Small cell lung cancer tumor antigen and application thereof

Abstract

Disclosed herein are high performance biomarkers and kits for the early detection of small cell lung cancer (SCLC), which can be used to identify, diagnose and treat SCLC patients in the early stage. Methods of diagnosing and treating SCLC in a subject can include detecting the presence of an autoantibody that specifically binds to one or more epitopes of an antigen in a biological sample obtained from the subject, wherein the one or more epitopes of the antigen comprise a post-translational modification; diagnosing the subject with SCLC when the presence of the autoantibody is detected in the biological sample; and treating the SCLC in the subject by administering a chemotherapy regimen, an immunotherapy regimen, an antibody, surgical and / or radiation therapy. In one example, post-translational modifications targeted by autoantibodies include citrullination, isoaspartic acidification, and / or cancer-specific glycosylation.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 415,143, filed on Oct. 11, 2022, which is hereby incorporated by reference in its entirety for all purposes.

[0003] Reference to a Sequence Listing

[0004] This application incorporates by reference a computer - readable sequence listing in ST.26 XML format, named 23 - 032 - WO - PCT_Sequence, created on Sep. 25, 2023, and containing 98,924 bytes.

[0005] Government Sponsorship Statement

[0006] This invention was made with government support under grants CA268066, CA243328, CA186157, and CA281801 awarded by the National Institutes of Health. The government has certain rights in the invention. Technical Field

[0007] The present disclosure relates to the medical field, and more particularly to autoantibodies and autoantibody - autoantigen complexes as biomarkers for small cell lung cancer (SCLC), and methods of using the same. Background Art

[0008] Small cell lung cancer (SCLC) is the sixth leading cause of cancer-related death in the United States, and less than 6% of patients survive 5 years after diagnosis (Siegel et al., 2022, A Cancer Journal for Clinicians 72, 7–33; Wang et al., 2017, Sci Rep 7, 1339). SCLC accounts for approximately 10%–15% of lung cancers and shares a very important feature with essentially all solid tumor malignancies: early detection of the cancer leads to improved survival metrics. However, the key obstacle to effective life-saving treatment is the lack of early detection, as 70% of SCLC patients are diagnosed with metastatic disease (Alvarado-Luna and Morales-Espinosa, 2016, Transl Lung Cancer Res 5, 26–38). This may be due to the aggressive nature and mediastinal location of SCLC, rendering annual imaging examinations insufficient. Currently, no blood test is recommended for the early detection of lung cancer. Since the majority of SCLC patients are diagnosed at the extensive stage, where current treatment options offer limited benefits, many researchers are not yet aware of the fact that nearly approximately 20% of patients with limited-stage SCLC can be cured with conventional cytotoxic chemotherapy. Additionally, for highly selected very early-stage patients, surgical resection, which is not typically considered for SCLC, can be curative when combined with chemotherapy. Low-dose CT screening programs that have proven effective for non-small cell lung cancer have not shown benefits in SCLC. Therefore, assays and methods for the early identification of SCLC are needed.

[0009] More than any other cancer type, SCLC has a strong association with a group of autoimmune diseases called paraneoplastic neurological syndromes (PNS) ( et al., 2015, Curr Opin Oncol 27, 489–495). Many PNS are thought to be the clinical manifestations resulting from autoantibodies (AAb) targeting neuroendocrine antigens expressed by tumor cells and cross-reacting in the central nervous system (Anwar et al., 2019, Ann Transl Med 7), but the mechanistic basis and extent of AAb production in SCLC remain poorly understood. Notably, PNS symptoms precede cancer diagnosis in 80% of affected patients and have led to increased early SCLC detection and improved overall survival (Sebastian et al., 2019, Journal of Thoracic Oncology 14, 1878–1880; Honnorat and Antoine, 2007, Orphanet Journal of Rare Diseases 2, 22). In addition, although PNS diagnosis is rare, AAb against PNS antigens can be found in the majority of SCLC patients who do not show autoimmune symptoms (Kazarian and Laird-Offringa, 2011, Mol Cancer 10, 33). This unique relationship of AAb production during SCLC tumorigenesis suggests that AAb against non-PNS antigens may exist and act more broadly as biomarkers for SCLC.

[0010] SCLC-specific AAb that do not cause overt autoimmune diseases such as PNS may be triggered by tumor alterations that increase the immunogenicity of self-antigens. Unfortunately, the ability of sequence- or structure-based methods for predicting immunogenic epitopes by in silico approaches is limited (Jespersen et al., 2019, Frontiers in Immunology 10), and current experimental methods for AAb discovery have several limitations. Many methods for AAb detection utilize capture reagents that have been immobilized or linearized, thus excluding AAb targeting conformational protein epitopes. Recombinant proteins produced in non-human species such as bacteria or yeast have been used as conformational capture antigens, but AAb targeting human-specific post-translational modifications (PTM) or disease-specific neoepitopes cannot be detected (Doyle and Mamula, 2001, Trends in Immunology 22, 443–449). Methods aimed at comprehensively understanding potential epitopes, such as serum epitope library analysis (Kamath et al., 2020, Sci Rep 10, 5294) or tumor proteome profiling (Qiu et al., 2008, J Clin Oncol 26, 5060–5066) can successfully identify disease-specific AAb, but require labor-intensive deciphering of homologous antigens at the backend. Therefore, alternative high-dimensional methods are needed to capture disease-related AAb.

[0011] Early diagnosis of SCLC can have a meaningful impact on current standards of care. Current treatment options include chemotherapy with platinum-etoposide, consideration of concurrent chest radiation for patients with limited-stage disease, and chemotherapy alone for those with extensive-stage disease (Corso et al., 2015, J Clin Oncol 33, 4240-4246). Prophylactic cranial radiation is recommended for patients who respond to initial treatment because it not only reduces the risk of brain metastases but also improves overall survival. Surgical resection of SCLC presenting as a solitary pulmonary nodule during early disease has shown promising results (Kreisman et al., 1992, Chest 101, 225-31).

[0012] Adoptive immunotherapy, which involves the transfer of ex vivo-generated self-antigen-specific T cells, is a promising strategy for treating cancer. Through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs), new specificities have been successfully generated in T cells (Jena et al., 2010, Blood 116(7), 1035-44). A CAR is a synthetic receptor composed of a targeting moiety associated with one or more signaling domains in a single fusion molecule. Typically, the binding moiety of a CAR consists of the antigen-binding domain of a single-chain antibody (scFv), which contains the variable fragments of the light and heavy chains of a monoclonal antibody linked by a flexible linker. The signaling domains of first-generation CARs are derived from the cytoplasmic regions of CD3ζ or the Fc receptor γ chain. Engineered cells expressing chimeric antigen receptors targeting SCLC can be used in therapeutic settings that require specific targeting of SCLC cells and T cell-mediated killing of SCLC cells. SUMMARY OF THE INVENTION

[0013] Methods for diagnosing and treating small cell lung cancer in a subject are disclosed herein. According to embodiments of the present disclosure, a large-format antibody array is disclosed that captures circulating AAb-antigen (AAb-Ag) complexes in human plasma. The unique isolation of AAb-Ag complexes directly from patients enables the ex vivo detection of AAbs that bind to native tumor-derived epitopes. In multiple independent SCLC cohorts, a set of antigens and their corresponding AAbs that are upregulated before diagnosis and in limited-stage and extensive-stage disease are identified and validated. These AAbs identify novel tumor-associated antigens, some of which are expressed on the cell surface in the majority of SCLC tumors examined. Detailed analysis reveals that some of these proteins contain immunogenic PTMs, including citrullination, isoaspartylation, and / or cancer-specific glycosylation, which are confirmed to bind to SCLC patient AAbs when mimicked with peptides containing synthetic PTMs.

[0014] Also disclosed herein is a unique high-throughput method for identifying tumor-specific AAbs that can be readily applied to AAb discovery in other diseases such as autoimmunity or infection. The validated AAb panel yields significant utility in a risk prediction model for SCLC, which can be further enhanced by incorporating pack-years of smoking. If applied during lung cancer screening, such a model has the potential to have a meaningful impact on overall survival of this lethal disease through early detection of SCLC and current standards of care treatment.

[0015] In one aspect, the present disclosure provides a method of treating a subject having small cell lung cancer (SCLC), the method comprising: (a) administering a chemotherapy regimen to the subject; (b) administering an immunotherapy regimen to the subject; (c) administering an antibody or an antigen-binding fragment thereof to the subject; (d) performing surgical resection of SCLC in the subject; and / or (e) exposing the subject to radiotherapy targeting SCLC, wherein the subject has autoantibodies that specifically bind one or more epitopes of one or more antigens selected from the group consisting of CDH5, CD133, SPINK1, CDH23, NLRP7, TFRC, SPINT2, NADSYN1, HIF1A, GRAP2, MAPRE1, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9, and wherein one or more epitopes of the antigen comprise a post-translational modification (PTM).

[0016] In some embodiments of the method, the subject has autoantibodies that specifically bind two or more epitopes of one or more antigens. In some cases, the subject has autoantibodies that specifically bind three or more epitopes of one or more antigens. In some cases, the subject has autoantibodies that specifically bind four or more epitopes of one or more antigens. In some cases, the subject has autoantibodies that specifically bind five or more epitopes of one or more antigens.

[0017] In some embodiments of the method, one or more antigens are selected from the group consisting of SPINK1, TFRC, GRAP2, TIMP2, and PLD3. In some cases, one or more antigens are selected from the group consisting of SPINK1, GRAP2, TIMP2, and PLD3. In some cases, one or more antigens are selected from the group consisting of SPINT2, SPINK1, TIMP2, and TFRC.

[0018] In some embodiments of the method, one or more epitopes are selected from the group consisting of SEQ ID NOs: 1-21. In some cases, the PTMs include citrullination, isoaspartylation, and / or glycosylation. In some cases, the PTMs include citrullination and / or isoaspartylation. In some cases, one or more epitopes are selected from the group consisting of SEQ ID NOs: 22-46.

[0019] In one aspect, the present disclosure provides a method for diagnosing and treating small cell lung cancer (SCLC) in a subject, the method comprising: (a) detecting the presence of one or more autoantibodies that specifically bind to one or more epitopes of one or more antigens in a biological sample obtained from the subject, wherein the one or more antigens are selected from the group consisting of: CDH5, CD133, SPINK1, CDH23, NLRP7, TFRC, SPINT2, NADSYN1, HIF1A, GRAP2, MAPRE1, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9, and wherein one or more epitopes of the antigen comprise post-translational modifications (PTMs); (b) diagnosing the subject with SCLC when the presence of one or more autoantibodies is detected in a biological sample obtained from the subject; and (c) treating the subject's SCLC by: (i) administering a chemotherapy regimen to the subject; (ii) administering an immunotherapy regimen to the subject; (iii) administering an antibody or an antigen-binding fragment thereof to the subject; (iv) performing surgical resection of SCLC in the subject; and / or (v) exposing the subject to radiotherapy targeting SCLC. In some embodiments, the method further comprises obtaining a biological sample from the subject. In some cases, the biological sample is serum or plasma.

[0020] In some embodiments of the method, diagnosing the subject comprises diagnosing the subject with SCLC when the presence of autoantibodies that bind to two or more epitopes of one or more antigens is detected in the biological sample. In some cases, diagnosing the subject comprises diagnosing the subject with SCLC when the presence of autoantibodies that bind to three or more epitopes of one or more antigens is detected in the biological sample. In some cases, diagnosing the subject comprises diagnosing the subject with SCLC when the presence of autoantibodies that bind to four or more epitopes of one or more antigens is detected in the biological sample. In some cases, diagnosing the subject comprises diagnosing the subject with SCLC when the presence of autoantibodies that bind to five or more epitopes of one or more antigens is detected in the biological sample.

[0021] In some embodiments of the methods discussed above, one or more antigens are selected from the group consisting of SPINK1, TFRC, GRAP2, TIMP2, and PLD3. In some cases, one or more antigens are selected from the group consisting of SPINK1, GRAP2, TIMP2, and PLD3. In some cases, one or more antigens are selected from the group consisting of SPINT2, SPINK1, TIMP2, and TFRC.

[0022] In some embodiments of the methods discussed above, one or more epitopes are selected from the group consisting of SEQ ID NO: 1-21. In some cases, the PTMs include citrullination, isoaspartylation, and / or glycosylation. In some cases, the PTMs include citrullination and / or isoaspartylation. In some cases, one or more epitopes are selected from the group consisting of SEQ ID NO: 22-46. In some cases, the subject is human.

[0023] In some embodiments of the methods discussed above, the chemotherapy regimen includes one or more cycles of cisplatin or carboplatin in combination with etoposide.

[0024] In some embodiments, the immunotherapy regimen includes chimeric antigen receptor T cells that specifically bind to one or more of the epitopes. In some cases, the immunotherapy regimen includes an antibody that binds to one or more of the epitopes. In some cases, the antibody is conjugated to a cytotoxic agent. In some embodiments, the immunotherapy regimen includes a bispecific antibody that binds to one or more of the epitopes and a T cell antigen. In some cases, the T cell antigen is CD3.

[0025] In one aspect, the treatment method includes administering to a subject an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or an antigen-binding fragment thereof specifically binds to one or more epitopes of one or more antigens, wherein the one or more antigens are selected from the group consisting of CDH5, CD133, SPINK1, CDH23, NLRP7, TFRC, SPINT2, NADSYN1, HIF1A, GRAP2, MAPRE1, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9, and wherein one or more epitopes of the antigen comprise a post-translational modification (PTM).

[0026] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs contained within the light-chain variable region (LCVR), wherein the amino acid sequences of the HCVR / LCVR respectively comprise amino acid sequences selected from the group consisting of: SEQ ID NO:47 / 51, 55 / 59, 63 / 67, 71 / 75, and 79 / 83.

[0027] In some embodiments, the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, which domain comprises amino acid sequences selected from the group consisting of: SEQ ID NO:48-49-50-52-53-54, 56-57-58-60-61-62, 64-65-66-68-69-70, 72-73-74-76-77-78, and 80-81-82-84-85-86, respectively.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy-chain variable region (HCVR) and a light-chain variable region (LCVR), wherein the amino acid sequences of the HCVR / LCVR are respectively selected from the group consisting of: SEQ ID NO:47 / 51, 55 / 59, 63 / 67, 71 / 75, and 79 / 83.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of TFRC-4 that contains a PTM, and wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), the heavy-chain variable region comprising the amino acid sequence of SEQ ID NO:79, and the light-chain variable region comprising the amino acid sequence of SEQ ID NO:83.

[0030] In some embodiments, the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, which domain respectively comprises the amino acid sequences of SEQ ID NO:80-81-82-84-85-86.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR containing the amino acid sequence of SEQ ID NO:79 and an LCVR containing the amino acid sequence of SEQ ID NO:83. In some embodiments, the epitope comprises the amino acid sequence of SEQ IDNO:39.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of CA9-3 that contains a PTM, and wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), the heavy-chain variable region comprising the amino acid sequence of SEQ ID NO:71 and the light-chain variable region comprising the amino acid sequence of SEQ ID NO:75.

[0033] In some embodiments, the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain that comprises the amino acid sequences of SEQ ID NO:72-73-74-76-77-78, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR that contains the amino acid sequence of SEQ ID NO:71 and an LCVR that contains the amino acid sequence of SEQ ID NO:75. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO:45.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of TFRC-5 that contains a PTM, and wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), the heavy-chain variable region comprising the amino acid sequence of SEQ ID NO:47 and the light-chain variable region comprising the amino acid sequence of SEQ ID NO:51.

[0035] In some embodiments, the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain that comprises the amino acid sequences of SEQ ID NO:48-49-50-52-53-54, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR that contains the amino acid sequence of SEQ ID NO:47 and an LCVR that contains the amino acid sequence of SEQ ID NO:51. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO:40.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of TFRC-1 or TFRC-2 that contains a PTM, and wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), the heavy-chain variable region comprising the amino acid sequence of SEQ ID NO:55 and the light-chain variable region comprising the amino acid sequence of SEQ ID NO:59.

[0037] In some embodiments, the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain that comprises the amino acid sequences of SEQ ID NO:56-57-58-60-61-62, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR that contains the amino acid sequence of SEQ ID NO:55 and an LCVR that contains the amino acid sequence of SEQ ID NO:59. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:37.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of SPINK1-1 that contains a PTM, and wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), the heavy-chain variable region comprising the amino acid sequence of SEQ ID NO:63 and the light-chain variable region comprising the amino acid sequence of SEQ ID NO:67.

[0039] In some embodiments, the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain that comprises the amino acid sequences of SEQ ID NO:64-65-66-68-69-70, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR that contains the amino acid sequence of SEQ ID NO:63 and an LCVR that contains the amino acid sequence of SEQ ID NO:67. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO:29.

[0040] In one aspect, the present disclosure provides a biomarker panel for small cell lung cancer (SCLC), the biomarker panel comprising one or more peptides comprising an epitope 7 to 50 amino acids in length containing one or more antigens, the one or more antigens selected from the group consisting of CDH5, CD133, SPINK1, CDH23, NLRP7, TFRC, SPINT2, NADSYN1, HIF1A, GRAP2, MAPRE1, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9, wherein the epitope comprises a post-translational modification (PTM) that is bound by an autoantibody.

[0041] In some embodiments, the biomarker panel is capable of detecting SCLC in a subject with at least 60% sensitivity and specificity. In some cases, the biomarker panel is capable of detecting SCLC in a subject with at least 70% or 75% sensitivity and specificity. In some cases, the biomarker panel is capable of detecting SCLC in a subject with at least 80% or 85% sensitivity and specificity. In some cases, the biomarker panel is capable of detecting SCLC in a subject with at least 90% or 95% sensitivity and specificity.

[0042] In some embodiments, the biomarker panel comprises two or more epitopes of one or more antigens. In some cases, the biomarker panel comprises three or more epitopes of one or more antigens. In some cases, the biomarker panel comprises four or more epitopes of one or more antigens. In some cases, the biomarker panel comprises five or more epitopes of one or more antigens.

[0043] In some embodiments of the biomarker panel discussed above, the one or more antigens are selected from the group consisting of SPINK1, TFRC, GRAP2, TIMP2, and PLD3. In some cases, the one or more antigens are selected from the group consisting of SPINK1, GRAP2, TIMP2, and PLD3. In some cases, the one or more antigens are selected from the group consisting of SPINT2, SPINK1, TIMP2, and TFRC.

[0044] In some embodiments of the biomarker panel discussed above, one or more epitopes are selected from the group consisting of SEQ ID NO: 1-21. In some cases, the PTM includes citrullination, isoaspartylation, and / or glycosylation. In some cases, the PTM includes citrullination and / or isoaspartylation. In some cases, one or more epitopes are selected from the group consisting of SEQ ID NO: 22-46.

[0045] In some embodiments, a biomarker panel is used to detect autoantibodies that bind to one or more epitopes of one or more antigens in a biological sample obtained from a subject.

[0046] In one aspect, the present disclosure relates to an isolated antibody or antigen-binding fragment thereof that binds to an epitope of TFRC-4 containing a PTM, wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), the heavy-chain variable region comprising the amino acid sequence of SEQ ID NO:79, and the light-chain variable region comprising the amino acid sequence of SEQ ID NO:83.

[0047] In some embodiments, the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, which domains comprise the amino acid sequences of SEQ ID NOs: 80-81-82-84-85-86, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR containing the amino acid sequence of SEQ ID NO:79 and an LCVR containing the amino acid sequence of SEQ ID NO:83. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO:39.

[0048] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to an epitope of CA9-3 containing a PTM, wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), the heavy-chain variable region comprising the amino acid sequence of SEQ ID NO:71, and the light-chain variable region comprising the amino acid sequence of SEQ ID NO:75.

[0049] In some embodiments, the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, which domains comprise the amino acid sequences of SEQ ID NOs: 72-73-74-76-77-78, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR containing the amino acid sequence of SEQ ID NO:71 and an LCVR containing the amino acid sequence of SEQ ID NO:75. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO:45.

[0050] In one aspect, the present disclosure relates to an antibody or an antigen-binding fragment thereof that binds to an epitope containing PTM of TFRC-5, wherein the antibody or the antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained in the light-chain variable region (LCVR), the heavy-chain variable region comprises the amino acid sequence of SEQ ID NO: 47, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO: 51.

[0051] In some embodiments, the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain that comprises the amino acid sequences of SEQ ID NO: 48-49-50-52-53-54, respectively. In some embodiments, the antibody or the antigen-binding fragment thereof comprises an HCVR containing the amino acid sequence of SEQ ID NO: 47 and an LCVR containing the amino acid sequence of SEQ ID NO: 51. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 40.

[0052] In one aspect, the present disclosure relates to an antibody or an antigen-binding fragment thereof that binds to an epitope containing PTM of TFRC-1 or TFRC-2, wherein the antibody or the antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained in the light-chain variable region (LCVR), the heavy-chain variable region comprises the amino acid sequence of SEQ ID NO: 55, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO: 59.

[0053] In some embodiments, the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain that comprises the amino acid sequences of SEQ ID NO: 56-57-58-60-61-62, respectively. In some embodiments, the antibody or the antigen-binding fragment thereof comprises an HCVR containing the amino acid sequence of SEQ ID NO: 55 and an LCVR containing the amino acid sequence of SEQ ID NO: 59. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 37.

[0054] In one aspect, the present disclosure relates to an antibody or an antigen-binding fragment thereof that binds to a post-translational modification (PTM)-containing epitope of SPINK1-1, wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), the heavy-chain variable region comprises the amino acid sequence of SEQ ID NO: 63, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO: 67.

[0055] In some embodiments, the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, which respectively comprises the amino acid sequences of SEQ ID NO: 64-65-66-68-69-70. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR containing the amino acid sequence of SEQ ID NO: 63 and an LCVR containing the amino acid sequence of SEQ ID NO: 67. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 29.

[0056] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof of the present disclosure and a pharmaceutically acceptable carrier or diluent.

[0057] In one aspect, the present disclosure relates to a unit dosage form comprising a container containing an antibody or an antigen-binding fragment thereof of any one of the antibodies of the present disclosure. In some embodiments, the container is a vial, syringe, pre-filled syringe, or auto-injector.

[0058] In one aspect, the present disclosure relates to a kit comprising a container containing an antibody or an antigen-binding fragment thereof of any one of the antibodies of the present disclosure, and instructions for treating small cell lung cancer using the antibody or antigen-binding fragment thereof.

[0059] In one aspect, the present disclosure relates to a nucleic acid molecule encoding any one of the antibodies or antigen-binding fragments thereof of the present disclosure. In some embodiments, the present disclosure relates to a recombinant expression vector comprising the nucleic acid molecule.

[0060] In one aspect, the present disclosure relates to a pair of nucleic acid molecules encoding the heavy-chain variable region and the light-chain variable region of any one of the antibodies or antigen-binding fragments of the present disclosure, wherein the pair of nucleic acid molecules comprises a first nucleic acid molecule encoding the heavy-chain variable region of the antibody or antigen-binding fragment thereof and a second nucleic acid molecule encoding the light-chain variable region of the antibody or antigen-binding fragment thereof. In some embodiments, the pair of nucleic acid molecules is contained in a recombinant expression vector.

[0061] In one aspect, the present disclosure relates to an isolated host cell comprising any antibody or antigen-binding fragment of the present disclosure, any nucleic acid molecule of the present disclosure, any recombinant expression vector of the present disclosure, any pair of nucleic acid molecules of the present disclosure, or any pair of recombinant expression vectors of the present disclosure. In some embodiments, the isolated host cell is a mammalian host cell.

[0062] In various embodiments, any feature or component of the embodiments discussed above or herein can be combined, and such combinations are within the scope of the present disclosure. Any specified value discussed above or herein can be combined with another related value discussed above or herein to enumerate a range, and the values represent the upper and lower limits of the range, and such ranges and all values falling within such ranges are within the scope of the present disclosure. Each value discussed above or herein can be represented with a variation of 1%, 5%, 10%, or 20%. Other embodiments will become apparent by referring to the following detailed description.

[0063] Other embodiments will become apparent by referring to the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figures 1A - 1D Shows the upregulation of antigen-complexed autoantibodies in small cell lung cancer (SCLC). The mean coefficient signals (one-way ANOVA) of IgG and IgM autoantibodies upregulated (p < 0.05) in SCLC were higher than those of any other cancer type analyzed on the antibody-antigen complex array ( Figure 1A ). N = 98 SCLC and 125 controls; N = 47 NSCLC and 47 controls; N = 86 pancreatic cancers and 86 controls; N = 139 colon cancers and 139 controls; N = 121 breast cancers and 121 controls. Figures 1B - 1D Shows that IgG and IgM autoantibodies were sequentially discovered in the Cardiovascular Health Study (CHS) pre-diagnosis cohort (1B), tested in the Fred Hutch diagnosis cohort (1C), and validated in a third cohort from Vanderbilt (1D). Expression in cases: ● IgG increased; ● IgM increased; - decreased; × negligible.

[0065] Figures 2A - 2B Shows the upregulation of antigens targeted by autoantibodies in SCLC. Figure 2A Shows representative immunohistochemical images of antigens targeted by peripheral autoantibodies on an SCLC tissue microarray (N = 45 - 62 cases). Scale bar, 50 μM. Figure 2B Shows the positive or negative scoring of tissue microarray cores. Positive scores were further classified by stage at diagnosis.

[0066] Figure 3 The expression of tumor-associated antigens in some normal tissues is shown. Representative normal tissue cores showing positive and negative expression for each immunohistochemical stain are shown, as well as the expression in normal lung cores. Scale bar is 500 μM.

[0067] Figures 4A - 4B It is shown that CD133 has sialylated Lewis A (sLeA) with cancer-specific glycosylation in H82 and H69 SCLC cell lines. Figure 4A An immunoblot of H82 lysate immunoprecipitated (IP) with CA9, SPINT2, PLD3, or CD133 antibody and probed with sLeA antibody is shown. Depleted (Dep), whole cell lysate (WCL), and target-specific IP are shown. The blue box indicates the expected target protein size. Figure 4B An immunoblot of H69 lysate immunoprecipitated (IP) with CD133 antibody and probed with anti-CD133 or anti-sLeA antibody is shown. Depleted (Dep), whole cell lysate (WCL), and target-specific IP are shown. The box indicates the expected target protein size.

[0068] Figures 5A - 5C It is shown that SCLC autoantibodies (AAb) against CD133 target glycosylation motifs. Figure 5A An immunoblot of H82 cell lysate (Lys) immunoprecipitated (IP) with CD133 antibody or isotype control and probed with anti-CD133 and anti-sLeA antibodies is shown. Dep is the depleted cell lysate after IP. Figure 5B An immunoblot of H82 lysate immunoprecipitated (IP) with CD133 antibody after treatment with PRIME deglycosylase and probed with anti-CD133 and anti-sLeA antibodies is shown. Whole cell lysate (WCL), control immunoprecipitation, and target-specific IP are shown. Figure 5C ELISA quantification of the level of autoantibodies in SCLC patient plasma that bind to CD133 in H82 cell lysate, which has been pretreated with or without PRIME deglycosylase. (N = 5 / group) Error bars show SEM *p < 0.05, **p < 0.005, ***p < 0.0005, Student's t-test.

[0069] Figures 6A - 6C It is shown that SCLC AAb can target post-translational modification of isoaspartylation. Figure 6AShows an immunoblot of GST-fusion protein bound to glutathione beads, which was treated with or without isoaspartylation induction buffer (IsoAsp), incubated with plasma from SCLC patients, and probed with anti-human IgG and anti-GST antibodies. Multiple quantitative experiments were performed in Figure 6B as shown. Figure 6C Shows IgG binding to IsoAsp in plasma from SCLC patients measured by ELISA compared to wild-type (WT) peptide (N = 8 cases).

[0070] Figures 7A - 7B Shows that TFRC is citrullinated. Figure 7A Shows an immunoblot of H82 and H69 whole cell lysates (WCL) probed with antibodies against citrulline, CD133, TFRC, PLD3, SPINT2, and CA9. Boxes indicate the expected target protein sizes. Figure 7B Shows an immunoblot of H82 lysate immunoprecipitated (IP) with citrulline antibody and probed with CD133, PLD3, CA9, or TFRC antibodies. The same blot was probed with CD133 and then PLD3 in sequence. Boxes indicate the expected target protein sizes.

[0071] Figures 8A - 8D Shows that SCLC AAb targets citrullinated TFRC. Figure 8A Shows an immunoblot of H82 or H69 whole cell lysates (WCL) probed with TFRC or citrulline antibodies. GAPDH was used as a loading control. Figure 8B Shows immunoblots of 3T3 or H82 WCL and H82 lysates immunoprecipitated (IP) with TFRC, citrulline (CIT), or isotype control antibody (CON) and probed with anti-TFRC or anti-citrulline antibodies. Figure 8C Shows IgG binding to citrullination (Cit) in plasma from SCLC patients measured by ELISA compared to wild-type (WT) peptide (N = 8). Figure 8D Shows IgG captured by anti-TFRC antibody in antigen complex (AAb-TAA), captured by TFRC recombinant protein without antigen (AAb), or captured by cit (PTM) / WT peptide in 10 SCLC patients and 10 controls, and quantified by ELISA. Anti-TFRC antibody was used as a positive control for TFRC recombinant protein. Error bars show SEM. P values were estimated by paired sample t-test.

[0072] Figures 9A - 9B Shows detection of SCLC by autoantibodies in the Figure 9AAmong them, autoantibodies of the group were analyzed according to the blood sampling time in CHS (before diagnosis, 1 - 2 hours before diagnosis (dx) or < 1 year from diagnosis) and FH or VB (limited or extensive stage of diagnosis). *p = < 0.05; **p = < 0.005, ***p = < 0.0005, unpaired t - test. In Figure 9B Among them, autoantibodies of the group were significantly higher in the plasma of SCLC cases (n = 98) compared to cases of other cancers (N = 75 NSCLC, N = 86 pancreatic cancers, N = 87 colon cancers) or controls (n = 175) from all cohorts. **p = < 0.001, unpaired t - test.

[0073] Figures 10A - 10F A weak association between the group of autoantibodies and pack - years of smoking was shown. Figure 10A It was shown that there was no differential expression of the group of autoantibodies between current smokers and former smokers (current N = 100, former N = 140). In Figures 10B - 10F Among them, the group of autoantibodies including PLD3 ( Figure 10B ), TIMP2 ( Figure 10C ), GRAP2 ( Figure 10D ), SPINK1 ( Figure 10E ) and TFRC ( Figure 10F ) was weakly correlated with pack - years of smoking. Pearson correlation coefficient, N = 240.

[0074] Figure 11A It was shown that the group of autoantibodies was not associated with chronic obstructive pulmonary disease (COPD) (N = 19 controls and 31 cases).

[0075] Figure 11B It was shown that the group of autoantibodies was not associated with autoimmunity (type 1 diabetes (N = 3), rheumatoid arthritis (N = 2), hypothyroidism (N = 2) or myasthenia gravis (N = 2). One case was diagnosed with both type 1 diabetes and myasthenia gravis (a total of N = 44 controls and 8 cases).

[0076] Figures 12A - 12C A risk prediction model that can accurately detect SCLC was shown. By maximizing the AUC ( Figure 12A ) in the Vanderbilt (VB) dataset, then fixing the coefficients and testing in the CHS cohort ( Figure 12B ) and the Fred Hutch (FH) cohort ( Figure 12C ), the receiver operating characteristic curve (ROC) curve of the 5 - autoantibody (AAb, dashed line) complex group was identified. The ROC curve of pack - years is shown as a dashed line, and the combination of pack - years and the 5 - AAb group is shown as a solid black line.

[0077] Figure 13Disclosed is a method for separating, sequencing, and cloning SCLC-related autoantibodies that bind to an identified PTM.

[0078] Figure 14 Near-infrared imaging is shown, which shows that the 770-labeled 2D2 antibody homed to H82 SCLC tumors in vivo.

[0079] Figure 15 Chemiluminescent ELISA is shown, which shows that the 2D2 antibody binds strongly to the specific TFRC-cit peptide.

[0080] Figure 16 The commercially available TFRC antibody prepared in rabbits is shown to recognize TFRC in a variety of normal human tissues and the H82 SCLC cell line. However, the 2D2 antibody isolated from SCLC patient PBMCs only recognizes TFRC in SCLC and not TFRC in normal tissues. Detailed Description

[0081] Before describing the present invention, it is to be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims. Any embodiment or feature of an embodiment may be combined with each other, and such combinations are expressly covered within the scope of the present invention. Any specified value discussed above or herein may be combined with another related value discussed above or herein to enumerate a range, and the values represent the upper and lower limits of the range, and such ranges are covered within the scope of the present disclosure.

[0082] 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 belongs. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0083] As used herein, when used in reference to a specifically recited numerical value, the term "about" means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0084] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned in this specification are hereby incorporated by reference in their entirety. In case of conflict, the present specification and the term interpretations included therein shall prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0085] Selected Abbreviations

[0086] SCLC: Small cell lung cancer

[0087] NSCLC: Non-small cell lung cancer

[0088] WT: Wild type

[0089] PTM: Post-translational modification

[0090] ELISA: Enzyme-linked immunosorbent assay

[0091] IgG: Immunoglobulin G

[0092] IgM: Immunoglobulin M

[0093] AAb: Autoantibody

[0094] AAb-Ag: Autoantibody-antigen complex

[0095] TMA: Tissue microarray

[0096] WCL: Whole cell lysate

[0097] PNS: Paraneoplastic neurological syndrome

[0098] CT: Computed tomography

[0099] CIT: Citrulline

[0100] PCR: Polymerase chain reaction

[0101] DNA: Deoxyribonucleic acid

[0102] cfDNA: Cell-free DNA

[0103] IP: Immunoprecipitation

[0104] SNP: Single Nucleotide Polymorphism

[0105] sLeA: Sialyl Lewis A

[0106] Definition

[0107] Administer: To provide or give an agent to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes or any technical combination thereof.

[0108] The disclosed compositions or other therapeutic agents of the present disclosure can be formulated into a pharmaceutically active pharmaceutical composition, which can be administered parenterally or orally to a subject. Parenteral routes of administration include, but are not limited to: transdermal, intra-arterial, intramuscular (IM and depot IM), intraperitoneal (IP), intravenous (IV), intracardiac injection or infusion techniques, intranasal (inhalation), intrathecal, injection into the stomach, subcutaneous injection (subcutaneous (SQ and depot SQ), transdermal, topical, and ophthalmic. The disclosed compositions or other therapeutic agents can be mixed or combined with suitable pharmaceutically acceptable excipients to prepare a pharmaceutical composition. For example, pharmaceutically acceptable excipients include, but are not limited to: aluminum oxide, aluminum stearate, buffers (such as phosphates), glycine, ion exchange agents (such as to assist in controlling the release of charged substances), lecithin, partial glyceride mixtures of saturated vegetable fatty acids, potassium sorbate, serum proteins (such as human serum albumin), sorbic acid, water, salts or electrolytes such as cellulose-based substances, silica gel, disodium hydrogen phosphate, magnesium trisilicate, polyacrylates, polyalkylene glycols (such as polyethylene glycol), polyethylene-polypropylene block polymers, polyvinylpyrrolidone, potassium hydrogen phosphate, protamine sulfate, Group 1 halide salts (such as sodium chloride), sodium carboxymethylcellulose, paraffin, lanolin, and zinc salts. Liposome suspensions are also suitable as pharmaceutically acceptable carriers.

[0109] After mixing or adding the disclosed compositions or other therapeutic agents, the resulting mixtures can be solids, solutions, suspensions, emulsions, etc. These can be prepared by methods known to those of ordinary skill in the art. The form of the resulting mixtures depends on many factors, including the intended mode of administration and the solubility of the agent in the selected carrier. Pharmaceutical carriers suitable for administering the disclosed compositions or other therapeutic agents include any such carriers known to be suitable for a particular mode of administration. Additionally, the disclosed compositions or other therapeutic substances can also be mixed with other inactive or active materials that do not impair the desired action, or with materials that supplement the desired action or have other actions. When the solubility of the agent in the carrier is insufficient, solubilization methods can be used. Such methods are known and include, but are not limited to, dissolving in an aqueous solution of sodium bicarbonate, using co-solvents such as dimethyl sulfoxide (DMSO), and using surfactants such as (ICI Americas, Inc., Wilmington, Del.).

[0110] The disclosed compositions or other therapeutic agents can be prepared with carriers that prevent their rapid clearance from the body, such as coatings or sustained-release formulations. Such carriers include controlled-release formulations, such as, but not limited to, microencapsulated delivery systems. The disclosed compositions or other therapeutic agents are included in a pharmaceutically acceptable carrier in an amount sufficient to produce a therapeutically useful effect on the subject being treated, generally in an amount that avoids undesirable side effects on the subject being treated. The therapeutically effective concentration can be determined empirically by testing the compound in in vitro and in vivo model systems for known therapeutic conditions. For example, an acceptable SCLC animal model can be used to determine the effective amount or effective concentration, which can then be translated to other subjects (such as humans), as is known in the art.

[0111] Injectable solutions or suspensions can be formulated using the following reagents: suitable non-toxic parenterally acceptable diluents or solvents such as 1,3-butanediol, isotonic sodium chloride solution, mannitol, Ringer's solution, saline solution or water; or suitable dispersing or wetting agents and suspending agents such as sterile, mild, non-volatile oils including synthetic mono- or diglycerides, and fatty acids including oleic acid; naturally occurring vegetable oils such as coconut oil, cottonseed oil, peanut oil, sesame oil, etc.; glycerol; polyethylene glycol; propylene glycol; or other synthetic solvents; antimicrobial agents such as benzyl alcohol and methylparaben; antioxidants such as ascorbic acid and sodium bisulfite; buffers such as acetate, citrate and phosphate; chelating agents such as ethylenediaminetetraacetic acid (EDTA); reagents for adjusting tonicity such as sodium chloride and dextrose; and combinations thereof. Parenteral preparations can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass, plastic or other suitable materials. Buffers, preservatives, antioxidants, etc. can be incorporated as needed. In the case of intravenous administration, suitable carriers include physiological saline, phosphate buffered saline (PBS) and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, propylene glycol and mixtures thereof. Liposome suspensions, including tissue-targeted liposomes, are also suitable as pharmaceutically acceptable carriers.

[0112] If the disclosed composition or other therapeutic agent is administered orally as a suspension, the pharmaceutical composition can be prepared according to techniques well known in the pharmaceutical formulation art and can contain suspending agents such as alginic acid or sodium alginate, fillers such as microcrystalline cellulose, thickening agents such as methyl cellulose, and sweetening / flavoring agents. Oral liquid preparations can contain conventional additives such as suspending agents, for example gelatin, glucose syrup, hydrogenated edible fats, methyl cellulose, sorbitol and syrup; emulsifying agents such as gum arabic, lecithin or sorbitan monooleate; non-aqueous carriers (including edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerol, propylene glycol or ethanol; preservatives such as methylparaben or propylparaben or sorbic acid; and, if desired, conventional flavoring or coloring agents. When formulated as immediate-release tablets, these compositions can contain dibasic calcium phosphate, lactose, magnesium stearate, microcrystalline cellulose and starch and / or other binders, diluents, disintegrants, excipients, swelling agents and lubricants.

[0113] If oral administration is desired, the disclosed compositions or other therapeutic substances can be provided in a composition that protects them from the acidic gastric environment. For example, the disclosed compositions or other therapeutic agents can be formulated with an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The disclosed compositions or other therapeutic agents can also be formulated in combination with an antacid or other such ingredients. Oral compositions generally include an inert diluent or an edible carrier and can be compressed into tablets or enclosed in gelatin capsules. For purposes of oral therapeutic administration, the disclosed compositions or other therapeutic substances can be admixed with excipients and used in the form of capsules, tablets, or lozenges. Pharmaceutically compatible adjuvant materials or binders can be included as part of the composition.

[0114] Capsules, pills, tablets, lozenges, etc. can contain any of the following ingredients or compounds having similar properties: binders such as, but not limited to, gum arabic, corn starch, gelatin, tragacanth, polyvinylpyrrolidone, or sorbitol; fillers such as calcium phosphate, glycine, lactose, microcrystalline cellulose, or starch; disintegrants such as, but not limited to, alginic acid and corn starch; lubricants such as, but not limited to, magnesium stearate, polyethylene glycol, silica, or talc; glidants such as, but not limited to, colloidal silica; sweeteners such as sucrose or saccharin; disintegrants such as potato starch; dispersing or wetting agents such as sodium lauryl sulfate; and flavoring agents such as peppermint, methyl salicylate, or fruit flavorings. When the dosage unit form is a capsule, in addition to the materials of the above type, it can also contain a liquid carrier such as a fatty oil. In addition, the dosage unit form can contain various other materials that modify the physical form of the dosage unit, such as coatings of sugar and other enteric reagents. The disclosed compositions or other therapeutic agents can also be administered as components of elixirs, suspensions, syrups, powder tablets, teas, chewing gums, etc. In addition to the active compound, the syrup can contain sucrose or glycerin as a sweetening agent and certain preservatives, coloring agents, and flavoring agents.

[0115] When administered orally, the compounds can be administered in common dosage forms for oral administration. These dosage forms include the common solid unit dosage forms of tablets and capsules, as well as liquid dosage forms such as solutions, suspensions, and elixirs. When solid dosage forms are used, they can be of the sustained release type, so that the compounds need to be administered less frequently.

[0116] Agent: Any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, antibody, small molecule, organic compound, inorganic compound, cell (such as a T cell), or other molecule of interest. An agent can include a therapeutic agent, a diagnostic agent, or a pharmaceutical agent. A therapeutic agent or pharmaceutical agent is an agent that induces a desired response (e.g., a therapeutic or prophylactic effect when administered to a subject, including a subject being treated for or at risk of developing SCLC) either alone or in combination with another compound.

[0117] In some instances, an agent can act directly or indirectly to alter the activity and / or expression of SCLC-related molecules such as early detection molecules for SCLC. In one specific instance, a therapeutic agent such as an antisense compound or an antibody significantly alters the expression and / or activity of SCLC-related molecules. An example of a therapeutic agent is a therapeutic agent that can reduce the activity of a gene or gene product associated with SCLC, such as as measured by a clinical response such as an increased survival time or a reduction in one or more signs or symptoms associated with SCLC. Therapeutic agents also include organic compounds or other compounds that mimic the action of a therapeutically effective peptide, antibody, or nucleic acid molecule.

[0118] An "agent" is a compound or composition that is capable of inducing a desired therapeutic or prophylactic effect when administered alone or in combination with another therapeutic agent or a pharmaceutically acceptable carrier to a subject. In one specific instance, the agent significantly reduces the expression and / or activity of SCLC-related molecules, thereby increasing the survival time of the subject, reducing the signs or symptoms associated with the disease, and prolonging the onset of SCLC signs or symptoms.

[0119] Antibody: A polypeptide that comprises at least a light chain or a heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen such as an SCLC-related molecule or a fragment thereof. An antibody is composed of a heavy chain and a light chain, each having a variable region, called the variable heavy chain (V H ) region and the variable light chain (V L ) region. The V H region and the V L region together are responsible for binding to the antigen recognized by the antibody. The antibodies of the present disclosure include those antibodies that are specific for the disclosed SCLC-related molecules.

[0120] As used herein, the term "antibody" means an immunoglobulin molecule, which consists of four polypeptide chains, namely two heavy (H) chains and two light (L) chains interconnected by disulfide bonds (i.e., "intact antibody molecule"); and its polymers (e.g., IgM) or its antigen-binding fragments. Each heavy chain consists of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (consisting of domains CH1, CH2, and CH3). Each light chain consists of a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The VH and VL regions can also be subdivided into hypervariable regions (called complementarity determining regions (CDRs)), which are interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of an antibody (or its antigen-binding fragment) can be identical to a human germline sequence or can be naturally or artificially modified. Amino acid consensus sequences can be defined based on a side-by-side analysis of two or more CDRs.

[0121] The term antibody includes intact immunoglobulins as well as variants and portions thereof, such as Fab′ fragments, F(ab)′2 fragments, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). An scFv protein is a fusion protein in which the variable region of the light chain of an immunoglobulin and the variable region of the heavy chain of an immunoglobulin are joined by a linker, and in dsFvs the chains have been mutated to introduce disulfide bonds to stabilize the association of the chains. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd ed., W.H. Freeman & Co., New York, 1997.

[0122] Generally, naturally occurring immunoglobulins have heavy chains (H) and light chains (L) that are linked to each other by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes), which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each heavy chain and light chain contains a constant region and a variable region (these regions are also referred to as "domains"). The variable regions of the heavy chain and the light chain combine to specifically bind an antigen. The variable regions of the light chain and the heavy chain contain "framework" regions interrupted by three hypervariable regions, also known as "complementary determining regions" or "CDRs". The ranges of the framework regions and the CDRs have been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody, that is, the combined framework regions that make up the light and heavy chains, are used to position and align the CDRs in three-dimensional space.

[0123] The CDRs are mainly responsible for binding to the epitope of an antigen. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are usually also identified by the chain in which the specific CDR is located. Thus, VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is present, while VL CDR1 is the CDR1 from the variable domain of the antibody light chain in which it is present. An antibody that binds RET will have specific VH and VL region sequences and thus specific CDR sequences. Antibodies with different specificities (such as different binding sites for different antigens) have different CDRs. Although the CDRs vary among different antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity-determining residues (SDRs). A reference to "V H " or "VH" refers to the variable region of an immunoglobulin heavy chain, including the variable regions of Fv, scFv, dsFv, or Fab. A reference to "V L " or "VL" refers to the variable region of an immunoglobulin light chain, including the variable regions of Fv, scFv, dsFv, or Fab.

[0124] A "monoclonal antibody" is an antibody produced by cloning a single B lymphocyte or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, such as by fusing myeloma cells with immune spleen cells to prepare hybrid antibody-forming cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0125] A "chimeric antibody" has framework residues from one species, such as a human, and CDRs from another species, which typically confer antigen binding, such as a murine antibody that specifically binds to an SCLC-related molecule.

[0126] The present disclosure also includes antibodies that comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein that have one or more conservative substitutions. For example, the present disclosure includes antibodies having HCVR and / or LCVR amino acid sequences that have, relative to any of the HCVR or LCVR amino acid sequences disclosed herein, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions. Also included are antibodies having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to one of the HCVR and / or LCVR sequences discussed herein.

[0127] As used herein, the term "recombinant" refers to an antibody or antigen-binding fragment thereof of the present disclosure that is produced, expressed, isolated, or obtained by techniques or methods referred to in the art as recombinant DNA techniques, which include, for example, DNA splicing and transgenic expression. The term refers to an antibody expressed in a non-human mammalian (including transgenic non-human mammals, such as transgenic mice), or cell (such as CHO cells) expression system, or an antibody isolated from a recombinant combinatorial human antibody library.

[0128] The terms "specifically binds" or "specifically binds to", etc., mean that an antibody or antigen-binding fragment thereof forms a relatively stable complex with an antigen under physiological conditions. The characteristics of specific binding may be characterized by a dissociation constant of at least about 1×10 -8 M or lower (a smaller K D indicating tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc.

[0129] The terms "antigen-binding portion of an antibody", "antigen-binding fragment of an antibody", etc., as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. As used herein, the term "antigen-binding fragment" or "antibody fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to bind the listed epitope.

[0130] As used herein, an "isolated antibody" is intended to mean an antibody that is substantially free of other antibodies (Abs) having different antigen specificities.

[0131] The polynucleotides discussed herein can encode all or a portion of an antibody or antigen-binding fragment as discussed throughout the present disclosure. In some cases, a single polynucleotide can encode both the HCVR and LCVR of an antibody or antigen-binding fragment (e.g., as defined by the CDRs contained within the HCVR and LCVR as defined with reference to their respective amino acid sequences, defined with reference to the amino acid sequences of the CDRs of the HCVR and LCVR, respectively, or defined with reference to the amino acid sequences of the HCVR and LCVR, respectively), or the HCVR and LCVR can be encoded by separate polynucleotides (i.e., a pair of polynucleotides). In the latter case (where the HCVR and LCVR are encoded by separate polynucleotides), the polynucleotides can be combined in a single vector or can be contained in separate vectors (i.e., a pair of vectors). In any case, the host cell used to express the polynucleotide or vector can contain the complete complementary sequences of the components to produce an antibody or its antigen-binding fragment. For example, the host cell can contain separate vectors, each encoding the HCVR and LCVR of an antibody or its antigen-binding fragment as discussed above or herein. Similarly, one or more polynucleotides and one or more vectors can be used to express the full-length heavy chain and full-length light chain of an antibody as discussed above or herein. For example, the host cell can contain a single vector having polynucleotides encoding both the heavy and light chains of the antibody, or the host cell can contain separate vectors having polynucleotides encoding the heavy and light chains of the antibody as discussed above or herein, respectively.

[0132] As used herein, the term "vector" means any molecule or particle used to deliver an exogenous nucleic acid into a cell. For example, the nucleic acid can be, for example, RNA or DNA. The molecule or particle can be, for example, a plasmid, cosmid, phage, or virus.

[0133] Autoantibody: An "autoantibody" is an antibody produced by the immune system against one or more of an individual's own proteins.

[0134] Epitope: The term "epitope" refers to the antigenic determinant that interacts with a specific antigen-binding site (called a paratope) in the variable region of an antibody molecule. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are produced by the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope can include a sugar, phosphoryl group, or sulfonyl group moiety on an antigen.

[0135] Altered or regulated expression: An alteration in the expression of a gene, gene product, or its regulatory factor (such as one or more SCLC-related molecules disclosed herein) refers to a change or difference in the level of a gene, gene product, or its regulatory factor detectable in a biological sample (such as a sample from a subject at risk of or suffering from SCLC) relative to a control (such as a sample from a subject without SCLC) or a known reference value indicative of the level of the gene, gene product, or its regulatory factor in the absence of disease, such as an increase or decrease. An "alteration" in expression includes an increase (upregulation) or a decrease (downregulation) in expression.

[0136] Array: An arrangement of molecules (such as biological macromolecules (such as peptide or nucleic acid molecules) or biological samples (such as tissue sections)) at addressable sites on or in a matrix. A "microarray" is a miniaturized array that requires or is evaluated or analyzed with the aid of microscopy. Arrays are sometimes referred to as antibody chips or biochips. Molecular arrays enable a large number of analyses of a sample to be performed at one time. In some exemplary arrays, one or more molecules (such as oligonucleotide probes or antibodies) will appear multiple times (such as twice) on the array, for example to provide an internal control. The number of addressable sites on the array can vary, for example from at least two to at least four, to at least 9, at least 10, at least 14, at least 15, at least 20, at least 30, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 500, at least 550, at least 600, at least 800, at least 1000, at least 10,000 or more. In one specific instance, the array includes 2 - 100 addressable sites, such as 2 - 40 addressable sites. In specific instances, the array consists essentially of probes or primers or antibodies specific for SCLC disclosed herein (such as those that allow amplification or detection).

[0137] Binding or stable binding: An association between two substances or molecules, such as the hybridization of one nucleic acid molecule to another (or itself), the association of an antibody with a peptide, or the association of a protein with another protein or nucleic acid molecule. An oligonucleotide molecule binds or stably binds to a target nucleic acid molecule if a sufficient amount of the oligonucleotide molecules form base pairs or hybridize to their target nucleic acid molecule to allow detection of the binding. "Preferential binding" means that one molecule binds to another molecule with high affinity and to a heterologous molecule with low affinity. Binding can be detected by any procedure known to those skilled in the art, such as by the physical or functional properties of the target complex. For example, binding can be detected functionally by determining whether the binding has an observable effect on a biosynthetic process (such as gene expression, DNA replication, transcription, translation, etc.). Methods for detecting the binding of an antibody to a protein are disclosed herein and can also include known protein detection methods, such as Western blotting.

[0138] Biological activity: The beneficial or adverse effect of an agent on living matter. When the agent is a complex chemical mixture, the activity is exerted by the active ingredient or pharmacophore of the substance, but can be modified by other components. Activity is usually dose-dependent, and it is not uncommon for a substance to have an effect that changes from beneficial to adverse as the dose increases from low to high. In one example, the agent significantly reduces the biological activity of one or more SCLC-related molecules disclosed herein, which reduces one or more SCLC-related signs or symptoms.

[0139] Biomarker: A molecular, biological, or physical property that characterizes a physiological state and can be objectively measured to detect or define disease progression or to predict or quantify a therapeutic response. For example, a substance that serves as an indicator of a biological state. It is characterized by being objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or the pharmacological response to a therapeutic intervention. In one example, the biomarker is the protein or nucleic acid sequence of the corresponding gene that serves as an indicator of SCLC.

[0140] Clinical outcome: Refers to the health status of a patient after treatment of a disease or disorder (such as SCLC) or in the absence of treatment. Clinical outcomes include, but are not limited to: an increase in the time before death, a decrease in the time before death, an increase in the chance of survival, an increase in the risk of death, survival period, disease-free survival period, chronic disease, metastasis, advanced or invasive disease, disease recurrence, death, and a favorable or adverse response to treatment.

[0141] Contact: Placing in a manner of direct physical association, including in solid and liquid forms. Contact of an agent with a cell can occur in vitro by adding the agent to isolated cells, or in vivo by administering the agent to a subject.

[0142] Control: A sample or standard used for comparison with a test sample, such as a biological sample obtained from a patient (or patients) without a specific disease or disorder (such as SCLC). In some embodiments, the control is a sample obtained from a healthy patient (or patients) (also referred to herein as a "normal" control), such as a normal biological sample or a non-cancerous biological sample from a patient with a specific disease or disorder (such as SCLC). In some embodiments, the control is a historical control or a standard value (e.g., a previously tested control sample or group of samples representing a baseline or normal value (e.g., an expression value), such as the baseline or normal value of a specific gene, gene product in a subject without SCLC). In some examples, the control is a standard value representing the average (or range of averages) obtained from multiple patient samples (such as the average or range of values of a gene or gene product in a subject without SCLC).

[0143] Detection: Identifying the presence, absence, or relative or absolute amount of an object to be detected.

[0144] Diagnosis: A method of identifying a disease (such as SCLC) through signs, symptoms, and various test results. The conclusion obtained through this method is also called a "diagnosis". Commonly performed test forms include blood tests, medical imaging, urine analysis, and biopsies.

[0145] Expression: A method of converting the coding information of a gene into a functional, non-functional, or structural part of a cell, such as protein synthesis. Gene expression can be affected by external signals. For example, a cell exposed to a hormone may stimulate the expression of a hormone-induced gene. Different types of cells can respond differently to the same signal. Gene expression can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation can include control of transcription, translation, RNA transport and processing, degradation of intermediate molecules (such as mRNA), or activation, inactivation, compartmentalization, or degradation of specific protein molecules after the production of a specific protein molecule. The expression of a nucleic acid molecule can be altered relative to a normal (wild-type) nucleic acid molecule. Alterations in gene expression, such as differential expression, include but are not limited to: (1) overexpression; (2) underexpression; or (3) suppressed expression. Alterations in the expression of a nucleic acid molecule may be related to changes in the expression of the corresponding protein and may actually cause changes in the expression of the corresponding protein.

[0146] Protein expression can also be altered in such a way that it differs from protein expression under normal (wild-type) conditions. This includes, but is not necessarily limited to: (1) mutation of the protein such that one or more amino acid residues are different; (2) deletion or addition of a short fragment of one or a few (such as no more than 10-20) amino acid residues in the protein sequence; (3) deletion or addition of a longer fragment of amino acid residues (such as at least 20 residues) such that an entire protein domain or subdomain is removed or added; (4) an increase in the amount of protein expression compared to a control / standard amount; (5) a decrease in the amount of protein expression compared to a control / standard amount; (6) alteration of the subcellular localization or targeting of the protein; (7) alteration of the temporal regulation of protein expression (such that protein expression occurs when the protein is not expressed under normal conditions, or alternatively such that protein expression does not occur when the protein is expressed under normal conditions); (8) alteration of protein stability by prolonging the time the protein remains in the cell; (9) alteration of the local (such as organ / tissue-specific or subcellular localization) expression of the protein (such that protein expression does not occur when the protein is expressed under normal conditions, or protein expression occurs when the protein is not expressed under normal conditions); and (10) alteration of the protein structure, stability or function by post-translational modification (PTM), each aspect being compared to a control / standard. Controls or standards for comparison with a sample to determine differential expression include samples considered normal (because their desired characteristics have not been altered, such as samples from subjects without SCLC) and laboratory values (such as ranges of values), even if arbitrarily set, but bearing in mind that such values may vary between laboratories.

[0147] Cytotoxic agent: As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell destruction. The term is intended to include radioactive isotopes (e.g., radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and Lu); chemotherapeutic agents such as methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunomycin or other intercalating agents, enzymes and fragments thereof (such as nuclease hydrolases), antibiotics and toxins (such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof) and various anti-tumor or anti-cancer agents described herein. An anti-tumor agent causes the destruction of tumor cells.

[0148] Toxin: A "toxin" is any substance capable of having a harmful effect on cell growth or proliferation.

[0149] Measurement: Detection, quantification or qualitative determination of the amount (including molar amount), concentration or mass of a physical entity or chemical composition, in the case of quantification, expressed as an absolute value or relative to a comparable physical entity or chemical composition.

[0150] Sample (or biological sample): A biological sample obtained from a subject and containing genomic DNA, RNA (including mRNA), protein, or a combination thereof. Examples include, but are not limited to, peripheral blood, plasma, serum, urine, saliva, tissue biopsy, surgical specimens, and autopsy materials.

[0151] Screening: As used herein, "screening" refers to a method for evaluating and identifying candidate agents that can be used to identify SCLC (such as early SCLC). In some cases, screening involves contacting a candidate agent (such as an antibody, small molecule, or cytokine) with SCLC cells and testing the effect of the agent on the expression of SCLC-related molecules. The expression of microRNA can be quantified using any of a number of techniques known in the art and described herein, such as by microarray analysis or by qRT-PCR.

[0152] Sensitivity: The percentage of diseased individuals (e.g., individuals with SCLC) in whom the biomarker of interest is detected (true positives / total diseased × 100). Testing non-diseased individuals diagnosed as diseased is a "false positive". In some instances, the sensitivity of an assay describes the ability of that assay, compared to another assay, to accurately predict the presence or absence of SCLC using the disclosed SCLC-related molecules. For example, a biomarker with a sensitivity of at least 60% (including 70%, 75%, 80%, 85%, 90%, 95% or higher sensitivity) is a biomarker capable of accurately predicting SCLC.

[0153] Specificity: The percentage of non-diseased individuals in whom the biomarker of interest is not detected (true negatives / total non-diseased × 100). Testing diseased individuals in whom the biomarker is not detected is a "false negative". Non-diseased subjects who test negative in the assay are called "true negatives".

[0154] Sign or symptom: Any subjective evidence of a disease or the condition of a subject, such as that perceived by the subject; a detectable change in the subject's condition indicating some physical or mental state. A "sign" is any abnormality indicative of a disease that can be detected upon examination or evaluation of the subject. Signs are generally objective indicators of disease.

[0155] Small cell lung cancer or carcinoma: A highly malignant cancer within the lung. Compared to non-small cell carcinoma, small cell lung cancer has a shorter doubling time, a higher growth fraction, and earlier metastatic development. Small cell lung cancer typically arises in the central airways, infiltrates the submucosa, and causes bronchial airway stenosis. Common symptoms include cough, dyspnea, weight loss, and weakness. Smoking is an important risk factor. More than 70% of patients with small cell lung cancer present with metastatic disease; common sites include the liver, adrenal glands, bone, and brain. Due to its highly neuroendocrine nature, small cell carcinoma can produce ectopic hormones, including adrenocorticotropic hormone (ACTH) and antidiuretic hormone (ADH). Ectopic production of large amounts of ADH results in the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Lambert-Eaton myasthenic syndrome (LEMS) is a well-known paraneoplastic disorder associated with small cell carcinoma. SCLC is also known as "oat cell carcinoma" because of the flattened cell shape and scant cytoplasm.

[0156] SCLC is thought to originate from neuroendocrine cells (APUD cells) in the bronchi, known as Feyrter cells. As such, they express various neuroendocrine markers and may result in the ectopic production of hormones such as ADH and ACTH, which can lead to paraneoplastic syndromes and Cushing's syndrome. Approximately half of all individuals diagnosed with Lambert-Eaton myasthenic syndrome (LEMS) are ultimately found to have small cell lung cancer. Concurrent small cell lung cancer may occur in combination with multiple other histological variants of lung cancer, including highly complex mixtures of malignant tissues. When a lung cancer is found to have one or more forms of differentiation (such as squamous cell carcinoma or adenocarcinoma), the malignancy is diagnosed and classified as combined small cell lung cancer (c-SCLC). C-SCLC is a recognized subtype of SCLC.

[0157] Subject: A living multicellular vertebrate organism, including classes of living multicellular vertebrate organisms, including the classes of human and non-human mammals. In one embodiment, the subject is a patient suffering from cancer (e.g., small cell lung cancer).

[0158] Tissue: Multiple functionally related cells. Tissue can be a suspension, semi-solid, or solid. Tissue includes cells collected from a subject (such as from the lung).

[0159] Treatment of diseases: A therapeutic intervention that improves the signs or symptoms of a disease or pathological condition associated with SCLC, such as the signs or symptoms of SCLC. Treatment can induce remission or cure of the condition or slow progression. For example, in some cases, it can include inhibiting the full development of the disease, such as preventing the development of SCLC. Prevention of a disease does not require the complete absence of the disease. For example, a reduction of at least 10%, such as at least 20%, at least 25%, at least 30%, at least 40%, at least 50% in the signs or symptoms associated with a condition or disease (such as SCLC) may be sufficient. As used herein, the term "improve" with respect to a disease or condition refers to any observable beneficial effect of the treatment. For example, the beneficial effect can be demonstrated, for example, by factors such as a delay in the onset of clinical symptoms of the disease or condition in a susceptible subject, a reduction in the severity of some or all of the clinical symptoms of the disease or condition, a slower progression of the disease or condition, a reduction in the number of recurrences of the disease or condition, an improvement in the overall health or well-being of the subject, other parameters specific to the particular disease or condition well known in the art, and combinations of such factors.

[0160] PTPRU: An enzyme encoded by the PTPRU gene, which is a member of the protein tyrosine phosphatase (PTP) family. PTPRU is also known as PTPπ, PTPλ, hPTP-J, PTPRO, and PTPψ. PTPs are known to be signaling molecules that regulate various cellular processes, including cell growth, differentiation, the mitotic cycle, and oncogenic transformation. This PTP has an extracellular region, a single transmembrane region, and two tandem intracellular catalytic tyrosine phosphatase domains and thus represents a receptor-type PTP (RPTP). RPTPs are capable of removing the phosphate moiety from tyrosine residues.

[0161] TFRC (transferrin receptor): The transferrin receptor, also known as cluster of differentiation 71 (CD71), is a protein encoded by the TFRC gene in humans.

[0162] Chimeric antigen receptor: The term "chimeric antigen receptor" (CAR) refers to a molecule that produces a chimeric protein exhibiting specific anti-target cell immune activity by combining a binding domain specific for a component present on a target cell (e.g., an antibody-based specificity for a desired antigen, such as a tumor antigen, such as BCMA) with an intracellular domain that activates the T cell receptor. Generally, a CAR consists of an extracellular single-chain antibody binding domain (scFv) fused to the intracellular signaling domain of the ζ chain of the T cell antigen receptor complex and, when expressed in a T cell, has the ability to redirect antigen recognition according to the specificity of the monoclonal antibody.

[0163] Polymerase Chain Reaction or PCR: As used herein, "PCR" refers to the polymerase chain reaction, a molecular biology technique used to amplify a single copy of a segment of DNA or RNA, thereby generating thousands to millions of copies of a specific DNA or RNA sequence. PCR is commonly used to amplify the copy number of a DNA or RNA segment for cloning or for other analytical procedures.

[0164] Nucleic acid: As used herein, the term "nucleic acid" refers to a polymeric form of nucleotides of any length, i.e., ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cfDNA, cDNA, DNA-RNA hybrids or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, unnatural or derivatized nucleobases. The backbone of a nucleic acid may contain sugar and phosphate groups (as may typically be present in RNA or DNA), or modified or substituted sugars or phosphate groups.

[0165] Vector: As used herein, "vector" refers to a recombinant plasmid containing a nucleic acid that will be delivered to a host cell in vitro or in vivo. A vector can be used as a vehicle to carry an exogenous nucleic acid sequence (usually DNA) into another cell, where the exogenous nucleic acid sequence can be replicated and / or expressed.

[0166] Methods for diagnosing and treating small cell lung cancer (SCLC)

[0167] Aspects of the present disclosure provide methods for diagnosing and treating small cell lung cancer (SCLC) in a subject. The disclosed methods include detecting the presence of one or more autoantibodies that specifically bind to one or more epitopes of one or more antigens in a biological sample obtained from the subject, wherein the one or more antigens are selected from the group consisting of CDH5, CD133, SPINK1, CDH23, NLRP7, TFRC, SPINT2, NADSYN1, HIF1A, GRAP2, MAPRE1, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9, and wherein the one or more epitopes of the antigen comprise a post-translational modification (PTM). The disclosed methods further include diagnosing the subject with SCLC when the presence of one or more autoantibodies is detected in a biological sample obtained from the subject. The disclosed methods further include treating the subject's SCLC by: (i) administering a chemotherapy regimen to the subject; (ii) administering an immunotherapy regimen to the subject; (iii) administering an antibody to the subject; (iv) performing surgical resection of SCLC in the subject; and / or (v) exposing the subject to radiation therapy targeting SCLC. As disclosed herein, the subject can include a human. In some embodiments, the subject can be a patient, such as a patient having or exhibiting one or more symptoms of SCLC, or a patient at risk of acquiring SCLC (e.g., a smoker or a patient with a smoking history). In some embodiments, the biological sample is serum or plasma obtained from the subject or patient. These patients or subjects can benefit from the methods of the present disclosure.

[0168] Small cell lung cancer (SCLC) elicits the production of autoantibodies, leading to a distinct paraneoplastic neurological syndrome. The mechanistic basis for the formation of such autoantibodies remains largely unclear, but it is crucial for understanding the etiology. A high-dimensional technology is disclosed herein that can directly detect autoantibodies complexed with native antigens from patient plasma. In a specific embodiment, this unique platform was used to screen 3,600 autoantibody-antigen complexes in 1,009 human plasma samples, and it was found that the disease-specific autoantibody signal in SCLC plasma was on average 4-fold higher compared to other cancers. In multiple independent SCLC cohorts (N = 240), a common but previously unknown set of autoantibodies was identified that were generated in response to intracellular and extracellular tumor antigens. The features of several disease-specific post-translational modifications within extracellular proteins were also characterized as being targeted by these autoantibodies, including citrullination, isoaspartylation, and cancer-specific glycosylation. Since most SCLC patients have metastatic disease at diagnosis, it was investigated whether these new autoantibodies could be used for the early detection and / or treatment of SCLC. In an embodiment, a risk prediction model was created using 5 autoantibodies, with an average area under the curve of 0.84, which was increased to 0.96 by introducing cigarette smoke consumption (in pack-years). Overall, the present disclosure provides a new method for identifying circulating autoantibodies in SCLC, with mechanistic insights into disease-specific immunogenicity and clinical utility.

[0169] In a specific embodiment, the AAb-Ag complex capture platform utilizes endogenous humoral immunity to capture disease-related epitopes in tumor-specific antigens, such as PTMs. By capturing the AAbs that bind to their immunogenic antigens, an AAb repertoire can be identified and validated that has clinical utility in an early detection risk prediction model. Additionally, in an embodiment, a subset of the AAbs can identify novel tumor cell surface antigens with targeting potential in a therapeutic setting. Although most of the antigens identified in this disclosure have not been previously described as being associated with SCLC, there are some exceptions. For example, CD133 has been reported to be overexpressed in SCLC stem cells (Sarvi et al., 2014, Cancer Research 74, 1554–1565), and TFRC expression is higher in SCLC plasma-derived microvesicles compared to controls (Pedersen et al., 2022, Clinical Proteomics 19, 2). Interestingly, regardless of disease stage, the expression of the antigens and the production of AAbs indicate that the tumor-associated antigens identified by the AAbs are not lost due to immune editing. In addition to the novel expression, the discovery of AAb targeting of PTM epitopes adds to the growing evidence that tumor-specific AAbs primarily recognize non-mutated self-proteins (Banville and Nelson, 2022, Cancer Cell 40, 356–358; Zaenker et al., 2016, Autoimmunity Reviews 15, 477–483). In an embodiment of the present disclosure, three immunogenic PTMs (i.e., citrullination, isoaspartylation, and cancer-specific glycosylation) of tumor-specific AAbs were identified based on the screening of PTM modifications and sites. However, in autoimmunity, AAbs have been identified against many types of PTMs, including phosphorylation, acetylation, hydroxylation, nitration, and carbamylation (Doyle and Mamula, 2012, Current Opinion in Immunology 24, 112–118). These PTM-AAbs can generate highly specific biomarkers.

[0170] AAbs are uniquely well-suited for early biomarker detection because they are obtained by simple blood draws, are produced in detectable amounts (albeit at low antigen concentrations), are upregulated years before clinical symptoms, and have a high degree of antigen specificity (Kobayashi et al., 2020, Seminars in Immunology 47, 101388). One of the main reasons for the proven success of early SCLC detection is the existence of a well-defined high-risk group: heavy cigarette smokers (Kenfield et al., 2008, Tobacco Control 17, 198). Since this consistent risk group is already eligible for low-dose CT lung cancer screening, in the current protocol, AAbs that identify unique molecular features of SCLC can be used to classify patients into high-risk or low-risk malignancy categories. A high-risk AAb result will signal the review or acquisition of diagnostic CT, while a low-risk AAb result will continue with annual lung cancer screening using LD-CT. There is also potential to add additional factors to further define the highest-risk patients for SCLC. AAbs against PNS antigens can pair well with the non-PNS AAbs disclosed herein.

[0171] In a specific embodiment, the 4-parameter risk prediction model generated according to the present disclosure can use one or more antigens selected from the group consisting of SPINK1, GRAP2, TIMP2, and PLD3, or it can use one or more antigens selected from the group consisting of SPINT2, SPINK1, TIMP2, and TFRC. In a specific embodiment, the 5-parameter risk prediction model generated according to the present disclosure can use one or more antigens selected from the group consisting of SPINK1, TFRC, GRAP2, TIMP2, and PLD3. Although in the examples shown below the antibody array contains over 3600 antibodies against approximately 2500 proteins selected for cancer relevance, other embodiments may identify other SCLC-specific autoantigens that may also perform well with the disclosed antibody array. Additionally, although the embodiments shown only probe three types of cancer-enriched PTMs, other embodiments may identify other cancer-enriched PTMs. It should also be noted that in some embodiments, the antibody-based nature of the disclosed method can be readily translated to higher-throughput platforms, such as multiple smaller arrays or ELISA on a single slide, without departing from the scope of the present disclosure.

[0172] Thus, the method disclosed herein identifies tumor-specific autoantigen-AAb complexes ex vivo. Separating tumor-targeting AAbs from their disease-specific antigens facilitates accurate detection of early SCLC. Autoantibody generation against non-PNS antigens is described herein, and SCLC-specific PTMs present in these antigens can lead to the generation of AAbs against epitopes that are suspected of being immunologically tolerant. The biomarker panel and method for diagnosing SCLC tumorigenesis according to the present disclosure may lead to the implementation of early detection strategies and antibody-based therapies. In a specific embodiment, the biomarker panel of the present disclosure may be capable of detecting SCLC in a subject or patient with a sensitivity and specificity of at least 60%, at least 70% or 75%, at least 80% or 85%, or at least 90% or 95%.

[0173] Small cell lung cancer (SCLC) is one of the few malignancies considered "refractory" cancers, highlighting the need for a better understanding of disease biology, more effective treatment strategies, and early intervention. Current treatment approaches for managing SCLC include chemotherapy, radiotherapy, surgical resection, immunotherapy, and / or combinations thereof. SCLC is highly sensitive to chemotherapy, and treatment typically elicits a rapid response. The current first-line treatment for SCLC (extensive stage) is platinum-based chemotherapy, consisting of four to six cycles of cisplatin or carboplatin in combination with etoposide (Mascaux et al., 2000, Lung Cancer 30, 23–36). Carboplatin is generally preferred over cisplatin because of its similar efficacy and lower toxicity (Rossi et al., 2012, J Clin Oncol 30, 1962). However, most patients relapse within the first year of treatment. In the case of platinum-sensitive relapse, rechallenge with first-line chemotherapy is preferred (Früh et al., 2013, Ann Oncol 24, Suppl. 6, vi99–vi105; Rudin et al., 2015, J Clin Oncol 33, 4106–4111). Topotecan is the only drug formally approved for second-line treatment of SCLC and remains the standard of care. The response rate (RR) for oral topotecan is 6%–17% (O'Brien et al., 2006, J Clin Oncol 24, 5441–5447). For limited-stage SCLC, standard treatment with curative intent consists of four cycles of platinum doublet chemotherapy combined with radiotherapy, which improves overall survival even in elderly patients compared with chemotherapy alone (Corso et al., 2015, J Clin Oncol 33, 4240-4246). The concurrent approach is preferred, and the timing of radiotherapy is important. Preferably, the start of radiotherapy (45 Gy, 30 fractions, twice daily) occurs concurrently with the first or second cycle of chemotherapy (De Ruysscher et al., 2006, J Clin Oncol 24, 1057–1063; De Ruysscher et al., 2016, Ann Oncol 27, 1818–1828).

[0174] Surgery for SCLC may be considered for very small tumors (very limited disease) cT1N0M0 (confirmed negative mediastinal staging) proven by biopsy. Most commonly, surgery

[0175] removal of a pulmonary nodule of unknown origin results in small cell lung cancer. There is a trend to offer surgery for very small SCLC with negative lymph nodes, but concurrent chemoradiotherapy is an alternative option (Hiddinga et al., 2021, Eur Respir Rev 30, 210079).

[0176] In addition, immunotherapy may play a role in the treatment of SCLC. The rationale for combining immunotherapy with chemotherapy in SCLC is the high mutational burden of this tumor, potentially enhancing immunogenicity. Chemotherapy may stimulate the expression of tumor antigens, triggering a response of the tumor to immunotherapy. In some instances, an immunotherapy regimen may include administering to a patient an antibody that binds one or more epitopes. As an example, the antibody may be a monoclonal antibody drug that activates the immune system by targeting CTLA-4 (e.g., ipilimumab, nivolumab) (Peters et al., 2020, Ann Oncol 31, LBA84). In some cases, the antibody may be conjugated to a cytotoxic agent (e.g., an anti-tumor agent or an anti-cancer agent). In some embodiments, for therapeutic purposes, a bispecific antibody capable of binding a target antigen and a T cell antigen (e.g., CD3) may be administered to a patient, involving targeting the T cell immune response to tissues / cells expressing the target antigen.

[0177] In an embodiment, an immunotherapy regimen may include chimeric antigen receptor T cells that specifically bind one or more epitopes. A chimeric antigen receptor (CAR) redirects T cell specificity to an antigen recognized by an antibody expressed on the surface of a cell (e.g., a cancer cell), while a T cell receptor expands the target range to include intracellular antigens (e.g., tumor antigens). One aspect of the present disclosure includes a CAR that is specific for an antigen expressed on the surface of SCLC tumor cells. In some instances, a method of treating a patient diagnosed with SCLC may include removing immune effector cells or T cells from a patient diagnosed with SCLC, genetically modifying the immune effector cells and T cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor, thereby generating a population of modified immune effector cells or T cells, and administering the population of modified immune effector cells or T cells to the same subject.

[0178] Circulating autoantibodies in SCLC have higher sensitivity compared to other cancers

[0179] To demonstrate that cancer-specific AAbs are produced abundantly in SCLC, the upregulated AAb-Ag content (i.e., the ratio between cases and controls) was measured in a total of 1009 plasma samples from SCLC, non-small cell lung cancer (NSCLC), breast cancer, colon cancer, and pancreatic cancer cohorts. It was found that both cancer-specific IgG and IgM levels were significantly higher in SCLC (p < 0.0001), on average 4-fold higher than in other cancers ( Figure 1A)。This indicates that the production of disease-specific AAbs in SCLC is more, and emphasizes the potential of AAbs as SCLC-specific biomarkers. Then, specific AAbs consistently present in 3 independent SCLC plasma cohorts were identified. First, pre-diagnostic plasma samples of 17 case-control pairs from the Cardiovascular Health Study (CHS, see Table 1) were analyzed for AAb-Ag complexes by an in-house discovery antibody array, which contained 3,600 antibody spots printed in triplicate (a total of 10,800 spots). 46 IgG and 219 IgM were observed to significantly (p<0.05) downregulate the AAb-Ag complex, and 24 IgG and 102 IgM significantly upregulated this complex ( Figure 1B )。The upregulated biomarkers were selected, and a new in-house antibody array for AAb-Ag complexes was designed, which contained 19 out of 24 IgG and 66 out of 102 IgM capture antibodies. After hybridization with 26 case-control pairs from the Fred Hutch Diagnostic Cohort (Table 1), compared with the controls, the AAb-Ag complexes of 14 out of 19 IgG (73.6% confirmed) and 15 out of 66 IgM (22.7% confirmed) were confirmed to be significantly upregulated in the cases ( Figure 1C )。To validate these results, 154 (55 SCLC cases and 99 matched controls) diagnostic plasma samples from Vanderbilt University (Table 1) were hybridized on a small in-house antibody array, and all AAb-Ag complexes remained upregulated, with 10 / 14 IgG (71.4% verified) and 12 / 13 IgM (92.3% verified) reaching statistical significance ( Figure 1D )。This high validation rate indicates that there are 22 promising SCLC-related AAb-Ag complexes for further study. The complete list of significantly upregulated and validated AAb-Ag complexes can be found in Table 2.

[0180] Table 1. Clinical characteristics of the SCLC cohorts. Age and pack-years are the means for each group. Gender and smoking status are counts.

[0181]

[0182] Table 2. Targets of upregulated AAb-Ag complexes validated in 3 independent SCLC cohorts (by stage).

[0183] Validated AAb - Ag Complex Target Target Strip Identifier CDH5 P33151 CD133 O43490 SPINK1 P00995 CDH23 Q9H251 NLRP7 Q8WX94 TFRC P02786 SPINT2 O43291 NADSYN1 Q6IA69 HIF1A Q16665 GRAP2 O75791 MAPRE1 Q15691 INHA P05111 PTEN P60484 CTSB P07858 B3GNT6 Q6ZMB0 PLD3 Q8IV08 TIMP2 P16035 NUDT2 P50583 ANAPC2 Q9UJX6 GPLD1 P80108 PTPRU Q92729 CA9 Q16790

[0184] Autoantibodies target tumor-associated antigens of SCLC

[0185] Assume 22 experimentally verified AAb - targeted tumor - associated antigens. To test if this is indeed the case, 5 AAb antigens with transmembrane domains were prioritized, which may be expressed on the cell surface of tumor cells and have potential translational relevance in future applications (e.g., imaging or antibody - drug conjugates). Immunohistochemistry was performed on an SCLC tissue microarray using antibodies against CD133, PLD3, TFRC, SPINT2, and CA9. CD133, PLD3, TFRC, and SPINT2 showed high and homogeneous expression in tumor cells but not in the adjacent stroma ( Figure 2A , Figure 3 staining control in Figure 2B ). In tumors expressing CA9, most showed a focal staining pattern, while approximately 20% showed homogeneous expression in tumor cells. Each core was scored as positive (>5% tumor cell staining) or negative (<5% tumor cell staining)( Figure 2B ), and it was found that >90% of tumor cores were positive for TFRC, SPINT2, and PLD3 staining, 60% were positive for CD133 staining, and 30% were positive for CA9 staining. Positive cores were further classified into stages at diagnosis, and it was demonstrated that tumor - associated antigens were expressed at similar frequencies in stage I, II, or III tumors. Cumulatively, this data validates the experimental approach of using AAb to discover tumor - associated antigens expressed early and throughout tumorigenesis.

[0186] Although SCLC has the highest tumor mutation burden, very few SCLC cell lines have non - silent mutations in the tumor - associated antigen list: among 51 SCLC cell lines with publicly available data, CD133 and CA9 each had mutations in 3, TFRC had a mutation in 1, and SPINT2 and PLD3 had no mutations in SCLC cell lines (Table 3). This low mutation rate (0% - 5.8%) suggests that the "neo - antigens" targeted by AAb may occur at the protein level. Since each of these proteins is exposed to the humoral immune system in normal tissues ( Figure 3 ), it is speculated that the immune system may be tolerant to their constitutive expression. Therefore, study tumor - associated PTMs, which may explain the observed AAb production.

[0187] Table 3. Antigens identified by autoantibodies are generally not mutated in SCLC cell lines. SNP = single - nucleotide polymorphism. HsCnt = hot - spot mutation count

[0188]

[0189]

[0190] Autoantibodies against CD133 target cancer - specific glycosylation motifs

[0191] Tumor-associated glycans can induce autoantibody formation (Tikhonov et al., 2020, Clinical Chemistry and Laboratory Medicine 58, 1611–1622), and most tumor biomarkers are glycoproteins (e.g., prostate-specific antigen, CA125 (ovary), CA19-9 (pancreas), α-fetoprotein (liver), and carcinoembryonic antigen (colon)). To determine whether the antigens identified by AAb have tumor-specific glycan modifications, they were screened for the cancer-enriched carbohydrate antigen sialyl Lewis A (sLeA, also known as CA19-9) after immunoprecipitating the target proteins from SCLC cell lines. In the H82 cell lysate, sLeA appeared on multiple proteins, but only the CD133 immunoprecipitation yielded a single sLeA-positive, CD133-positive band( Figure 4A ). Immunoprecipitation of CD133 in a second SCLC cell line, H69, also showed a band positive for both SleA and CD133( Figure 4B ). Additionally, this sLeA-positive, CD133-positive band was absent when precipitation was performed using an anti-mouse antibody (isotype control)( Figure 5A ). To confirm the glycosylation of CD133, CD133 was immunoprecipitated from the H82 cell line and treated with PRIME deglycosylase to remove N-linked glycans and complex sialylated structures such as sLeA. PRIME treatment disrupted the migration of CD133, confirming the removal of glycan residues and eliminating the detection of sLeA on the CD133 protein( Figure 5B ). The specificity of autoantibodies present in the plasma of SCLC patients for glycan-modified CD133 was evaluated by creating a modified sandwich ELISA on a replicated array platform. Commercial CD133 antibody was seeded to capture CD133 protein in the H82 SCLC cell line lysate, and after incubation with plasma from SCLC patients with known high or low levels of CD133 AAb (determined by the array platform in Figure 1C ), a significant difference in the level of bound IgG was verified by ELISA( Figure 5C ). Treatment of the H82 lysate with PRIME deglycosylase reduced the autoantibody signal to the same level as that of CD133-low plasma, suggesting that glycan modification is required for increased autoantibody binding.

[0192] Autoantibodies bind to post-translational modifications of aspartic acid in SPINK1

[0193] There is a prior report that the isoaspartic acid PTM (IsoAsp) present in ELAVL4, a PNS antigen, can be immunogenic in SCLC-related autoimmunity (Pulido et al., 2016, Journal of Neuroimmunology 299, 70–78). IsoAsp is a disease-enriched naturally occurring PTM formed by asparagine deamidation or aspartic acid isomerization. To search for AAbs targeting IsoAsp, asparagine was converted to IsoAsp residues in the extracellular region of the minimal antigen as a GST fusion using a published in vitro method (Geiger and Clarke, 1987, J Biol Chem 262, 785–794; Curnis et al., 2006, Journal of Biological Chemistry 281, 36466–36476). The IsoAsp-modified protein was used to pull down AAbs from the SCLC plasma pool compared to the unmodified protein, and immunoblotting qualitatively showed more IgG-AAb binding to isoAsp SPINK1, but not to isoAsp SPINT2 or TIMP2( Figures 6A - 6B ). To determine the specific substitution sites, peptides of all asparagine residues in the extracellular regions of SPINT2 (6 sites), SPINK1 (2 sites), and TIMP2 (4 sites) were synthesized. Wild-type (WT) asparagine or isoAsp peptides were coated on assay plates, incubated with SCLC plasma samples, and AAb binding was detected using anti-human IgG( Figure 6C ). Consistent with the immunoblot data, it was found that AAbs bound to IsoAsp at two positions of SPINK1 were at least 2-fold more than the corresponding WT peptides. Thus, this demonstrated that some AAbs in SCLC plasma recognize neoepitope PTM sites on SPINK1.

[0194] SCLC AAbs recognize citrullinated TFRC not present in the recombinant protein

[0195] Citrulline, a unique amino acid produced by spontaneous arginine deamination, has also been shown to be immunogenic in cancer but has not been described in SCLC (Brentville et al., 2016, Cancer Res 76, 548–560; Pulido et al., 2016, J Neuroimmunol 299, 70–78). Therefore, lysates of human SCLC H82 and H69 cells were screened with antibodies against citrulline, TFRC, PLD3, CD133, SPINT2, and CA9, and any overlapping band patterns of similar sizes between citrulline and the target proteins were looked for. TFRC had potential overlapping bands with citrulline, which were evident at the expected protein sizes ( Figure 7A and Figure 8A ). To increase the sensitivity of citrulline detection, citrulline was immunoprecipitated with the citrulline antibody and the expression of the target antibody was blotted, and TFRC was found to be the only target protein detected ( Figure 7B ). These observations were extended to show whole-cell lysates of the immortalized fibroblast cell line 3T3, which had low levels of TFRC expression but no corresponding bands were detected after probing with the citrulline antibody ( Figure 8B ). In addition, immunoprecipitation with the anti-TFRC antibody and blotting for citrulline or vice versa showed matching bands in H82 cells but not in the controls, indicating that TFRC has arginine residues that are specifically converted to citrulline in H82 cells. Thirty-two arginines in the extracellular domain of TFRC were considered, and 7 arginines that might undergo deamidation to citrulline were selected using a publicly available consensus motif (Ju and Wang, 2018, Gene 664, 78–83). Peptides representing TFRC fragments (with wild-type arginine or citrullinated amino acids in the appropriate sequences) were coated on assay plates, and 5 of the 7 peptides with citrullinated residues pulled down more AAb from SCLC plasma than the corresponding WT peptides ( Figure 8C ). TFRC AAb complexed with or without antigen was studied in a subset of plasma samples from the Fred Hutch cohort. By ELISA, it was confirmed that the level of the AAb-TFRC complex detected in SCLC plasma was significantly (p < 0.03) higher compared to the controls. In contrast, using the TFRC recombinant protein as the capture antigen, no significant difference in AAb was detected, although the anti-TFRC antibody readily detected the recombinant TFRC as a positive control ( Figure 8D)。Finally, compared to controls, the inclusion of 2 citrullinated TFRC peptides as capture antigens bound more AAbs in SCLC plasma, indicating that TFRC-AAbs recognize neoantigens of TFRC citrullination not found on recombinant proteins or arginine-containing (wild-type) peptides. Collectively, the data support the AAb-Ag complex platform as a method for identifying tumor-specific AAbs targeting post-translationally modified "neoantigens" that are difficult to detect using other methods.

[0196] Construction of an SCLC risk prediction model

[0197] The current annual low-dose computed tomography screening protocol, which is actually used for early detection of NSCLC, is not effective for SCLC (Thomas et al., 2018, Chest 154, 1284–1290). However, the clinical benefits of early detection of SCLC are clear: when detected in the limited stage, conventional chemotherapy is curative for nearly 20% of patients, and surgical resection, which is not usually considered, can be curative when combined with chemotherapy for very early patients (Lassen et al., 1995, J Clin Oncol 13, 1215–1220; Winer et al., 2011, Nature Medicine 17, 610–617). After demonstrating that AAbs generated in SCLC target specific PTMs of SCLC antigens, it was explored whether these unique AAbs could be used to generate a risk prediction model for early detection of SCLC. Therefore, the ability of all 22 validated AAb-Ag complexes to reliably detect the presence of SCLC in plasma samples was examined. Since the Vanderbilt (VB) cohort had the largest sample size, this set was used as training data, and the best combination of 4 or 5 markers was calculated by maximizing the AUC based on logistic regression. To account for the class switch from IgM to IgG during the immune response, each marker included two AAb isotypes. This identified a 4-marker group with a maximum total AUC of 0.872, composed of AAbs against PLD3, TIMP2, GRAP2, and SPINK1, and adding TFRC gave a 5-marker group with an AUC = 0.874 (Table 4). After fixing the coefficients using data from the Vanderbilt set, the 5-marker risk prediction model was tested in the CHS cohort, yielding an AUC = 0.700, and in the Fred Hutch (FH) cohort an AUC = 0.950.

[0198] Table 4. AUC maximization analysis in the VB training cohort to develop an SCLC risk prediction model.

[0199]

[0200] A multivariate logistic regression model was fitted to the VB data by including the 5 AAb markers that produced the highest AUC in the model. The prediction model was applied to the CHS and FH test datasets (Table 5) by classifying cases and controls into high-risk or low-risk categories according to positive or negative risk prediction scores, respectively. The risk prediction model was found to perform well, with a positive predictive value of 76.9% in VB, 64.7% in CHS, and 79.3% in FH, and a negative predictive value of 85.3% in VB, 64.7% in CHS, and 86.9% in FH. When pre-diagnostic samples from CHS were grouped by diagnostic time, the accuracy of marking high-risk samples within one year before diagnosis (7 / 11, 63.6%) was higher than that within 1-2 years before diagnosis (3 / 6, 50%). In addition, in the FH and VB cohorts, it was able to correctly identify 23 / 26 (88.5%) of the limited-stage SCLC and 18 / 24 (75%) of the extensive-stage SCLC in the high-risk pool.

[0201] Table 5. The 5-AAb group risk prediction model can accurately classify patient samples into high-risk and low-risk categories. Samples in each cohort were scored as high-risk or low-risk according to positive or negative risk prediction scores, respectively.

[0202]

[0203] The 5 AAbs were studied more specifically and individually in each cohort. When blood was drawn within one year before diagnosis, the AAbs targeting PLD3, TIMP2, GRAP2, and SPINK1 were significantly upregulated (p = 0.05), while the AAb targeting TFRC showed an upward trend (p = 0.15)( Figure 9A ). PLD3 and TIMP2 were still significantly upregulated even up to two years before diagnosis (p < 0.005). In diagnostic samples, all 5 components were highly significantly upregulated in both limited-stage and extensive-stage diseases (p < 0.0005). In cases of current smokers compared to former smokers, there was no differential expression of the group markers( Figure 10A ), and PLD3 was not associated with pack-years of smoking( Figure 10B ). TIMP2, GRAP2, SPINK1, and TFRC were weakly correlated with pack-years of smoking (r < 0.25)( Figures 10C - 10F ). The group AAbs had no differential expression in the diagnosis of chronic obstructive pulmonary disease( Figure 11A ) or autoimmunity( Figure 11B ). The individual performance of these markers was examined in the plasma of other cancers (including non-SCLC, pancreatic cancer, and colon cancer), and it was found that these markers were not significantly upregulated in other diseases compared to controls or SCLC( Figure 9B), indicating SCLC specificity. Cumulatively, these AAbs are specifically upregulated early in SCLC tumorigenesis and remain elevated throughout the disease course.

[0204] The single greatest risk factor for SCLC is current heavy smoking, which can increase the likelihood of developing SCLC in women and men by 60- to 100-fold, respectively (Wang et al., 2017, Sci Rep 7, 1339). Since the smoking histories (current, former, never smokers) were matched across all three cohorts, pack-year smoking history was incorporated into the 5-group model. The continuous variable of pack-years of smoking alone had an AUC of 0.57 in VB, 0.72 in CHS, and 0.612 in FH ( Figures 12A - 12C ). The combination of pack-years of smoking and the AAb risk prediction model had little impact on the Vanderbilt cohort (AUC remained at 0.87), while the AUC increased to 1.0 in both the CHS and Fred Hutch cohorts.

[0205] Experimental Methods and Materials

[0206] The following paragraphs provide additional details regarding the experiments discussed above. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and biases should be considered. Unless otherwise specified, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are atmospheric or near atmospheric.

[0207] Study Design

[0208] All human plasma samples were obtained after institutional review board approval and informed consent. The study used three independent plasma cohorts: 1) The Cardiovascular Health Study (CHS) is a population-based longitudinal study of coronary heart disease and stroke (Fried et al., 1991, Ann Epidemiol 1, 263–276). Clinical examinations and plasma samples were completed annually over a 10-year period. Seventeen cases were newly diagnosed with SCLC within 24 months of blood draw. These 17 cases were individually matched to controls based on age, sex, body mass index, and smoking history. 2) The Fred Hutchinson Early Detection and Prevention Clinic for Lung Nodule Evaluation (Fred Hutch) consisted of N = 26 SCLC patients and N = 26 patients with benign lung nodules (current IRB protocol number 6663). Corresponding clinicopathological data were retained in a highly annotated database. Controls were matched to cases based on age, sex, and smoking history. 3) Fifty-five diagnostic plasma samples and 99 unmatched control plasma samples were matched based on age, sex, and smoking history and were generously provided by Vanderbilt University in Nashville, Tennessee.

[0209] Detection of AAb-Ag complex

[0210] The detection of AAb-Ag complexes using antibody arrays has been previously described (Winer et al., 2011, Nature Medicine 17, 610–617). Briefly, antibody microarrays were printed in-house, incubated with 1:80 diluted patient plasma, and probed with anti-human IgG-SeTau647 or IgM-DyLight550 antibodies. The microarrays were imaged on a GenePix 4000B scanner, and spot intensities were measured in GenePix Pro 6.0 (Molecular Devices).

[0211] Immunohistochemistry

[0212] Tissue microarrays (TMAs) containing 62 individual cases of SCLC and 6 individual lung tissue controls (RLN681A, USBiolab) were stained for CD133 (#64326, 1:1000 Cell Signaling Technologies) or an anti-rabbit IgG isotype antibody at the same concentration using an automated Leica Bond RX system and detected with rabbit HRP and DAB from Leica. After heat-induced epitope retrieval in citrate buffer pH 6, the same TMAs were also stained for TFRC (HPA028598, 1:500 Sigma), SPINT2 (HPA011101, 1:200 Sigma), and CA9 (HPA055207, 1:1000 Sigma). The sections were probed with biotinylated pan-specific universal antibody for 10 minutes, streptavidin-HRP for 5 minutes (PK-8800, Vector Laboratories), and counterstained with hematoxylin, with washing steps in between. After RLN681A was discontinued, different TMAs were purchased from USBiomax (BS04116a), containing 45 duplicate SCLC cores and 10 lung tissue controls, and stained for PLD3 (HPA012800, 1:200 Sigma) using the Vector protocol described above. Each antibody was also stained on a TMA containing 33 normal tissues (3 individuals per organ, FDA999-1, USBiolab) as positive or negative controls. The stained slides were digitized using a Leica Aperio whole-slide scanner at 20× magnification. Scoring of TMA staining was performed blindly, where each biopsy was determined to be positive (>5% of tumor cells stained) or negative (<5% of tumor cells stained).

[0213] ELISA

[0214] A plate-based assay was developed to quantify autoantibody levels for different capture methods. WT and PTM peptides were purchased from CHIScientific (sequences are listed in Table 6), and 10 μg of the peptides were plated in each well of a maleimide 96-well plate (#15152, Pierce). A white 96-well plate (#15042 Pierce) was coated with 1 μg / well of α-CD133 antibody (HB#7, Developmental Studies Hybridoma Bank), and CD133 was captured from H82 cell lysates treated with or without PRIME glycosylase (#50-999-475, Fisher Scientific) at 1 U / 50 μg cell lysate. The α-TFRC antibody (HPA028598, Sigma) or TFRC recombinant protein (89-760aa, #11020-H07H, Sinobiological) was plated at 0.5 μg / well. After binding the capture methods, all plates were washed and used as bait for autoantibodies in human plasma (1:500). All autoantibody signals were quantified using an α-human IgG-HRP secondary antibody (I:5000, #709-065-149, Jackson ImmunoResearch), SuperSignal ELISA Femto (#37074, ThermoFisher), and a SpectroMax L microplate reader (Molecular Devices).

[0215] Table 6. Peptide sequences. Wild-type (WT) or post-translationally modified (PTM) peptide sequences are listed below.

[0216]

[0217]

[0218] Immunoblot

[0219] To induce isoaspartylation, GST-fusion proteins were mixed with magnetic glutathione beads (#78601, ThermoFisher) at 1:20 in NH4HCO3 (100 mM, pH 8.0) or PBS containing 0.05% sodium azide and incubated at 37 °C for 48 h. After washing, the treated GST-fusion proteins were incubated with human plasma at 1:10,000 overnight at 4 °C. Samples were directly lysed in Laemmli sample buffer containing 1x complete protease inhibitor cocktail, 1x phosphatase inhibitor cocktail, and 2 mM phenylmethylsulfonyl fluoride (all from MilliporeSigma) and sonicated. Samples were separated by SDS-PAGE, transferred to nitrocellulose, the loading amount was checked with Ponceau S, blocked with 1% non-fat milk powder, probed with 1:5000 α-human IgG-AF680 (#709-625-149, ImmunoResearch) and 1:500 mouse α-GST (FHCC Antibody Technology Core), and detected by α-mouse IgG-AF680 (1:10,000, A32729, ThermoFisher). Immunoblots were imaged with ImageStudio v5 on an Odyssey CLx (LiCor; Lincoln, NE). CD133 protein was immunoprecipitated from H82 cells and treated with or without PRIME deglycosylase (#50-999-475, Fisher Scientific) at 1 U / 50 μg cell lysate for 30 min at 37 °C, then immunoblotted as described above, and any differences are noted below. Immunoblots were probed with antibodies against sLeA (1:2000, #10-C04E, Fitzgerald Industries) and CD133 (1:1000, #6436, Cell Signaling Technologies) and detected with α-mouse IgM-AlexaFluor680 and α-rabbit IgG-AlexaFluor790 (1:10,000, #A10038, #A10043, ThermoFisher), respectively. Lysates from H82 or 3T3 cell lines were immunoprecipitated with 1 μg of αTFRC (#13-6800 Invitrogen), α-citrulline (ab100932, AbCam), or isotype control antibody (FHCC Antibody Technology Core) and protein G magnetic beads (#88847, ThermoFisher).Probe immunoblots with antibodies against TFRC (1:1000, HPA028598, Sigma) or citrulline (1:500, MA5-27573, Invitrogen), and detect them with α-mouse IgG-AlexaFluor680 and α-rabbit IgG-AlexaFluor790 (1:10000, A32729, A10043, ThermoFisher), respectively.

[0220] Cell culture

[0221] The NCI-H82 and NCI-H69 cell lines were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium supplemented with 4 mM L-glutamine, 10% fetal bovine serum, and 1% penicillin-streptomycin in an incubator at 37 °C and 5% CO2, and were routinely tested mycoplasma negative by PCR.

[0222] Statistical analysis

[0223] The array data were in the same format as the dual-channel gene expression arrays, and the analysis was performed similarly as described previously (Rho and Lampe, 2013, J Proteome Res 12, 2311–2320). In GraphPad Prism, two groups were compared by t-test (the type of t-test is indicated in the figure legend), and one-way ANOVA along with Tukey's test was used to compare three or more groups. Logistic regression was used to identify combinations of multiple autoantibodies that best distinguished cases from controls. Receiver operating characteristic curves were created, and the area under the curve was calculated to evaluate the predictive ability. All relevant statistical analyses were performed using R statistical software (R Core Team (2021)).

[0224] Identification of autoantibodies

[0225] Once the immunogenic PTM peptides targeted by autoantibodies were identified, PTM-specific autoantibodies were directly isolated from SCLC patients and sequenced ( Figure 13) To achieve this, PTM peptide tetramers were used to enrich PTM-binding B cells from the peripheral blood mononuclear cells of SCLC patients. The PTM peptide tetramers were generated by conjugating PE or APC-labeled magnetic tetramers containing a streptavidin core with biotinylated PTM peptides and were identified to bind more AAbs in the plasma of SCLC patients compared to wild-type peptides. Each PTM tetramer contains five different PTM peptides; 5 citrullinated TFRC peptides were conjugated to the PE tetramer and 5 isoaspartylated peptides (3 CA9, 1 PLD3, and 1 SPINK1) were conjugated to the APC tetramer. These PTM tetramers were then incubated with pooled peripheral blood mononuclear cells (PBMCs) from 3 different SCLC patients and the tetramer-binding cells were isolated using magnetic beads. This PTM tetramer "enriched" fraction was labeled with fluorescent antibodies and fluorescence-activated cell sorting (FACS) was performed into single cells. PTM tetramer + B cells were sorted, the heavy and light chain antibody sequences of each B cell were amplified by RT-PCR, and the heavy and heavy chain pairs were successfully sequenced. These sequences were cloned into a human IgG expression vector for antibody production, shown in Table 7, where the positions of the CDRs are underlined. The PTM modifications of the autoantigens to which the antibodies each bind are also shown in Table 7. The sequences of the CDRs were determined using the ImMunoGeneTics (IMGT) information system domain gap alignment. Table 8 lists the CDR sequences and their corresponding SEQ ID NOs.

[0226] Table 7. Heavy and light chain variable region sequences of antibodies

[0227]

[0228]

[0229] Table 8. Antibody complementarity-determining regions (CDRs)

[0230]

[0231]

[0232] A PTM-specific antibody 2D2 was isolated from SCLC PBMCs and further characterized. The 2D2 antibody has been successfully used for specific imaging of tumor TFRC in female athymic nude mice bearing subcutaneous flank H82 tumors ( Figure 14 ). For this, the 2D2 antibody was labeled with CF770 and injected retroorbitally into the mice. NIRF imaging was performed at 3, 4, and 5 days. Figure 14 Scans starting from day 5 are shown, with essentially all the signal concentrated in the tumor. Figure 15It is shown that the 2D2 antibody binds mainly to the specific TFRC sequence representing citrullinated peptide 13, to a lesser extent to other TFRC citrullinated peptides, but not to isoaspartylated or WT CA9. These results indicate that the 2D2 antibody is specific for the specific TFRC sequence with citrulline at a specific position. Figure 16 It is shown that the 2D2 antibody does not bind to TFRC in normal uterus, small intestine, lung or placenta, while the commercial TFRC antibody (WT TFRC) binds extensively.

[0233] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0234] *******

Claims

1. A method of treating a subject with small cell lung cancer (SCLC), the method comprising: (a) administering a chemotherapy regimen to the subject; (b) administering an immunotherapy regimen to the subject; (c) administering an antibody or an antigen-binding fragment thereof to the subject; (d) performing surgical resection of the SCLC in the subject; and / or (e) exposing the subject to radiotherapy targeting the SCLC, wherein the subject has autoantibodies that specifically bind one or more epitopes of one or more antigens selected from the group consisting of CDH5, CD133, SPINK1, CDH23, NLRP7, TFRC, SPINT2, NADSYN1, HIF1A, GRAP2, MAPRE1, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9, and wherein the one or more epitopes of the antigen comprise a post-translational modification (PTM).

2. The method of claim 1, wherein the subject has autoantibodies that specifically bind two or more epitopes of the one or more antigens.

3. The method of claim 1, wherein the subject has autoantibodies that specifically bind three or more epitopes of the one or more antigens.

4. The method of claim 1, wherein the subject has autoantibodies that specifically bind four or more epitopes of the one or more antigens.

5. The method of claim 1, wherein the subject has autoantibodies that specifically bind five or more epitopes of the one or more antigens.

6. The method of any one of claims 1-5, wherein the one or more antigens are selected from the group consisting of SPINK1, TFRC, GRAP2, TIMP2, and PLD3.

7. The method of any one of claims 1-5, wherein the one or more antigens are selected from the group consisting of SPINK1, GRAP2, TIMP2, and PLD3.

8. The method of any one of claims 1-5, wherein the one or more antigens are selected from the group consisting of SPINT2, SPINK1, TIMP2, and TFRC.

9. The method of any one of claims 1-8, wherein the one or more epitopes are selected from the group consisting of SEQ ID NO: 1-21.

10. The method of any one of claims 1-9, wherein the PTM comprises citrullination, isoaspartylation, and / or glycosylation.

11. The method of claim 10, wherein the PTM comprises citrullination and / or isoaspartylation.

12. The method of any one of claims 1-11, wherein the one or more epitopes are selected from the group consisting of SEQ ID NO: 22-46.

13. A method of diagnosing and treating a subject with small cell lung cancer (SCLC), the method comprising: (a) Detect the presence of one or more autoantibodies that specifically bind to one or more epitopes of one or more antigens in a biological sample obtained from the subject, wherein the one or more antigens are selected from the group consisting of: CDH5, CD133, SPINK1, CDH23, NLRP7, TFRC, SPINT2, NADSYN1, HIF1A, GRAP2, MAPRE1, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9, and wherein the one or more epitopes of the antigen comprise post-translational modification (PTM); (b) When the presence of the one or more autoantibodies is detected in the biological sample obtained from the subject, diagnose the subject with SCLC; and (c) Treat the subject's SCLC by: (i) Administering a chemotherapy regimen to the subject; (ii) Administering an immunotherapy regimen to the subject; (iii) Administering an antibody or an antigen-binding fragment thereof to the subject; (iv) Performing surgical resection of the SCLC in the subject; and / or (v) Exposing the subject to radiotherapy targeting the SCLC.

14. The method according to claim 13, further comprising obtaining the biological sample from the subject.

15. The method according to claim 13 or 14, wherein the biological sample is serum or plasma.

16. The method according to any one of claims 13-15, wherein diagnosing the subject comprises diagnosing the subject with SCLC when the presence of autoantibodies that bind to two or more epitopes of the one or more antigens is detected in the biological sample.

17. The method according to any one of claims 13-15, wherein diagnosing the subject comprises diagnosing the subject with SCLC when the presence of autoantibodies that bind to three or more epitopes of the one or more antigens is detected in the biological sample.

18. The method according to any one of claims 13-15, wherein diagnosing the subject comprises diagnosing the subject with SCLC when the presence of autoantibodies that bind to four or more epitopes of the one or more antigens is detected in the biological sample.

19. The method according to any one of claims 13-15, wherein diagnosing the subject comprises diagnosing the subject with SCLC when the presence of autoantibodies that bind to five or more epitopes of the one or more antigens is detected in the biological sample.

20. The method according to any one of claims 13-19, wherein the one or more antigens are selected from the group consisting of SPINK1, TFRC, GRAP2, TIMP2, and PLD3.

21. The method according to any one of claims 13-19, wherein the one or more antigens are selected from the group consisting of SPINK1, GRAP2, TIMP2, and PLD3.

22. The method according to any one of claims 13-19, wherein the one or more antigens are selected from the group consisting of SPINT2, SPINK1, TIMP2, and TFRC.

23. The method according to any one of claims 13-22, wherein the one or more epitopes are selected from the group consisting of SEQ ID NO: 1-21.

24. The method according to any one of claims 13-23, wherein the PTM comprises citrullination, isoaspartylation, and / or glycosylation.

25. The method according to claim 24, wherein the PTM comprises citrullination and / or isoaspartylation.

26. The method according to any one of claims 13-25, wherein the one or more epitopes are selected from the group consisting of SEQ ID NO: 22-46.

27. The method according to any one of claims 1-26, wherein the subject is a human.

28. The method according to any one of claims 1-27, wherein the chemotherapy regimen comprises one or more cycles of cisplatin or carboplatin in combination with etoposide.

29. The method according to any one of claims 1-28, wherein the immunotherapy regimen comprises chimeric antigen receptor T cells that specifically bind to one or more of the epitopes.

30. The method according to any one of claims 1-28, wherein the immunotherapy regimen comprises an antibody that binds to one or more of the epitopes.

31. The method according to claim 30, wherein the antibody is conjugated to a cytotoxic agent.

32. The method according to any one of claims 1-28, wherein the immunotherapy regimen comprises a bispecific antibody that binds to one or more of the epitopes and a T cell antigen.

33. The method according to claim 32, wherein the T cell antigen is CD3.

34. The method according to any one of claims 1-33, wherein treating the subject's SCLC is by administering to the subject an antibody or an antigen-binding fragment thereof.

35. The method according to claim 34, wherein the antibody or an antigen-binding fragment thereof specifically binds to one or more epitopes of one or more antigens, wherein the one or more antigens are selected from the group consisting of: CDH5, CD133, SPINK1, CDH23, NLRP7, TFRC, SPINT2, NADSYN1, HIF1A, GRAP2, MAPRE1, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9, and wherein the one or more epitopes of the antigen comprise a post-translational modification (PTM).

36. The method according to claim 35, wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) contained in the heavy-chain variable region (HCVR) and three light-chain CDRs contained in the light-chain variable region (LCVR), and wherein the amino acid sequences of the HCVR / LCVR respectively comprise amino acid sequences selected from the group consisting of: SEQ ID NO: 47 / 51, 55 / 59, 63 / 67, 71 / 75, and 79 / 83.

37. The method according to claim 35, wherein the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, and the domain comprises amino acid sequences selected from the group consisting of: SEQ ID NO: 48-49-50-52-53-54, 56-57-58-60-61-62, 64-65-66-68-69-70, 72-73-74-76-77-78, and 80-81-82-84-85-86.

38. The method according to claim 35, wherein the antibody or antigen-binding fragment thereof comprises a heavy-chain variable region (HCVR) and a light-chain variable region (LCVR), and wherein the amino acid sequences of the HCVR / LCVR are respectively selected from the group consisting of: SEQ ID NO: 47 / 51, 55 / 59, 63 / 67, 71 / 75, and 79 / 83.

39. The method according to any one of claims 34-38, wherein the antibody or antigen-binding fragment thereof binds to an epitope of TFRC-4 comprising a PTM, and wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained in the light-chain variable region (LCVR), the heavy-chain variable region comprises the amino acid sequence of SEQ ID NO: 79, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO:

83.

40. The method according to claim 39, wherein the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, and the domain respectively comprises the amino acid sequences of SEQ ID NO: 80-81-82-84-85-86.

41. The method according to claim 39 or 40, wherein the antibody or antigen-binding fragment thereof comprises an HCVR containing the amino acid sequence of SEQ ID NO: 79 and an LCVR containing the amino acid sequence of SEQ ID NO:

83.

42. The method according to any one of claims 39-41, wherein the epitope comprises the amino acid sequence of SEQ ID NO:

39.

43. The method according to any one of claims 34-38, wherein the antibody or antigen-binding fragment thereof binds to an epitope of CA9-3 comprising a PTM, and wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), the heavy-chain variable region comprising the amino acid sequence of SEQ ID NO:71, and the light-chain variable region comprising the amino acid sequence of SEQ ID NO:

75.

44. The method according to claim 43, wherein the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, the domain comprising the amino acid sequences of SEQ ID NO:72-73-74-76-77-78, respectively.

45. The method according to claim 43 or 44, wherein the antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO:71 and an LCVR comprising the amino acid sequence of SEQ ID NO:

75.

46. The method according to any one of claims 43-44, wherein the epitope comprises the amino acid sequence of SEQ ID NO:

45.

47. The method according to any one of claims 34-38, wherein the antibody or antigen-binding fragment thereof binds to an epitope of TFRC-5 comprising a PTM, and wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), the heavy-chain variable region comprising the amino acid sequence of SEQ ID NO:47, and the light-chain variable region comprising the amino acid sequence of SEQ ID NO:

51.

48. The method according to claim 47, wherein the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, the domain comprising the amino acid sequences of SEQ ID NO:48-49-50-52-53-54, respectively.

49. The method according to claim 47 or 48, wherein the antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO:47 and an LCVR comprising the amino acid sequence of SEQ ID NO:

51.

50. The method according to any one of claims 47-49, wherein the epitope comprises the amino acid sequence of SEQ ID NO:

40.

51. The method according to any one of claims 34-38, wherein the antibody or antigen-binding fragment thereof binds to an epitope of TFRC-1 or TFRC-2 that contains a PTM, and wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), the heavy-chain variable region comprising the amino acid sequence of SEQ ID NO:55, and the light-chain variable region comprising the amino acid sequence of SEQ ID NO:

59.

52. The method according to claim 51, wherein the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, the domains comprising the amino acid sequences of SEQ ID NO:56-57-58-60-61-62, respectively.

53. The method according to claim 51 or 52, wherein the antibody or antigen-binding fragment thereof comprises an HCVR containing the amino acid sequence of SEQ ID NO:55 and an LCVR containing the amino acid sequence of SEQ ID NO:

59.

54. The method according to any one of claims 51-53, wherein the epitope comprises the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:

37.

55. The method according to any one of claims 34-38, wherein the antibody or antigen-binding fragment thereof binds to an epitope of SPINK1-1 that contains a PTM, and wherein the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), the heavy-chain variable region comprising the amino acid sequence of SEQ ID NO:63, and the light-chain variable region comprising the amino acid sequence of SEQ ID NO:

67.

56. The method according to claim 55, wherein the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, the domains comprising the amino acid sequences of SEQ ID NO:64-65-66-68-69-70, respectively.

57. The method according to claim 55 or 56, wherein the antibody or antigen-binding fragment thereof comprises an HCVR containing the amino acid sequence of SEQ ID NO:63 and an LCVR containing the amino acid sequence of SEQ ID NO:

67.

58. The method according to any one of claims 55-57, wherein the epitope comprises the amino acid sequence of SEQ ID NO:

29.

59. A biomarker detection panel for small cell lung cancer (SCLC), the biomarker detection panel comprising one or more peptides having an epitope of 7 to 50 amino acids in length containing one or more antigens, the one or more antigens selected from the group consisting of: CDH5, CD133, SPINK1, CDH23, NLRP7, TFRC, SPINT2, NADSYN1, HIF1A, GRAP2, MAPRE1, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9, wherein the epitope contains a post-translational modification (PTM) bound by an autoantibody.

60. The biomarker detection panel according to claim 59, wherein the panel is capable of detecting SCLC in a subject with at least 60% sensitivity and specificity.

61. The biomarker detection panel according to claim 60, wherein the panel is capable of detecting SCLC in a subject with at least 70% or 75% sensitivity and specificity.

62. The biomarker detection panel according to claim 60, wherein the panel is capable of detecting SCLC in a subject with at least 80% or 85% sensitivity and specificity.

63. The biomarker detection panel according to claim 60, wherein the panel is capable of detecting SCLC in a subject with at least 90% or 95% sensitivity and specificity.

64. The biomarker detection panel according to any one of claims 59 - 63, wherein the panel comprises two or more epitopes of the one or more antigens.

65. The biomarker detection panel according to any one of claims 59 - 63, wherein the panel comprises three or more epitopes of the one or more antigens.

66. The biomarker detection panel according to any one of claims 59 - 63, wherein the panel comprises four or more epitopes of the one or more antigens.

67. The biomarker detection panel according to any one of claims 59 - 63, wherein the panel comprises five or more epitopes of the one or more antigens.

68. The biomarker detection panel according to any one of claims 59 - 67, wherein the one or more antigens are selected from the group consisting of SPINK1, TFRC, GRAP2, TIMP2, and PLD3.

69. The biomarker detection panel according to any one of claims 59 - 67, wherein the one or more antigens are selected from the group consisting of SPINK1, GRAP2, TIMP2, and PLD3.

70. The biomarker detection panel according to any one of claims 59 - 67, wherein the one or more antigens are selected from the group consisting of SPINT2, SPINK1, TIMP2, and TFRC.

71. The biomarker detection panel according to claim 70, wherein the one or more epitopes are selected from the group consisting of SEQ ID NO: 1 - 21.

72. A biomarker detection panel according to any one of claims 59 - 71, wherein the PTM comprises citrullination, isoaspartylation, and / or glycosylation.

73. A biomarker detection panel according to claim 72, wherein the PTM comprises citrullination and / or isoaspartylation.

74. A biomarker detection panel according to claim 45, wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 22 - 46.

75. A biomarker detection panel according to any one of claims 34 - 49, wherein the panel is for detecting autoantibodies that bind to the one or more epitopes of the one or more antigens in a biological sample obtained from a subject.

76. An isolated antibody or antigen - binding fragment thereof that binds to an epitope of TFRC - 4 comprising a PTM, wherein the antibody or antigen - binding fragment comprises three heavy - chain complementarity - determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy - chain variable region (HCVR) and three light - chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light - chain variable region (LCVR), the heavy - chain variable region comprises the amino acid sequence of SEQ ID NO: 79, and the light - chain variable region comprises the amino acid sequence of SEQ ID NO:

83.

77. The antibody or antigen - binding fragment according to claim 76, wherein the antibody or antigen - binding fragment comprises an HCDR1 - HCDR2 - HCDR3 - LCDR1 - LCDR2 - LCDR3 domain, and the domain comprises the amino acid sequences of SEQ ID NOs: 80 - 81 - 82 - 84 - 85 - 86, respectively.

78. The antibody or antigen - binding fragment according to claim 76 or 77, wherein the antibody or antigen - binding fragment comprises an HCVR containing the amino acid sequence of SEQ ID NO: 79 and an LCVR containing the amino acid sequence of SEQ ID NO:

83.

79. The antibody or antigen - binding fragment according to any one of claims 76 - 79, wherein the epitope comprises the amino acid sequence of SEQ ID NO:

39.

80. An antibody or antigen - binding fragment thereof that binds to an epitope of CA9 - 3 comprising a PTM, wherein the antibody or antigen - binding fragment comprises three heavy - chain complementarity - determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy - chain variable region (HCVR) and three light - chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light - chain variable region (LCVR), the heavy - chain variable region comprises the amino acid sequence of SEQ ID NO: 71, and the light - chain variable region comprises the amino acid sequence of SEQ ID NO:

75.

81. The antibody or antigen-binding fragment according to claim 80, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, and the domains respectively comprise the amino acid sequences of SEQ ID NO:72-73-74-76-77-78.

82. The antibody or antigen-binding fragment according to claim 80 or 81, wherein the antibody or its antigen-binding fragment comprises an HCVR containing the amino acid sequence of SEQ ID NO:71 and an LCVR containing the amino acid sequence of SEQ ID NO:

75.

83. The antibody or antigen-binding fragment according to any one of claims 80-82, wherein the epitope comprises the amino acid sequence of SEQ ID NO:

45.

84. An antibody or its antigen-binding fragment that binds to an epitope of TFRC-5 containing PTM, wherein the antibody or its antigen-binding fragment comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained in the light-chain variable region (LCVR), the heavy-chain variable region comprises the amino acid sequence of SEQ ID NO:47, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO:

51.

85. The antibody or antigen-binding fragment according to claim 84, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, and the domains respectively comprise the amino acid sequences of SEQ ID NO:48-49-50-52-53-54.

86. The antibody or antigen-binding fragment according to claim 84 or 85, wherein the antibody or its antigen-binding fragment comprises an HCVR containing the amino acid sequence of SEQ ID NO:47 and an LCVR containing the amino acid sequence of SEQ ID NO:

51.

87. The antibody or antigen-binding fragment according to any one of claims 84-86, wherein the epitope comprises the amino acid sequence of SEQ ID NO:

40.

88. An antibody or its antigen-binding fragment that binds to an epitope of TFRC-1 or TFRC-2 containing PTM, wherein the antibody or its antigen-binding fragment comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained in the light-chain variable region (LCVR), the heavy-chain variable region comprises the amino acid sequence of SEQ ID NO:55, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO:

59.

89. The antibody or antigen-binding fragment according to claim 88, wherein the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, and the domains respectively comprise the amino acid sequences of SEQ ID NO:56-57-58-60-61-62.

90. The antibody or antigen-binding fragment according to claim 88 or 89, wherein the antibody or its antigen-binding fragment comprises an HCVR containing the amino acid sequence of SEQ ID NO:55 and an LCVR containing the amino acid sequence of SEQ ID NO:

59.

91. The antibody or antigen-binding fragment according to any one of claims 88-90, wherein the epitope comprises the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:

37.

92. An antibody or its antigen-binding fragment that binds to an epitope of SPINK1-1 containing a PTM, wherein the antibody or its antigen-binding fragment comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in the heavy-chain variable region (HCVR) and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained in the light-chain variable region (LCVR), the heavy-chain variable region comprises the amino acid sequence of SEQ ID NO:63, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO:

67.

93. The antibody or antigen-binding fragment according to claim 92, wherein the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain, and the domains respectively comprise the amino acid sequences of SEQ ID NO:64-65-66-68-69-70.

94. The antibody or antigen-binding fragment according to claim 92 or 93, wherein the antibody or its antigen-binding fragment comprises an HCVR containing the amino acid sequence of SEQ ID NO:63 and an LCVR containing the amino acid sequence of SEQ ID NO:

67.

95. The antibody or antigen-binding fragment according to any one of claims 92-94, wherein the epitope comprises the amino acid sequence of SEQ ID NO:

29.

96. A pharmaceutical composition comprising the antibody or its antigen-binding fragment according to any one of claims 76-95 and a pharmaceutically acceptable carrier or diluent.

97. A unit dosage form comprising a container containing the antibody or its antigen-binding fragment according to any one of claims 76-95.

98. The unit dosage form according to claim 97, wherein the container is a vial, syringe, pre-filled syringe, or auto-injector.

99. A kit comprising a container containing the antibody or its antigen-binding fragment according to any one of claims 76-95 and instructions for treating small cell lung cancer using the antibody or antigen-binding fragment.

100. A nucleic acid molecule encoding an antibody or antigen-binding fragment as claimed in any one of claims 76-95.

101. A recombinant expression vector comprising the nucleic acid molecule as claimed in claim 100.

102. A pair of nucleic acid molecules encoding the heavy chain variable region and the light chain variable region of an antibody or antigen-binding fragment as claimed in any one of claims 76-95, wherein the pair of nucleic acid molecules comprises a first nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment and a second nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment.

103. A pair of recombinant expression vectors respectively comprising the pair of nucleic acid molecules as claimed in claim 102.

104. An isolated host cell comprising an antibody or antigen-binding fragment as claimed in any one of claims 76-95, the nucleic acid molecule as claimed in claim 100, the recombinant expression vector as claimed in claim 101, the pair of nucleic acid molecules as claimed in claim 102, or the pair of recombinant expression vectors as claimed in claim 103.

105. The isolated host cell as claimed in claim 104, which is a mammalian host cell.

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