Small cell lung cancer tumor antigens and uses thereof

Abstract

Disclosed herein are high-performance biomarkers and panels for the early detection of small cell lung cancer (SCLC), which are useful for identifying, diagnosing, and treating SCLC patients at an early stage. A method for diagnosing and treating SCLC in a subject includes 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, where the one or more epitopes of the antigen comprise a post-translational modification; diagnosing the subject as having SCLC if 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, performing surgery, and / or performing radiation therapy. In one example, the post-translational modifications targeted by the autoantibody include citrullination, isoaspartylation, and / or cancer-specific glycosylation.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 415,143, filed October 11, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing Reference This application incorporates by reference a computer readable sequence listing in ST.26 XML format entitled 23-032-WO-PCT_Sequence, created on September 25, 2023, and containing 98,924 bytes.

[0003] Government support approval 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 this invention.

[0004] FIELD OF THE DISCLOSURE This disclosure relates to the field of medicine, specifically to autoantibodies and autoantibody-autoantigen complexes as biomarkers for small cell lung cancer (SCLC), and methods of use thereof. [Background technology]

[0005] Small cell lung cancer (SCLC) is the sixth leading cause of cancer-related deaths in the United States, with fewer than 6% of patients surviving five 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 one very important characteristic with essentially all solid tumor malignancies: early detection leads to improved survival outcomes. However, a major barrier to effective and life-saving treatment is the lack of early detection, with 70% of SCLC patients being diagnosed with metastatic disease (Alvarado-Luna and Morales-Espinosa, 2016, Transl Lung Cancer Res 5, 26–38). This is likely a result of insufficient annual imaging due to the invasive nature and mediastinal location of SCLC. Currently, there are no recommended blood tests for early detection of lung cancer. Because the majority of SCLC patients are diagnosed with extensive disease, where current treatment options offer limited benefit, many researchers are unaware of the fact that nearly 20% of patients with limited-stage SCLC can be cured with conventional cytotoxic chemotherapy. Furthermore, surgical resection, which is not generally considered for SCLC, can be curative when combined with chemotherapy in highly selected patients and those with very early-stage disease. Low-dose CT screening protocols, which have proven effective in non-small cell lung cancer, have not shown benefit in SCLC. Therefore, assays and methods for identifying SCLC at an early stage are needed.

[0006] SCLC, more than any other cancer type, is strongly associated with a group of autoimmune disorders called paraneoplastic neurological syndromes (PNS) (Hoftberger, et al., 2015, Curr Opin Oncol 27, 489–495). Many PNS are considered clinical manifestations of autoantibody (AAb) production against neuroendocrine antigens expressed by tumor cells and cross-reactive in the central nervous system (CNS) (Anwar, et al., 2019, Ann Transl Med 7). However, the mechanistic basis and extent of AAb production in SCLC remain largely unknown. Strikingly, PNS symptoms precede cancer diagnosis in 80% of affected patients, leading to increased early detection of SCLC 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). Furthermore, although PNS diagnoses are rare, AAb against PNS antigens can be found in a large proportion of SCLC patients who do not exhibit 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 exist and may play a broader role as biomarkers for SCLC.

[0007] SCLC-specific AAbs, which do not cause overt autoimmune diseases such as PNS, are likely initiated by tumor alterations that increase the immunogenicity of autoantigens. Unfortunately, sequence- or structure-based methods for predicting immunogenic epitopes in silico are limited (Jespersen, et al., 2019, Frontiers in Immunology 10), and current experimental methods for AAb discovery have several limitations. Most approaches for AAb detection utilize immobilized or linearized capture reagents, thus excluding AAbs targeting conformational protein epitopes. Recombinant proteins produced in non-human species, such as bacteria or yeast, have been used as conformational capture antigens, but they are unable to detect AAbs targeting human-specific post-translational modifications (PTMs) or disease-specific neoepitopes (Doyle and Mamula, 2001, Trends in Immunology 22, 443–449). Methods that aim to comprehensively map potential epitopes, such as serum epitope repertoire analysis (Kamath, et al., 2020, Sci Rep 10, 5294) or tumor proteome fractionation (Qiu, et al., 2008, J Clin Oncol 26, 5060-5066), can be successful in identifying disease-specific AAbs but require labor-intensive elucidation of cognate antigens on the backend. Therefore, alternative high-dimensional approaches to capture disease-associated AAbs are needed.

[0008] Early diagnosis of SCLC can have a significant impact on current standard of care. Current treatment options include platinum-etoposide chemotherapy, with patients with limited-stage disease receiving concurrent thoracic irradiation and those with extensive-stage disease receiving chemotherapy alone (Corso, et al., 2015, J Clin Oncol 33, 4240-4246). Prophylactic cranial irradiation is recommended for patients who respond to initial treatment, as it not only reduces the risk of brain metastases but also improves overall survival. Surgical resection of SCLC, which presents as a solitary pulmonary nodule in early-stage disease, has shown promising results (Kreisman, et al., 1992, Chest 101, 225-31).

[0009] Adoptive immunotherapy, involving the transfer of ex vivo-generated autoantigen-specific T cells, is a promising strategy for treating cancer. Novel specificities in T cells have been successfully generated through gene transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, et al., 2010, Blood 116(7), 1035-44). CARs are synthetic receptors consisting of a targeting moiety associated with one or more signaling domains in a single fusion molecule. Generally, the binding portion of a CAR consists of the antigen-binding domain of a single-chain antibody (scFV), which contains the light-chain variable fragment and heavy-chain variable fragment of a monoclonal antibody connected by a flexible linker. The signaling domain of first-generation CARs originates from the cytoplasmic region of CD3zeta or the Fc receptor gamma chain. Engineered cells expressing chimeric antigen receptors targeting SCLC are useful in therapeutic situations where specific targeting and T cell-mediated killing of SCLC cells is desired. Summary of the Invention

[0010] Disclosed herein are methods for diagnosing and treating small cell lung cancer in subjects. According to embodiments of the present disclosure, a large-scale antibody array is disclosed that captures circulating AAb-antigen (AAb-Ag) complexes from human plasma. Direct isolation of AAb-Ag complexes from patients uniquely enables ex vivo detection of AAbs bound to their native tumor-derived epitopes. In multiple independent cohorts of SCLC, a group of antigens, along with their corresponding AAbs, were upregulated prediagnostically, identifying and validating the disease in both limited-stage and extensive-stage disease. These AAbs identified novel tumor-associated antigens, some of which were expressed on the cell surface in most SCLC tumors examined. Detailed analysis revealed that some of these proteins contained immunogenic PTMs, including citrullination, isoaspartylation, and cancer-specific glycosylation, and binding to AAbs from SCLC patients was validated when mimicked by synthetic PTM-containing peptides.

[0011] Additionally, a unique high-throughput approach for identifying tumor-specific AAbs is disclosed herein that can be easily applied to AAb discovery in other diseases, such as autoimmune or infectious diseases. The validated AAb combinations provide significant utility in SCLC risk prediction models, which can be further improved by incorporating pack-years of smoking. When applied during lung cancer screening, this model could meaningfully impact overall survival from this deadly disease through early detection and current standard treatment of SCLC.

[0012] In one aspect, the disclosure provides a method of treating small cell lung cancer (SCLC) in a subject, the method comprising: (a) administering a chemotherapy regimen to the subject; (b) administering an immunotherapy regimen to the subject; (c) administering an antibody or antigen-binding fragment thereof to the subject; (d) performing surgical resection of the SCLC in the subject; and / or (e) subjecting the subject to radiation therapy targeted to the SCLC, wherein the subject has autoantibodies that specifically bind to 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, MAPREL, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9, and wherein the one or more epitopes of the antigens comprise a post-translational modification (PTM).

[0013] In some embodiments of the method, the subject has autoantibodies that specifically bind to two or more epitopes of one or more antigens. In some cases, the subject has autoantibodies that specifically bind to three or more epitopes of one or more antigens. In some cases, the subject has autoantibodies that specifically bind to four or more epitopes of one or more antigens. In some cases, the subject has autoantibodies that specifically bind to five or more epitopes of one or more antigens.

[0014] In some embodiments of the method, the one or more antigens are selected from the group consisting of SPINK1, TFRC, GRAP2, TIMP2, and PLD3. Optionally, the one or more antigens are selected from the group consisting of SPINK1, GRAP2, TIMP2, and PLD3. Optionally, the one or more antigens are selected from the group consisting of SPINT2, SPINK1, TIMP2, and TFRC.

[0015] In some embodiments of the method, the one or more epitopes are selected from the group consisting of SEQ ID NOs: 1-21. Optionally, the PTM comprises citrullination, isoaspartylation, and / or glycosylation. Optionally, the PTM comprises citrullination and / or isoaspartylation. Optionally, the one or more epitopes are selected from the group consisting of SEQ ID NOs: 22-46.

[0016] In one aspect, the disclosure provides a method of 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, MAPREL, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9. The present invention provides a method for treating SCLC in a subject, the method comprising: (a) selecting an antigen from a subject, wherein one or more epitopes of the antigen comprise a post-translational modification (PTM); (b) diagnosing the subject as having SCLC when the presence of one or more autoantibodies is detected in a biological sample obtained from the subject; and (c) treating SCLC in the subject by (i) administering a chemotherapy regimen to the subject, (ii) administering an immunotherapy regimen to the subject, (iii) administering an antibody or antigen-binding fragment thereof to the subject, (iv) performing surgical resection of the SCLC in the subject, and / or (v) subjecting the subject to radiation therapy targeted to the 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.

[0017] In some embodiments of the method, diagnosing the subject includes diagnosing the subject as having SCLC when the presence of autoantibodies binding to two or more epitopes of one or more antigens is detected in the biological sample. Optionally, diagnosing the subject includes diagnosing the subject as having SCLC when the presence of autoantibodies binding to three or more epitopes of one or more antigens is detected in the biological sample. Optionally, diagnosing the subject includes diagnosing the subject as having SCLC when the presence of autoantibodies binding to four or more epitopes of one or more antigens is detected in the biological sample. Optionally, diagnosing the subject includes diagnosing the subject as having SCLC when the presence of autoantibodies binding to five or more epitopes of one or more antigens is detected in the biological sample.

[0018] In some embodiments of the methods discussed immediately above, the one or more antigens are selected from the group consisting of SPINK1, TFRC, GRAP2, TIMP2, and PLD3. Optionally, the one or more antigens are selected from the group consisting of SPINK1, GRAP2, TIMP2, and PLD3. Optionally, the one or more antigens are selected from the group consisting of SPINT2, SPINK1, TIMP2, and TFRC.

[0019] In some embodiments of the methods discussed immediately above, the one or more epitopes are selected from the group consisting of SEQ ID NOs: 1-21. Optionally, the PTM comprises citrullination, isoaspartylation, and / or glycosylation. Optionally, the PTM comprises citrullination and / or isoaspartylation. Optionally, the one or more epitopes are selected from the group consisting of SEQ ID NOs: 22-46. Optionally, the subject is a human.

[0020] In some embodiments of the methods discussed herein, the chemotherapy regimen comprises one or more cycles of cisplatin or carboplatin and etoposide.

[0021] In some embodiments, the immunotherapy regimen comprises chimeric antigen receptor T cells that specifically bind to one or more epitopes. Optionally, the immunotherapy regimen comprises an antibody that binds to one or more epitopes. Optionally, the antibody is conjugated to a cytotoxic agent. In some embodiments, the immunotherapy regimen comprises a bispecific antibody that binds to one or more epitopes and a T cell antigen. Optionally, the T cell antigen is CD3.

[0022] In one aspect, a method of treatment comprises administering an antibody or antigen-binding fragment thereof to a subject. In some embodiments, the antibody or 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, MAPREL, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9, and wherein the one or more epitopes of the antigens comprise a post-translational modification (PTM).

[0023] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) contained within a heavy chain variable region (HCVR) and three light chain CDRs contained within a light chain variable region (LCVR), wherein the amino acid sequences of the HCVR / LCVR comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 47 / 51, 55 / 59, 63 / 67, 71 / 75, and 79 / 83, respectively.

[0024] In some embodiments, the antibody or antigen-binding fragment comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.

[0025] 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 selected from the group consisting of SEQ ID NOs: 47 / 51, 55 / 59, 63 / 67, 71 / 75, and 79 / 83, respectively.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of TFRC-4 that contains a PTM, and the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) that comprises the amino acid sequence of SEQ ID NO: 79, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) that comprises the amino acid sequence of SEQ ID NO: 83.

[0027] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino sequences of SEQ ID NOs: 80-81-82-84-85-86, respectively.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 79 and an LCVR comprising the amino acid sequence of SEQ ID NO: 83. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 39.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of CA9-3 that contains a PTM, and the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) that comprises the amino acid sequence of SEQ ID NO: 71, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) that comprises the amino acid sequence of SEQ ID NO: 75.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising 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 a HCVR comprising the amino acid sequence of SEQ ID NO: 71 and a LCVR comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 45.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of TFRC-5 that contains a PTM, and the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) that comprises the amino acid sequence of SEQ ID NO: 47, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) that comprises the amino acid sequence of SEQ ID NO: 51.

[0032] In some embodiments, the antibody or antigen-binding fragment comprises a THCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain comprising the amino acid sequence of SEQ ID NOs: 48-49-50-52-53-54, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 47 and a LCVR comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 40.

[0033] 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 the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) that comprises the amino acid sequence of SEQ ID NO: 55, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) that comprises the amino acid sequence of SEQ ID NO: 59.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 56-57-58-60-61-62, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 55 and a LCVR comprising 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.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of SPINK1-1 that contains a PTM, and the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) that comprises the amino acid sequence of SEQ ID NO: 63, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) that comprises the amino acid sequence of SEQ ID NO: 67.

[0036] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 64-65-66-68-69-70, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 63 and a LCVR comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 29.

[0037] In one aspect, the disclosure provides a biomarker detection panel for small cell lung cancer (SCLC), comprising one or more peptides comprising an epitope, 7 to 50 amino acids in length, from one or more antigens selected from the group consisting of CDH5, CD133, SPINK1, CDH23, NLRP7, TFRC, SPINT2, NADSYN1, HIF1A, GRAP2, MAPREL, 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.

[0038] In some embodiments, the biomarker detection panel is capable of detecting SCLC in a subject with at least 60% sensitivity and specificity. In some cases, the biomarker detection panel is capable of detecting SCLC in a subject with at least 70% or 75% sensitivity and specificity. In some cases, the biomarker detection panel is capable of detecting SCLC in a subject with at least 80% or 85% sensitivity and specificity. In some cases, the biomarker detection panel is capable of detecting SCLC in a subject with at least 90% or 95% sensitivity and specificity.

[0039] In some embodiments, a biomarker detection panel includes two or more epitopes from one or more antigens. In some cases, a biomarker detection panel includes three or more epitopes from one or more antigens. In some cases, a biomarker detection panel includes four or more epitopes from one or more antigens. In some cases, a biomarker detection panel includes five or more epitopes from one or more antigens.

[0040] In some embodiments of the biomarker detection panels 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.

[0041] In some embodiments of the biomarker detection panels discussed above, one or more epitopes are selected from the group consisting of SEQ ID NOs: 1-21. Optionally, the PTM comprises citrullination, isoaspartylation, and / or glycosylation. Optionally, the PTM comprises citrullination and / or isoaspartylation. Optionally, the one or more epitopes are selected from the group consisting of SEQ ID NOs: 22-46.

[0042] In some embodiments, the biomarker detection panel is for detecting autoantibodies that bind to one or more epitopes of one or more antigens in a biological sample obtained from a subject.

[0043] In one aspect, the disclosure relates to an isolated antibody or antigen-binding fragment thereof that binds to an epitope of TFRC-4 that contains a PTM, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 79, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 83.

[0044] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising 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 a HCVR comprising the amino acid sequence of SEQ ID NO: 79 and a LCVR comprising the amino acid sequence of SEQ ID NO: 83. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 39.

[0045] In one aspect, the 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 a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 71, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 75.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising 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 a HCVR comprising the amino acid sequence of SEQ ID NO: 71 and a LCVR comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 45.

[0047] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to an epitope of TFRC-5 that contains a PTM, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 47, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 51.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 48-49-50-52-53-54, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 47 and a LCVR comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 40.

[0049] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to an epitope of TFRC-1 or TFRC-2 comprising a PTM, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 55, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 59.

[0050] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 56-57-58-60-61-62, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 55 and a LCVR comprising 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.

[0051] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to an epitope of SPINK1-1 comprising a PTM, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 63, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 67.

[0052] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 64-65-66-68-69-70, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 63 and a LCVR comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 29.

[0053] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the present disclosure and a pharmaceutically acceptable carrier or diluent.

[0054] In one aspect, the disclosure relates to a unit dosage form comprising a container containing any one of the antibodies or antigen-binding fragments thereof of the present disclosure. In some embodiments, the container is a vial, a syringe, a pre-filled syringe, or an autoinjector.

[0055] In one aspect, the present disclosure relates to a kit comprising a container containing an antibody or antigen-binding fragment thereof, or any one of the antibodies of the present disclosure, and instructions for using the antibody or antigen-binding fragment thereof for the treatment of small cell lung cancer.

[0056] 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.

[0057] In one aspect, the present disclosure relates to a pair of nucleic acid molecules encoding the heavy and light chain variable regions of any of the antibodies or antigen-binding fragments of the present disclosure, the pair of nucleic acid molecules comprising 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. In some embodiments, the pair of nucleic acid molecules is comprised within a recombinant expression vector.

[0058] In one aspect, the present disclosure relates to an isolated host cell comprising any one of the antibodies or antigen-binding fragments of the present disclosure, any one of the nucleic acid molecules of the present disclosure, any one of the recombinant expression vectors of the present disclosure, any pair of the nucleic acid molecules of the present disclosure, or any pair of the recombinant expression vectors of the present disclosure. In some embodiments, the isolated host cell is a mammalian host cell.

[0059] In various embodiments, any of the features or components of the embodiments described above or discussed herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value described above or discussed herein may be combined with another related value described above or discussed herein to recite a range with values ​​representing the upper and lower limits of the range, and such ranges and all values ​​contained within such ranges are encompassed within the scope of the present disclosure. Each of the values ​​described above or discussed herein may be expressed with a variance of 1%, 5%, 10%, or 20%. Other embodiments will be apparent from consideration of the detailed description that follows.

[0060] Other embodiments will be apparent from consideration of the detailed description that follows. [Brief explanation of the drawings]

[0061] [Figure 1-1]Figures 1A-1D show the upregulation of antigen-conjugated autoantibodies in small cell lung cancer (SCLC). The mean index signal of upregulated (p<0.05) IgG and IgM autoantibodies in SCLC was higher than that of any other cancer type analyzed on the antibody-antigen conjugate array (one-way ANOVA) (Figure 1A). N = 98 (SCLC) and 125 (control), N = 47 (NSCLC) and 47 (control), N = 86 (pancreatic cancer) and 86 (control), N = 139 (colon cancer) and 139 (control), and N = 121 (breast cancer) and 121 (control). Figures 1B-1D show that IgG and IgM autoantibodies were consecutively discovered in the Cardiovascular Health Study (CHS) prediagnostic cohort (1B), tested in the Fred Hutch diagnostic cohort (1C), and validated in a third cohort from Vanderbilt (1D). Expression in cases: ● IgG increase, ● IgM increase, - decrease, × not significant. [Figure 1-2] Same as above. [Figure 2A] Figure 2A shows the upregulation of antigens targeted by peripheral autoantibodies in SCLC. Representative immunohistochemical images of antigens targeted by peripheral autoantibodies on SCLC tissue microarrays (N = 45-62 cases) are shown. Scale bar, 50 μM. Figure 2B shows the positive or negative scoring of tissue microarray cores. Positive scores are further classified by stage at diagnosis. [Figure 2B] Figure 2A shows the upregulation of antigens targeted by peripheral autoantibodies in SCLC. Representative immunohistochemical images of antigens targeted by peripheral autoantibodies on SCLC tissue microarrays (N = 45-62 cases) are shown. Scale bar, 50 μM. Figure 2B shows the positive or negative scoring of tissue microarray cores. Positive scores are further classified by stage at diagnosis. [Figure 3] Figure 1 illustrates the expression of tumor-associated antigens in some normal tissues. Representative normal tissue cores showing both positive and negative expression for each immunohistochemical stain, as well as expression in normal lung cores, are shown. The scale bar is 500 μM. [Figure 4A]Figure 4 illustrates that CD133 has cancer-specific glycosylation with sialyl-Lewis A (sLeA) in H82 and H69 SCLC cell lines. Figure 4A shows immunoblots from H82 lysates immunoprecipitated (IP) with CA9, SPINT2, PLD3, or CD133 antibodies and probed with sLeA antibodies. Depleted lysates (Dep), whole cell lysates (WCL), and target-specific IPs are shown. Blue boxes indicate the predicted target protein sizes. [Figure 4B] Figure 4 illustrates that CD133 has cancer-specific glycosylation with sialyl-Lewis A (sLeA) in H82 and H69 SCLC cell lines. Figure 4B shows immunoblots from H69 lysates immunoprecipitated (IP) with CD133 antibody and probed with anti-CD133 or anti-sLeA antibodies. Depleted lysate (Dep), whole cell lysate (WCL), and target-specific IP are shown. Boxes indicate the predicted target protein sizes. [Figure 5] Figures 5A-5C illustrate SCLC autoantibodies (AAbs) against CD133-targeted glycosylation motifs. Figure 5A shows immunoblots from H82 cell lysates (Lys) immunoprecipitated (IP) with CD133 antibody or an isotype control and probed with anti-CD133 and anti-sLeA antibodies. Dep indicates degraded cell lysates after IP. Figure 5B shows immunoblots from H82 lysates treated with PRIME deglycosylase, immunoprecipitated (IP) with CD133 antibody, and probed with anti-CD133 and anti-sLeA antibodies. Whole cell lysates (WCL), control IP, and target-specific IP are shown. Figure 5C shows ELISA quantification of autoantibody levels from SCLC patient-derived plasma bound to CD133 from H82 cell lysates pretreated or not with PRIME deglycosylase. (N=5 / group) Error bars indicate SEM by Student's t-test (*p<0.05, **p<0.005, ***p<0.0005). [Figure 6-1]Figures 6A-6C illustrate that SCLC AAbs can target the isoaspartylation post-translational modification. Figure 6A shows immunoblots from GST fusion proteins bound to glutathione beads, treated with or without isoaspartylation induction buffer (IsoAsp), incubated with plasma from an SCLC patient, and probed with anti-human IgG and anti-GST antibodies. Quantitation of multiple experiments is shown in Figure 6B. Figure 6C shows IgG from plasma from an SCLC patient bound to IsoAsp compared to the wild-type (WT) peptide as measured by ELISA (N = 8 cases). [Figure 6-2] Same as above. [Figure 7] Figures 7A-7B illustrate that TFRC is citrullinated. Figure 7A shows immunoblots from H82 and H69 whole cell lysates (WCLs) probed with antibodies against citrulline, CD133, TFRC, PLD3, SPINT2, and CA9. Boxes indicate the expected target protein sizes. Figure 7B shows immunoblots from H82 lysates immunoprecipitated (IP) with citrulline antibodies and probed with CD133, PLD3, CA9, or TFRC antibodies. The same blot was sequentially probed with CD133 and then PLD3. Boxes indicate the expected target protein sizes. [Figure 8-1]Figures 8A-8D illustrate SCLC AAbs targeting citrullinated TFRC. Figure 8A shows immunoblots from H82 or H69 whole cell lysates (WCLs) probed with TFRC or citrulline antibodies. GAPDH was used as a loading control. Figure 8B shows immunoblots from 3T3 or H82 WCLs and H82 lysates immunoprecipitated (IP) with TFRC, citrulline (CIT), or an isotype control antibody (CON) and probed with anti-TFRC or anti-citrulline antibodies. Figure 8C shows IgG from plasma derived from SCLC patients bound to citrullinated (Cit) peptide compared to the wild-type (WT) peptide, as measured by ELISA (N=8). Figure 8D shows IgG from 10 SCLC patients and 10 controls captured in complexes with antigen (AAb-TAA) by anti-TFRC antibody, in complexes without antigen (AAb) by TFRC recombinant protein, or by cit(PTM) / WT peptide, quantified by ELISA. Anti-TFRC antibody was used as a positive control for TFRC recombinant protein. Error bars indicate SEM. P values ​​were estimated by paired-sample t-test. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 9A] Illustrating the detection of SCLC by panel autoantibodies. In Figure 9A, panel autoantibodies are analyzed as a function of time of blood draw in CHS (prediagnostic, 1-2 hours before diagnosis (dx), or <1 year until dx) and FH or VB (diagnostic, limited stage, or extensive stage). *p=<0.05; **p=<0.005, ***p=<0.0005 unpaired t-test. [Figure 9B] 9 illustrates the detection of SCLC by panel autoantibodies. In Figure 9B, panel autoantibodies are significantly higher in plasma from SCLC cases (n=98) compared to cases from other cancers (N=75 (NSCLC), N=86 (pancreas), N=87 (colon)) or controls (n=175) from the entire cohort. **p=<0.001 unpaired t-test. [Figure 10]Figures 10A-10F illustrate the weak association between panel autoantibodies and pack-years of smoking. Figure 10A shows that panel autoantibodies are not differentially expressed between current and former smokers (current N = 100, former N = 140). Figures 10B-10F show that panel autoantibodies, including PLD3 (Figure 10B), TIMP2 (Figure 10C), GRAP2 (Figure 10D), SPINK1 (Figure 10E), and TFRC (Figure 10F), are loosely correlated with pack-years of smoking. Pearson correlation, N = 240. [Figure 11A] Panel autoantibodies are not associated with chronic obstructive pulmonary disease (COPD) (N=19 controls and 31 cases). [Figure 11B] Panel autoantibodies are not associated with autoimmunity (type 1 diabetes (N = 3), rheumatoid arthritis (N = 2), hypothyroidism (N = 2), or myasthenia gravis (N = 2). One case had a diagnosis of both type 1 diabetes and myasthenia gravis (total N = 44 controls and 8 cases). [Figure 12] Figures 12A-12C illustrate risk prediction models that can accurately detect SCLC. The receiver operating characteristic (ROC) curve for the 5-autoantibody (AAb, dashed line) complex panel was identified by maximizing the AUC in the Vanderbilt (VB) dataset (Figure 12A), then fixing the coefficients and testing in the CHS cohort (Figure 12B) and the Fred Hutch (FH) cohort (Figure 12C). The ROC curve for pack-year is shown as a dotted line, and the combination of pack-year and 5-AAb panel is shown as a solid black line. [Figure 13] FIG. 13 shows the process for isolating, sequencing, and cloning SCLC-associated autoantibodies that bind to the identified PTMs. [Figure 14] FIG. 14 shows near-infrared imaging demonstrating that 770-labeled 2D2 antibody is directed to H82 SCLC tumors in vivo. [Figure 15] FIG. 15 shows a chemiluminescent ELISA demonstrating that the 2D2 antibody strongly binds to the specific TFRC-cit peptide. [Figure 16]Figure 16 illustrates that a commercially available TFRC antibody produced in rabbits recognizes TFRC in multiple normal human tissues and the H82 SCLC cell line. However, the 2D2 antibody isolated from PBMCs from an SCLC patient recognizes TFRC only in SCLC, but not in normal tissues. DETAILED DESCRIPTION OF THE INVENTION

[0062] Before describing the present invention, it should be understood that the present invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Any embodiments or features of the embodiments may be combined with each other, and such combinations are expressly encompassed within the scope of the present invention. Any specific value described above or discussed herein may be combined with another related value described above or discussed herein to recite a range having values ​​representing the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Definitions of common terms in molecular biology can be found in publications such as Benjamin Lewin, Genes V, 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).

[0064] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.). 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 herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0065] Selected Abbreviations SCLC: small cell lung cancer NSCLC: non-small cell lung cancer WT: wild type PTM: Post-translational modification ELISA: enzyme-linked immunosorbent assay IgG: immunoglobulin G IgM: immunoglobulin M AAb: autoantibody AAb-Ag: autoantibody-antigen complex TMA: tissue microarray WCL: whole cell lysate PNS: Paraneoplastic neurological syndrome CT: Computed tomography CIT: Citrulline PCR: polymerase chain reaction DNA: deoxyribonucleic acid cfDNA: cell-free DNA IP: immunoprecipitation SNP: Single Nucleotide Polymorphism sLeA: sialyl Lewis A

[0066] definition Administration: Providing or giving a drug 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, intravaginal, and inhalation routes, or any combination of these techniques.

[0067] The disclosed compositions or other therapeutic agents of the present disclosure can be formulated into therapeutically active pharmaceutical compositions that can be administered parenterally or orally to a subject. Parenteral routes of administration include, but are not limited to, intraepidermal, intraarterial, intramuscular (IM and depo-IM), intraperitoneal (IP), intravenous (IV), intrasternal injection or infusion techniques, intranasal (inhalation), intrathecal, intragastric injection, subcutaneous injection (subcutaneous (SQ and depo-SQ), transdermal, topical, and ophthalmic. The disclosed compositions or other therapeutic agents can be mixed or combined with suitable pharmaceutically acceptable excipients to prepare pharmaceutical compositions. Pharmaceutically acceptable excipients include, for example, alumina, aluminum stearate, buffers (such as phosphates), glycine, ion exchangers (such as to help control the release of charged substances), lecithin, and portions of saturated vegetable oil fatty acids. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, glyceride mixtures, potassium sorbate, serum proteins (such as human serum albumin), sorbic acid, water, salts or electrolytes such as cellulosic materials, colloidal silica, disodium hydrogen phosphate, magnesium trisilicate, polyacrylates, polyalkylene glycols such as polyethylene glycol, polyethylene-polyoxypropylene block polymers, polyvinylpyrrolidone, potassium hydrogen phosphate, protamine sulfate, Group 1 halide salts such as sodium chloride, sodium carboxymethylcellulose, waxes, wool fat, and zinc salts. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers.

[0068] Upon mixing or addition of the disclosed compositions or other therapeutic agents, the resulting mixture may be a solid, solution, suspension, emulsion, or the like. These can be prepared according to methods known to those skilled in the art. The form of the resulting mixture 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 the particular mode of administration. In addition, the disclosed compositions or other therapeutic agents can be mixed with other inactive or active materials that do not impair the desired action, or with materials that complement the desired action, or with materials that have a different action. Solubilization methods can be used when the agent exhibits insufficient solubility in the carrier. Such methods are known and include, but are not limited to, dissolution in aqueous sodium bicarbonate, the use of cosolvents such as dimethyl sulfoxide (DMSO), and the use of surfactants such as TWEEN® (ICI Americas, Inc., Wilmington, Del.).

[0069] The disclosed compositions or other therapeutic agents can be prepared with carriers that protect them from rapid elimination from the body, such as coatings or sustained-release formulations. Such carriers include, but are not limited to, controlled-release formulations, such as microencapsulated delivery systems. The disclosed compositions or other therapeutic agents are included in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically beneficial effect, typically in an amount that avoids undesirable side effects in the treated subject. Therapeutically effective concentrations can be empirically determined by testing the compounds in known in vitro and in vivo model systems for the treated condition. For example, acceptable SCLC animal models can be used to determine effective amounts or concentrations that can be translated to other subjects, such as humans, as is known in the art.

[0070] Injectable solutions or suspensions can be formulated using suitable non-toxic parenterally acceptable diluents or solvents, such as 1,3-butanediol, isotonic saline, mannitol, Ringer's solution, saline, or water; or suitable dispersing or wetting agents and suspending agents, such as sterile, non-irritating fixed oils including synthetic mono- or diglycerides, and fatty acids including oleic acid; natural vegetable oils such as coconut oil, cottonseed oil, peanut oil, and sesame oil; glycerin; polyethylene glycol; propylene glycol; or other synthetic solvents; antibacterial agents such as benzyl alcohol and methylparabens; antioxidants such as ascorbic acid and sodium sulfite; buffers such as acetate, citrate, and phosphate; chelating agents such as ethylenediaminetetraacetic acid (EDTA); agents for adjusting tonicity such as sodium chloride and dextrose; and combinations thereof. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as needed. For intravenous administration, suitable carriers include saline, phosphate buffered saline (PBS), and solutions containing viscosity enhancers and solubilizers, such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.

[0071] When the disclosed compositions or other therapeutic agents are orally administered as a suspension, the pharmaceutical composition can be prepared according to techniques well known in the art of pharmaceutical formulation and can contain suspending agents such as alginic acid or sodium alginate, bulking agents such as microcrystalline cellulose, viscosity enhancing agents such as methylcellulose, and sweeteners / flavoring agents. Oral liquid preparations can contain conventional additives such as suspending agents, e.g., gelatin, glucose syrup, hydrogenated edible fats, methylcellulose, sorbitol, and syrup; emulsifiers, e.g., acacia, lecithin, or sorbitan monooleate; non-aqueous carriers (including edible oils), e.g., almond oil, fractionated coconut oil, oily esters such as glycerin, propylene glycol, or ethyl alcohol; preservatives, such as methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavorings or coloring agents. When formulated as immediate-release tablets, these compositions may contain dicalcium phosphate, lactose, magnesium stearate, microcrystalline cellulose, and starch and / or other binders, diluents, disintegrants, excipients, fillers, and lubricants.

[0072] If oral administration is desired, the disclosed compositions or other therapeutic agents can be provided in a composition that protects them from the acidic environment of the stomach. For example, the disclosed compositions or other therapeutic agents can be formulated with an enteric coating that maintains their 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 ingredient. Oral compositions generally include an inert diluent or an edible carrier and can be compressed into tablets or enclosed in a gelatin capsule. For oral therapeutic administration, the disclosed compositions or other therapeutic agents can be incorporated 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.

[0073] Capsules, pills, tablets, troches, etc. may contain any of the following ingredients or compounds of similar nature: binders such as, but not limited to, acacia, corn starch, gelatin, tragacanth gum, polyvinylpyrrolidone, or sorbitol; fillers such as calcium phosphate, glycine, lactose, microcrystalline cellulose, or starch; disintegrating agents 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 silicon dioxide; sweeteners such as sucrose or saccharin; disintegrating agents such as potato starch; dispersing or wetting agents such as sodium lauryl sulfate; and flavoring agents such as peppermint, methyl salicylate, or fruit flavors. When the dosage unit form is a capsule, in addition to the above-mentioned materials, a liquid carrier such as fatty oil may be contained. In addition, dosage unit forms may contain various other materials that modify the physical form of the dosage unit, for example, sugar coatings and other enteric agents. The disclosed compositions or other therapeutic agents may also be administered as a component of an elixir, suspension, syrup, wafer, tea, chewing gum, or the like. A syrup may contain, in addition to the active compound, sucrose or glycerin as a sweetening agent, certain preservatives, dyes and colorings, and flavorings.

[0074] When orally administered, compound can be administered in the usual dosage form for oral administration.These dosage forms include the usual solid unit dosage forms of tablets and capsules, and liquid dosage forms such as solution, suspension and elixir.When solid dosage forms are used, they can be of sustained release type, so that compound needs to be administered less frequently.

[0075] Agent: Any protein, nucleic acid molecule (including chemically modified nucleic acids), chemical compound, antibody, small molecule, organic compound, inorganic compound, cell such as a T cell, or other molecule of interest. Agents can include therapeutic agents, diagnostic agents, or pharmaceutical agents. A therapeutic agent or pharmaceutical agent is one that, alone or in combination with additional compounds, elicits a desired response (such as eliciting a therapeutic or prophylactic effect when administered to a subject, including treating a subject with or at risk of developing SCLC).

[0076] In some examples, an agent can act directly or indirectly to alter the activity and / or expression of an SCLC-associated molecule, such as a molecule for the early detection of SCLC. In particular examples, a therapeutic agent (such as an antisense compound or an antibody) significantly alters the expression and / or activity of an SCLC-associated molecule. Examples of therapeutic agents are those that can decrease the activity of a gene or gene product associated with SCLC, as measured, for example, by a clinical response (e.g., increased survival or a decrease in one or more signs or symptoms associated with SCLC). Therapeutic agents also include organic or other compounds that mimic the effects of therapeutically effective peptides, antibodies, or nucleic acid molecules.

[0077] A "pharmaceutical agent" is a compound or composition that can elicit a desired therapeutic or prophylactic effect when administered to a subject, alone or in combination with another therapeutic agent(s) or a pharmaceutically acceptable carrier. In certain examples, the pharmaceutical agent significantly reduces the expression and / or activity of an SCLC-associated molecule, thereby increasing the subject's survival time, reducing signs or symptoms associated with the disease, or prolonging the onset of signs or symptoms of SCLC.

[0078] Antibody: A polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen, such as an SCLC-associated molecule or fragment thereof. Antibodies are composed of heavy and light chains, each of which contains a variable heavy chain (V H ) region and the variable light chain (V L ) region. H Area and V LBoth regions are involved in binding the antigen recognized by the antibody. Antibodies of the present disclosure include those specific for the disclosed SCLC-associated molecules.

[0079] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule (i.e., a "full antibody molecule") consisting of four polypeptide chains, two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain consists of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (consisting of a CH1 domain, a CH2 domain, and a CH3 domain). 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 be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences, or may be naturally occurring or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0080] The term antibody includes intact immunoglobulins and variants and portions thereof, such as Fab' fragments, F(ab)' fragments, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). scFv proteins are fusion proteins in which an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region are joined by a linker; in dsFv, the chains have been mutated to introduce a disulfide bond 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), and the like. 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.

[0081] Typically, native immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (the regions are also known as "domains"). In combination, the heavy and light chain variable regions specifically bind to antigens. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework regions and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, USDapartment 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 region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0082] CDRs are primarily responsible for binding to the epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, are numbered sequentially starting from the N-terminus, and are typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it resides, while a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it resides. Antibodies that bind to RET have specific VH and VL region sequences, and therefore specific CDR sequences. Antibodies with different specificities (such as different combining sites for different antigens) have different CDRs. Although CDRs differ from antibody to antibody, 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). "V HReferences to "V" or "VH" refer to the variable region of an immunoglobulin heavy chain, including that of an FV, scFV, dsFv or Fab. L References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

[0083] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes 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, for example, by creating hybrid antibody-producing cells from the fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0084] A "chimeric antibody" has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds to an SCLC-associated molecule.

[0085] The present disclosure also includes antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes antibodies having HCVR and / or LCVR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative to any of the HCVR or LCVR amino acid sequences disclosed herein. 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.

[0086] The term "recombinant," as used herein, refers to an antibody or antigen-binding fragment thereof of the present disclosure that is made, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term also refers to antibodies that are expressed in a non-human mammal (including, for example, a transgenic non-human mammal such as a transgenic mouse) or cell (e.g., a CHO cell) expression system, or isolated from a recombinant combinatorial human antibody library.

[0087] The terms "specifically bind" or "specifically binds to" and the like mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 x 10 -8 can be characterized by an equilibrium dissociation constant equal to or less than M (e.g., a smaller K D indicates 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, and the like.

[0088] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody, or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to bind to the recited epitope.

[0089] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities.

[0090] The polynucleotides discussed herein may encode all or part of the antibodies or antigen-binding fragments discussed throughout this disclosure. In some cases, a single polynucleotide may encode both the HCVR and LCVR of an antibody or antigen-binding fragment (e.g., defined in terms of the CDRs contained within the HCVR and LCVR, each of whose amino acid sequences is defined, defined in terms of the amino acid sequences of the CDRs of the HCVR and LCVR, respectively, or defined via the amino acid sequences of the HCVR and LCVR, respectively), or the HCVR and LCVR may 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 may be combined in a single vector or may be contained in separate vectors (i.e., a pair of vectors). In either case, the host cell used to express the polynucleotide or vector may contain the full complement of component parts to produce the antibody or antigen-binding fragment thereof. For example, a host cell may contain separate vectors, each encoding the HCVR and LCVR of an antibody or antigen-binding fragment thereof described above or discussed herein. Similarly, one or more polynucleotides and one or more vectors can be used to express the full-length heavy and full-length light chains of an antibody described above or discussed herein. For example, a host cell can contain a single vector having polynucleotides encoding both the heavy and light chains of an antibody, or the host cell can contain separate vectors having polynucleotides encoding the heavy and light chains, respectively, of an antibody described above or discussed herein.

[0091] The term "vector," as used herein, refers to any molecule or particle used to deliver 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.

[0092] Autoantibodies: "Autoantibodies" are antibodies produced by the immune system that are directed against one or more of an individual's own proteins.

[0093] Epitope: The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen can have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes can include sugar, phosphoryl, or sulfonyl moieties on the antigen.

[0094] Altered or Modulated Expression: Altered expression of a gene, gene product, or regulator thereof, such as one or more SCLC-associated molecules disclosed herein, refers to a change or difference, e.g., an increase or decrease, in the level of the gene, gene product, or regulator thereof detectable in a biological sample (such as a sample from a subject at risk for or with SCLC) relative to a control (such as a sample from a subject without SCLC) or a reference value known to represent the level of the gene, gene product, or regulator thereof in the absence of disease. An "altered" expression includes an increase in expression (upregulation) or a decrease in expression (downregulation).

[0095] Array: An arrangement of molecules, such as biopolymers (such as peptides or nucleic acid molecules) or biological samples (such as tissue sections), at addressable locations on or within a substrate. A "microarray" is an array that is so miniaturized that it requires or is aided by microscopy for evaluation or analysis. Arrays are sometimes called antibody chips or biochips. Arrays of molecules allow for a large number of analyses to be performed on a single sample at a time. In certain exemplary arrays, one or more molecules (such as oligonucleotide probes or antibodies) occur multiple times (e.g., twice) on the array, for example, to provide an internal standard. The number of addressable locations on the array can vary, for example, from at least two, to at least four, to at least nine, 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 certain examples, the array comprises 2 to 100 addressable locations, e.g., 2 to 40 addressable locations. In certain examples, the array consists essentially of SCLC-specific probes or primers or antibodies (e.g., those that enable amplification or detection) disclosed herein.

[0096] Binding or stable binding: An association between two substances or molecules, such as hybridization of one nucleic acid molecule to another (or to itself), association between an antibody and a peptide, or association of one protein with another protein or nucleic acid molecule. An oligonucleotide molecule binds to or is stably bound to a target nucleic acid molecule when a sufficient amount of the oligonucleotide molecule base pairs or hybridizes to its target nucleic acid molecule, allowing for detection of the binding. "Preferentially binds" indicates that one molecule binds with high affinity to the other and with lower affinity to a heterologous molecule. Binding can be detected by any method known to those skilled in the art, for example, by physical or functional properties of the target complex. For example, binding can be functionally detected by determining whether binding has an observable effect on biosynthetic processes such as gene expression, DNA replication, transcription, and translation. Methods for detecting antibody binding to proteins are disclosed herein and may include known methods of protein detection, such as Western blotting.

[0097] Biological activity: The beneficial or adverse effect of a drug on an organism. When a drug is a complex chemical mixture, this activity is exerted by the active ingredient or pharmacophore of the substance, but can be modified by other components. Activity is generally dose-dependent, and it is not uncommon for low to high doses of a single substance to have effects ranging from beneficial to harmful. In one example, the drug significantly reduces the biological activity of one or more SCLC-associated molecules disclosed herein, which reduces one or more signs or symptoms associated with SCLC.

[0098] Biomarker: A molecular, biological, or physical attribute that can be objectively measured to characterize a physiological state, detect or define disease progression, or predict or quantify therapeutic response. For example, a substance used as an indicator of a biological state. It is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological response to a therapeutic intervention. In one example, a biomarker is a protein or nucleic acid sequence of a corresponding gene that is indicative of SCLC.

[0099] Clinical outcome: Refers to a patient's health status after or in the absence of treatment for a disease or disorder, such as SCLC. Clinical outcomes include, but are not limited to, increased time to death, decreased time to death, increased chance of survival, increased risk of death, survival, disease-free survival, chronic disease, metastasis, progressive or aggressive disease, disease recurrence, death, and good or bad response to treatment.

[0100] Contacting: Placement in direct physical association, including both solid and liquid forms. Contacting an agent with a cell can occur in vitro by adding the agent to an isolated cell, or in vivo by administering the agent to a subject.

[0101] Control: A sample or standard used for comparison with a test sample, such as a biological sample obtained from a patient (or patients) who does not have a particular disease or condition, such as SCLC. In some embodiments, a control is a sample obtained from a healthy patient (or patients) (also referred to herein as a "normal" control), e.g., a normal biological sample, or a sample obtained from a non-cancerous biological sample from a patient with a particular disease or condition, such as SCLC. In some embodiments, a control is a historical control or reference value (e.g., a control sample tested in the past, or a group of samples representing baseline or normal values ​​(e.g., expression values), e.g., baseline or normal values ​​of a particular gene, gene product in subjects who do not have SCLC). In some examples, a control is a reference value that represents an average value (or average range of values) obtained from samples from multiple patients (e.g., the average value or range of values ​​of a gene or gene product in subjects who do not have SCLC).

[0102] Detection: To determine the presence, absence, or relative or absolute amount of a detected object.

[0103] Diagnosis: The process of identifying a disease, such as SCLC, through its signs, symptoms, and the results of various tests. The conclusion reached through this process is also called a "diagnosis." Commonly performed forms of testing include blood tests, medical imaging, urine tests, and biopsies.

[0104] Expression: The process by which a gene's coded information is converted into the operative, non-operative, or structural parts of a cell, such as the synthesis of a protein. Gene expression can be influenced by external signals. For example, exposure of a cell to a hormone can stimulate the expression of hormone-induced genes. Different types of cells can respond differently to the same signal. Gene expression can also be regulated anywhere along the pathway from DNA to RNA to protein. Regulation can include control over transcription, translation, RNA transport and processing, degradation of intermediate molecules such as mRNA, or by its activation, inactivation, compartmentalization, or degradation after a specific protein molecule is produced. 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) suppression of expression. Changes in the expression of a nucleic acid molecule can be associated with and actually cause changes in the expression of the corresponding protein.

[0105] Protein expression can also be altered in some way to differ from that of the protein under normal (wild-type) conditions, including (1) a mutation in the protein that results in one or more different amino acid residues, (2) a short deletion or addition of one or a few (e.g., 10-20 or fewer) amino acid residues to the protein's sequence, (3) a longer deletion or addition of amino acid residues (e.g., at least 20 residues) that results in the removal or addition of an entire protein domain or subdomain, (4) an increased amount of protein expressed compared to a control or standard amount, (5) a decreased amount of protein expressed compared to a control or standard amount, (6) a change in the subcellular localization or targeting of the protein, or (7) a change in the temporal regulation of the protein. These include, but are not necessarily limited to, (8) changes in localized expression (such as changes in the protein's expression when it is not normally expressed, or changes in its stability due to an increase in the lifespan of the protein, i.e., the time it remains localized within the cell), (9) changes in the protein's localized (such as organ- or tissue-specific or subcellular localization) expression (such that the protein is not expressed where it is normally expressed, or is expressed where it is not normally expressed), and (10) changes in the protein's structure, stability, or function (compared to a control or standard, respectively) via post-translational modifications (PTMs). Controls or standards for comparison with samples to determine differential expression include samples considered normal (in that they are not altered for the desired characteristic, e.g., samples from subjects without SCLC) and clinical test values ​​(e.g., ranges of values) (although such values ​​may be arbitrarily set, keeping in mind that such values ​​may vary between laboratories).

[0106] Cytotoxic Agent: As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. This term is intended to include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu), chemotherapeutic agents such as methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastone, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents, enzymes and fragments thereof, such as nucleases, antibiotics, and toxins (including fragments and / or variants thereof), such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, as well as various antitumor or anticancer agents described herein. Tumoricidal agents cause the destruction of tumor cells.

[0107] Toxin: A "toxin" is any substance that can have a deleterious effect on cell growth or proliferation.

[0108] Measurement: To detect, quantify, or determine the amount (including molar amount), concentration, or mass of a physical entity or chemical composition, either absolutely in the case of quantification, or relative to an equivalent physical entity or chemical composition.

[0109] Sample (or biological sample): A biological specimen containing genomic DNA, RNA (including mRNA), protein, or a combination thereof obtained from a subject. Examples include, but are not limited to, peripheral blood, plasma, serum, urine, saliva, tissue biopsy, surgical specimen, and autopsy material.

[0110] Screening: As used herein, "screening" refers to a process used to evaluate and identify candidate agents that can be used to identify SCLC, such as early-stage 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-associated molecules. MicroRNA expression can be quantified using any one of a number of techniques known in the art and described herein, such as microarray analysis or qRT-PCR.

[0111] Sensitivity: The proportion of diseased individuals (e.g., individuals with SCLC) in which a target biomarker is detected (true positives / total disease × 100). Non-diseased individuals diagnosed with the disease by the test are "false positives." In some examples, the sensitivity of an assay describes the ability of the assay to accurately predict whether a patient has SCLC using the disclosed SCLC-associated molecules, compared to another assay method. For example, a marker with a sensitivity of at least 60%, including a sensitivity of 70%, 75%, 80%, 85%, 90%, 95% or more, is a marker that can accurately predict SCLC.

[0112] Specificity: The proportion of disease-free individuals in whom the target biomarker is not detected (true negatives / total disease-free × 100). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and test negative in the assay are referred to as "true negatives."

[0113] Signs and symptoms: Any subjective evidence of a disease or a subject's condition, e.g., evidence as perceived by the subject, a noticeable change in the subject's condition that is indicative of some physical or mental state, etc. A sign is any abnormal manifestation of a disease that is detectable on examination or evaluation of a subject. A sign is generally an objective manifestation of a disease.

[0114] Small cell lung cancer or carcinoma: A type of highly malignant cancer within the lung. Compared to non-small cell lung cancer, small cell lung cancer has a shorter doubling time, a higher proliferation fraction, and an earlier onset of metastasis. Small cell lung cancer usually presents in the central airways, invading the submucosa and causing bronchial airway narrowing. Common symptoms include cough, dyspnea, weight loss, and weakness. Smoking is a significant risk factor. More than 70% of patients with small cell lung cancer develop metastatic disease, with common sites including the liver, adrenal glands, bone, and brain. Due to its highly neuroendocrine nature, small cell lung cancer can produce ectopic hormones, including adrenocorticotropic hormone (ACTH) and antidiuretic hormone (ADH). Ectopic production of large amounts of ADH leads to the syndrome of inappropriate antidiuretic hormone hypersecretion (SIADH). Lambert-Eaton myasthenic syndrome (LEMS) is a well-known paraneoplastic condition associated with small cell lung cancer. SCLC is also called "oat cell carcinoma" due to its squamous cell shape and small amount of cytoplasm.

[0115] SCLC is thought to originate from bronchial neuroendocrine cells (APUD cells) called Feyrter cells. Therefore, they express various neuroendocrine markers and can lead to ectopic production of hormones such as ADH and ACTH, which can result in paraneoplastic syndrome and Cushing's syndrome. Approximately half of all individuals diagnosed with Lambert-Eaton myasthenic syndrome (LEMS) are ultimately found to have small cell carcinoma of the lung. Mixed small cell lung cancer can occur in combination with a wide variety of other histological variants of lung cancer, including highly complex malignant histological mixtures. When found in one or more differentiated types of lung cancer, such as squamous cell carcinoma or adenocarcinoma, the malignant tumor is diagnosed and classified as mixed small cell lung cancer (c-SCLC). C-SCLC is a recognized subtype of SCLC.

[0116] Subject: A living multicellular organism with a spinal column, a category that includes living multicellular organisms with a spinal column, a category that includes both human and non-human mammals. In one embodiment, the subject is a patient with cancer (e.g., small cell lung cancer).

[0117] Tissue: A plurality of functionally related cells. A tissue can be a suspension, semi-solid, or solid. Tissue includes cells taken from a subject, for example, cells taken from the lungs.

[0118] Treatment of disease: 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 delay its progression, and may, for example, in some cases, include inhibiting the complete progression of the disease, e.g., preventing the development of SCLC. Prevention of disease does not require complete disease freedom. For example, a reduction of at least 10%, e.g., at least 20%, at least 25%, at least 30%, at least 40%, or at least 50%, of the signs or symptoms associated with a condition or disease, such as SCLC, may be sufficient. As used herein, the term "amelioration" with respect to a disease or condition refers to any observable beneficial effect of treatment. A beneficial effect can be evidenced, for example, by 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 clinical symptoms of the disease or condition, a delay in the progression of the disease or condition, a reduction in the number of recurrences of the disease or condition, an improvement in the subject's overall health or well-being, other parameters specific to a particular disease or condition known in the art, and combinations of such factors.

[0119] PTPRU: An enzyme encoded by the PTPRU gene, a member of the protein tyrosine phosphatase (PTP) family. PTPRU is also known as PTP-pi, PTP lambda, hPTP-J, PTPRO, and PTP psi. PTPs are known to be signaling molecules that regulate various cellular processes, including cell proliferation, differentiation, the mitotic cycle, and oncogenic transformation. This PTP has an extracellular domain, a single transmembrane domain, and two tandem intracellular catalytic tyrosine phosphatase domains, and therefore represents a receptor-type PTP (RPTP). RPTPs can remove phosphate moieties from tyrosine residues.

[0120] TFRC (Transferrin Receptor): Transferrin receptor, also known as cluster of differentiation 71 (CD71), is a protein that in humans is encoded by the TFRC gene.

[0121] Chimeric Antigen Receptor: The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a binding domain for a component present on a target cell, e.g., an antibody specific for a desired antigen (e.g., a tumor antigen such as BCMA), with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific anti-target cellular immune activity. Generally, CARs consist of an extracellular single-chain antibody binding domain (scFv) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain, and when expressed in T cells, have the ability to redirect antigen recognition based on the specificity of a monoclonal antibody.

[0122] 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, producing thousands to millions of copies of a particular DNA or RNA sequence. PCR is commonly used to amplify the number of copies of a DNA or RNA segment for cloning or other analytical methods.

[0123] Nucleic Acid: As used herein, the term "nucleic acid" refers to a polymeric form of nucleotides of any length, either 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, non-natural, or derivatized nucleotide bases. The backbone of a nucleic acid can comprise sugars and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups.

[0124] Vector: As used herein, a "vector" refers to a recombinant plasmid containing a nucleic acid that is delivered into a host cell either in vitro or in vivo. A vector may be used as a vehicle to transport a foreign nucleic acid sequence, usually DNA, into another cell where it can be replicated and / or expressed.

[0125] Diagnosis and treatment of small cell lung cancer (SCLC) 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, where the one or more antigens are selected from the group consisting of CDH5, CD133, SPINK1, CDH23, NLRP7, TFRC, SPINT2, NADSYN1, HIF1A, GRAP2, MAPREL, INHA, PTEN, CTSB, B3GNT6, PLD3, TIMP2, NUDT2, ANAPC2, GPLD1, PTPRU, and CA9, and where the one or more epitopes of the antigens comprise a post-translational modification (PTM). The disclosed methods further include diagnosing the subject as having SCLC when the presence of the one or more autoantibodies is detected in the biological sample obtained from the subject. The disclosed methods further include treating SCLC in the subject 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 the SCLC in the subject, and / or (v) subjecting the subject to radiation therapy targeted to the SCLC. As disclosed herein, the subject can include a human. In some embodiments, the subject can be a patient, for example, a patient suffering from SCLC or exhibiting one or more symptoms of SCLC, or a patient at risk of developing SCLC (e.g., a patient who is a smoker or has a history of smoking). 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.

[0126] SCLC induces the production of autoantibodies that result in unique paraneoplastic neurological syndromes. The underlying mechanisms of such autoantibody formation are largely unknown but are crucial for understanding their pathogenesis. Disclosed herein is a high-dimensional technology that enables the detection of autoantibodies complexed with native antigens directly from patient-derived plasma. In a specific embodiment, this unique platform was used to screen 1,009 human plasma samples for 3,600 autoantibody-antigen complexes, and SCLC plasma was found to have an average of four-fold higher disease-specific autoantibody signal compared to other cancers. Across multiple independent SCLC cohorts (N=240), a common yet previously unknown set of autoantibodies produced in response to both intracellular and extracellular tumor antigens was identified. Several disease-specific post-translational modifications within extracellular proteins, including citrullination, isoaspartylation, and cancer-specific glycosylation, were further characterized and targeted by these autoantibodies. Because most SCLC patients have metastatic disease at the time of diagnosis, these novel autoantibodies are investigated for potential use in early detection and / or treatment of SCLC. In embodiments, a risk prediction model was developed using five autoantibodies with a mean area under the curve of 0.84 (improved to 0.96 by incorporating pack-year cigarette consumption). Collectively, this disclosure provides a novel approach to identify circulating autoantibodies in SCLC with mechanistic insights into disease-specific immunogenicity and clinical utility.

[0127] In certain embodiments, the AAb-Ag complex capture platform utilizes endogenous humoral immunity to capture disease-associated epitopes, such as PTMs, in tumor-specific antigens. By capturing AAbs bound to their immunogenic antigens, a set of AAbs can be identified and validated, which has clinical utility in early detection risk prediction models. Furthermore, in embodiments, a subset of AAbs can identify novel tumor cell surface antigens that may be targeted in therapeutic settings. While most of the antigens identified in this disclosure have not previously been described as associated with SCLC, there have been some exceptions. For example, CD133 overexpression has been reported in SCLC stem cells (Sarvi, et al., 2014, Cancer Research 74, 1554-1565), and TFRC expression was higher in SCLC plasma-derived vesicles compared to controls (Pedersen, et al., 2022, Clinical Proteomics 19, 2). Interestingly, regardless of disease stage, antigen expression and AAb production suggest that tumor-associated antigens identified by AAbs are not lost to immunoediting. In addition to de novo expression, the finding that AAbs target PTM epitopes adds to 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 embodiments of the present disclosure, based on screening of PTM modifications and sites, immunogenic PTMs (i.e., citrullination, isoaspartylation, and cancer-specific glycosylation) for three tumor-specific AAbs are identified. 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.

[0128] AAb are uniquely well suited as early detection biomarkers because they can be obtained via a simple blood draw, are produced in detectable amounts (despite low antigen concentrations), are upregulated years before clinical symptoms, and are highly antigen-specific (Kobayashi, et al., 2020, Seminars in Immunology 47, 101388). One of the key reasons that early detection of SCLC has proven successful is the existence of a clearly defined high-risk population: heavy smokers (Kenfield, et al., 2008, Tobacco Control 17, 198). Because this homogeneous risk population is already eligible for low-dose CT lung cancer screening, AAb that recognize the unique molecular features of SCLC can be utilized to stratify patients into high- or low-risk categories of malignancy during current protocols. High-risk AAb results would flag the acquisition of a review or diagnostic CT scan, while low-risk AAb results would support continued annual lung cancer screening using LD-CT. Additional factors could also be added to further define patients at highest risk for SCLC. AAbs against PNS antigens can be successfully paired with the non-PNS AAbs disclosed herein.

[0129] In certain embodiments, a four-parameter risk prediction model generated according to the present disclosure may use one or more antigens selected from the group consisting of SPINK1, GRAP2, TIMP2, and PLD3, or may use one or more antigens selected from the group consisting of SPINT2, SPINK1, TIMP2, and TFRC. In certain embodiments, a five-parameter risk prediction model generated according to the present disclosure may use one or more antigens selected from the group consisting of SPINK1, TFRC, GRAP2, TIMP2, and PLD3. In the illustrative examples below, the antibody array contains over 3,600 antibodies against approximately 2,500 proteins selected for cancer association, although other examples may indicate other SCLC-specific autoantigens that may perform equally well with the disclosed antibody array. Furthermore, while the illustrated embodiments probe only three types of PTMs enriched in cancer, other embodiments may indicate other cancer-enriched PTMs. Additionally, it should be noted that in some embodiments, the antibody-based nature of the disclosed methods may be readily translated to higher-throughput platforms, such as multiple smaller arrays on a single slide or ELISA, without departing from the scope of the present disclosure.

[0130] Thus, the methods disclosed herein identify tumor-specific autoantigen AAb complexes ex vivo. Isolating tumor-targeted AAb against disease-specific antigens contributes to the accurate detection of early-stage SCLC. Autoantibody production against non-PNS antigens is described herein, and SCLC-specific PTMs present in these antigens may trigger AAb production to epitopes otherwise suspected of immune tolerance. The biomarker detection panel and method for diagnosing SCLC tumorigenesis disclosed herein may lead to the implementation of early detection strategies and antibody therapy. In certain embodiments, the biomarker detection panel of the present disclosure can detect SCLC in subjects or patients 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%.

[0131] SCLC is one of the few malignancies considered a "refractory" cancer, highlighting the need for a deeper understanding of disease biology, more effective treatment strategies, and earlier intervention. Current treatments for the management of SCLC include chemotherapy, radiation therapy, surgical resection, immunotherapy, and / or combinations thereof. SCLC is highly sensitive to chemotherapy, and treatment usually induces rapid responses. The current first-line treatment for extensive-stage SCLC 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 due to its similar efficacy and lower toxicity (Rossi, et al., 2012, J Clin Oncol 30, 1962). However, the majority of patients experience recurrence within one year of treatment. In the case of platinum-sensitive recurrence, a rechallenge trial with first-line chemotherapy is preferred (Fruh et al., 2013, Ann Oncol 24, Suppl. 6, pp. 699-6105; Rudin et al., 2015, J Clin Oncol 33, pp. 4106-4111). Topotecan is the only agent formally approved for second-line treatment of SCLC and remains the standard of care. Oral topotecan has demonstrated a response rate (RR) of 6-17% (O'Brien et al., 2006, J Clin Oncol 24, pp. 5441-5447). In limited-stage SCLC, the standard of care with curative intent consists of four cycles of platinum-doublet chemotherapy combined with radiation therapy, which improves overall survival compared with chemotherapy alone, even in elderly patients (Corso et al., 2015, J Clin Oncol 33, pp. 4240-4246). A combined approach is preferred, and the timing of radiation therapy is important, with the initiation of radiation therapy (45 Gy in 30 fractions twice daily) preferably coinciding 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). Surgery in SCLC may be considered for biopsy-confirmed very small tumors (very localized disease), cT1N0M0, with negative mediastinal staging.

[0132] Resection of pulmonary nodules of unknown origin results in small SCLC. There is a trend towards surgery for very small, node-negative SCLC, but concurrent radiochemotherapy is an alternative option (Hiddinga, et al., 2021, Eur Respir Rev 30, 210079).

[0133] Furthermore, immunotherapy may play a role in the treatment of SCLC. The rationale for combining immunotherapy with chemotherapy in SCLC is the high mutational burden in these tumors, potentially enhancing immunogenicity. Chemotherapy may stimulate tumor antigen expression, priming the tumor in response to immunotherapy. In some examples, immunotherapy regimens may include administering to patients antibodies that bind to one or more epitopes. As an example, the antibody may be a monoclonal antibody drug (e.g., ipilimumab, nivolumab) that activates the immune system by targeting CTLA-4 (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 or anti-cancer agent). In some embodiments, a bispecific antibody capable of binding to a target antigen and a T cell antigen (e.g., CD3) may be administered to patients for therapeutic purposes involving targeting T cell immune responses to tissues / cells expressing the target antigen.

[0134] In embodiments, immunotherapy regimens may include chimeric antigen receptor T cells that specifically bind one or more epitopes. Chimeric antigen receptors (CARs) redirect T cell specificity to antibody-recognized antigens expressed on the surface of cells (e.g., cancer cells), while T cell receptors broaden the range of targets to include intracellular antigens (e.g., tumor antigens). One aspect of the present disclosure includes a CAR specific for an antigen expressed on the surface of SCLC tumor cells. In some examples, a method of treating a patient diagnosed with SCLC may include depleting immune effector cells or T cells from a patient diagnosed with SCLC, genetically modifying the immune effector cells or T cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor, thereby producing 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.

[0135] Circulating autoantibodies in SCLC are more sensitive than in other cancers To demonstrate that cancer-specific AAbs were abundantly produced in SCLC, we measured the upregulated AAb-Ag content (i.e., the ratio between cases and controls) in a total of 1,009 plasma samples from cohorts of SCLC, non-small cell lung cancer (NSCLC), breast cancer, colon cancer, and pancreatic cancer. Both cancer-specific IgG and IgM levels were found to be significantly higher in SCLC (p<0.0001), on average 4-fold higher than in other cancers (Figure 1A). This indicated greater production of disease-specific AAbs in SCLC and highlighted the potential of AAbs as SCLC-specific biomarkers. Next, we identified specific AAbs consistently present across three independent cohorts of SCLC plasma. First, 17 case-control pairs of prediagnostic plasma samples from the Cardiovascular Health Study (CHS, see Table 1) were analyzed for AAb-Ag complexes via an in-house discovery antibody array containing 3,600 antibody spots (total of 10,800 spots) printed in triplicate. Significantly downregulated 46 IgG and 219 IgM AAb-Ag complexes (p<0.05) and significantly upregulated 24 IgG and 102 IgM AAb-Ag complexes were observed (Figure 1B). We selected upregulated biomarkers of AAb-Ag complexes and designed a novel in-hospital antibody array containing 19 of the 24 IgG and 66 of the 102 IgM capture antibodies. After hybridizing 26 case-control pairs (Table 1) from the Fred Hutch diagnostic cohort, significantly upregulated 14 of the 19 IgG (73.6% confirmed) and 15 of the 66 IgM (22.7% confirmed) AAb-Ag complexes were identified in cases compared with controls (Figure 1C). To validate these results, 154 diagnostic plasma samples (55 SCLC cases and 99 paired controls) from Vanderbilt University (Table 1) were hybridized on a small in-house antibody array, and all AAb-Ag complexes remained upregulated, reaching statistical significance in 10 / 14 IgG (71.4% validation) and 12 / 13 IgM (92.3% validation) (Figure 1D). Such a high validation rate indicated that 22 promising SCLC-associated AAb-Ag complexes existed for further investigation.A complete list of significantly upregulated and validated AAb-Ag complexes can be found in Table 2.

[0136] [Table 1]

[0137] [Table 2]

[0138] Autoantibodies target tumor-associated antigens in SCLC We hypothesized that the 22 validated AAbs targeted tumor-associated antigens. To test whether this was indeed the case, we prioritized five AAb antigens with transmembrane domains that are likely expressed on the cell surface of tumor cells and have potential translational relevance for future applications (e.g., imaging or antibody-drug conjugates). We performed immunohistochemistry on SCLC tissue microarrays using antibodies against CD133, PLD3, TFRC, SPINT2, and CA9. CD133, PLD3, TFRC, and SPINT2 showed high levels of uniform expression within tumor cells but not in the adjacent stroma (Figure 2A, staining control in Figure 3). Of the tumors that expressed CA9, most showed a focal staining pattern, while approximately 20% showed uniform expression in tumor cells. Each core was scored as positive (>5% of tumor cells stained) or negative (<5% of tumor cells stained) (Figure 2B), and it was found that >90% of the stained tumor cores were positive for TFRC, SPINT2, and PLD3, 60% for CD133, and 30% for CA9. Positive cores were further classified by stage at diagnosis, demonstrating that tumor-associated antigens were expressed with similar frequencies in stage I, II, or III tumors. Cumulatively, this data validates the experimental approach of utilizing AAbs to discover tumor-associated antigens expressed early and throughout tumorigenesis.

[0139] Despite SCLC having the highest tumor mutation burden, very few SCLC cell lines harbored non-silent mutations in the list of tumor-associated antigens. Among 51 SCLC cell lines with publicly available data, three each of CD133 and CA9 were mutated, one of TFRC, and none of SPINT2 or PLD3 were mutated (Table 3). This low mutation rate (0-5.8%) suggested that the "neoantigens" targeted by AAbs likely originate at the protein level. Because each of these proteins is exposed to the humoral immune system in normal tissues (Figure 3), it was reasoned that the immune system is likely tolerant to their homeostatic expression. Therefore, we investigated tumor-associated PTMs that could explain the observed production of AAbs.

[0140] [Table 3]

[0141] Autoantibodies to CD133 target cancer-specific glycosylation motifs 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 (ovarian), CA19-9 (pancreatic), α-fetoprotein (liver), and carcinoembryonic antigen (colon)). To determine whether the antigens identified by AAbs possess tumor-specific glycan modifications, we immunoprecipitated target proteins from SCLC cell lines and then screened for the carbohydrate antigen sialyl Lewis A (sLeA, also known as CA19-9), which is enriched in cancer. In H82 cell lysates, sLeA appeared on multiple proteins, but only CD133 immunoprecipitation yielded a single sLeA-positive, CD133-positive band (Figure 4A). Immunoprecipitation of CD133 in a second SCLC cell line, H69, also showed positive bands for both sLeA and CD133 (Figure 4B). Furthermore, when an anti-mouse antibody (isotype control) was used for precipitation, this sLeA-positive, CD133-positive band was absent (Figure 5A). To confirm the glycosylation of CD133, it was immunoprecipitated from the H82 cell line and treated with PRIME deglycosylase to remove complex sialylated structures, such as N-linked glycans and sLeA. PRIME treatment disrupted CD133 migration, confirming the removal of glycan residues and the detection of deleted sLeA on the CD133 protein (Figure 5B). The specificity of autoantibodies present in plasma from SCLC patients was assessed against glycan-modified CD133 by creating a modified sandwich ELISA that replicated the array platform. After seeding a commercially available CD133 antibody to capture CD133 protein from H82 SCLC cell line lysates and incubating with plasma from SCLC patients with known high or low levels of CD133 AAb (as determined by the array platform in Figure 1C), ELISA confirmed that the levels of bound IgG differed significantly ( Figure 5C ).Treatment of H82 lysates with PRIME deglycosylase reduced autoantibody signals to levels similar to those of CD133-low plasma, arguing that glycan modification was required for increased autoantibody binding.

[0142] Autoantibodies bind to isoaspartic acid post-translational modifications in SPINK1 Previously, one study reported that the isoaspartic acid PTM (IsoAsp) present in the PNS antigen ELAVL4 may be immunogenic in SCLC-associated autoimmunity (Pulido et al., 2016, Journal of Neuroimmunology 299, 70–78). IsoAsp is a naturally occurring PTM enriched in disease and formed by asparagine deamidation or aspartic acid isomerization. To identify IsoAsp-targeting AAbs, we converted asparagine to IsoAsp residues in GST fusions of the extracellular domains of minimal antigens using published in vitro methods (Geiger and Clarke, 1987, Biol Chem 262, 785–794; Curnis et al., 2006, Journal of Biological Chemistry 281, 36466–36476). We elicited AAb from pooled SCLC plasma using modified IsoAsp compared with unmodified protein, which qualitatively demonstrated by immunoblotting that more IgG AAb bound to isoAsp SPINK1 but not to isoAsp SPINT2 or TIMP2 (Figures 6A-6B). To determine the specific sites of substitution, we synthesized peptides directed against all asparagine residues in the extracellular regions of SPINT2 (6 sites), SPINK1 (2 sites), and TIMP2 (4 sites). Both wild-type (WT) asparagine peptides and IsoAsp peptides were coated on assay plates and incubated with SCLC plasma samples, and AAb binding was detected using anti-human IgG (Figure 6C). Consistent with the immunoblotting data, IsoAsp was found at both positions of SPINK1 bound by at least two-fold more AAb than the corresponding WT peptide. Therefore, this demonstrated that some AAb in SCLC plasma recognize neoepitope PTM sites on SPINK1.

[0143] SCLC AAb recognizes citrullinated TFRC that is not present in the recombinant protein Citrulline, a unique amino acid generated by spontaneous arginine deamination, has also been shown to be immunogenic in cancer but has not yet been described in SCLC (Brentville, et al., 2016, Cancer Res 76, 548–560; Pulido, et al., 2016, J Neuroimmunol 299, 70–78). Therefore, lysates from human SCLC H82 and H69 cells were screened with antibodies against citrulline, TFRC, PLD3, CD133, SPINT2, and CA9 to explore any overlapping band patterns between citrulline and the target proteins. TFRC likely had an overlapping band with citrulline that was evident at the predicted protein size (Figures 7A and 8A). To increase the sensitivity of citrulline detection, we immunoprecipitated with citrulline antibodies and blotted back for expression of the target antibodies, and found that TFRC was the only target protein detected (Figure 7B). These observations were expanded to show that whole-cell lysates from the immortalized fibroblast cell line 3T3, which had low levels of TFRC expression, did not show a corresponding band after probing with a citrulline antibody (Figure 8B). Furthermore, immunoprecipitation with either anti-TFRC antibody and blotting back or reverse for citrulline showed a corresponding band in H82 cells but not in controls, indicating that TFRC has an arginine residue that is specifically converted to citrulline in H82 cells. The 32 arginines in the extracellular domain of TFRC were considered, and seven were selected that were likely to undergo deamidation to citrulline using a published consensus motif (Ju and Wang, 2018, Gene 664, 78–83). Peptides representing fragments of TFRC with either wild-type arginine or citrullinated amino acids within the appropriate sequence were coated onto assay plates, and five of the seven peptides with citrullinated residues pulled down more AAb from SCLC plasma than the corresponding WT peptides (Figure 8C).We investigated either antigen-complexed or antigen-free TFRC AAbs in a subset of plasma samples from the Fred Hutch cohort. Significantly higher levels of AAb-TFRC complexes (p<0.03) could be detected in SCLC plasma compared to controls, as confirmed via ELISA. Conversely, when TFRC recombinant protein was used as the capture antigen, no significant differences in AAb were detected, but anti-TFRC antibodies readily detected recombinant TFRC as a positive control (Figure 8D). Finally, including two citrullinated TFRC peptides as capture antigens resulted in greater binding to AAb from SCLC plasma compared to controls, suggesting that TFRC-AAbs recognized citrullinated neoantigens of TFRC that were not found in the recombinant protein or the arginine-containing (wild-type) peptide. Collectively, this data supports the AAb-Ag conjugate platform as a method to identify tumor-specific AAbs targeting post-translational "neoantigens" that have been difficult to detect using other approaches.

[0144] Construction of a SCLC risk prediction model Current annual low-dose computed tomography screening protocols for the early detection of NSCLC are ineffective for SCLC (Thomas, et al., 2018, Chest 154, 1284-1290). However, the clinical benefit of early detection of SCLC is clear: when detected at limited stage, conventional chemotherapy is curative in nearly 20% of patients. Surgical resection is generally not considered, and for patients with very early stage disease, combined chemotherapy can be curative (Lassen, et al., 1995, J Clin Oncol 13, 1215-1220; Winer, et al., 2011, Nature Medicine 17, 610-617). Having demonstrated that AAb were generated by SCLC target-specific PTMs in SCLC antigens, we investigated whether these unique AAb could be utilized to generate risk prediction models for early detection of SCLC. Therefore, all 22 validated AAb-Ag conjugates were investigated for their ability to reliably detect the presence of SCLC in plasma specimens. Because the Vanderbilt (VB) cohort had the largest sample size, this set was used as training data. The best four- or five-marker combinations were calculated by maximizing the AUC based on logistic regression. To account for IgM-to-IgG class conversion during the course of an immune response, both AAb isotypes were included in each marker. This identified a four-marker panel consisting of AAbs against PLD3, TIMP2, GRAP2, and SPINK1, with the highest overall AUC of 0.872, and a five-marker panel with the addition of TFRC, with an AUC of 0.874 (Table 4). After fixing the coefficients using data from the Vanderbilt set, the five-marker risk prediction model was tested in the CHS cohort, yielding an AUC of 0.700, and in the Fred Hutch (FH) cohort, yielding an AUC of 0.950.

[0145] [Table 4]

[0146] Multiple logistic regression models were fitted to the VB data by including the five AAb markers that yielded the highest AUC in the model. The prediction model was applied to the CHS and FH study datasets by dividing cases and controls into high-risk or low-risk categories based on positive or negative risk prediction scores, respectively (Table 5). The risk prediction model was found to perform well, with positive predictive values ​​of 76.9% for VB, 64.7% for CHS, and 79.3% for FH, and negative predictive values ​​of 85.3% for VB, 64.7% for CHS, and 86.9% for FH. When prediagnostic samples from CHS were grouped by time to diagnosis, samples taken within the first year before diagnosis more accurately flagged high-risk samples (7 / 11, 63.6%) than those taken 1-2 years before diagnosis (3 / 6, 50%). Furthermore, we were able to correctly identify 23 / 26 (88.5%) limited-stage SCLC in both the FH and VB cohorts and 18 / 24 (75%) extensive-stage SCLC in the high-risk pool.

[0147] [Table 5]

[0148] The five AAbs were investigated in more detail within each cohort individually. When blood samples were collected within the year before diagnosis, AAbs targeting PLD3, TIMP2, GRAP2, and SPINK1 were significantly upregulated (p = 0.05), while AAbs against TFRC showed an upward trend (p = 0.15) (Figure 9A). PLD3 and TIMP2 remained significantly upregulated up to two years before diagnosis (p < 0.005). In diagnostic samples, all five panel members were highly upregulated in both limited and extensive disease (p < 0.0005). None of the panel markers were differentially expressed in current and former smoker cases (Figure 10A), and PLD3 was not associated with pack-years of smoking (Figure 10B). TIMP2, GRAP2, SPINK1, and TFRC were weakly associated with pack-years of smoking (r<0.25) (Figures 10C-10F). Panel AAbs were not differentially expressed with a diagnosis of chronic obstructive pulmonary disease (Figure 11A) or autoimmunity (Figure 11B). The individual performance of these markers was examined in plasma from other cancers, including non-SCLC, pancreatic, and colon cancer, and found to be not significantly upregulated in other diseases compared with controls or SCLC (Figure 9B), suggesting SCLC specificity. Cumulatively, these AAbs suggest that they are specifically upregulated early in SCLC tumorigenesis and remain elevated throughout the disease process.

[0149] The single largest risk factor for SCLC is current heavy smoking, which can increase the likelihood of developing SCLC by 60-100-fold for women and men, respectively (Wang, et al., 2017, Sci Rep 7, 1339). Because all three cohorts were matched for smoking history (current smokers, former smokers, and never smokers), we incorporated pack-year smoking history into the five-panel model. The continuous variable of pack-years smoking had an AUC equal to 0.57 in VB, 0.72 in CHS, and 0.612 in FH (Figures 12A-12C). Combining pack-years smoking with the AAb risk prediction model had minimal impact in the Vanderbilt cohort (AUC remained at 0.87), while the AUC increased to 1.0 in both the CHS and Fred Hutch cohorts.

[0150] Experimental methods and materials Additional details regarding the experiments discussed above are provided in the following paragraphs. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0151] Study design All human plasma samples were obtained after institutional review board approval and informed consent. This study used three independent plasma cohorts: 1) the Cardiovascular Health Study (CHS), a population-based longitudinal study of coronary heart disease and stroke (Fried et al., 1991, Ann Epidemiol 1, 263–276). Laboratory tests and plasma samples were completed annually for up to 10 years. Seventeen cases of SCLC were newly diagnosed within 24 months of blood collection. 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 (Fred Hutch) for pulmonary nodule evaluation consisted of N = 26 SCLC patients and N = 26 patients with benign pulmonary nodules (active IRB protocol number 6663). Corresponding clinicopathological data were maintained in a highly annotated database. Controls were matched to cases based on age, sex, and smoking history. 3) 55 diagnostic plasma samples and 99 unmatched control plasma samples, matched for age, sex, and smoking history, were generously provided by Vanderbilt University, Nashville, TN.

[0152] Detection of AAb-Ag complexes Detection of AAb-Ag complexes using antibody arrays has been previously reported (Winer, et al., 2011, Nature Medicine 17, 610-617). Briefly, antibody microarrays were printed in-house, incubated with patient plasma diluted 1:80, and probed with anti-human IgG-SeTau647 or IgM-DyLight550 antibodies. Microarrays were imaged using a GenePix 4000B scanner, and spot intensities were measured using GenePix Pro 6.0 (Molecular Devices).

[0153] immunohistochemistry Tissue microarrays (TMAs) containing 62 individual SCLC cases and 6 individual lung tissue controls (RLN681A, US Biolab) were stained for CD133 (#64326, 1:1000 Cell Signaling Technologies) or an equivalent concentration of anti-rabbit IgG isotype antibody using an automated Leica Bond RX system, detected via Leica rabbit-HRP and DAB. The same TMAs were also used to stain for TFRC (HPA028598, 1:500 Sigma), SPINT2 (HPA011101, 1:200 Sigma), and CA9 (HPA055207, 1:1000 Sigma) after heat-induced epitope retrieval in citrate pH 6 buffer. Sections were probed with biotinylated panspecific antibodies for 10 minutes, streptavidin-HRP for 5 minutes (PK-8800, Vector Laboratories), and counterstained with hematoxylin, washing between each step. After RLN681A was discontinued, a separate TMA containing 45 duplicate SCLC cores and 10 lung tissue controls was purchased from US Biomax (BS04116a) and stained for PLD3 (HPA012800, 1:200 Sigma) using the Vector protocol described above. TMAs containing 33 normal tissues (3 per organ, FDA999-1, US Biolab) were also stained for each antibody as positive or negative controls. Stained slides were digitized using a Leica Aperio whole slide scanner at 20x magnification. Scoring of TMA staining was performed in a blinded fashion where each biopsy was determined to be either positive (>5% tumor cells stained) or negative (<5% tumor cells stained).

[0154] ELISA A plate-based assay was developed to quantify autoantibody levels for different capture approaches. WT and PTM peptides were purchased from CHI Scientific (sequences are listed in Table 6). 10 μg of peptide was seeded per well in a maleimide 96-well plate (#15152, Pierce). White 96-well plates (#15042, Pierce) were coated with 1 μg / well of α-CD133 antibody (HB#7, Developmental Studies Hybridoma Bank) and 1 U / 50 μg of cell lysate was used to capture CD133 from H82 lysates treated with or without PRIME glycosylase (#50-999-475, Fisher Scientific). α-TFRC antibody (HPA028598, Sigma) or TFRC recombinant protein (89-760aa, #11020-H07H, Sinobiological) was seeded at 0.5 μg / well. After the binding capture approach, all plates were washed and used as bait for autoantibodies from human plasma (1:500). Total autoantibody signals were quantified using an α-human IgG-HRP secondary (1:5000, #709-065-149, Jackson ImmunoResearch) and SuperSignal ELISA Femto (#37074, ThermoFisher) on a SpectroMax L Microplate reader (Molecular Devices).

[0155] [Table 6]

[0156] Immunoblotting To induce isoaspartylation, GST-fusion proteins were combined with magnetic glutathione beads (#78601, ThermoFisher) at a 1:20 ratio with 100 mM NH4HCO3 (pH 8.0) or PBS containing 0.05% sodium azide and incubated at 37°C for 48 hours. After washing, the treated GST-fusion proteins were incubated with human plasma at a 1:10,000 ratio 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, blotted onto nitrocellulose, checked for loading with Ponceau S, blocked in 1% nonfat dry milk, and probed with α-human IgG-AF680 (#709-625-149, Jackson ImmunoResearch) at 1:5000 and mouse α-GST (FHCC Antibody Technology Core) at 1:500, detected with α-mouse IgG-AF680 (1:10000, A32729, ThermoFisher). Immunoblots were imaged on an Odyssey CLx (LiCor, Lincoln, NE) using ImageStudio v5. CD133 protein was immunoprecipitated from H82 cells, treated with PRIME deglycosylase (#50-999-475, Fisher Scientific) at 1 U / 50 μg for 30 min at 37°C, and immunoblotted as described above (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 (1:10000, #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). Immunoblots were probed with antibodies against TFRC (1:1000, HPA028598, Sigma) or citrulline (1:500, MA5-27573, Invitrogen) and detected with α-mouse IgG-AlexaFluor680 and α-rabbit IgG-AlexaFluor790 (1:10000, A32729, A10043, ThermoFisher), respectively.

[0157] cell culture The NCI-H82 and NCI-H69 cell lines were obtained from ATCC, grown 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% CO , and were routinely tested negative for mycoplasma by PCR.

[0158] statistical analysis The array data contained the same format as the two-channel gene expression array, and analysis proceeded as previously described (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 described in the figure brief), and three or more groups were compared using one-way analysis of variance with Tukey's test. Logistic regression was used to identify the combination of multiple autoantibodies that best distinguished cases from controls. Receiver operating characteristic curves were generated, and the area under the curve was calculated to assess predictive ability. All relevant statistical analyses were performed using R statistical software (R Core Team (2021)).

[0159] Identification of autoantibodies Once the immunogenic PTM peptides targeted by the autoantibodies were identified, PTM-specific autoantibodies were isolated directly from SCLC patients and sequenced (Figure 13). To accomplish this, PTM-peptide tetramers were used to enrich PTM-binding B cells from peripheral blood mononuclear cells (PBMCs) of SCLC patients. PTM-peptide tetramers were generated by combining PE- or APC-labeled magnetic tetramers containing a streptavidin core with biotinylated PTM peptides identified to bind more strongly to AAbs from plasma derived from SCLC patients compared to the wild-type peptide. Five different PTM peptides were included in each PTM-tetramer: five citrullinated TFRC peptides were conjugated to the PE-tetramer, and five isoaspartylated peptides (three CA9, one PLD3, and one SPINK1) were conjugated to the APC-tetramer. These PTM-tetramers were then incubated with pooled peripheral blood mononuclear cells (PBMCs) from three different SCLC patients, and tetramer-binding cells were isolated with magnetic microbeads. This PTM-tetramer "enriched" fraction was labeled with fluorescent antibodies and subjected to fluorescence-activated cell sorting (FACS) to single cells. PTM-tetramer+ B cells were sorted, and heavy and light chain antibody sequences from each B cell were amplified by RT-PCR. The heavy and light chain pairs were successfully sequenced. These sequences were cloned into a human IgG expression vector for antibody production, and the CDR positions are underlined in Table 7. The autoantigen PTM modifications to which each antibody binds are also listed in Table 7. The CDR sequences were determined using the ImMunoGeneTics (IMGT) information system domain gap align. Table 8 lists the CDR sequences and their respective SEQ ID NOs.

[0160] [Table 7]

[0161] [Table 8]

[0162] One PTM-specific antibody, 2D2, was isolated from SCLC PBMCs and further characterized. The 2D2 antibody was successfully used to specifically image tumor TFRC in female athymic nude mice bearing subcutaneous flank H82 tumors (Figure 14). To do this, the 2D2 antibody was labeled with CF770 and injected retroorbitally into the mice. NIRF imaging was performed 3, 4, and 5 days later. Figure 14 shows the scan from day 5, in which essentially all signal was focused on the tumor. Figure 15 shows that the 2D2 antibody primarily binds to a specific TFRC sequence representing citrullinated peptide 13, binds to a lesser extent to other TFRC-citrullinated peptides, but does not bind to CA9 (isoaspartylated or WT). These results demonstrate that the 2D2 antibody has specificity for both specific TFRC sequences with citrullines at specific positions. FIG. 16 shows that the 2D2 antibody does not bind to TFRC in normal uterus, small intestine, lung, or placenta, whereas the commercially available TFRC antibody (WT TFRC) binds broadly.

[0163] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, 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 be within the scope of the appended claims.

Claims

1. 1. A method of treating small cell lung cancer (SCLC) in a subject, comprising: (a) administering to said subject a chemotherapy regimen; (b) administering to said subject an immunotherapeutic regimen; (c) administering the antibody or antigen-binding fragment thereof to the subject; (d) performing a surgical resection of said SCLC in said subject; and / or (e) subjecting the subject to radiation therapy targeted to the SCLC; 10. The method of claim 1, wherein the subject has autoantibodies that specifically bind to 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 antigens comprise a post-translational modification (PTM).

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

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

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

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

6. The method of any one of claims 1 to 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 to 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 to 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 to 8, wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 1 to 21.

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

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

12. The method of any one of claims 1 to 11, wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 22 to 46.

13. 1. A method of diagnosing and treating small cell lung cancer (SCLC) in a subject, 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 the one or more epitopes of the antigens comprise a post-translational modification (PTM); (b) diagnosing the subject as having SCLC if the presence of the one or more autoantibodies is detected in the biological sample obtained from the subject; and (c) determining the SCLC in the subject; (i) administering to said subject a chemotherapy regimen; (ii) administering to said subject an immunotherapeutic regimen; (iii) administering the antibody or antigen-binding fragment thereof to the subject; (iv) performing a surgical resection of said SCLC in said subject; and / or (v) treating said subject by subjecting said subject to radiation therapy targeted to said SCLC.

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

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

16. 16. The method of any one of claims 13 to 15, wherein diagnosing the subject comprises diagnosing the subject as having 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. 16. The method of any one of claims 13 to 15, wherein diagnosing the subject comprises diagnosing the subject as having 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. 16. The method of any one of claims 13 to 15, wherein diagnosing the subject comprises diagnosing the subject as having 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. 16. The method of any one of claims 13 to 15, wherein diagnosing the subject comprises diagnosing the subject as having 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 of any one of claims 13 to 19, wherein the one or more antigens are selected from the group consisting of SPINK1, TFRC, GRAP2, TIMP2, and PLD3.

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

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

23. The method of any one of claims 13 to 22, wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 1 to 21.

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

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

26. The method of any one of claims 13 to 25, wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 22 to 46.

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

28. 28. The method of any one of claims 1 to 27, wherein the chemotherapy regimen comprises one or more cycles of cisplatin or carboplatin and etoposide.

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

30. The method of any one of claims 1 to 28, wherein the immunotherapeutic regimen comprises antibodies that bind to one or more of said epitopes.

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

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

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

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

35. 35. The method of claim 34, wherein the antibody or 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 antigens comprise a post-translational modification (PTM).

36. 36. The method of claim 35, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) contained within a heavy chain variable region (HCVR) and three light chain CDRs contained within a light chain variable region (LCVR), and the amino acid sequences of the HCVR / LCVR comprise amino acid sequences selected from the group consisting of SEQ ID NOs: 47 / 51, 55 / 59, 63 / 67, 71 / 75, and 79 / 83, respectively.

37. 36. The method of claim 35, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 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.

38. 36. The method of 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 the amino acid sequences of the HCVR / LCVR are selected from the group consisting of SEQ ID NOs: 47 / 51, 55 / 59, 63 / 67, 71 / 75, and 79 / 83, respectively.

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

83.

40. 40. The method of claim 39, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino sequences of SEQ ID NOs: 80-81-82-84-85-86, respectively.

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

83.

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

39.

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

75.

44. 44. The method of claim 43, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino sequences of SEQ ID NOs: 72-73-74-76-77-78, respectively.

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

45.

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

51.

48. 48. The method of claim 47, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino sequences of SEQ ID NOs: 48-49-50-52-53-54, respectively.

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

40.

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

59.

52. 52. The method of claim 51, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino sequences of SEQ ID NOs: 56-57-58-60-61-62, respectively.

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

59.

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

37.

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

67.

56. 56. The method of claim 55, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino sequences of SEQ ID NOs: 64-65-66-68-69-70, respectively.

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

67.

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

29.

59. 1. A biomarker detection panel for small cell lung cancer (SCLC), comprising one or more peptides comprising an epitope of 7 to 50 amino acids in length from 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.

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

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

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

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

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

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

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

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

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

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

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

71. 71. The biomarker detection panel of claim 70, wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 1-21.

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

73. 73. The biomarker detection panel of claim 72, wherein the PTM comprises citrullination and / or isoaspartylation.

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

75. 50. The biomarker detection panel of any one of claims 34 to 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 thereof comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 79, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

83.

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

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

83.

79. 80. The antibody or antigen-binding fragment of any one of claims 76 to 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 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 a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 71, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

75.

81. 81. The antibody or antigen-binding fragment of claim 80, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 72-73-74-76-77-78, respectively.

82. 82. The antibody or antigen-binding fragment thereof of claim 80 or 81, 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.

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

45.

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

51.

85. 85. The antibody or antigen-binding fragment of claim 84, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 48-49-50-52-53-54, respectively.

86. The antibody or antigen-binding fragment thereof of claim 84 or 85, 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.

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

40.

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

59.

89. 89. The antibody or antigen-binding fragment of claim 88, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 56-57-58-60-61-62, respectively.

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

59.

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

37.

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

67.

93. 93. The antibody or antigen-binding fragment of claim 92, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 64-65-66-68-69-70, respectively.

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

67.

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

29.

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

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

98. 98. The unit dosage form of claim 97, wherein the container is a vial, a syringe, a pre-filled syringe, or an autoinjector.

99. 96. A kit comprising a container containing the antibody or antigen-binding fragment thereof according to any one of claims 76 to 95, and instructions for use of the antibody or antigen-binding fragment for the treatment of small cell lung cancer.

100. A nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 76 to 95.

101. A recombinant expression vector comprising the nucleic acid molecule of claim 100.

102. A pair of nucleic acid molecules encoding the heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment of any one of claims 76 to 95, comprising: 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 each comprising a pair of nucleic acid molecules according to claim 102.

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

105. 105. The isolated host cell of claim 104, which is a mammalian host cell.

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