 3. Rheumatology patient sera used in this study (Sera with live cell surface staining)

[0158] Table 4. Rheumatology patient sera used in this study (Sera without live cell surface staining)

[0159] "Live cytoplasmic binding refers to a minority (~5%) of cells in the sample

[0160] Example 6 - Rheumatology patient sera mediate cytotoxic clearance of breast cancer cells

[0161] Having established that patient autoantibodies can bind to the surface of live breast cancer cells, it was next sought to determine if binding mediates a cytotoxic immune response. Two major antibody dependent pathways of cellular toxicity are the complement pathway and natural killer (NK) cell-mediated antibody dependent cellular cytotoxicity (ADCC). First, the ability of serum autoantibodies to induce complement activation against MCF7 breast cancer cells was evaluated. It was found that both cell surface-binding and junction-binding patient sera potently activated complement, within an hour of incubation with live cells (FIGs. 11A- 1 IB). Sera from healthy control patients did not activate complement, and sera lacking live cell surface binding was a poor inducer of complement (FIG. 1 IB).

[0162] It was previously shown that the outgrowth of clusters of 2-10 cancer cells in Matrigel predicts the results of metastatic outgrowth in the lungs following tail vein injection. A coculture variant of the assay was also developed that includes NK cells to study immune-cancer interactions in an ex vivo setting. Of note, the media in the colony forming assays does not support complement activation. Sera from seven myositis patients and eleven scleroderma patients were evaluated for their ability to reduce colony forming ability of patient-derived xenograft (PDX) triple negative breast cancer (TNBC) cells, in monoculture or in co-culture with NK cells (FIG. 11C). There was no significant reduction in colony forming in the monocultures in responseto treatment with any of the patient sera (FIGs. 11D-11E). In contrast, a two-fold reduction in colony formation was observed following treatment with eight of the nineteen patient sera in NK cell co-culture assays (FIGs. 11D and 1 IF). These results indicate that the IgG fraction of rheumatic patient sera can activate complement and induce ADCC against cancer cells.

[0163] Example 7 - Anti-Ro52 antibodies bind the surface of breast cancer cells in vitro and metastases in vivo

[0164] Rheumatic patients are known to raise a diverse repertoire of autoantibodies, to a variety of host antigens and proteins. Knowing this, it was sought to resolve the individual antigens responsible for cell surface binding in these patient sera, using a candidate approach (FIG. 12A). Nine antigens that are frequently targeted by rheumatic patient autoantibodies were selected and commercial antibodies raised against these antigens were acquired (FIG. 12B). For reference, the level of staining observed with anti-ERBBl (Cetuximab biosimilar) and anti-ERBB2 (Herceptin biosimilar) in MCF7 cells was determined (FIG. 13A). It should be noted that antibody drug conjugates (ADCs) targeting ERBB1 are in clinical development and ADCs targeting ERBB2 are in clinical use. The pattern of antibody binding in live cells were then compared (FIG. 13B) versus fixed cells (FIG. 13C). Fixed staining patterns recapitulated the expected cytoplasmic (Ro52) and nuclear localizations (POLR3A, CCAR1, TIFlg, and Mi2b) of each antigen. Robust live cell binding to the plasma membrane was only observed with Ro52 (FIG. 12C and 13B). It was noted that intensity and uniformity of staining was comparable to that observed with the Herceptin biosimilar (FIG. 12C vs. FIG. 13A).

[0165] Fortunately, the anti-Ro52 monoclonal antibody recognizes both murine and human proteins and so were able to test the ability of anti-Ro52 antibodies to recognize metastatic cancer cells in the lungs of live mice. To do so, either fluorescently conjugated isotype control IgG or fluorescently conjugated monoclonal anti-Ro52 IgG antibody were injected into the tail vein of mice bearing spontaneous murine tumors and their corresponding lung metastases (FIG. 12D). After one hour, the lungs were harvested, counterstained for the middle T oncogene, and quantified fluorescence intensity. Strong and selective binding of anti-Ro52 antibody to middle T+ cancer cells was observed, while isotype control antibody showed no specific staining in the lungs, (FIGs. 12E-12F). There was essentially no background staining of normal cells by the anti-Ro52 antibody, suggesting that it is highly selective for cell surface Ro52 and that normal lung cells rarely localize Ro52 to the cell surface. Since Ro52 is a widely expressed cytoplasmic FC receptor, it was anticipated that fixed cell staining would show widespread binding.

[0166] Example 8 - Interferons induce surface presentation of the Ro52 antigen selectively in cancer cells

[0167] It was next sought to assess the differential in live surface binding of anti-Ro52 antibodies to luminal breast cancer cells (MCF7), TNBC cells (MDA-MB-231), and normal mammary epithelial cells (MCF10A). Robust binding to MCF7 cells was observed and no live staining of either MDA-MB-231 or MCF10A cells was observed (FIG. 14A). As Ro52 is part of the host defense against viral infection, it was hypothesized that negative Ro52 surface expression could be due to the absence of immune tumor microenvironment (TME) signals in the simplified 2D culture context. Such signals include interferons, a class of cytokines released by the innate immune system and host epithelial cells in response to viral infection. It was tested whether addition of interferon-a 2A, interferon-a 2B, interferon-b, or interferon-g could regulate expression levels or subcellular localization of Ro52 (FIG. 14B). Upregulation of TRIM21 / Ro52 RNA was observed, in concert with a suite of interferon-induced transcripts (IFITs; FIG. 14C), and upregulation of Ro52 protein (FIGs. 14D, 15E), in each of the three cell lines. Interferon treatment induced a large increase in surface localized Ro52 in MDA- MB-231 cells; in contrast, the normal MCF10A cells still had no detectable Ro52 in live antibody binding assays (FIG. 14E-14E). It was confirmed that MDA-MB-231 and MCF10A cells have substantial cytoplasmic pools of Ro52 at baseline through fixed cell staining (FIG. 15A). Taken together, the data revealed that cancer cells can have either constitutive or interferon-dependent surface localization of Ro52 protein, while normal mammary epithelial cells do not exhibit surface localization even in response to interferon treatment. Furthermore, the data suggested that cancer cells frequently localize Ro52 to the cell surface, likely in inflamed TMEs, and that there could be a substantial therapeutic index between surface- available Ro52 in cancer cells vs. normal cells.

[0168] Next, molecular insight was sought into why exogenous interferon is required for Ro52 surface localization in MDA-MB-231 cells and not in MCF7 cells, using qRT-PCR. It was first confirmed that both TRIM21 / Ro52 and the interferon receptors are expressed in all three cell lines (FIGs. 15B-15C). The expression of five interferon ligands were then assayed and significant expression of four interferons in MCF7 cells and no expression of interferon ligands in MDA-MB-231 cells was observed (FIG. 15D). Therefore, it was anticipated that surface localization of Ro52 is dependent on autocrine interferon signaling in MCF7, while MDA-MB- 231 cell surface localization is dependent on interferon from the TME. There must be additional regulatory signals, as expression of TRIM21 / Ro52, interferon receptors, and interferon ligands is not sufficient to induce surface localization of Ro52 in normal MCF10A cells. It was speculated that this molecular logic could increase the therapeutic index of Ro52 targeting strategies, as it suggests that adjacent normal cells may be spared, consistent with the robust live labeling of metastatic breast cancer cells in murine lungs, without detectable labeling of lung epithelium or stroma (FIG. 12F).

[0169] Example 9 - Live binding anti-Ro52 antibodies recognize an epitope from the fourth exon oi TRIM21

[0170] The next challenge was to understand precisely what the anti-Ro52 antibodies were recognizing at the surface of breast cancer cells. There were two elements here: determining which domains of the protein are surface localized and using biochemical techniques to demonstrate the specificity of the antigen target. Analysis was guided by prior molecular analyses of protein variants of Ro52 that are observed in rheumatic patients. Briefly, Ro52 is expressed in cells in full length form (Ro52a), with exon 4 missing (Ro52b), and with exon 6 and part of exon 7 missing (Ro52g; FIG. 16A). Protein was expressed and purified from each of these naturally occurring Ro52 variants and an engineered construct containing only exons 3 and 4 (Ro52Ex3-4). Then, gels were run with each lane loaded with a single purified protein. The ability of different commercial and patient-derived serum antibodies to bind to the different Ro52 variants was evaluated. Evaluation started by comparing commercial antibodies raised to the c-terminus or to Exon 4, to validate the approach. A C-terminus targeted commercial anti-Ro52 antibody bound only to Ro52a and Ro52b, while an exon 4-targeted commercial anti-Ro52 antibody bound Ro52a, Ro52g, and Ro52Ex3-4 (FIG. 16B). Next, the binding to Ro52 variants of two patient derived sera was evaluated, one from a patient with scleroderma and one from a patient with lupus. Both sera had clinical reactivity to Ro52 (Tables 3 and 4) and both displayed live labeling of the surface of cancer cells (FIG. 10A). Both patient sera bound Ro52a, Ro52g, and Ro52Ex3-4, consistent with selective binding to the fourth exon (FIG. 16C)

[0171] Since the sera from these patients bound Ro52’s fourth exon in protein lysates and the surface of live cells, it was next tested whether it would be possible to compete away binding with addition of Ro52 protein variants. To validate this approach, gels were run again with the Ro52 variants and blotted with commercial exon 4-specific antibody alone, with recombinant Ro52b (lacking exon 4), and with Ro52Ex3-4. Antibody alone recapitulated the staining pattern seen earlier, addition of Ro52b did not affect binding, while addition of Ro52Ex3-4 prevented binding of the Exon 4-specific antibody to any of the lanes (FIG. 16D). Next, an analogous approach was taken to test whether it would be possible to compete live cell surface binding with addition of Ro52 variants, in MCF7 cells (FIG. 16E). First, commercial exon 4-specific antibody was tested alone, antibody plus recombinant Ro52b, or antibody plus recombinant Ro52Ex3-4 in the live cell binding assay. Addition of Ro52b had no significant effect on cell surface staining, while addition of Ro52Ex3-4 reduced fluorescence levels by more than 60% (FIG. 16F). Having established the logic of the assay, it was demonstrated that cell surface binding could be outcompeted for two patient sera with clinically validated Ro52 specificity and not for two patient sera known to be reactive to other antigens (FIG. 16G). Taken together, these data demonstrate that sera from patients with rheumatic disease can selectively bind cancer cells based on interactions with the fourth exon of Ro52. They also reveal that there are additional, Ro52-independent cancer cell surface antigens that are recognized by rheumatic autoantibodies, suggesting a broader class of therapeutic targets.

[0172] Example 10 - Anti-Ro52 antibody drug conjugates selectively kill cancer cells

[0173] Definitionally, if cancer is progressing in a patient, it has found some way to evade or coopt the immune system. The data suggests that cancer cells selectively localize conserved antigens to their cell surface and that the immune system generates antibodies that recognize these antigens. Advances in immune engineering enable therapeutic targeting of cancer based on these antigens. Antibody drug conjugates (ADCs) are a particularly attractive technology because they can kill cancer cells directly through selective delivery of chemotherapy to antigen positive cells. To test this concept, isotype control and commercial fourth exon-specific anti-Ro52 antibodies were conjugated with the pHrodo pH sensitive dye. The pHrodo dye is selectively fluorescent at the low pH of late endosomes and so enables direct assessment of antibody internalization kinetics (FIG. 17A). MCF7 cells were treated with labeled antibodies and rapid and efficient internalization of anti-Ro52 antibody was observed, relative to isotype control (FIGs. 17B-17C). Next, these same reagents were tested in MCF10A cells and minimal internalization of either antibody was observed (FIGs. 17D-17E), demonstrating that there is significantly greater binding to and internalization into target antigen positive cancer cells than target antigen negative normal epithelial cells. As direct proof of concept, MCF7 and MCF10A cells were treated with free chemo (VcMMAE or DM1), isotype control antibody plus and minus payload, and commercial Exon 4-specific anti-Ro52 antibody plus and minus payload. Selective killing of cancer cells was observed in the anti-Ro52 plus payload conditions (FIG. 17F).

[0174] Example 11 - Ro52 antigen can be recognized on the surface of diverse cancer types

[0175] It was then sought to test the generality of Ro52 as a therapeutically targetable cell surface antigen by assaying cell surface staining in live cancer cells from diverse cancer types and sources. It was first verified that a panel of human gastrointestinal cancer cell lines and a normal human mesothelial cell line were all positive for Ro52 in fixed cell assays (FIG. 19). It was then tested in live cells and observed uniform cell surface binding to pancreatic, colorectal, and gastric cancer cell lines, heterogeneous surface binding to an ovarian cancer cell line, and no binding to a second, independent colorectal cancer cell line or to the normal mesothelial cells (FIG. 18A). Finally, fresh primary human tumor tissue was obtained from breast tumors, cultured in 3D collagen I gels, and observed strong binding of anti-Ro52 antibody to live cancer cells from multiple cases (FIG. 18B).

Claims

WHAT IS CLAIMED IS:

1. A method of treating a cancer in a subject, wherein the cancer expresses a Ro52 antigen, the method comprising administering an anti-Ro52 binding agent to the subject, thereby treating the cancer in the subject.

2. The method of claim 1, wherein the Ro52 antigen is expressed on a cancer cell surface.

3. The method of claim 1 or 2, wherein the anti-Ro52 binding agent comprises a Fab, Fab’, F(ab’)2, Fab^SH, Fv, diabody, linear antibody or single-chain variable fragment (scFv).

4. The method of any one of claims 1-3, wherein the anti-Ro52 binding agent is a humanized or chimeric antibody.

5. The method of any one of claims 1-4, wherein the anti-Ro52 binding agent is conjugated to a therapeutic agent.

6. The method of claim 5, wherein the therapeutic agent comprises a chemotherapeutic agent.

7. The method of claim 6, wherein the chemotherapeutic agent comprises DM1, MMAE, MMAF, mertansine, calicheamicin, SN-38, or exatecan.

8. The method of claim 6, wherein the chemotherapeutic agent mediates cytotoxicity of the cancer cell.

9. The method of any one of claims 1-8, wherein the cancer is a metastatic cancer.

10. The method of claim 9, wherein the metastatic cancer is a breast cancer, colon cancer, gastric cancer, pancreatic cancer, or ovarian cancer.

11. The method of any one of claims 1-10, wherein prior to administration, the Ro52 antigen is present on the cancer cell surface.

12. The method of claim 11, wherein the cancer cell is from a primary tumor sample or a metastatic tumor sample.

13. The method of claim 12, wherein the primary tumor sample or the metastatic tumor sample is from a fresh biopsy sample.

14. The method of claim 12, wherein the primary tumor sample or the metastatic tumor sample is from a fixed biopsy sample.

15. The method of claim 11, wherein the cancer cell comprises circulating tumor cells.

16. A method for identifying a tumor in a subject, the method comprising:(a) administering an anti-Ro52 binding agent to the subject; and(b) detecting the anti-Ro52 binding agent on a cell of the tumor in the subject, thereby identifying the tumor in the subject.

17. The method of claim 16, wherein the anti-Ro52 binding agent is bound to a cell surface of the cell.

18. The method of claim 16 or 17, wherein the anti-Ro52 binding agent is conjugated to a label.

19. The method of claim 18, wherein the label is a detectable label.

20. The method of claim 19, wherein the label is a fluorophore.

21. The method of claim 20, wherein the detecting step (b) comprises fluorescence imaging.

22. The method of claim 16, wherein the anti-Ro52 binding agent is conjugated to an imaging agent.

23. The method of claim 22, wherein the detecting step (b) comprises magnetic resonance imaging (MRI) or positron emission tomography (PET) imaging.

24. The method of any one of claims 16-23, wherein the administering step (a) comprises intravenous, intrathecal, intra-thoracic, or intraperitoneal administration.

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