Anti-dll3 antibody and method of making and using thereof

Abstract

A delta-like ligand 3 (DLL3) binding peptide having binding specificity for human DLL3 comprising an amino acid sequence having at least 98%, 95% or 92% sequence identity to SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480076149.X (22) Application Date 2024.11.14 (30) Priority Data 63 / 599,433 2023.11.15 US (85) PCT International Application Entering National Phase Date 2026.06.01 (86) PCT International Application Application Data PCT / US2024 / 055803 2024.11.14 (87) PCT International Application Publication Data WO2025 / 106600 EN 2025.05.22 (71) Applicant: Seattle Immuno Inc. Address: 15318 N95th Street, Redmond, Washington, USA (72) Inventors: Kelsey D. Dinkl, Jahan Khalili, Zhu Yi (74) Patent Agency: Beijing Jingwantong Intellectual Property Agency Co., Ltd. 11440 Patent Attorneys: Xu Tianyi, Xu Xiaoqin (51) Int.Cl. C07K 16 / 28 (2006.01) A61K 47 / 68 (2006.01) (54) Invention Title: Anti-DLL3 Antibody and its Preparation and Use Method (57) Abstract: A δ-like ligand 3 (DLL3) binding peptide with binding specificity to human DLL3, comprising SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, Amino acid sequences 32, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 have at least 98%, 95%, or 92% sequence identity. Claims (3 pages), Description (37 pages), Sequence Listing (electronic publication), Drawings (11 pages) CN 122374334 A 2026.07.10 CN 1 22 37 43 34 A 1. A δ-like ligand 3 (DLL3) binding peptide with binding specificity to human DLL3, comprising a variable heavy (VH) chain and a variable light (VL) chain, wherein the VH chain comprises: CDR H1 with amino acid sequence SEQ ID: 104, CDR H2 with amino acid sequence SEQ ID: 105, CDR H3 with amino acid sequence SEQ ID: 106, CDR H1 with amino acid sequence SEQ ID: 110, CDR H2 with amino acid sequence SEQ ID: 111, CDR H3 with amino acid sequence SEQ ID: 112, CDR H1 with amino acid sequence SEQ ID: 116, CDR H2 with amino acid sequence SEQ ID: 117, and CDR H3 with amino acid sequence SEQ ID: 118. H3, or a CDR with the amino acid sequence SEQ ID: 120H1, CDR H2 with amino acid sequence SEQ ID: 105, and CDR H3 with amino acid sequence SEQ ID: 119, wherein the VL chain comprises: CDR L1 with amino acid sequence SEQ ID: 101, CDR L2 with amino acid sequence SEQ ID: 102, CDR L3 with amino acid sequence SEQ ID: 103, CDR L1 with amino acid sequence SEQ ID: 107, CDR L2 with amino acid sequence SEQ ID: 108, CDR L3 with amino acid sequence SEQ ID: 109, CDR L1 with amino acid sequence SEQ ID: 113, CDR L2 with amino acid sequence SEQ ID: 114, and CDR L3 with amino acid sequence SEQ ID: 115, or CDR L1 with amino acid sequence SEQ ID: 101, CDR L2 with amino acid sequence SEQ ID: 102, and CDR L3 with amino acid sequence SEQ ID: 121. 2. The DLL3-binding peptide of claim 1, wherein the VH chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 17, 19, 21, 23, 25, 27, 28, or 29; and wherein the VL chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 16, 18, 20, 22, 24, 26, or 30. 3. The DLL3-binding peptide of claim 1, wherein the amino acid sequences of the VH chain and the VL chain are selected from SEQ ID NO: 17 and 16; 19 and 18; 21 and 20; 23 and 22; 25 and 24; 27 and 26; 28 and 22; 29 and 22; 29 and 30; or 28 and 30. 4. The DLL3-binding peptide of claim 1, comprising: an scFv domain, wherein the scFv domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32; and a Fab domain, wherein the Fc domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 31, 32, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. 5. The DLL3-binding peptide of claim 4, comprising at least one histidine residue connected to the end of the scFv domain. 6. The DLL3-binding peptide of claim 4, further comprising an Fc domain connected to the Fab domain to provide a Fab-monoFc fusion protein. 7. The DLL3-binding peptide of claim 1, comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.NO: Amino acid sequences having at least 98% sequence identity in numbers 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46. 8. An scFv domain comprising the DLL3-binding peptide of claim 1. (Claims 1 / 3 page 2 CN 122374334 A) 9. A Fab domain comprising the DLL3-binding peptide of claim 1. 10. An antibody with binding specificity to human DLL3 comprising the DLL3-binding peptide of claim 1. 11. The antibody of claim 10, wherein the VH chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 17, 19, 21, 23, 25, 27, 28, or 29, and wherein the VL chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 16, 18, 20, 22, 24, 26, or 30. 12. The antibody of claim 10, comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 37, 39, 41, 43, 45, or 31; and wherein the LC comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 32, 38, 40, 42, 44, or 46. 13. The antibody of claim 10, comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or a combination thereof. 14. The antibody of claim 10, wherein the antibody comprises a monospecific antibody, a bispecific antibody, or a multispecific antibody, a purified antibody, a humanized antibody, IgG, or a combination thereof. 15. An isolated nucleic acid sequence encoding the antibody of claim 10. 16. An expression vector or host cell comprising the isolated nucleic acid sequence of claim 15. 17. An immunoconjugate comprising the antibody of claim 10 conjugated to a pharmaceutical unit via a linker, wherein the linker comprises a covalent bond selected from ester bonds, ether bonds, amine bonds, amide bonds, disulfide bonds, imide bonds, sulfone bonds, phosphate bonds, phosphate ester bonds, peptide bonds, hydrazone bonds, or combinations thereof. 18. The immunoconjugate of claim 17, wherein the pharmaceutical unit comprises a unit derived from a therapeutic agent, imaging agent, diagnostic agent, radioisotope, or a combination thereof. 19. The immunoconjugate of claim 18, wherein the therapeutic agent comprises a cytotoxic agent, a chemotherapeutic agent, an enzyme, etc.Anti-estrogens, receptor tyrosine kinase inhibitors, kinase inhibitors, cell cycle inhibitors, DNA, RNA, or protein synthesis inhibitors, RAS inhibitors, or combinations thereof. 20. The immunoconjugate of claim 18, wherein the therapeutic agent comprises capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, nitrogen mustard, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, leucovorin, gemcitabine, oxaliplatin, irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testrolide, vorazole, formetatan, fadroxil, erlotinib, lafatinib, dasatinib, gefitinib. Osimertinib, vandetanib, afatinib, imatinib, pazopanib, lapatinib, sunitinib, nilotinib, sorafenib, albumin-bound paclitaxel, everolimus, sirolimus esterified, dabrafenib, vemurafenib, trametinib, vinpocetine, apatinib, crizotinib, perifolliculin, olaparib, bortezomib, tofacitinib, trastuzumab, or derivatives or combinations thereof. 21. The immunoconjugate of claim 18, wherein the radioisotope comprises deuterium, 131I, 32P, 90Sr, 90Y, 89Zr, 177Lu, or combinations thereof. 22. A pharmaceutical composition comprising the antibody of claim 10 or the immunoconjugate of claim 17, and a pharmaceutically acceptable carrier. 23. A method of producing the antibody of claim 10, comprising culturing host cells such that a DNA sequence encoding the antibody of claim 10 is expressed, and purifying the antibody. (Claims 2 / 3, Page 3, CN 122374334 A) 24. A method of producing the immunoconjugate of claim 17, comprising conjugating the antibody of claim 7 to the pharmaceutical unit. 23. A method of treating or preventing cancer, autoimmune disease, or infectious disease in a subject, comprising administering to the subject an effective amount of the antibody of claim 10 or the immunoconjugate of claim 17. 25. The method of claim 25, wherein the cancer comprises lung cancer, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), medullary thyroid carcinoma (MTC), cervical neuroendocrine carcinoma (CNEC), gastrointestinal pancreatic neuroendocrine tumor (GEP-NEN), bladder cancer, bladder NEN (BMEN), prostate cancer, NE prostate cancer (NEPC), Merkel cell carcinoma (MCC), breast cancer, liver cancer, hepatocellular carcinoma (HCC), glioma, pancreatic cancer, or combinations thereof. 26. The method of claim 25, further comprising co-administering an effective amount of the therapeutic agent, wherein the therapeutic agent comprises…Including antibodies, chemotherapeutic agents, enzymes, anti-estrogens, receptor tyrosine kinase inhibitors, kinase inhibitors, cell cycle inhibitors, DNA, RNA, or protein synthesis inhibitors, RAS inhibitors, or combinations thereof. 27. The method of claim 27, wherein the therapeutic agent comprises capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, nitrogen mustard, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, leucovorin, gemcitabine, oxaliplatin, irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant. Tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testosterone, vorazole, formestane, fadroxil, erlotinib, lafatinib, dasatinib, gefitinib, osimertinib, vandetanib, afatinib, imatinib, pazopanib, lapatinib, sunitinib, nilotinib, sorafenib, albumin-bound paclitaxel, everolimus, sirolimus esterified, dabrafenib, vemurafenib, trametinib, vinpocetide, apatinib, crizotinib, perifolium, olaparib, bortezomib, tofatinib, trastuzumab, or derivatives or combinations thereof. 28. The method of claim 25, wherein the subject is a human. 29. A solution comprising an effective concentration of the antibody of claim 10 or the immunoconjugate of claim 17, wherein the solution is the plasma of a subject. Claims 3 / 3 Page 4 CN 122374334 A Anti-DLL3 antibody and its preparation and use

[0001] Cross-Reference to Related Applications This application claims the benefit of U.S. Provisional Application Serial No. 63 / 599,433, filed November 15, 2023, under 35 USC 119(e), the entire disclosure of which is incorporated herein by reference. Technical Field

[0002] This disclosure generally relates to the technical field of antibodies, antibody therapies, cancer immunotherapy, pharmaceutical compositions, and methods of treating diseases. Background Art

[0003] Globally, lung cancer is the leading cause of death for both men and women compared to any other cancer. The main types of lung cancer are non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Approximately 80% to 85% of lung cancers are NSCLC, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma subtypes. Although these subtypes originate from different types of lung cells, they are generally grouped together as NSCLC due to similar treatment and prognosis (prospects).

[0004] Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, accounting for 15% of all lung cancers [1]. An estimated 250,000 new cases of SCLC are diagnosed globally each year [2]. Of these, 30,000–35,000 are diagnosed in the United States [3]. Globally,It is estimated that at least 200,000 people die from SCLC[4]. The number of cases and deaths varies annually due to the close association between smoking and SCLC and the 30-year lag between smoking and cancer progression[5]. SCLC diagnoses are classified into two categories based on the severity and stage of the cancer: limited-stage SCLC (LS-SCLC) and extensive-stage SCLC (ES-SCLC). Patients diagnosed with LS-SCLC respond well to chemotherapy, with 75% showing a complete response (CR). In contrast, patients diagnosed with ES-SCLC rarely show a complete response rate of more than 30%[3]. In 2022, 80–85% of SCLC diagnoses were classified as ES-SCLC[6]. Even when patients respond to treatment, ES-SCLC is associated with high recurrence rates, rapid proliferation, intracranial metastases, and the development of chemotherapy resistance[6]. Therefore, patients diagnosed with ES-SCLC have a poor prognosis.

[0005] δ-like canonical Notch ligand 3 (also known as δ-like ligand 3, DLL3) is a member of the δ / Serrate / Lag2 (DSL) family of Notch receptor ligands. Other members in mammals include DLL1, DLL4, JAG1, and JAG2

[10] . Notch signaling is a highly conserved intercellular signaling pathway that influences a number of cellular processes, including differentiation, proliferation, survival, and apoptosis. Notch signaling has been shown to play an important role in the development of pulmonary neuroendocrine cells

[11] . DLL3 is an inhibitory ligand of the Notch receptor and plays a key role in Notch signaling. DLL3 binds to the Notch receptor via cis-interaction, producing highly specific regulatory effects

[12] . DLL3 is a 619-amino acid single transmembrane protein consisting of a DSL domain, an intracellular domain, and six epidermal growth factor-like domains. The extracellular N-terminal DSL domain is highly conserved and is essential for binding to the Notch receptor. Normally, DLL3 is expressed in the Golgi apparatus; however, during pathogenesis, DLL3 is overexpressed and appears on the cell membrane

[10] .

[0006] DLL3 is rarely detected in normal tissues but is highly expressed in SCLC. This highly specific expression profile has led to the development of a series of DLL3-specific therapeutic agents targeting SCLC tumors. Indications for anti-DLL3 therapeutic agents also include other neuroendocrine tumors, including melanoma, glioblastoma multiforme, and small cell bladder cancer

[11] . In addition, DLL3 has been suggested as a potential biomarker for better prognosis in patients with resectable pancreatic ductal adenocarcinoma (PDAC)

[16] . DLL3 expression is regulated by the transcription factor achaete-scute homolog 1 (ASCL1), which is a key component of lung neuroendocrine cells.ASCL1 is required for normal development. It has been identified as the oncogenic driver in approximately 60% of small cell lung cancers

[11] . In addition, the Notch signaling pathway in SCLC is known to lead to the upregulation of pro-oncogenic processes, including chemotherapy resistance, differentiation and proliferation [2]. DLL3 is an atypical ligand of the Notch receptor, which is overexpressed on the surface of cancer cells in 80% of SCLCs, but is expressed in limited or no way in normal lung tissue [7]. DLL3 blocks cell-cell interactions and triggers Notch internalization by binding to Notch in a cis-regulatory manner [8, 9]. The selective expression profile of DLL3 in neuroendocrine tumors and its low cytoplasmic expression in some normal tissues make it a promising therapeutic target for treating a variety of cancer types, including SCLC.

[0007] Several immunotherapies targeting DLL3 for advanced SCLC are under development, including bispecific antibodies (BsAbs), chimeric antigen receptor T cells (CAR-T), and antibody-drug conjugates (ADCs). Talatumab is a first-in-class, long-lasting bispecific T-cell connective (HLE BiTE) that binds to DLL3 and CD3 to induce T-cell-mediated lysis [13, 14]. Talatumab was the first DLL3-targeted therapy to be clinically evaluated in SCLC, demonstrating manageable safety in a Phase I study and promising activity in previously treated SCLC patients. However, larger, randomized studies with longer follow-up are needed to precisely determine safety and efficacy

[13] . Nevertheless, talatumab (IMDELLTRA™, Amgen) received accelerated approval in 2024 for ES-SCLC. A CAR-T, AMG119 (Amgen), has also shown potent and durable antitumor activity

[15] . Teslin-lovatozumab (ROVA-T) is a first-in-class antibody-drug conjugate (ADC) from Abbvie / StemCentRx. ROVA-T has shown regression of SCLC; however, clinical activity is modest, and overall survival has not exceeded that of the current standard treatment, topotecan

[15] . For these reasons, development of ROVA-T has been terminated. Although most anti-DLL3 antibody-based therapies or cell therapies have shown promising preclinical and clinical data, no ADC has yet been approved for clinical use

[10] . Therefore, there is still a need to develop effective therapeutic agents targeting neuroendocrine tumors expressing DLL3, including ES-SCLC. Summary of the Invention

[0008] The following summary of the invention is illustrative only and is not intended to be limiting in any way. Other aspects, embodiments, and features will become apparent from the accompanying drawings and the following detailed description, in addition to the illustrative aspects, embodiments, and features described above.

[0009] On one hand, this application provides a delta-like ligand 3 (DLL3) binding peptide. In one embodiment, the binding peptide has binding specificity for human DLL3. In one embodiment, the DLL-binding peptide comprises a variable heavy chain (VH) chain and a variable light chain (VL) chain.

[0010] In one embodiment, the VH chain comprises: CDR H1 with the amino acid sequence SEQ ID: 104 (TYYMT), CDR H2 with the amino acid sequence SEQ ID: 105 (VIYASGGTYYATWAKG), and CDR H3 with the amino acid sequence SEQ ID: 106 (AYPDNGDGLDI); CDR H1 with the amino acid sequence SEQ ID: 110 (RNVIN), CDR H2 with the amino acid sequence SEQ ID: 111 (IIATAGDTYYANWAKG), and CDR H3 with the amino acid sequence SEQ ID: 112 (KYGDTFDL); CDR H1 with the amino acid sequence SEQ ID: 116 (SHYFN), CDR H2 with the amino acid sequence SEQ ID: 117 (IVYASGSTYYASWAKG), and CDR H3 with the amino acid sequence SEQ ID: 118 (DRSVAYSNI); or CDR H3 with the amino acid sequence SEQ ID: 120 (TYGMT). H1, CDR with amino acid sequence SEQ ID: 105 (VIYASGGTYYATWAKG), H2, and CDR with amino acid sequence SEQ ID: 119 (AYPDSGDGLDI).

[0011] In one embodiment, the VL chain comprises: CDR L1 with the amino acid sequence SEQ ID: 101 (QASEDISGWLA), CDR L2 with the amino acid sequence SEQ ID: 102 (WASNLAS), and CDR L3 with the amino acid sequence SEQ ID: 103 (QSTFYGTSDVAA); CDR L1 with the amino acid sequence SEQ ID: 107 (QASQSISSYLS), CDR L2 with the amino acid sequence SEQ ID: 108 (QASTLAS), and CDR L3 with the amino acid sequence SEQ ID: 109 (QGYDSNSVENA); CDR L1 with the amino acid sequence SEQ ID: 113 (QASQSIGGNLA), CDR L2 with the amino acid sequence SEQ ID: 114 (SASKLAS), and CDR L3 with the amino acid sequence SEQ ID: 115 (QQAWSYSNVDNT); or the amino acid sequence SEQ ID: SEQ ID: 103 (QASQSIGGNLA), CDR L2 with the amino acid sequence SEQ ID: 102 (WASNLAS), and CDR L3 with the amino acid sequence SEQ ID: 103 (QSTFYGTSDVAA); or CDR L3 with the amino acid sequence SEQ ID: SEQ ID: 103 (QASQSISSYLS), CDR L3 with the amino acid sequence SEQ ID: 104 (QASQSIGGNLA), CDR L2 with the amino acid sequence SEQ ID: ... The CDR L1 and amino acid sequence of ID: 101 (QASEDISGWLA) is SEQ ID NO.CDR L2 of SEQ ID: 102 (WASNLAS) and CDR L3 of SEQ ID: 121 (QSTFGGTSDVAA).

[0012] In one embodiment, the VH chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 17, 19, 21, 23, 25, 27, 28 or 29. In one embodiment, the VL chain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 16, 18, 20, 22, 24, 26 or 30.

[0013] In one embodiment, the amino acid sequences of the VH and VL chains are selected from SEQ ID NO: 17 and 16; 19 and 18; 21 and 20; 23 and 22; 25 and 24; 27 and 26; 28 and 22; 29 and 22; 29 and 30; or 28 and 30.

[0014] In one embodiment, the DLL3-binding peptide comprises an scFv domain. In one embodiment, the scFv domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32. In one embodiment, the DLL3-binding peptide comprises a histidine residue connected to at least one end of the scFv domain.

[0015] In one embodiment, the DLL3-binding peptide comprises a Fab domain. In one embodiment, the Fc domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 31, 32, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47.

[0016] In one embodiment, the DLL3-binding peptide further comprises an Fc domain linked to a Fab domain to provide a Fab-monoFc fusion protein.

[0017] In one embodiment, the DLL3-binding peptide comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29. In one embodiment, the DLL3-binding peptide comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 30, 31, 32, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46.

[0018] In one aspect, this application provides an antibody or its binding domain having binding specificity to human DLL3. This structureThe domain may be an scFv domain or a Fab domain. In some embodiments, the antibody or binding domain may include the DLL3 binding peptide disclosed herein.

[0019] In one embodiment, the antibody comprises a VH chain and a VL chain. In one embodiment, the VH chain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 17, 19, 21, 23, 25, 27, 28, or 29. In one embodiment, the VL chain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16, 18, 20, 22, 24, 26, or 30.

[0020] In one embodiment, the antibody comprises a heavy chain (HC) and a light chain (LC). In one embodiment, HC comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 37, 39, 41, 43, 45, or 31. In one embodiment, LC comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 32, 38, 40, 42, 44, or 46.

[0021] In one embodiment, the antibody comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0022] In one embodiment, the antibody includes a monospecific antibody, a bispecific antibody, a multispecific antibody, a purified antibody, a humanized antibody, IgG, or a combination thereof.

[0023] In one aspect, this application provides one or more isolated nucleic acid sequences encoding the antibody, binding domain, and DLL3-binding peptide disclosed herein. Specification 3 / 37 pages 7 CN 122374334 A

[0024] In one aspect, this application provides an expression vector or host cell comprising an isolated nucleic acid sequence encoding the antibody, binding domain, and DLL3-binding peptide disclosed herein.

[0025] In one aspect, this application provides an immunoconjugate. In one embodiment, the immunoconjugate comprises an antibody or DLL3-binding peptide conjugated to a pharmaceutical unit via a linker. Example linkers that can be used for such conjugation include, but are not limited to, covalent bonds selected from ester bonds, ether bonds, amine bonds, amide bonds, disulfide bonds, imide bonds, sulfone bonds, phosphate bonds, phosphate ester bonds, peptide bonds, hydrazone bonds, or combinations thereof.

[0026] In one embodiment, the pharmaceutical unit comprises a unit derived from a therapeutic agent, an imaging agent, a diagnostic agent, a radioisotope, or a combination thereof.

[0027] In one embodiment, the therapeutic agent comprises a cytotoxic agent, a chemotherapeutic agent, an enzyme, an anti-estrogenic agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA, or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof.

[0028] In one embodiment, the therapeutic agent comprises capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, nitrogen mustard, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, leucovorin, gemcitabine, oxaliplatin, irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testrolide, vorazole, and thiamethoxam. Tanshinone, faldazole, erlotinib, lafatinib, dasatinib, gefitinib, osimertinib, vandetanib, afatinib, imatinib, pazopanib, lapatinib, sunitinib, nilotinib, sorafenib, albumin-bound paclitaxel, everolimus, sirolimus esterified, dabrafenib, vemurafenib, trametinib, vintafolide, apatinib, crizotinib, perifolfen, olaparib, bortezomib, tofacitinib, trastuzumab, or derivatives or combinations thereof.

[0029] In one embodiment, the radioisotope includes deuterium, 131I, 32P, 90Sr, 90Y, 89Zr, 177Lu, or combinations thereof.

[0030] In one aspect, this application provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises an antibody or a DLL3-binding peptide. In one embodiment, the pharmaceutical composition comprises an immunoconjugate. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0031] In one aspect, this application provides a method for generating the antibody of claim 8, comprising culturing host cells such that a DNA sequence encoding the antibody of claim 8 is expressed, and purifying the antibody.

[0032] In one aspect, this application provides a method for generating the immunoconjugate of claim 15, comprising conjugating the antibody of claim 7 to a pharmaceutical unit.

[0033] In one aspect, this application provides a method for treating or preventing a subject from cancer, an autoimmune disease, or an infectious disease. In one embodiment, the method comprises administering an effective amount of an antibody or a DLL3-binding peptide to the subject. In one embodiment, the method comprises administering an effective amount of an immunoconjugate to the subject.

[0034] The cancer can be any cancer expressing DLL3. In one embodiment, the cancer includes lung cancer, small cell lung cancer.(SCLC), large cell neuroendocrine carcinoma (LCNEC), medullary thyroid carcinoma (MTC), cervical neuroendocrine carcinoma (CNEC), gastrointestinal pancreatic neuroendocrine tumor (GEP-NEN), bladder cancer, bladder NEN (BMEN), prostate cancer, NE prostate cancer (NEPC), Merkel cell carcinoma (MCC), breast cancer, liver cancer, hepatocellular carcinoma (HCC), glioma, pancreatic cancer, or combinations thereof.

[0035] In one embodiment, the method further includes co-administering an effective amount of the therapeutic agent. In one embodiment, the therapeutic agent includes antibodies, chemotherapeutic agents, enzymes, anti-estrogens, receptor tyrosine kinase inhibitors, kinase inhibitors, cell cycle inhibitors, DNA, RNA, or protein synthesis inhibitors, RAS inhibitors, or combinations thereof. In one embodiment, the therapeutic agent includes the product described on page 4 / 37 of the specification, CN 122374334 A. Capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, nitrogen mustard, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, leucovorin, gemcitabine, oxaliplatin, irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen Letrozole, exemestane, anastrozole, aminoglutethimide, testosterone, vorazole, formestane, fadroxil, erlotinib, lafatinib, dasatinib, gefitinib, osimertinib, vandetanib, afatinib, imatinib, pazopanib, lapatinib, sunitinib, nilotinib, sorafenib, albumin-bound paclitaxel, everolimus, sirolimus esterified, dabrafenib, vemurafenib, trametinib, vinpocetide, apatinib, crizotinib, perifolfen, olaparib, bortezomib, tofatinib, trastuzumab, or derivatives or combinations thereof.

[0036] In one embodiment, the subject is a human.

[0037] In one aspect, this application provides a solution containing an effective concentration of an antibody, DLL3-binding peptide, or immunoconjugate. In one embodiment, the solution is the plasma of a subject.

[0038] The foregoing and other features of this disclosure will become more apparent from the accompanying drawings, the following description, and the appended claims. It should be understood that these drawings depict only a few embodiments arranged according to this disclosure and should not be considered as limiting its scope. Further specific and detailed description of the disclosure will be given below using the drawings, in which: Figure 1 shows the results of a direct sandwich ELISA used for screening and identifying anti-DLL3 antibodies; Figure 2 depicts FACS results showing the cross-species binding of the chimeric antibody to CHO cells transfected with human and cynomolgus monkey DLL3 compared to the background binding of the negative control; Figure 3 shows the biolayer interference affinity of the chimeric antibody binding with recombinant human and recombinant cynomolgus monkey DLL3 with high affinity, with KD values ​​ranging from 0.1 to 1.Within the nM range; Figure 4 shows the OCTET results, indicating that, as detected by the biolayer interferometry, the humanized anti-DLL3 clone 5018A1 GS binds to recombinant mouse and rat DLL3 with high affinity across species, with a KD value less than 1 nM; Figure 5 shows the OCTET results, indicating that the humanized anti-DLL3 clones SI-83M10 (5018A1 GS VH-N99S) and SI-83M8 (5029C10 FRS) bind to human DLL3 with high affinity and cohesion across species, with a KD value within the picomolar range; Figure 6 shows the flow cytometry histogram of the screened purified antibodies, indicating that the humanized anti-DLL3 clones SI-83M10 (5018A1 GS VH-N99S) and SI-83M8 (5029C10 FRS) bind to human DLL3 with high affinity and cohesion across species, with a KD value within the picomolar range; FRS) and anti-DLL3 reference antibodies SI-83C1 (lovatozumab) and SI-83C2 (talatumab) were used to stain the surface and intracellular cells of human cancer cell lines transiently transfected with a eukaryotic expression vector encoding the human DLL3-EpCAM fusion construct (SEQ ID NO: 33). These human cancer cell lines included (6A) ExpiCHO cells, (6B) CORL279, (6C) NCI-H82, and (6D) MIA PaCa-2, where black and gray lines represent stained and unstained cells, respectively. Figure 7 depicts the hierarchical clustering results of the antibodies (5029C10, 5018A1, and 5257C8) and positive control antibodies (SI-83C1 and SI-83C2) disclosed in the cluster matrix table, which shows that 5018A1 recognizes a novel epitope. Figure 8 depicts the anti-DLL3 antibody SI-83M10 (also known as 5018A1 GS). Antibody-dependent cell-mediated cytotoxicity (ADCC) of VH-N99S and controls was performed using 3-fold serial dilutions ranging from 33 nM to 0.04 nM. Controls included buffer only, anti-DLL3 reference antibodies (i.e., SI-83C1 and lovatozumab), rituximab (i.e., anti-CD20 antibody), and cetuximab (i.e., anti-EGFR antibody). The experiments used NK effector cells and riboglobin (NR)-labeled target cells: (8A) Burkitt lymphoma cells expressing DLL3 (Daudi-NR), (8B) small cell lung cancer cells (COR-L279), (8C) CHO cells, and (8D) CHO cells expressing DLL3. Figure 9 depicts the internalization and lysosomal transport of the anti-DLL3 antibody. The experiments were performed using time-series live-cell fluorescence microscopy quantitative red calibration units (RCU), which represent a concentration of 10... nM anti-DLL3 antibody (SI-83M10, 5018A1 GS VH-N99S), anti-DLL3 reference antibody (SI-83C1, lovatozumab), and anti-CD20 antibody (rituximab) were used in (9A) COR-L279 cells, a small cell lung cancer cell line expressing DLL3; (9B) SHP-77 cells, an epithelial cell line derived from non-encapsulated primary lung tumors expressing DLL3; and (9C) internalization and transport signals in NUGC-4, a DLL3-negative gastric cancer cell line. The results showed that SI-83M10 had higher internalization efficiency and more signals for transport to lysosomes (indicating cytotoxic load in the case of ADC).

[0039] Figure 10 shows the heavy chain (10A) and light chain (10B) sequence alignments of the lead and reference anti-DLL3 antibodies, including SI-83M10, SI-83M8, 5257C8 BSM, FZ-AD005, GenSun mAb, Gocatamig, Obrixtamig, PT217, lovatozumab (SI-83C1), and talatumab (SI-83C2); and Figure 11 depicts the sequence similarity between SI-83M10, SI-83M8, or 5257C8 BSM and the average of the remaining reference antibodies, showing the percentage of identity for CDR (11A), frame-only region (11B), and variable region (CDR + frame region) (11C). Detailed Description

[0040] In the following detailed description, reference is made to the accompanying drawings, which form a part thereof. In the drawings, similar symbols generally identify similar components unless the context otherwise requires. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments and changes may be utilized without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that various aspects of this disclosure (as shown in the general description and drawings herein) can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0041] This disclosure provides, among other things, peptides, proteins (including, for example, antibody-like proteins), isolated antibodies or their binding fragments; methods for preparing such peptides, proteins, or antibodies; monoclonal and / or recombinant monospecific antibodies or their binding fragments, multispecific antibodies or their binding fragments; peptide-drug conjugates, protein-drug conjugates, antibody-drug conjugates, and / or immunoconjugates composed of such peptides, proteins, antibodies, or antigen-binding fragments; pharmaceutical compositions containing peptides, proteins, antibodies, monoclonal and / or recombinant monospecific antibodies or their binding fragments, multispecific antibodies or their binding fragments, peptide-drug conjugates, protein-drug conjugates, antibody-drug conjugates, and / or immunoconjugates; methods for preparing peptides, proteins, antibodies, and compositions; and methods for treating cancer using the peptides, proteins, antibodies, and compositions disclosed herein.

[0042] As used herein, the terms “a,” “an,” and “the” are defined to mean “one or more” and include the plural unless the context is inappropriate.

[0043] As used herein, the terms “polypeptide,” “peptide,” and “protein” are interchangeable and are defined to mean a biomolecule composed of amino acids linked by peptide bonds.

[0044] The term “antigen” refers to an entity or fragment thereof that can induce an immune response in an organism, particularly animals, and more particularly mammals, including humans. This term includes immunogens and the regions responsible for antigenicity or antigenic determinants.

[0045] The terms “antigen or epitope binding portion or fragment,” “variable region,” “variable region sequence,” or “binding domain” refer to antibody fragments capable of binding to antigens (such as EGFR in this application). An antigen-binding fragment (Fab) is a region (Fab region) on an antibody that binds to an antigen. These fragments may have the full antigen-binding function of the antibody and additional functions. Examples of conjugated fragments include, but are not limited to, single-chain Fv fragments (scFv), which consist of variable light chain (VL) and variable heavy chain (VH) domains of an antibody single arm linked to a single polypeptide chain via synthetic linkers; or Fab fragments, which are monovalent fragments consisting of VL, constant light chain (CL), VH, and constant heavy chain 1 (CH1) domains.

[0046] Antibody fragments can even be smaller subfractions and can consist of domains as small as a single CDR domain, particularly the CDR3 region from the VL and / or VH domains (see, for example, Beiboer et al., J. Mol. Biol. 296:833-49 (2000)). Antibody fragments are generated using conventional methods known to those skilled in the art. The utility of antibody fragments can be screened using the same techniques employed with whole antibodies.

[0047] The terms “antigen or epitope binding portion or fragment,” “variable region,” “variable region sequence,” or “binding domain” can be obtained from the antibodies of this disclosure using a variety of techniques known in the art. For example, purified monoclonal antibodies can be lysed with an enzyme such as pepsin and subjected to HPLC gel filtration. Papain digestion of the antibody produces two identical antigen-binding fragments (referred to as “Fab” fragments) and a residual “Fc” fragment, each Fab fragment having a single antigen-binding site, the name Fc fragment reflecting its ease of crystallization. Pepsin treatment produces an F(ab’)2 fragment having two antigen-binding sites and still capable of cross-linking the antigen. The appropriate fraction containing the Fab fragments can then be collected and concentrated by membrane filtration, etc. For a further description of general techniques for isolating active antibody fragments, see, for example, Khaw, BA et al., J. Nucl. Med. 23:1011-1019.(1982); Rousseaux et al., Methods Enzymology, 121:663-69, Academic Press, 1986.

[0048] The term “antibody” is used in the broadest sense, specifically covering single monoclonal antibodies and / or recombinant antibodies (including agonist and antagonist antibodies), antibody compositions having multi-epitope specificity, and antibody fragments (e.g., Fab, F(ab')2, and Fv), provided they exhibit the desired biological activity. In some embodiments, antibodies may be monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single-chain antibodies, multispecific or pleiotropic antibodies, human antibodies, and humanized antibodies, as well as their active fragments. Examples of molecularly active fragments that bind to known antigens include Fab, F(ab')2, scFv, and Fv fragments, including products of Fab immunoglobulin expression libraries and epitope-binding fragments of any of the antibodies and fragments described above.

[0049] The term “Fv” refers to the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a tightly, non-covalently associated dimer of one heavy chain variable domain and one light chain variable domain. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. The six CDRs collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific CDRs) can recognize and bind antigens, although with less affinity than the entire binding site.

[0050] In some embodiments, the antibody may include an immunoglobulin molecule and the immunoactive portion of an immunoglobulin molecule, i.e., a molecule containing a binding site and immune-specifically binding antigens. A typical antibody is a heterotetrameric protein that typically comprises two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain consists of a light chain variable domain (abbreviated as VL) and a light chain constant domain. Based on the amino acid sequence of constant domains, the light chains of antibodies (immunoglobulins) from any vertebrate species can be divided into one of two distinct types, called κ and λ. The VH and VL regions can be further subdivided into highly variable complementarity-determining region (CDR) domains and more conserved regions called framework regions (FRs). Each variable domain (VH or VL) typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Binding regions that interact with antigens exist within the variable regions of both the light and heavy chains.

[0051] Immunoglobulins can be classified into different categories based on the amino acid sequence of the heavy chain constant domain.There are five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0052] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for the possible small number of naturally occurring mutations. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations that typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized from hybridoma cultures and are not contaminated by other immunoglobulins. The modifier “monoclonal” indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to this disclosure can be prepared by the hybridoma method first described in Kohler & Milstein, Nature, 256:495 (1975), or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). “Recombinant” means that the antibody is generated in a foreign host cell using recombinant nucleic acid technology.

[0053] Monoclonal antibodies can be produced by a variety of methods, including but not limited to mouse hybridoma, phage display, recombinant DNA, molecular cloning of antibodies directly from primary B cells, and antibody discovery methods (see Siegel. Transfus. Clin. Biol. 2002; Tiller. New Biotechnol. 2011; Seeber et al., PLOS One. 2014). Monoclonal antibodies may include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, provided they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855

[1984] ).

[0054] The term “humanized antibody” refers to a class of engineered antibodies having a CDR derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portion of the molecule derived from one (or more) human immunoglobulins. Additionally, the framework region support residues may be altered to maintain binding affinity. Methods for obtaining “humanized antibodies” are well known to those skilled in the art (see, for example, Queen et al., Proc. Natl Acad Sci USA, 86:10029–10032 (1989); Hodgson et al., Bio / Technology, 9:421 (1991)). Humanization of antibodies found in non-human species is a routine practice that not only reduces immunogenicity but also improves stability and removes sequence defects.

[0055] The terms “isolated” or “purified” mean that a biomolecule does not contain at least some of its naturally occurring components. When used to describe the various polypeptides disclosed herein, “isolated” or “purified” means a polypeptide that has been identified and isolated and / or recovered from the cells or cell cultures in which it is expressed. Typically, purified peptides are prepared through at least one purification step. "Isolated" or "purified" antibodies refer to antibodies that are substantially free of other antibodies with different antigen-binding specificities.

[0056] The term "immunogenic" refers to a substance that induces or enhances the production of antibodies, T cells, or other reactive immune cells against an immunogenic agent and contributes to an immune response in a human or animal. An immune response occurs when an individual produces sufficient antibodies, T cells, and other reactive immune cells in response to the administered immunogenic composition of this disclosure to reduce or alleviate the condition to be treated. Although an immunogenic response typically includes both cellular (T cell) and humoral (antibody) arms of the immune response, antibodies against therapeutic proteins (antidrug antibodies, ADA) may consist of IgM, IgG, IgE, and / or IgA isotypes.

[0057] The terms "specific binding," "specifically bound to," or "specific to a particular antigen or epitope" mean that the binding is measurably different from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule to a control molecule (which is typically a molecule with a similar structure that does not have binding activity). For example, specific binding can be determined by competition with a control molecule similar to the target.

[0058] The term "affinity" refers to a measure of the attractive force between two peptides, such as antibody / antigen, receptor / ligand, etc. The inherent attractive force between two peptides can be expressed as the binding affinity equilibrium dissociation constant (KD) of a particular interaction. The KD binding affinity constant can be measured, for example, by biolayer interferometry, where KD is the ratio of kdis (dissociation rate constant) to kon (association rate constant), such as KD = kdis / kon. Specification 8 / 37 pages 12 CN 122374334 A

[0059] Specific binding to a particular antigen or epitope can be manifested, for example, by antibodies with a KD of at least about 10⁻⁴ M, at least about 10⁻⁵ M, at least about 10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, at least about 10⁻⁹ M, or alternatively at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, at least about 10⁻¹² M, or greater, where KD refers to the equilibrium dissociation constant of the specific antibody-antigen interaction. Typically, the KD of an antibody that specifically binds to an antigen is 20, 50, 100, 500, 1000, 5000, 10000, or more times greater than that of the control molecule relative to the antigen or epitope.

[0060] Furthermore, specific binding to a particular antigen or epitope can be manifested, for example, by an antibody whose KA or Ka is at least 20, 50, 100, 500, 1000, 5000, 10000 or more of the epitope relative to the control, wherein KA or Ka refers to the association rate of a specific antibody-antigen interaction.

[0061] This disclosure can be more readily understood by referring to the following detailed description of specific embodiments and examples included herein. Although specific details of this disclosure have been described with reference to certain embodiments, such details are not intended to be construed as limiting the scope of this disclosure.

[0062] Examples Example 1. Generation of anti-DLL3 antibody Rabbit antibody immunization For immunization, New Zealand white rabbits were divided into two groups (see Tables 1A and 1A). Group 1 received 100 μg of DLL3-His protein (catalog number DL3-H52H4) formulated with Freund's complete adjuvant from Acro for primary immunization. Rabbits were then given four booster immunizations at 7-day intervals, consisting of one 50 μg immunization and two 25 μg immunizations, with Freund's incomplete adjuvant and Alum CpG2007 alternating as adjuvants. Following the fourth immunization, rabbits received three booster immunizations at 21-day intervals with 25 μg protein, with Freund's incomplete adjuvant and Alum CpG2007 alternating as adjuvants. Twenty-eight days after the third booster immunization, another round of booster immunizations at 21-day intervals began, consisting of three 25 μg booster immunizations and two 10 μg booster immunizations, with Freund's incomplete adjuvant and Alum CpG2007 alternating as adjuvants. Sixty-three days after the final booster immunization, another round of booster immunizations at 21-day intervals began. These booster immunizations followed the following strategy: one 10 μg immunization and four 5 μg immunizations, with Freund's incomplete adjuvant and Alum CpG2007 alternating as adjuvants. The second group used the same immunization strategy, except that the rabbits received DLL3-Fc protein (catalog number DL3-H5255) purchased from Acro. For both groups, peripheral whole blood was collected from day 28 to day 329 (day 98, ...).No blood was collected on days 245 and 308), and PBMCs were isolated and stored in liquid nitrogen for subsequent sorting.

[0063] PBMC isolation: Blood diluted with DPBS at a 1:1 ratio was spread onto the top layer of 15 mL Lympholyte cell separation medium (Cedarlane, catalog number CL5050) in a 50 mL centrifuge tube. To obtain the PBMC layer, the centrifuge tube was centrifuged at 2500 rpm for 30 minutes at room temperature without braking. The white PBMC layer was then carefully removed and placed in a clean 50 mL centrifuge tube, diluted with DPBS to 50 mL. The tube was then centrifuged at 2500 rpm for 10 minutes at room temperature with braking to allow the cells to precipitate. Any remaining red blood cells (RBCs) were then lysed with RBC lysis buffer (Qiagen, catalog number 158904). In short, the cells were resuspended in 10 mL of RBC lysis buffer and incubated at room temperature for 5 minutes. The cells were then neutralized and lysed with 40 mL of DPBS. Centrifuge at 2500 rpm for 10 minutes at room temperature to reprecipitate the cells. Then resuspend the PBMCs in FBS containing 10% DMSO for storage, with a final concentration ≤5×10^7 cells / mL.

[0064] FACS isolation of rabbit B cells Rabbit PBMCs were revived in immunocellular culture medium (ICM). Once revved, the cells were stained in an antibody staining mixture containing specific rabbit B cell markers. B cells were enriched from the revved PBMCs by magnetically activated cell sorting (MACS) using a QuadroMACs magnetic column separator (Miltenyi Biotec, catalog number 130-090-976). Instructions 9 / 37, page 13, CN 122374334 A. After MACS separation, cells were stained with live / dead cell efluor780 active dye (ebioscience, catalog number 65-0865-14), washed, resuspended in buffer, and kept at 4°C until cells were sorted by fluorescence activated cell sorting (FACS).

[0065] Before sorting B cells, appropriate compensation was made through sorter software and manual adjustment. To obtain live IgG positive rabbit B cells, the sorting gate was manually adjusted. Single B cells were sorted from the final sorting gate into 96-well plates containing pre-prepared B cell culture medium containing ICM, rabbit spleen cell conditioned medium, and feeder cells. A total of 7 rounds of sorting were completed, with a maximum of 33 plates sorted. Before screening, the plates were incubated at 37°C and 5% CO2 for 12 days.

[0066] ELISA Screening Antibody: Before ELISA screening, B cell culture supernatant was harvested from each culture plate and stored at 4°C for screening. The remaining PBMCs in the culture plates were stored in RNA Later (Invitrogen, catalog number AM7021) at -80°C for use in...Subsequent RT-PCR amplification of the antibody variable region was performed. IgG secretion and DLL3 binding in B cell cultures were screened using a direct sandwich ELISA. Flat-bottom ELISA plates were coated with 1 μg / mL anti-rabbit IgG Fc fragment (Jackson ImmunoResearch, catalog number 111-005-046) or human DLL3-His (Acro, catalog number DL3-H52H4) and incubated overnight at 4°C. The plates were then washed three times with 1X PBST and blocked with DPBS containing 2% BSA at room temperature for 1 hour. After blocking, the plates were washed as described above, and a 1:10 dilution (with DPBS containing 2% BSA) of B cell supernatant was added to each plate, and incubated at 4°C for 1 hour. After primary antibody incubation, the plates were washed again as described above. Then, add HRP-conjugated anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, catalog number 111-035-046) diluted 1:15,000 (with DPBS containing 2% BSA) and incubate at 4°C for 30 minutes. Wash the plate and detect the binding antibody using TMB substrate (ThermoFisher Scientific, catalog number SS04). Incubate the plate at room temperature for 10 minutes and terminate the reaction with commercially available stop solution (ThermoFisher Scientific, catalog number SS04). Read the plate at 450 nm using a spectrophotometer. Based on the OD value being higher than that of the negative control with secondary antibody only, DLL3-specific rabbit IgG antibody was detected.

[0067] The results of the direct sandwich ELISA used for screening and identification of anti-DLL3 antibodies are shown in Figure 1. For sorting #1, 11.1% of the sorted B cells were IgG positive. A total of 2 clones bound DLL3-His. For sorting #2, 16.7% of the sorted B cells were IgG positive. A total of 2 clones bound DLL3-His. For sorting #3, a total of 4 clones bound DLL3-His. For sorting #6, 10.1% of the sorted B cells were IgG positive. A total of 6 clones bound DLL3-His. All clones that bound DLL3-His protein were used for amplification of the antibody variable region to identify heavy and light chain sequences.

[0068] Example 2. Amplification of the variable region of rabbit / human chimeric monoclonal antibody B cell clones obtained by ELISA screening were taken out and thawed from storage conditions at -80°C. Heavy and light chain variable sequences were amplified by multiplex RT-PCR using leader sequences and specific primers for the constant region of rabbit IgG and rabbit κ sequences. In the secondary PCR, nested primers containing restriction sites were used to further amplify the amplicons. The heavy and light chain amplicons were cloned into expression vectors containing human IgG1 or human IgK, respectively. Transiently co-transfect the sequencing-validated expression plasmid into...In 293 HEK cells, rabbit / human chimeric antibodies were generated.

[0069] Characterization of chimeric anti-DLL3 antibodies—Flow cytometry: The binding of supernatant containing recombinant antibodies to surface-expressed DLL3 was screened by flow cytometry. Under normal circumstances, DLL3 is located in the Golgi apparatus within the cell. To facilitate expression on the surface of transfected cells, a DLL3-EpCAM fusion construct was used. This DLL3-EpCAM fusion construct contains the extracellular domain of DLL3; however, the transmembrane domain of DLL3 is replaced by the membrane domain of EpCAM. The eukaryotic expression vector encoding the human or cynomolgus monkey DLL3-EpCAM fusion construct (SEQ ID NO: 33-34) was transiently transfected into ExpiCHO cells. ExpiCHO cells transfected with unrelated DNA were used as a negative control. Two days after transfection, cells were collected (see page 14 of the instruction manual, CN 122374334 A) and stained with a cell-tracking dye (ThermoFisher Scientific, catalog number C34565). Cells were then seeded at approximately 1.5 × 10⁵ cells / well. Cells were stained with a chimeric antibody serially diluted from 10 μg / mL to 40 ng / mL at 4°C for 1 hour. Cells were washed and then stained with PE-conjugated anti-rabbit IgG Fc secondary antibody (Jackson ImmunoResearch, catalog number 111-605-008) at 4°C for 30 minutes. Cells were washed, resuspended in FACS buffer, and binding was determined by flow cytometry using a BD Fortessa flow cytometer. Clones expressing antibodies that bind to human and cynomolgus monkey DLL3 were selected for further screening. For example, 5018A1 and 5029C10 are exemplary antibodies that can bind to CHO cells transfected with human and cynomolgus monkey DLL3, but not to negative controls (see Figure 2).

[0070] Characterization of chimeric anti-DLL3 antibodies—OCTET affinity was verified using a ForteBio Octet Red 384 instrument via biolayer interferometry (also referred to as OCTET in the patent) to verify the binding of the supernatant containing the recombinant antibody to DLL3. The antibody was diluted to 10 μg / mL and captured onto an anti-human Fc biosensor. The antibody-coated sensor was bound to the extracellular domain of recombinant human DLL3 (Acro Biosystems, catalog number DL3-H52H4), which was initially diluted at a concentration of 300 nM in a 1:3 gradient. After establishing a baseline in kinetic buffer, the dissociation of DLL3 from the antibody was measured by biolayer interferometry. Curve fitting was performed using the manufacturer's software, and affinity was calculated. The biolayer interference affinity results of the disclosed antibodies indicate that the chimeric antibodies bind to recombinant human DLL3 with high affinity, with KD values ​​ranging from 0.1 to 1.Within the nM range (Figure 3). All antibodies bound recombinant human and cynomolgus monkey DLL3.

[0071] Cross-species binding with mouse and rat DLL3 was performed using the method described in the previous embodiment, and the cross-species binding of humanized anti-DLL3 clone 5018A1 GS with mouse and rat DLL3 was analyzed by OCTET. As shown in Figure 4, by biolayer interference method, humanized 5018A1 GS bound recombinant mouse and rat DLL3 with high affinity, and its KD value was less than 1 nM. However, after affinity modification, the OCTET response value and affinity decreased.

[0072] Example 3. Humanization of the variable regions of the heavy and light chains of humanized anti-DLL3 antibody The "Predict Humanizing Mutations" algorithm in Discovery Studio was used to humanize the variable regions of the heavy and light chains of the antibody selected as the lead antibody. The selected humanization methods included best single mutation (BSM), high frequency residue substitution (FRS), or germline transplantation (GS). For 5029C10, a computational extrapolation method was used to obtain the humanized sequence. Using Genewiz, the DNA sequence encoding the VH and VL regions of the humanized antibody was cloned into a mammalian expression vector containing the human IgG1 / κ constant region. Sequence analysis revealed the presence of an NG motif in the heavy chain CDR-H3 of clone 5018A1. To eliminate the risk of deamidation, a series of mutants were generated targeting asparagine or glycine residues, which had no significant effect on expression, aggregation, or binding. Clone 5018A1 was further engineered to modify affinity. Mutations were introduced into the heavy chain and / or light chain CDRs to obtain lower affinity. The characteristic profiles of the engineered clones are shown in Table 2A, including ExpiCHO transfection titers and the percentage of target protein after purification (% POI).

[0073] Antibody clustering was performed using a ForteBio OCTET Red 384 instrument, and the epitopes of the antibodies were clustered by the biological layer interference (i.e., OCTET) tandem assay. Biotinylated recombinant human DLL3 full-length protein (Acro Biosystems, catalog number DL3-H82E4, 10 μg / mL) was captured onto a streptavidin-conjugated biosensor and then bound to 10 μg / mL of antibody (i.e., first binding). The sensor was then moved to wells containing different antibodies (i.e., second binding). Using instrument software, antibodies were clustered based on hierarchical clustering of response values ​​from the first binding to the second binding. To determine blocking or non-blocking antibody pairs, a threshold was set with an appropriate self-binding signal. Antibodies that blocked binding were clustered into the same group and considered to have similar epitopes. Antibodies that did not block binding were clustered into separate groups and considered to have different epitopes.

[0074] (Instructions for Use, 11 / 37 pages, 15 CN 122374334 A)OCTET affinity was characterized using humanized antibody supernatant. The binding affinity of engineered clones is summarized in Table 2B. High binding affinity of the humanized clone supernatant was detected by biolayer interferometry, ranging from 0.2 to 4 nM. The N99S mutation of 5018A1 did not significantly alter the affinity. Further mutation of 5018A1 to modify affinity successfully reduced the binding affinity from 0.5 nM to 30 nM.

[0075] OCTET affinity biolayer interferometry was also used to evaluate the affinity of engineered antibodies for recombinant human DLL3. Biotinylated recombinant human DLL3 (10 μg / mL) was captured onto a streptavidin biosensor and then bound to an antibody diluted 1:3 at a starting concentration of 300 nM (Table 2C). The sensor was transferred to wells containing kinetic buffer to analyze the dissociation of the antibody / DLL3 complex. Curve fitting and affinity calculations were analyzed using software provided by the manufacturer.

[0076] After purification of the OCTET affinity and affinity protein A of the purified antibody, the binding affinity and affinity of the disclosed humanized antibody to human DLL3 were characterized by OCTET using the method described in the previous embodiment. The purified disclosed antibodies SI-83M10 (also referred to herein as 5018A1 GS VH-N99S) and SI-83M8 (also referred to herein as 5029C10 FRS) bound to human DLL3 with high affinity and affinity, with KD values ​​in the picomolar range (Figure 5). Table 2D summarizes the affinity of the lead clone to recombinant human DLL3 in the range of 0.2 to 12 nM. The high affinity detected by 5018A1-GS-N99S was not calculated because the value exceeded the limits of the program software. However, further engineering to modify the affinity reduced it to a detectable value of 0.5 nM. Mutations in the heavy and light chains further reduced the affinity to 153 nM.

[0077] Flow cytometry was used to detect the cell binding of the disclosed humanized antibody using the aforementioned flow cytometry method (Figure 2). This humanized antibody bound human and cynomolgus monkey DLL3-EpCAM on transiently transfected ExpiCHO cells. However, the N99S mutation in 5018A1 GS was insufficient to alter cell binding activity; additional mutations were required to modify affinity in order to successfully reduce binding activity. Binding was eliminated in 5018A1-N99S containing heavy and light chain affinity-modifying mutations, as shown in Table 2D.

[0078] Using CORL279 (small cell lung cancer), NCI-H82 (lung cancer), MIA Paca-2 (pancreatic cancer), and ExpiCHO cells transiently transfected with a eukaryotic expression vector encoding a human DLL3-EpCAM fusion construct (SEQ ID NO: 33), purified...The binding of the humanized antibody to human cancer cells was characterized (Figure 6). Due to the low surface expression and intracellular localization of DLL3, surface binding and intracellular binding were analyzed (Figure 6, top and bottom, respectively). As previously mentioned, ExpiCHO cells transiently transfected with DLL3-EpCAM were used as a positive control.

[0079] Flow cytometry surface staining was performed using the methods described in the preceding sections. For intracellular staining, cells were incubated with BD fixation buffer (BD, catalog number 554655) at 4°C for 1 hour. Cells were then washed twice with 1X BD permeabilization wash buffer (BD, catalog number 554723) and incubated at 4°C for 15 minutes in BD permeabilization wash buffer (BD, catalog number 554723). Cells were then centrifuged to pellet, diluted to approximately 1.5 × 10⁵ cells / well, and stained at 4°C for 1 hour with antibody serially diluted from 10 μg / mL to 40 ng / mL. After washing the cells, they were stained with FITC-conjugated anti-human IgG Fc secondary antibody (Jackson ImmunoResearch, catalog number 10-096-003) at 4°C for 30 minutes. After incubation, the cells were washed, resuspended in FACS buffer, and binding was measured by flow cytometry using a BD Fortessa flow cytometer. As expected, binding was detected on the surface of transiently transfected ExpiCHO cells (Fig. 6, top).

[0080] Intracellular staining resulted in a >10-fold increase in binding (Fig. 6, bottom). Very weak surface staining was detected for all three cell lines. However, after intracellular staining, a detectable shift in binding was detected compared to the unstained (i.e., secondary antibody only) control (Fig. 6B-6D). This is consistent with the low surface expression and high intracellular expression detected in DLL3-expressing cell lines. Further testing was conducted on the binding of reference clones SI-83C1 and SI-83C2, page 12 / 37 of the instruction manual, CN 122374334 A. Data showed that the disclosed clones SI-83M8 and SI-83M10 bound to a neuroendocrine tumor-derived cell model.

[0081] Example 4. Comparative Advantages of Anti-DLL3 Antibodies as Therapeutic Agent Candidates When targeting tumors with heterogeneous antigen expression, antibody-dependent cytotoxicity (ADCC) against tumors with low-density antigen expression is generally more effective with high-affinity antibodies. Conversely, high-affinity antibodies, especially those with slow dissociation rates, can induce unintended toxicity. For antibody-drug conjugates, reducing antibody affinity can optimize the therapeutic window. Furthermore, antibody binding sites can alter antibody internalization and downstream biological activity. Therefore, antibodies targeting novel epitopes with different binding properties can improve therapeutic efficacy.

[0082] The disclosed antibodies (5018A1, 5029C10, and 5257C8) are humanized monoclonal antibodies that bind to DLL3 with high affinity and have KD values ​​ranging from 0.2 to 4.Within the nM range. An affinity-modified antibody from parental clone 5018A1 is also disclosed. It includes a low-affinity variant with a KD of 30 nM and relatively weak binding. Binding affinity can significantly alter the efficacy and safety profile of therapeutic antibodies. Compared to the clinical antibodies talatumab (Amgen, also referred to as SI-83C1 in this paper) and tesculin-lovatuzumab (Abbvie / StemCentRx, also referred to as SI-83C2 in this paper), the affinity of the disclosed clones is within a similar range (Table 2B).

[0083] The hierarchical clustering results of the disclosed antibodies and positive control reference antibodies SI-83C1 (lovatuzumab) and SI-83C2 (talatumab) are shown in the cluster matrix table (see Figure 7). One clone 5257C8 is unidirectionally blocked by clone 5029C10, indicating that there is partial overlap of epitopes between the two (circled in Figure 7). The candidate antibodies neither blocked the binding of the reference antibody nor were blocked by the reference antibody, indicating that the epitopes bound by these antibodies are different from those of the antibodies currently under investigation.

[0084] However, the clustering results of the biolayer interferometry showed that the disclosed clones bind to a unique epitope of DLL3 (see clone 5018A1 in Figure 7).

[0085] Example 5. Antibody-dependent cell-mediated cytotoxicity can be compared in vitro with the Fc-mediated ADCC activity of antibody therapeutic agents to determine whether the antibody has the potential to enhance the mechanism of action of the therapeutic. In this test system, the low ADCC response may be due to the low level of expression of the bindable antigen during the assay. Therefore, different binding characteristics or antibody internalization may affect ADCC activity

[17] .

[0086] To test this, human primary NK (natural killer) cells were enriched from whole blood by negative screening and then subjected to density gradient centrifugation to make them “effective” cells. Cells used as target cells were transduced to express riboglobin (NR) and included Daudi (B lymphoblastic cell line of Burkitt lymphoma), COR-L279 (small cell lung cancer cell line), CHO cells transduced to express DLL3, and untransduced CHO cells. Target cells were treated with SI-83M10 (5018A1 GS VH N99S) and controls, including buffer only, lovatozumab (SI-83C1) as a reference antibody against DLL3, and rituximab (anti-CD20) and cetuximab (anti-EGFR) as control systems, all serially diluted 3-fold from 33 nM to 0.04 nM. NK cells were added at a 5:1 E:T (effective cell:target cell) ratio to assess ADCC over the assay period. ADCC was assessed using time-series fluorescence microscopy by observing the attenuation of NR signal in target cells.

[0087] As shown in Figure 8A, after 36 hours, CD20-specific rituximab was detected in the Daudi-NR target cell line.ADCC activity was demonstrated, proving the effectiveness of the assay system. As expected, no cytotoxic activity was observed in 5018A1 GS or lovatozumab. Alternatively, after 45 hours, ADCC activity of SI-83M10 was detected in the COR-L279-NR cell line expressing DLL3 (Fig. 8B). This activity was significantly higher than that of lovatozumab, demonstrating that the latter had no cytotoxic effect on COR-L279 target cells. In transduced CHO cells, as expected, no ADCC was detected in CHO-NR (Fig. 8C). However, after 60 hours, both SI-83M10 and lovatozumab showed significant cytotoxic activity against CHO-NR-DLL3. The system controls, cetuximab and lovatozumab, showed negative results, as expected (Fig. 8D). In summary, the data indicate that SI-83M10 exhibits enhanced ADCC activity in small cell lung cancer cell lines compared to lovatozumab.

[0088] Example 6. Internalization and Lysosomal Transport of Anti-DLL3 Antibodies For many antibody-drug conjugates (ADCs), a key step in the mechanism of action involves internalization

[18] . After antibody binding, internalization facilitates the uptake of cytotoxic loads and their transport to cancer cell lysosomes, which act as centers of material circulation within the cell. Cell lysis follows different mechanisms of action depending on the type of cytotoxic load. Furthermore, the mechanisms of internalization and transport to lysosomes of anti-DLL3 antibodies may be the same, but efficacy may depend on antibody binding.

[0089] Anti-DLL3 antibody SI-83M10, reference antibody lovatozumab, and experimental control rituximab were labeled with FabFluor-pH red fluorescent dye (an acid-sensitive fluorescent reagent for quantifying the transport of internalized proteins into acidic lysosomes). The fluorescence signal of FabFluor-pH red fluorescent dye is dependent on the low pH environment of the lysosome. In this way, the accumulation of internalized antibodies in the lysosomal compartments over time can be quantified using time-series fluorescence microscopy. The cell lines were revived and cultured in RPMI medium supplemented with penicillin / streptomycin (Pen / Strep) and 10% FBS. Cells were seeded at a density of 10,000 cells / well in 50 μL of culture medium in 96-well round-bottom ULA plates (Corning). FabFluor-pH labeled medium, anti-DLL3 antibody, and control were used according to the manufacturer's instructions. Add 50 μL of 20 nM FabFluor-pH labeled antibody or control solution to the plated cells. The final assay volume per well is 100 μL, and the final concentration is 10 nM. Incubate the cells in an Incucyte S3 live cell imaging system (Sartorius) at 37°C and 5% CO2 humidification for 20 hours.

[0090] Internalization and transport signals of SI-83M10 at a concentration of 10 nM were readily detected using time-series live-cell fluorescence microscopy (Fig. 9). Signals were measured in red calibration units (RCU), automatically counted, and plotted over time. In the DLL3-expressing small cell lung cancer cell line COR-L279, SI-83M10 showed higher internalization efficiency and more signals transported to lysosomes (indicating cytotoxic load in the ADC case) compared to lovastatinumab (Fig. 9A). In the DLL3-expressing epithelial cell line SHP-77, derived from non-encapsulated primary lung tumors, the internalization levels of lovastatinumab and SI-83M10 were similar (Fig. 9B). Finally, no internalization of SI-83M10 or the control was observed in the DLL3-negative gastric cancer cell line NUGC-4 (Fig. 9C).

[0091] Example 7. Sequence Alignment of Leader and Reference Antibodies To investigate the structural basis of unique binding properties, the coding sequences of the disclosed antibodies (SI-83M10, SI-83M8, 5257C8 BSM) and reference antibodies (see Table 3) were aligned using Geneious Prime bioinformatics software according to the Kabat numbering rules, as shown in Figure 10. The heavy and light chain complementarity-determining regions (CDRs) of all antibodies were aligned (boxed in the upper and lower figures of Figures 10A and 10B). In Figures 11A, 11B, and 11C, the percentage of identity for CDR-only, frame region-only, and complete variable region (CDR+FR) was calculated for comparison. In the aligned CDR sequences, the identity between SI-83M10 and each reference antibody was 13-46%, while the identity between the reference antibodies was 25-45%. A special case in this comparison was Gocatamig and Obrixtamig, with an identity of only 5.6%. Similarly, SI-83M8 showed 24-45% identity with each reference antibody, and 5257C8 BSM showed 14-67% identity with each reference antibody. The wide range of sequence identity between the disclosed sequences and the reference antibodies demonstrates the variability of therapeutic antibodies in a competitive landscape.

[0092] A two-tailed one-sample t-test was used to compare the percentage of identity between SI-83M10, SI-83M8, or 5257C8 BSM and a particular reference antibody with the mean percentage of identity among the remaining reference antibodies. Although sequence differences were observed among all antibodies, significant differences in the percentage of identity between the reference antibodies and the disclosed clones were observed only when compared with a few clinical antibodies (P<0.05). For example, the percentage of identity between SI-83M10 and FZ-AD005 was significantly lower than the pooled percentage of identity among the listed reference antibodies (P<0.0001 for CDR only). The same sequence identity was also detected between SI-83M10 and Gocatamig.Results (P=0.013 for CDR only). The identity percentage of SI-83M8 was significantly lower than that of the pooled reference antibodies compared to talatumab (P=0.05 for CDR only). The sequence diversity between 5257C8 BSM and the reference antibodies was highest, with its identity percentage significantly lower than FZ-AD005 (P=0.0093 for CDR only), Gensun (P=0.0331 for CDR only), and lovatozumab (P=0.0046 for CDR only). While not all differences detected in the amino acid sequences encoding the primary structure of the CDR were statistically significant, they form the basis for various characteristics of the disclosed antibodies, including epitope clustering (Figure 7), and thus constitute the basis for biological activity.

[0093] References (each of these references is incorporated into this paper in its entirety by reference) 1. Oronsky, B., Reid, TR, Oronsky, A., & Carter, CA (2017). What's New in SCLC? A Review. Neoplasia (New York, NY), 19(10), 842–847. https: / / doi.org / 10.1016 / j.neo.2017.07.007 2. Gazdar, AF, Bunn, PA, & Minna, J. D. (2017). Small-cell lung cancer: what we know, what we need to know and the path forward. Nature reviews. Cancer, 17(12), 725–737. https: / / doi.org / 10.1038 / nrc.2017.87 3. Drapkin, B. J., & Rudin, CM (2021). Advances in Small-Cell Lung Cancer (SCLC) Translational Research. Cold Spring Harbor perspectives in medicine, 11(4), a038240. https: / / doi.org / 10.1101 / cshperspect.a038240 4. Bray, F., Ferlay, J ., Laversanne, M.,Brewster , D . H ., Gombe Mbalawa , C., Kohler, B., Piñeros , M., Steliarova‑Foucher, E., Swaminathan , R ., Antoni , S., Soerjomataram , I., & Forman , D. (2015) .Cancer Incidence in Five Continents: Inclusion criteria, highlights from Volume X and the global status of cancer registration.International journal of cancer, 137(9) , 2060– 2071.https: / / doi.org / 10.1002 / ijc.29670 5.Megyesfalvi, Z., Gay , C. M., Popper, H., Pirker, R ., Ostoros , G., Heeke , S ., Lang , C ., Hoetzenecker , K ., Schwendenwein , A ., Boettiger , K ., Bunn , P . A ., Jr , Renyi‑Vamos , F ., Schelch , K ., Prosch , H ., Byers , L . A ., Hirsch , F . R ., & Dome , B . (2023) .Clinical insights into small cell lung cancer: Tumor heterogeneity, diagnosis, therapy, and future directions.CA: a cancer journal for clinicians , 10 .3322 / caac .21785 .Advance online publication.https: / / doi.org / 10.3322 / caac.21785 6.Schwendenwein, A., Megyesfalvi, Z., Barany, N., Valko, Z., Bugyik, E., Lang , C., Ferencz, B., Paku, S.,Lantos, A., Fillinger, J., Rezeli, M., Marko‑Varga , G ., Bogos , K ., Galffy , G ., Renyi‑Vamos , F ., Hoda , M . A ., Klepetko , W ., Hoetzenecker , K ., Laszlo , V ., & Dome , B . (2021) .Molecular profiles of small cell lung cancer subtypes: therapeutic implications .Molecular therapy oncolytics , 20 , 470–483 .https: / / doi .org / 10.1016 / j.omto.2021.02.004 7.Saunders , L. R ., Bankovich, A. J ., Anderson, W. C., Aujay , M. A., Bheddah, S., Black , K ., Desai , R ., Escarpe , P. A., Hampl , J ., Laysang , A., Liu , D ., Lopez‑Molina , J ., Milton , M ., Park , A ., Pysz , M . A ., Shao , H ., Slingerland , B ., Torgov , M ., Williams , S . A ., Foord , O ., … Dylla , S . J . (2015) .A DLL3‑targeted antibody‑drug conjugate eradicates high‑grade DESCRIPTION Page 15 of 37 19 CN 122374334 A pulmonary neuroendocrine tumor‑initiating cells in vivo.Science translational medicine, 7(302) , 302ra136.https: / / doi.org / 10.1126 / scitranslmed.aac9459 8 .Kim , J . W ., Ko, J . H ., & Sage , J . (2022) .DLL3 regulates Notch signaling in small cell lung cancer . iScience , 25(12) , 105603 .https: / / doi.org / 10.1016 / j.isci.2022.105603 9.Steinbuck, M. P., & Winandy, S. (2018) .A Review of Notch Processing With New Insights Into Ligand‑Independent Notch Signaling in T‑ Cells .Frontiers in immunology , 9 , 1230 .https: / / doi .org / 10 .3389 / fimmu.2018.01230 10.Cortinovis , D. L., Colonese, F., Abbate, M. I., Sala , L., Meazza Prina , M ., Cordani , N ., Sala , E ., & Canova , S . (2022) .Harnessing DLL3 inhibition: From old promises to new therapeutic horizons .Frontiers in medicine, 9, 989405.https: / / doi.org / 10.3389 / fmed.2022.989405 11 .Owen H.D., Giffin M.J ., Bailis J .M., Damiette Smit M.A., Carbone D .P ., & He K . (2019) .DLL3: an emerging target in small cell lung cancer . Journal of Hematology & Oncology. 12(61) .https: / / doi.org / 10.1186 / s13045‑019‑ 0745‑2 12 .Matsuo K ., Taniguchi K ., Hamamoto H ., Inomata Y ., Komura K ., Tanaka T., Lee S.W., UchiyamaK. (2021) .Delta‑like canonical Notch ligand 3 as a potential therapeutic target in malignancies: A brief overview. Cancer Science.112, 2984‑2992.https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC8353941 / pdf / CAS‑112‑2984.pdf 13 .Paz‑Ares L ., et . al .(2023) .Tarlatamab , a First‑in‑Class DLL3‑ Targeted Bispecific T‑Cell Engager, in Recurrent Small‑Cell Lung Cancer: An Open‑Label , Phase I Study . Journal of Clinical Oncology .41(16) , 2893‑ 2903.https: / / ascopubs.org / doi / full / 10.1200 / JCO.22.02823 14.Tang D., Kang R. (2023) . Tarlatamab: the promising immunotherapy on its way from the lab to the clinic. Translational Lung Cancer Research.12 (6) , 1355‑1357.https: / / tlcr.amegroups.org / article / view / 73451 / html 15.Rudin C.M., Reck M., Johnson M.L., Blackhall F., Hann C.L., Yang J .C., Bailis J .M., Bebb G ., Goldrick A., Umejiego J ., & Paz‑Ares L. (2023) Emerging therapies targeting the delta‑like ligand 3 (DLL3) in small cell lung cancer . Journal of Hematology & Oncology .16(66) , https: / / doi.org / 10.1186 / s13045‑023‑01464‑y 16. Lacalle‑Gonzales, Carlos, et. al. (2023) . DLL3 Is a Prognostic and Potentially Predictive Biomarker for Immunotherapy Linked to PD / PD‑L Axis and NOTCH1 in Pancreatic Cancer . Biomed icines , 11 , 2812.https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC10604228 / pdf / biomedicines‑11‑02812.pdf 17.Garvin D, Stecha P, Gilden J, Wang J, Grailer J, Hartnett J, Fan Instructions 16 / 37 Page 20 CN 122374334 AF, Cong M, Cheng ZJ. Determining ADCC Activity of Antibody-Based Therapeutic Molecules using Two Bioluminescent Reporter-Based Bioassays. Curr Protoc. 2021 Nov;1(11):e296. 18. Yu et al. Frontiers Molecular Biosciences 2022 Table 1A. Immunotherapy regimens for anti-DLL3 antibody generation - Group 1 Table 1B. Immunotherapy regimens for anti-DLL3 antibody generation - Group 2 Instructions 17 / 37 pages 21 CN 122374334 A Table 2A. Characteristic profiles of engineered clones and controls targeting DLL3 Instructions 18 / 37 pages 22 CN 122374334 A Table 2B. Binding affinity of engineered clones and controls targeting DLL3 Table 2C. Binding affinity of engineered clones and controls targeting DLL3 Instructions 19 / 37 pages 23 CN 122374334 A Table 2D. Engineered clones targeting DLL3 and control cell binding*. *Transfected ExpiCHO cells (MFI at 1 μg / mL) Table 3. Summary of anti-DLL3 antibodies. Instructions for use 20 / 37, page 24. CN 122374334 A Sequence listing. Instructions for use 21 / 37, page 25.CN 122374334 A Specification 22 / 37 Page 26 CN 122374334 A Specification 23 / 37 Page 27 CN 122374334 A *CDR regions in the amino acid sequence are indicated in bold>Seq ID 1:5018A1 Light chain variable region nucleic acid sequence GATGTTGTGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGC CAGGCCAGTGAGGATATTAGCGGTTGGTTGGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTCATCTA CTGGGCATCCAATCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTAGATCTGGGACAGAGTTCACTCTCACCA TCAGCGACCTGGAGTGTGCCGATTCTGCCACTTACTACTGTCAATCTACTTTTTATGGTACTAGTGATGTTGCTGCT TTCGGCGGAGGGACCGAGGTGGTGNTCAAA >Seq ID 2: 5018A1 heavy chain variable region nucleic acid sequence CAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGC ACAGCCTCTGGATTCGACTTCAATAACCTACTACATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGAT CGGAGTCATTTATGCTAGTGGTGGCACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGA CCACGATGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGCCTATCCTGAT AATGGTGATGGATTGGACATCTGGGGCCCAGGGACCCTCGTCACCGTCTCGAGC >Seq ID 3:5029C10 light chain variable region nucleic acid sequence: GATGTTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTAGTTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTA TCAGGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCA TCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAGGCTATGATTCTAATAGTGTTGAGAATGCTTTC GGCGGAGGGACCGAGGTGGAGTTCAAA >Seq ID 4: 5029C10 heavy chain variable region nucleic acid sequence CAGTCTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGAGGATCCCTGACACTCACCTGC ACAGTCTCTGGAATCGACTTCAGTAGGAATGTAATTAATTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGAT CGGAATCATTGCTACTGCTGGTGACACATACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGT CGACCACGGTGGATCTGAAAATGACCAGTCTGACACCCGAGGACACGGCCACCTATTTCTGTGCCGGAAAATATGGT GATACTTTTGATCTCTGGGGCCCGGGCACCCTGGTCACCGTCTCGAGC >Seq ID 5: 5257C8 light chain variable region nucleic acid sequence DESCRIPTION Page 24 / 37 28 CN 122374334 A GCCTATGATCTGACCCAGACTCCAGCCTCTGTGGAGGTAGATGTGGGAGGCACAGTCACCATCAAGTGC CAGGCCAGTCAGAGCATTGGTGGTAATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTA TTCTGCATCCAAATTGGCAAGTGGGGTCCCATCGCGGTTCAGAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAACAGGCTTGGAGTTATAGTAATGTTGATAATACT TTCGGCGGAGGCACCGAGGTGGTGTTCAAA >Nucleic acid sequence of heavy chain variable region of 5257C8 Seq ID 6 CAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGT ACAGTCTCTGGAATCGACCTCAGTAGTCACTATTTCAATTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGAT CGGGATCGTTTATGCTAGTGGTAGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGA CCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCTGGAGATCGGAGTGTT GCCTATTCAAACATCTGGGGCCAGGGGACCCTCGTCACCGTCTCGAGC >Nucleic acid sequence of light chain variable region of 501A8-GS Seq ID 7 GACGTGGTGATGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTGACCATCACCTGC CAGGCCTCCGAGGACATCTCCGGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTA CTGGGCCTCCAACCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCA TCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGTCCACCTTCTACGGCACCTCCGACGTGGCCGCC TTCGGCGGCGGCACCAAGGTGGAGATCAAG >Nucleic acid sequence of heavy chain variable region of 501A8-GS Seq ID 8 CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTTCGACTTCAACACCTACTACATGACCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGG CGTGATCTACGCCTCCGGCGGCACCTACTACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACA CCATGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGCCTACCCCGACAAC GGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 9: 5029C10-FRS light chain variable region nucleic acid sequence GACGTGGTGATGACCCAGTCCCCCTCCTCCCTGTCCGCCTCCGTGGGCGACAGGGTGACCATCTCCTGC CAGGCCTCCCAGTCCATCTCCTCCTACCTGTCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTA CCAGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCA TCTCCTCCCTGGAGCCCGAGGACTTCGCCACCTACTACTGCCAGGGCTACGACTCCAACTCCGTGGAGAACGCCTTC GGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID 10: 5029C10-FRS heavy chain variable region nucleic acid sequence CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCC GCCTCCGGCATCGACTTCTCCAGGAACGTGATCAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGG CATCATCGCCACCGCCGGCGACACCTACTACGCCAACTGGGCCAAGGGCAGGTTCACCATCTCCAAGACCTCCAAGA ACACCGTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCGGCAAGTACGGCGACACCTTCGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 11: 5257C8‑BSM light chain variable region nucleic acid sequence GACTACCAGCTGACCCAGTCCCCCTCCTCCGTGGCCCTGACCGTGGGCCAGAGGGTGACCATCAACTGC CAGGCCTCCCAGTCCATCGGCGGCAACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGGCCCAAGCTGCTGATCTA CTCCGCCTCCAAGCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCA Specification 25 / 37 Page 29 CN 122374334 A TCTCCTCCCTGCAGTCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTGGTCCTACTCCAACGTGGACAACACC TTCGGCGGCGGCACCAGGGTGGACATCAAG >Seq ID 12: 5257C8‑BSM heavy chain variable region nucleic acid sequence CAGCAGCTGCAGGAGTCCGGCGGCAGGCTGATCAAGCCCGGCGAGCCCCTGAGGCTGACCTGCAAGACC TCCGGCATCGACCTGTCCTCCCACTACTTCAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCAT CGTGTACGCCTCCGGCTCCACCTACTACGCCTCCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCACCAACACCG TGTTCCTGCAGATGAGGTCCCTGAGGTCCGAGGACACCGCCGTGTACTACTGCGCCGGCGACAGGTCCGTGGCCTAC TCCAACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 13: 5018A1‑GS VH‑N99S heavy chain variable region nucleic acid sequence CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTTCGACTTCAACACCTACTACATGACCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGG CGTGATCTACGCCTCCGGCGGCACCTACTACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACA CCATGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGCCTACCCCGACAGC GGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCC >Seq ID 14: 5018A1‑GS VH‑N99S VH‑Y33G Heavy chain variable region nucleic acid sequence CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCC GCCTCCGGCTTCGACTTCAACACCTACGGCATGACCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGG CGTGATCTACGCCTCCGGCGGCACCTACTACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACA CCATGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGCCTACCCCGACAGC GGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCC >Seq ID 15: 5018A1‑GS VL‑Y93G Light chain variable region nucleic acid sequence GACGTGGTGATGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTGACCATCACCTGC CAGGCCTCCGAGGACATCTCCGGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTA CTGGGCCTCCAACCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGTCCACCTTCGGCGGCACCTCCGACGTGGCCGCC TTCGGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID 16: 5018A1 Light chain variable region amino acid sequence DVVMTQTPSSVSAAVGGTVTIKCQASEDISGWLAWYQQKPGQPPKLLIYWASNLASGVSSRFKGSRSGT EFTLTISDLECADSATYYCQSTFYGTSDVAAFGGGTEVVXK >Seq ID 17: 5018A1 Heavy chain variable region amino acid sequence QSLEESGGRLVTPGTPLTLTCTASGFDFNTYYMTWVRQAPGKGLEWIGVIYASGGTYYATWAKGRFTIS KTSTTMDLKITSPTTEDTATYFCARAYPDNGDGLDIWGPGTLVTVSS >Seq ID 18: 5029C10 Light chain variable region amino acid sequence DVVMTQTPASVEAAVGGTVTIKCQASQSISSYLSWYQQKPGQRPKLLIYQASTLASGVPSRFKGSGSGT QFTLTISDLECADAATYYCQGYDSNSVENAFGGGTEVEFK >Seq ID 19: 5029C10 Heavy chain variable region amino acid sequence QSQSVEESGGRLVTPGGSLTLTCTVSGIDFSRNVINWVRQAPGKGLEWIGIIATAGDTYYANWAKGRFT ISKTSSTTVDLKMTSLTPEDTATYFCAGKYGDTFDLWGPGTLVTVSS >Seq ID 20: 5257C8 Light chain variable region amino acid sequence Specification page 26 / 37 30 CN 122374334 A AYDLTQTPASVEVDVGGTVTIKCQASQSIGGNLAWYQQKPGQRPKLLIYSASKLASGVPSRFRGSGSGT EFTLTISDLECDDAATYYCQQAWSYSNVDNTFGGGTEVVFK >Seq ID 21: 5257C8 heavy chain variable region amino acid sequence QSVEESGGRLVTPGTPLTLTCTVSGIDLSSHYFNWVRQAPGKGLEWIGIVYASGSTYYASWAKGRFTIS KTSTTVDLKMTSLTTEDTATYFCAGDRSVAYSNIWGQGTLVTVSS>Seq ID 22: 5018A1-GS light chain variable region amino acid sequence DVVMTQSPSSVSASVGDRVTITCQASEDISGWLAWYQQKPGQAPKLLIYWASNLASGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQSTFYGTSDVAAFGGGTKVEIK >Seq ID 23: 5018A1-GS heavy chain variable region amino acid sequence QVQLVESGGGLVQPGGSLRLSCAASGFDFNTYYMTWVRQAPGKGLEWVGVIYASGGTYYATWAKGRFTI SKSKNTMYLQMNSLRAEDTAVYYCARAYPDNGDGLDIWGQGTLVTVSS >Seq ID 24: 5029C10-FRS light chain variable region amino acid sequence DVVMTQSPSSLSASVGDRVTISCQASQSISSYLSWYQQKPGQAPKLLIYQASTLASGVPSRFSGSGSGT DFTLTISSLEPEDFATYYCQGYDSNSVENAFGGGTKVEIK >Seq ID 25: 5029C10-FRS heavy chain variable region amino acid sequence QVQLVESGGGLVKPGGSLRLSCAASGIDFSRNVINWVRQAPGKGLEWVGIIATAGDTYYANWAKGRFTI SKTSKNTVYLQMNSLRAEDTAVYYCAGKYGDTFDLWGQGTLVTVSS >Seq ID 26: 5257C8-BSM light chain variable region amino acid sequence DYQLTQSPSSVALTVGQRVTINCQASQSIGGNLAWYQQKPGQRPKLLIYSASKLASGVPSRFSGSGSGT DFTLTISSLQSEDFATYYCQQAWSYSNVDNTFGGGTRVDIK >Seq ID 27: 5257C8-BSM heavy chain variable region amino acid sequence QQLQESGGRLIKPGEPLRLTCKTSGIDLSSHYFNWVRQAPGKGLEWIGIVYASGSTYYASWAKGRFTIS KSTNTVFLQMRSLRSEDTAVYYCAGDRSVAYSNIWGQGTLVTVSS >Seq ID 28: 5018A1-GS VH-N99S Heavy Chain Variable Region Amino Acid Sequence: QVQLVESGGGLVQPGGSLRLSCAASGFDFNTYYMTWVRQAPGKGLEWVGVIYASGGTYYATWAKGRFTISKSKNTMYLQMNSLRAEDTAVYYCARAYPDSGDGLDIWGQGTLVTVSS >Seq ID 29: 5018A1‑GS VH‑N99S VH‑Y33G Heavy chain variable region amino acid sequence QVQLVESGGGLVQPGGSLRLSCAASGFDFNTYGMTWVRQAPGKGLEWVGVIYASGGTYYATWAKGRFTI SKSKNTMYLQMNSLRAEDTAVYYCARAYPDSGDGLDIWGQGTLVTVSS >Seq ID 30: 5018A1‑GS VL‑Y93G Light chain variable region amino acid sequence DVVMTQSPSSVSASVGDRVTITCQASEDISGWLAWYQQKPGQAPKLLIYWASNLASGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQSTFGGTSDVAAFGGGTKVEIK >Seq ID 31: 5018A1‑GS null Fc Heavy chain amino acid sequence QVQLVESGGGLVQPGGSLRLSCAASGFDFNTYYMTWVRQAPGKGLEWVGVIYASGGTYYATWAKGRFTI SKSKNTMYLQMNSLRAEDTAVYYCARAYPDSGDGLDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG DESCRIPTION Page 27 of 37 31 CN 122374334 A >Seq ID 32: 5018A1‑GS null Fc Light chain amino acid sequence DVVMTQSPSSVSASVGDRVTITCQASEDISGWLAWYQQKPGQAPKLLIYWASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSTFYGTSDVAAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVCTKSFNRGEC >Seq ID 33: Human DLL3 ECD nucleic acid sequence comprising EpCAM transmembrane domain and cytoplasmic domain ATGGTGAGCCCTAGAATGAGCGGCCTGCTGTCTCAGACCGTGATCCTGGCCCTGATCTTCCTGCCTCAG ACAAGACCCGCCGGCGTGTTCGAGCTGCAAATCCATAGCTTCGGTCCCGGCCCCGGCCCTGGTGCACCTAGAAGCCC CTGCAGCGCTAGACTGCCCTGCAGACTGTTCTTCAGAGTATGCCTGAAACCCGGGCTGAGCGAAGAGGCCGCCGAAA GTCCATGTGCCCTGGGAGCCGCCCTATCCGCGAGGGGCCCCGTGTATACCGAACAACCGGGCGCACCCGCCCCCGAC TTACCACTCCCCGACGGGTTGTTGCAAGTGCCCTTCAGAGACGCCTGGCCCGGCACCTTCAGCTTCATCATCGAAAC CTGGAGAGAAGAGCTGGGTGATCAGATCGGTGGCCCGGCCTGGAGCCTTCTAGCACGGGTGGCCGGCAGAAGAAGAC TGGCAGCCGGCGGCCCGTGGGCAAGAGACATTCAGAGAGCCGGCGCCTGGGAGCTGAGATTCAGCTACAGAGCTAGA TGCGAACCCCCGGCCGTGGGCACCGCCTGCACAAGACTGTGTAGACCTAGAAGCGCCCCGTCCCGATGCGGCCCCGG TCTTAGACCCTGCGCCCCACTGGAAGACGAGTGTGAAGCCCCCCTGGTTTGCCGTGCCGGTTGCAGCCCCGAGCACG GCTTCTGTGAACAGCCCGGGGAATGCAGATGCCTGGAAGGGTGGACTGGTCCCCTGTGCACCGTGCCCGTGAGCACCTCGTCGTGCCTCTCTCCTAGAGGACCTAGCAGCGCAACGACCGGCTGCCTGGTGCCCGGCCCCGGCCCTTGTGATGG CAACCCGTGCGCAAACGGCGGCTCATGCAGCGAGACCCCTAGAAGCTTCGAGTGCACCTGCCCTAGAGGCTTCTACG GCCTGAGATGCGAGGTATCGGGGGTGACCTGTGCCGACGGCCCTTGTTTTAATGGCGGGTTGTGCGTGGGAGGCGCC GATCCCGACAGCGCCTACATCTGCCACTGTCCCCCCGGCTTCCAAGGCAGCAACTGCGAGAAGAGAGTGGACAGATG CAGCCTGCAGCCCTGCAGAAACGGCGGTCTATGCCTCGACCTGGGCCACGCCCTGAGATGCAGATGCAGAGCGGGCT TCGCGGGCCCTAGATGTGAACATGATCTGGACGACTGTGCCGGACGAGCATGCGCTAACGGAGGTACGTGTGTCGAA GGGGGAGGAGCGCATCGATGCAGCTGCGCACTGGGCTTCGGAGGCAGAGACTGCAGAGAGAGAGCCGATCCGTGTGC CGCACGGCCTTGTGCCCACGGCGGCAGATGCTACGCCCACTTCAGCGGCCTGGTGTGCGCCTGTGCCCCCGGCTACA TGGGCGCGAGGTGCGAATTTCCCGTGCACCCCGATGGAGCATCGGCATTGCCCGCAGCACCTCCCGGCCTGCGGCCC GGTGACCCTCAGAGGTATTTATCTGGCGGAGGCGGCTCTGGCGCAGGCGTGATCGCCGTGATCGTGGTGGTGGTCAT TGCCATCGTGGCTGGCATCGTGGTGCTGGTCATCTCCAGAAAGAAGCGGATGGCCAAGTACGAGAAGGCCGAAATCA AAGAGATGGGCGAGATGCACAGAGAACTGAACGCCTAG >Seq ID 34: Cynomolgus DLL3 ECD nucleic acid sequence comprising the EpCAM transmembrane domain and cytoplasmic domainATGGTGAGCCCTAGAATGAGCAGACTGCTGAGCCAAACCGTGATCCTGGCCCTGATCTTCATCCCCCAA GCTAGACCCGCCGGCGTATTCGAACTTCAAATTCACAGCTTTGGCCCTGGTCCCGGCCCGGGCGCTCCAAGAAGCCC CTGCAGCGCTAGAGGCCCCTGCCGACTGTTCTTTAGAGTGTGCCTGAAGCCCGGACTGAGCGAGGAGGCGGCCGAGT CTCCCTGTGCGCTGGGCGCCGCATTGAGTGCGAGAGGCCCCGTCTATACCGAACAACCTGAGGCTCCTGCCCCCGAT CTGCCCCTGCCCAACGGCCTGCTGCAAGTGCCCTTCAGAGACGCCTGGCCCGGCACCTTCAGCCTGATCATCGAGAC CTGGAGAGAGGAGCTGGGCGATCAGATCGGCGGCCCCGCCTGGAGCCTGCTGGCTAGAGTGACAAGAAGAAGAAGAC TGGCCGCCGGAGGCCCCTGGGCTAGAGACATTCAGAGAGCCGGCGCCTGGGAGCTGAGATTCAGCTACAGAGCTAGA TGCGAACTTCCGGCCGTGGGCACCGCCTGCACAAGATTATGCAGACCTAGAAGTGCTCCTAGCCGGTGTGGCCCTGG CTTGCGCCCCTGCGCACCCTTGGAAGATGAGTGTGAAGCGCCCCCGGTGTGTAGGGCCGGCTGTAGCCTGGAGCACG GCTTCTGCGAGCAGCCCGGCGAGTGCAGATGCCTGGAGGGCTGGACCGGCCCCCTGTGCATGGTGCCTGTTTCCACA AGCAGCTGCCTGGGCCTGAGGGGGCCTAGCTCAACTACGACCGGATGTCTGGTGCCCGGGCCCGGCCCATGCGACGG DESCRIPTION Page 28 / 37 32 CN 122374334 A CAACCCATGTGCCAACGGCGGCTCATGCTCGGAGACCCCCGGCAGCTTCGAGTGCACCTGCCCTAGAGGCTTCTATGGTCTGAGATGCGAAGTGAGCGGCGTGACCTGCGCAGACGGCCCCTGTTTTAATGGCGGCTTGTGTGTGGGCGGCGCG GATCCCGATTCGGCCTACATCTGCCACTGCCCCCCCGGCTTCCAAGGCAGCAACTGCGAGAAGAGAGTGGACAGATG CAGCCTGCAGCCCTGCAGAAACGGGGGGCTGTGCCTGGACCTGGGCCACGCCCTGAGATGCAGATGCAGAGCCGGCT TCGCCGGCCCTAGATGCGAGCACGACCTGGACGATTGTGCCGGAAGGGCTTGCGCTAACGGAGGTACTTGTGTTGAG GGCGGCGGGGCTCACAGATGCAGCTGCGCCCTGGGCTTCGGAGGGAGAAACTGCAGAGAAAGAGCTGACCCGTGCGC CGCGCGCCCCTGCGCCCACGGAGGCAGATGCTATGCCCATTTCAGCGGCCTGGTTTGCGCCTGCGCCCCCGGCTATA TGGGTGCTAGATGCGAGTTCCCCGTACACCCGGATGGCGTGTCAGCTCTCCCCGCAGCCCCACCTGGCCTGAGGCCC GGAGATCCCCAAAGATATCTG >Seq ID 35: Human IgG1 amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 36: Human CK amino acid sequenceRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 37: 5018A1‑GS Heavy chain amino acid sequence QVQLVESGGGLVQPGGSLRLSCAASGFDFNTYYMTWVRQAPGKGLEWVGVIYASGGTYYATWAKGRFTI SKSKNTMYLQMNSLRAEDTAVYYCARAYPDNGDGLDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 38: 5018A1‑GS Light chain amino acid sequence DVVMTQSPSSVSASVGDRVTITCQASEDISGWLAWYQQKPGQAPKLLIYWASNLASGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQSTFYGTSDVAAFGGGTKVEIKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 39: 5029C10‑FRS Heavy chain amino acid sequence QVQLVESGGGLVKPGGSLRLSCAASGIDFSRNVINWVRQAPGKGLEWVGIIATAGDTYYANWAKGRFTISKTSKNTVYLQMNSLRAEDTAVYYCAGKYGDTFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 40: 5029C10-FRS light chain amino acid sequence DVVMTQSPSSLSASVGDRVTISCQASQSISSYLSWYQQKPGQAPKLLIYQASTLASGVPSRFSGSGSGT DFTLTISSLEPEDFATYYCQGYDSNSVENAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA DESCRIPTION Page 29 / 37 33 CN 122374334 A KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 41: 5257C8-BSM heavy chain amino acid sequence QQLQESGGRLIKPGEPLRLTCKTSGIDLSSHYFNWVRQAPGKGLEWIGIVYASGSTYYASWAKGRFTIS KSTNTVFLQMRSLRSEDTAVYYCAGDRSVAYSNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 42: Amino acid sequence of 5257C8‑BSM light chain DYQLTQSPSSVALTVGQRVTINCQASQSIGGNLAWYQQKPGQRPKLLIYSASKLASGVPSRFSGSGSGT DFTLTISSLQSEDFATYYCQQAWSYSNVDNTFGGGTRVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 43: Amino acid sequence of 5018A1‑GS VH‑N99S heavy chain QVQLVESGGGLVQPGGSLRLSCAASGFDFNTYYMTWVRQAPGKGLEWVGVIYASGGTYYATWAKGRFTI SKSKNTMYLQMNSLRAEDTAVYYCARAYPDSGDGLDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 44: Amino acid sequence of 5018A1‑GS VH‑N99S light chain DVVMTQSPSSVSASVGDRVTITCQASEDISGWLAWYQQKPGQAPKLLIYWASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSTFYGTSDVAAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 45: 5018A1‑GS VH‑N99S VH‑Y33G Heavy chain amino acid sequence QVQLVESGGGLVQPGGSLRLSCAASGFDFNTYGMTWVRQAPGKGLEWVGVIYASGGTYYATWAKGRFTI SKSKNTMYLQMNSLRAEDTAVYYCARAYPDSGDGLDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 46: 5018A1‑GS VH‑N99S VL‑Y93G Light chain amino acid sequence DVVMTQSPSSVSASVGDRVTITCQASEDISGWLAWYQQKPGQAPKLLIYWASNLASGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQSTFGGTSDVAAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 47: Human IgG1 null amino acid sequenceAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAK >Seq ID 48: human CK inserted amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS DESCRIPTION Page 30 / 37 34 CN 122374334 A STLTLSKADYEKHKVYACEVTHQGLSSPVCTKSFNRGEC >Seq ID 49: 5018A1 BSM heavy chain variable region nucleic acid sequence CAGGTGCAGCTGCAGGAGTCCGGCGGCAGGCTGATCAAGCCCGGCGCCCCCCTGAGGCTGTCCTGCAAG GCCTCCGGCTTCGACTTCAACACCTACTACATGACCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGG CGTGATCTACGCCTCCGGCGGCACCTACTACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCACCAACA CCATGTACCTGCAGATCAGGTCCCCCAGGTCCGAGGACACCGCCATCTACTACTGCGCCAGGGCCTACCCCGACAAC GGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 50: 5018A1 BSM heavy chain variable region amino acid sequence QVQLQESGGRLIKPGAPLRLSCKASGFDFNTYYMTWVRQAPGKGLEWIGVIYASGGTYYATWAKGRFTI SKSTNTMYLQIRSPRSEDTAIYYCARAYPDNGDGLDIWGQGTLVTVSS >Seq ID 51: 5018A1 BSM heavy chain amino acid sequence QVQLQESGGRLIKPGAPLRLSCKASGFDFNTYYMTWVRQAPGKGLEWIGVIYASGGTYYATWAKGRFTI SKSTNTMYLQIRSPRSEDTAIYYCARAYPDNGDGLDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 52: Nucleic acid sequence of 5018A1 BSM light chain variable region GACGTGCAGATGACCCAGTCCCCCTCCTCCGTGTCCGCCACCGTGGGCCAGAGGGTGACCATCAAGTGC CAGGCCTCCGAGGACATCTCCGGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGCCCCCCAAGCTGCTGATCTA CTGGGCCTCCAACCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCA TCTCCTCCCTGCAGTGCGACGACTTCGCCACCTACTACTGCCAGTCCACCTTCTACGGCACCTCCGACGTGGCCGCC TTCGGCGGCGGCACCAAGGTGGACGTGAAG >Seq ID 53: Amino acid sequence of 5018A1 BSM light chain variable region DVQMTQSPSSVSATVGQRVTIKCQASEDISGWLAWYQQKPGKPPKLLIYWASNLASGVPSRFSGSGSGT DFTLTISSLQCDDFATYYCQSTFYGTSDVAAFGGGTKVDVK >Seq ID 54: Amino acid sequence of 5018A1 BSM light chain DVQMTQSPSSVSATVGQRVTIKCQASEDISGWLAWYQQKPGKPPKLLIYWASNLASGVPSRFSGSGSGT DFTLTISSLQCDDFATYYCQSTFYGTSDVAAFGGGTKVDVKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 55: 5018A1 FRS heavy chain variable region nucleic acid sequence CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCC GCCTCCGGCTTCGACTTCAACACCTACTACATGACCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGG CGTGATCTACGCCTCCGGCGGCACCTACTACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACA CCATGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGCCTACCCCGACAAC GGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 56: 5018A1 FRS heavy chain variable region amino acid sequence QVQLVESGGGLVKPGGSLRLSCAASGFDFNTYYMTWVRQAPGKGLEWVGVIYASGGTYYATWAKGRFTI SKSKNTMYLQMNSLRAEDTAVYYCARAYPDNGDGLDIWGQGTLVTVSS DESCRIPTION Page 31 of 37 35 CN 122374334 A >Seq ID 57: 5018A1 FRS heavy chain amino acid sequence QVQLVESGGGLVKPGGSLRLSCAASGFDFNTYYMTWVRQAPGKGLEWVGVIYASGGTYYATWAKGRFTI SKSKNTMYLQMNSLRAEDTAVYYCARAYPDNGDGLDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 58: 5018A1 FRS light chain variable region nucleic acid sequence GACGTGGTGATGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTGACCATCTCCTGC CAGGCCTCCGAGGACATCTCCGGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTA CTGGGCCTCCAACCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCA TCTCCTCCCTGGAGCCCGAGGACTTCGCCACCTACTACTGCCAGTCCACCTTCTACGGCACCTCCGACGTGGCCGCC TTCGGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID 59: 5018A1 FRS light chain variable region amino acid sequence DVVMTQSPSSVSASVGDRVTISCQASEDISGWLAWYQQKPGQAPKLLIYWASNLASGVPSRFSGSGSGT DFTLTISSLEPEDFATYYCQSTFYGTSDVAAFGGGTKVEIK >Seq ID 60: 5018A1 FRS light chain amino acid sequence DVVMTQSPSSVSASVGDRVTISCQASEDISGWLAWYQQKPGQAPKLLIYWASNLASGVPSRFSGSGSGT DFTLTISSLEPEDFATYYCQSTFYGTSDVAAFGGGTKVEIKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 61: 5029C10 BSM heavy chain variable region nucleic acid sequenceCAGGTGCAGCTGGTGGAGTCCGGCGGCAGGCTGGTGAAGCCCGGCGGCTCCCTGAAGCTGACCTGCAAG ACCTCCGGCATCGACTTCTCCAGGAACGTGATCAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGG CATCATCGCCACCGCCGGCGACACCTACTACGCCAACTGGGCCAAGGGCAGGTTCACCATCTCCAAGACCTCCACCA ACACCGTGTTCCTGCAGATGAGGTCCCTGAAGTCCGAGGACACCGCCATCTACTACTGCGCCGGCAAGTACGGCGAC ACCTTCGACCTGTGGGGCCCCGGCACCCTGGTGACCGTGTCCTCC >Seq ID 62: 5029C10 BSM heavy chain variable region amino acid sequence QVQLVESGGRLVKPGGSLKLTCKTSGIDFSRNVINWVRQAPGKGLEWIGIIATAGDTYYANWAKGRFTI SKTSTNTVFLQMRSLKSEDTAIYYCAGKYGDTFDLWGPGTLVTVSS >Seq ID 63: 5029C10 BSM heavy chain amino acid sequence QVQLVESGGRLVKPGGSLKLTCKTSGIDFSRNVINWVRQAPGKGLEWIGIIATAGDTYYANWAKGRFTI SKTSTNTVFLQMRSLKSEDTAIYYCAGKYGDTFDLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 64: 5029C10Nucleic acid sequence of BSM light chain variable region GACGTGCAGATGACCCAGTCCCCCTCCTCCCTGGCCGCCTCCGTGGGCCAGAGGGTGACCATCAACTGC CAGGCCTCCCAGTCCATCTCCTCCTACCTGTCCTGGTACCAGCAGAAGCCCGGCCAGAGGCCCAAGCTGCTGATCTA DESCRIPTION Page 32 / 37 36 CN 122374334 A CCAGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCAAGGGCTCCGGCTCCGGCACCCAGTTCACCCTGACCA TCTCCTCCCTGCAGTGCGACGACTTCGCCACCTACTACTGCCAGGGCTACGACTCCAACTCCGTGGAGAACGCCTTC GGCGGCGGCACCAGGGTGGAGATCAAG >Seq ID 65: 5029C10 Amino acid sequence of BSM light chain variable region DVQMTQSPSSLAASVGQRVTINCQASQSISSYLSWYQQKPGQRPKLLIYQASTLASGVPSRFKGSGSGT QFTLTISSLQCDDFATYYCQGYDSNSVENAFGGGTRVEIK >Seq ID 66: 5029C10 Amino acid sequence of BSM light chain DVQMTQSPSSLAASVGQRVTINCQASQSISSYLSWYQQKPGQRPKLLIYQASTLASGVPSRFKGSGSGT QFTLTISSLQCDDFATYYCQGYDSNSVENAFGGGTRVEIKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 67: 5029C10 Nucleic acid sequence of GS heavy chain variable region CAGGTGCAGCTGCTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCC GCCTCCGGCATCGACTTCTCCAGGAACGTGATCAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCATCATCGCCACCGCCGGCGACACCTACTACGCCAACTGGGCCAAGGGCAGGTTCACCATCTCCAAGACCTCCAAGA ACACCGTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCGGCAAGTACGGCGAC ACCTTCGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 68: 5029C10 GS heavy chain variable region amino acid sequence QVQLLESGGGLVQPGGSLRLSCAASGIDFSRNVINWVRQAPGKGLEWVGIIATAGDTYYANWAKGRFTI SKTSKNTVYLQMNSLRAEDTAVYYCAGKYGDTFDLWGQGTLVTVSS >Seq ID 69: 5029C10 GS heavy chain amino acid sequence QVQLLESGGGLVQPGGSLRLSCAASGIDFSRNVINWVRQAPGKGLEWVGIIATAGDTYYANWAKGRFTI SKTSKNTVYLQMNSLRAEDTAVYYCAGKYGDTFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 70: 5029C10 GS light chain variable region nucleic acid sequence GACGTGGTGATGACCCAGTCCCCCTCCTCCCTGTCCGCCTCCGTGGGCGACAGGGTGACCATCACCTGCCAGGCCTCCCAGTCCATCTCCTCCTACCTGTCCTGGTACCAGCAGAAGCCCGGCCAGGTGCCCAAGCTGCTGATCTA CCAGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCA TCTCCTCCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGGGCTACGACTCCAACTCCGTGGAGAACGCCTTC GGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID 71: 5029C10 GS light chain variable region amino acid sequence DVVMTQSPSSLSASVGDRVTITCQASQSISSYLSWYQQKPGQVPKLLIYQASTLASGVPSRFSGSGSGT DFTLTISSLQPEDVATYYCQGYDSNSVENAFGGGTKVEIK >Seq ID 72: 5029C10 GS light chain amino acid sequence DVVMTQSPSSLSASVGDRVTITCQASQSISSYLSWYQQKPGQVPKLLIYQASTLASGVPSRFSGSGSGT DFTLTISSLQPEDVATYYCQGYDSNSVENAFGGGTKVEIKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC DESCRIPTION Page 33 of 37 37 CN 122374334 A >Seq ID 73: 5257C8 FRS heavy chain variable region nucleic acid sequence CAGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCC TCCGGCATCGACCTGTCCTCCCACTACTTCAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCAT CGTGTACGCCTCCGGCTCCACCTACTACGCCTCCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCGGCGACAGGTCCGTGGCCTAC TCCAACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 74: 5257C8 FRS heavy chain variable region amino acid sequence QQLVESGGGLVKPGGSLRLSCAASGIDLSSHYFNWVRQAPGKGLEWVGIVYASGSTYYASWAKGRFTIS KSKNTVYLQMNSLRAEDTAVYYCAGDRSVAYSNIWGQGTLVTVSS >Seq ID 75: 5257C8 FRS heavy chain amino acid sequence QQLVESGGGLVKPGGSLRLSCAASGIDLSSHYFNWVRQAPGKGLEWVGIVYASGSTYYASWAKGRFTIS KSKNTVYLQMNSLRAEDTAVYYCAGDRSVAYSNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 76: 5257C8 FRS light chain variable region nucleic acid sequence GACTACGTGCTGACCCAGTCCCCCTCCTCCGTGTCCGTGTCCGTGGGCGACAGGGTGACCATCTCCTGC CAGGCCTCCCAGTCCATCGGCGGCAACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACTCCGCCTCCAAGCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCA TCTCCTCCCTGGAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTGGTCCTACTCCAACGTGGACAACACC TTCGGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID 77: 5257C8 FRS light chain variable region amino acid sequence DYVLTQSPSSVSVSVGDRVTISCQASQSIGGNLAWYQQKPGQAPKLLIYSASKLASGVPSRFSGSGSGT DFTLTISSLEPEDFATYYCQQAWSYSNVDNTFGGGTKVEIK >Seq ID 78: 5257C8 FRS light chain amino acid sequence DYVLTQSPSSVSVSVGDRVTISCQASQSIGGNLAWYQQKPGQAPKLLIYSASKLASGVPSRFSGSGSGT DFTLTISSLEPEDFATYYCQQAWSYSNVDNTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 79: 5257C8 GS heavy chain variable region nucleic acid sequence CAGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCC TCCGGCATCGACCTGTCCTCCCACTACTTCAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCAT CGTGTACGCCTCCGGCTCCACCTACTACGCCTCCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACACCG TGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCGGCGACAGGTCCGTGGCCTAC TCCAACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 80: 5257C8 GS heavy chain variable region amino acid sequenceQQLVESGGGLVQPGGSLRLSCAASGIDLSSHYFNWVRQAPGKGLEWVGIVYASGSTYYASWAKGRFTIS KSKNTVYLQMNSLRAEDTAVYYCAGDRSVAYSNIWGQGTLVTVSS >Seq ID 81: 5257C8 GS Heavy Chain Amino Acid Sequence Description Page 34 of 37 38 CN 122374334 A QQLVESGGGLVQPGGSLRLSCAASGIDLSSHYFNWVRQAPGKGLEWVGIVYASGSTYYASWAKGRFTIS KSKNTVYLQMNSLRAEDTAVYYCAGDRSVAYSNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 82: 5257C8 GS Light Chain Variable Region Nucleic Acid Sequence GACTACCAGCTGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTGACCATCACCTGC CAGGCCTCCCAGTCCATCGGCGGCAACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTA CTCCGCCTCCAAGCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCA TCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTGGTCCTACTCCAACGTGGACAACACC TTCGGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID83: 5257C8 GS light chain variable region amino acid sequence DYQLTQSPSSVSASVGDRVTITCQASQSIGGNLAWYQQKPGQAPKLLIYSASKLASGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQAWSYSNVDNTFGGGTKVEIK>Seq ID 84: 5257C8 GS light chain amino acid sequence DYQLTQSPSSVSASVGDRVTITCQASQSIGGNLAWYQQKPGQAPKLLIYSASKLASGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQAWSYSNVDNTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 85: 5029C10 AP01 Heavy chain variable region amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNVINWVRQAPGKGLEWVGIIATAGDTYYANWAKGRFTI SKDSSKNTVYLQMNSLRAEDTAVYYCARKYGDTFDLWGQGTLVTVSS >Seq ID 86: 5029C10 AP01 Light chain variable region amino acid sequence DIQMTQSPSSLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGT EFTLTISSLQPDDFATYYCQGYDSNSVENAFGQGTKVEIK >Seq ID 87: 5029C10 OAS01 Heavy Chain Variable Region Amino Acid Sequence: QVHLVESGGGVVQPGKSLRLSCAASGIDFSRNVINWVRQAPGKGLEWIGIIATAGDTYYANWAKGRFTI SKDSSRNTVYLQMDSLRAEDTAMYYCAGKYGDTFDLWGQGTLVTVSS >Seq ID 88: 5029C10 OAS01 Light Chain Variable Region Amino Acid Sequence: DVQMTQSPSTLSASVGDSVTITCQASQSISSYLSWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGT EFTLAISNLQPDDFATYYCQGYDSNSVENAFGQGTEVEIK >Seq ID 89: 5029C10OAS02 Heavy chain variable region amino acid sequence EVQLVESGGGLVQPGRSLTVSCTTSGIDFSRNVINWVRQAPGKGLEWIGIIATAGDTYYANWAKGRFTI SKDSSKNTVYLQMNSLTNEDTAVYFCAGKYGDTFDLWGQGTLVTVSS >Seq ID 90:5029C10 OAS02 Light chain variable region amino acid sequence DVQMTQSPSSLSASVGDRVTITCQASQSISSYLSWYQQKPGKRPKLLIYQASTLASGVPSRFSGSGSGT DFTLTISSLQPEDVATYYCQGYDSNSVENAFGGGTKVEIK >Seq ID 91:5029C10 OAS03 Heavy chain variable region amino acid sequence EVQLVESGGGLVQPGGSLRLSCEASGIDFSRNVINWVRQAPGKGLEWVGIIATAGDTYYANWAKGRFTI Instruction manual, pages 35 / 37, 39, CN 122374334 A SKTSKNTVYLQMNSLKTEDTAVYYCARKYGDTFDLWGKGTTVTVSS >Seq ID 92: 5029C10 OAS03 Light chain variable region amino acid sequence DIQMTQSPSSLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQGYDSNSVENAFGGGTKVEIK >Seq ID 101: 5018A1 CDR L1 QASEDISGWLA >Seq ID 102: 5018A1 CDR L2 WASNLAS >Seq ID 103: 5018A1 CDR L3 QSTFYGTSDVAA >Seq ID 104: 5018A1 CDR H1 TYYMT >Seq ID 105: 5018A1 CDR H2 VIYASGGTYYATWAKG >Seq ID 106: 5018A1 CDR H3 AYPDNGDGLDI >Seq ID 107: 5029C10 CDR L1 QASQSISSYLS >Seq ID 108: 5029C10 CDR L2 QASTLAS >Seq ID 109: 5029C10 CDR L3 QGYDSNSVENA >Seq ID 110:5029C10 CDR H1 RNVIN >Seq ID 111:5029C10CDR H2 IIATAGDTYYANWAKG >Seq ID 112: 5029C10 CDR H3 KYGDTFDL >Seq ID 113: 5257C8 CDR L1 QASQSIGGNLA >Seq ID 114: 5257C8 CDR L2 SASKLAS >Seq ID 115: 5257C8 CDR L3 QQAWSYSNVDNT >Seq ID 116: 5257C8 CDR H1 SHYFN >Seq ID 117: 5257C8 CDR H2 IVYASGSTYYASWAKG >Seq ID 118: 5257C8 CDR H3 Specification 36 / 37 Page 40 CN 122374334 A DRSVAYSNI >Seq ID 119: 5018A1‑GS VH‑N99S CDR H3 AYPDSGDGLDI >Seq ID 120: 5018A1-GS VH-Y33G CDR H1 TYGMT >Seq ID 121: 5018A1-GS VL-Y93G CDR L1 QSTFGGTSDVAA Instruction Manual 37 / 37 Page 41 CN 122374334 A Figure 1 Instruction Manual Figure 1 / 11 Page 42 CN 122374334 A Figure 2 Instruction Manual Figure 2 / 11 Page 43 CN 122374334 A Figure 3 Instruction Manual Figure 3 / 11 Page 44 CN 122374334 A Figure 4 Instruction Manual Figure 4 / 11 Page 45 CN 122374334 A Figure 5 Instruction Manual Figure 5 / 11 Page 46 CN 122374334 A Figure 6 Instruction Manual Figure 6 / 11 Page 47 CN 122374334 A Figure 7 Figure 8 of the instruction manual, page 48 of page 7 / 11, CN 122374334 A; Figure 9 of the instruction manual, page 49 of page 8 / 11, CN 122374334 A; Figure 10 of the instruction manual, page 50 of page 9 / 11, CN 122374334 A; Figure 11 of the instruction manual, page 51 of page 10 / 11, CN 122374334 A; Figure 52 of the instruction manual, page 11 / 11, CN 122374334 A.

Claims

1. A delta-like ligand 3 (DLL3)-binding peptide with binding specificity to human DLL3, comprising a variable heavy (VH) chain and a variable light (VL) chain, The VH chain mentioned therein includes: The amino acid sequences are CDR H1 (SEQ ID: 104), CDR H2 (SEQ ID: 105), and CDR H3 (SEQ ID: 106). The amino acid sequences are CDR H1 of SEQ ID: 110, CDR H2 of SEQ ID: 111, and CDR H3 of SEQ ID:

112. The amino acid sequences are CDR H1 of SEQ ID: 116, CDR H2 of SEQ ID: 117, and CDR H3 of SEQ ID: 118, or The amino acid sequences are CDR H1 (SEQ ID: 120), CDR H2 (SEQ ID: 105), and CDR H3 (SEQ ID: 119). The VL chain mentioned therein includes: The amino acid sequences are CDR L1 of SEQ ID: 101, CDR L2 of SEQ ID: 102, and CDR L3 of SEQ ID:

103. The amino acid sequences are CDR L1 of SEQ ID: 107, CDR L2 of SEQ ID: 108, and CDR L3 of SEQ ID:

109. The amino acid sequence is CDR L1 of SEQ ID: 113, CDR L2 of SEQ ID: 114, or CDR L3 of SEQ ID:

115. The amino acid sequences are SEQ ID: 101, CDR L1, SEQ ID: 102, and CDR L3, respectively.

2. The DLL3-binding peptide according to claim 1, wherein the VH chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 17, 19, 21, 23, 25, 27, 28 or 29; and wherein the VL chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 16, 18, 20, 22, 24, 26 or 30.

3. The DLL3-binding peptide according to claim 1, wherein the amino acid sequences of the VH chain and the VL chain are selected from SEQ ID NO: 17 and 16; 19 and 18; 21 and 20; 23 and 22; 25 and 24; 27 and 26; 28 and 22; 29 and 22; 29 and 30; or 28 and 30.

4. The DLL3-binding peptide according to claim 1, comprising: The scFv domain, wherein the scFv domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32, and The Fab domain, wherein the Fc domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 31, 32, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47.

5. The DLL3-binding peptide of claim 4, comprising at least one histidine residue connected to at least one end of the scFv domain.

6. The DLL3-binding peptide of claim 4, further comprising an Fc domain connected to the Fab domain to provide a Fab-monoFc fusion protein.

7. The DLL3-binding peptide according to claim 1, comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46.

8. An scFv domain comprising the DLL3-binding peptide of claim 1.

9. A Fab domain comprising the DLL3-binding peptide of claim 1.

10. An antibody with binding specificity to human DLL3, comprising the DLL3-binding peptide of claim 1.

11. The antibody of claim 10, wherein the VH chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 17, 19, 21, 23, 25, 27, 28 or 29, and wherein the VL chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 16, 18, 20, 22, 24, 26 or 30.

12. The antibody according to claim 10, comprising a heavy chain (HC) and a light chain (LC). The HC contains an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 37, 39, 41, 43, 45 or 31; and The LC contains an amino acid sequence that has at least 98% sequence identity with SEQ ID NO: 32, 38, 40, 42, 44 or 46.

13. The antibody of claim 10, comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, or a combination thereof.

14. The antibody of claim 10, wherein the antibody comprises a monospecific antibody, a bispecific antibody or a multispecific antibody, a purified antibody, a humanized antibody, IgG or a combination thereof.

15. An isolated nucleic acid sequence encoding the antibody of claim 10.

16. An expression vector or host cell comprising the isolated nucleic acid sequence of claim 15.

17. An immunoconjugate comprising the antibody of claim 10 conjugated to a pharmaceutical unit via a linker, wherein the linker comprises a covalent bond selected from ester bonds, ether bonds, amine bonds, amide bonds, disulfide bonds, imide bonds, sulfone bonds, phosphate bonds, phosphate ester bonds, peptide bonds, hydrazone bonds, or combinations thereof.

18. The immunoconjugate of claim 17, wherein the pharmaceutical unit comprises a unit derived from a therapeutic agent, an imaging agent, a diagnostic agent, a radioisotope, or a combination thereof.

19. The immunoconjugate of claim 18, wherein the therapeutic agent comprises a cytotoxic agent, a chemotherapeutic agent, an enzyme, an anti-estrogenic agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof.

20. The immunoconjugate of claim 18, wherein the therapeutic agent comprises capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, nitrogen mustard, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, leucovorin, gemcitabine, oxaliplatin, irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, and aminophylline. Lumet, testosterone, vorazole, formestane, faldroxil, erlotinib, lafatinib, dasatinib, gefitinib, osimertinib, vandetanib, afatinib, imatinib, pazopanib, lapatinib, sunitinib, nilotinib, sorafenib, albumin-bound paclitaxel, everolimus, sirolimus esterified, dabrafenib, vemurafenib, trametinib, vinpocetine, apatinib, crizotinib, perifolfen, olaparib, bortezomib, tofacitinib, trastuzumab, or their derivatives or combinations thereof.

21. The immunoconjugate of claim 18, wherein the radioisotope comprises deuterium, 131I, 32P, 90Sr, 90Y, 89Zr, 177Lu or a combination thereof.

22. A pharmaceutical composition comprising the antibody of claim 10 or the immunoconjugate of claim 17, and a pharmaceutically acceptable carrier.

23. A method for producing the antibody of claim 10, comprising culturing host cells such that a DNA sequence encoding the antibody of claim 10 is expressed, and purifying the antibody.

24. A method of producing the immunoconjugate of claim 17, comprising conjugating the antibody of claim 7 to the pharmaceutical unit.

25. A method of treating or preventing cancer, autoimmune disease, or infectious disease in a subject, comprising administering to the subject an effective amount of the antibody of claim 10 or the immunoconjugate of claim 17.

26. The method of claim 25, wherein the cancer comprises lung cancer, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), medullary thyroid carcinoma (MTC), cervical neuroendocrine carcinoma (CNEC), gastrointestinal pancreatic neuroendocrine tumor (GEP-NEN), bladder cancer, bladder NEN (BMEN), prostate cancer, NE prostate cancer (NEPC), Merkel cell carcinoma (MCC), breast cancer, liver cancer, hepatocellular carcinoma (HCC), glioma, pancreatic cancer, or combinations thereof.

27. The method of claim 25, further comprising co-administering an effective amount of a therapeutic agent, wherein the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, an anti-estrogenic agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof.

28. The method of claim 27, wherein the therapeutic agent comprises capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, nitrogen mustard, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, leucovorin, gemcitabine, oxaliplatin, irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, and ampicillin. Mite, testosterone, vorozo, formestane, faldrozol, erlotinib, lafatinib, dasatinib, gefitinib, osimertinib, vandetanib, afatinib, imatinib, pazopanib, lapatinib, sunitinib, nilotinib, sorafenib, albumin-bound paclitaxel, everolimus, sirolimus esterified, dabrafenib, vemurafenib, trametinib, vinpocetine, apatinib, crizotinib, perifolfen, olaparib, bortezomib, tofacitinib, trastuzumab, or their derivatives or combinations thereof.

29. The method of claim 25, wherein the subject is a human.

30. A solution comprising an effective concentration of the antibody of claim 10 or the immunoconjugate of claim 17, wherein the solution is plasma of a subject.

Images