Anti-dll3 antibodies and methods of making and using the same

Abstract

A delta-like ligand 3 (DLL3) binding peptide with binding specificity to human DLL3, comprising an amino acid sequence having at least 98%, 95%, or 92% 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.

Description

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

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

[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.

[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] An estimated 200,000 people die from SCLC worldwide.[4] Case numbers and deaths vary annually due to the strong 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 exceeding 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 relapse 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 therapy also include other neuroendocrine-derived 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 required for the normal development of pulmonary neuroendocrine cells. ASCL1 has been identified as an oncogenic driver in approximately 60% of small cell lung cancers

[11] . Furthermore, 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, while expression is limited or absent in normal lung tissue[7]. DLL3 binds to Notch in a cis-regulatory manner, blocking cell-cell interactions and triggering Notch internalization [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, half-life-extended 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 for SCLC, showing 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] . Terslin-lovatozumab (ROVA-T) is a first-in-class antibody-drug conjugate (ADC) from Abbvie / StemCentRx. ROVA-T has shown regression in SCLC; however, its clinical activity is modest, and overall survival has not exceeded that of the current standard of care, 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 that target neuroendocrine tumors expressing DLL3, including ES-SCLC. Summary of the Invention

[0008] The following description 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 H1 with the amino acid sequence SEQ ID: 120 (TYGMT), and CDR H3 with the amino acid sequence SEQ ID: 105 (VIYASGGTYYATWAKG). CDR H2 with amino acid sequence ID: 105 (VIYASGGTYYATWAKG) and CDR H3 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 CDR L1 with the amino acid sequence SEQ ID: 101 (QASEDISGWLA), and CDR L3 with the amino acid sequence SEQ ID: 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); or CDR L1 with the amino acid sequence SEQ ID: 101 (QASEDISGWLA), and CDR L3 with the amino acid sequence SEQ ID: SEQ ID: 109 (QGYDSNSVENA). CDR L2 with ID: 102 (WASNLAS) and CDR L3 with amino acid sequence 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 terminal 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 includes 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, 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] On one hand, this application provides an antibody or its binding domain that has binding specificity to human DLL3. This 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, the 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, the 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] On the one hand, this application provides one or more isolated nucleic acid sequences that encode the antibodies, binding domains, and DLL3 binding peptides disclosed herein.

[0024] On the one hand, this application provides an expression vector or host cell containing an isolated nucleic acid sequence encoding an antibody, a binding domain, and a DLL3 binding peptide disclosed herein.

[0025] On one hand, 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 includes cytotoxic agents, 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.

[0028] In one implementation, the therapeutic agents include 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, perifolfenib, olaparib, bortezomib, tofatinib, trastuzumab, or their derivatives or combinations thereof.

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

[0030] On one hand, 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] On one hand, this application provides a method for generating the antibody of claim 8, comprising culturing host cells such that the DNA sequence encoding the antibody of claim 8 is expressed, and purifying the antibody.

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

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

[0034] Cancer can be any cancer that expresses DLL3. In one implementation, 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 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, and testrolide. Vorozolol, formetastatin, faldazole, 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, perifolfenib, olaparib, bortezomib, tofacitinib, trastuzumab, or their derivatives or combinations thereof.

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

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

[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 are therefore not intended to limit its scope. Further specific and detailed description of the disclosure will follow below using the drawings, in which: Figure 1 The results of a direct sandwich ELISA used for screening and identification of anti-DLL3 antibodies are shown. Figure 2 FACS results were depicted, 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 The biolayer interference affinity of chimeric antibodies binding with recombinant human and recombinant cynomolgus monkey DLL3 with high affinity is shown, with KD values ​​ranging from 0.1 to 1 nM. Figure 4 The OCTET results show that, as detected by the biolayer interferometry method, the humanized anti-DLL3 clone 5018A1 GS binds to recombinant mouse and rat DLL3 with high affinity across species, with a KD value of less than 1 nM. Figure 5 The OCTET results show that the humanized anti-DLL3 clones SI-83M10 (5018A1 GS VH-N99S) and SI-83M8 (5029C10 FRS) bind to human DLL3 across species with high affinity and co-affinity, with KD values ​​in the picomolar range. Figure 6 The flow cytometry histograms of the screened purified antibodies are shown, indicating that humanized anti-DLL3 clones SI-83M10 (5018A1 GS VH-N99S) and SI-83M8 (5029C10 FRS), as well as 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 eukaryotic expression vectors encoding the human DLL3-EpCAM fusion construct (SEQ ID NO: 33). These human cancer cell lines include (6A) ExpiCHO cells, (6B) CORL279, (6C) NCI-H82, and (6D) MIA PaCa-2, where black lines and gray lines represent stained and unstained cells, respectively. Figure 7 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 are depicted, which shows that 5018A1 recognizes a novel epitope; Figure 8 Antibody-dependent cell-mediated cytotoxicity (ADCC) of anti-DLL3 antibody SI-83M10 (also known as 5018A1 GS VH-N99S) and controls was depicted 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) DLL3-expressing Burkitt lymphoma cells (Daudi-NR), (8B) small cell lung cancer cells (COR-L279), (8C) CHO cells, and (8D) DLL3-expressing CHO cells. Figure 9 The internalization and lysosomal transport of anti-DLL3 antibodies were depicted using time-series live-cell fluorescence microscopy quantitative red calibration units (RCU), which represent the internalization and transport signals of 10 nM anti-DLL3 antibody (SI-83M10, 5018A1 GS VH-N99S), anti-DLL3 reference antibody (SI-83C1, lovatozumab), and anti-CD20 antibody (rituximab) in (9A) DLL3-expressing small cell lung cancer cell line COR-L279; (9B) DLL3-expressing epithelial cell line SHP-77 derived from non-encapsulated primary lung tumors; and (9C) DLL3-negative gastric cancer cell line NUGC-4. The results showed that SI-83M10 had higher internalization efficiency and more signals for lysosomal transport (indicating cytotoxic load in the case of ADC).

[0039] Figure 10 The heavy chain (10A) and light chain (10B) sequences of the lead and reference anti-DLL3 antibodies are shown, including SI-83M10, SI-83M8, 5257C8 BSM, FZ-AD005, GenSun mAb, Gocatamig, Obrixtamig, PT217, lovatozumab (SI-83C1), and talatumab (SI-83C2); and Figure 11 Sequence similarity between SI-83M10, SI-83M8, or 5257C8 BSM and the mean of the remaining reference antibodies was depicted, showing the percentage of identity of CDR (11A), frame-only region (11B), and variable region (CDR+frame region) (11C). Detailed Implementation

[0040] In the following detailed description, reference is made to the accompanying drawings, which form a part of the description. 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 is readily understood that aspects of this disclosure (as shown in the general description and drawings) 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 plural forms, unless the context is inappropriate.

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

[0044] The term "antigen" refers to an entity or segment 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 possess the full antigen-binding function of the antibody as well as additional functions. Examples of binding 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 a single arm of the antibody 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 disclosed herein 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 reflects 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 its broadest sense, specifically encompassing single monoclonal antibodies and / or recombinant antibodies (including agonist and antagonist antibodies), antibody compositions with 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 can 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 aforementioned antibodies and fragments.

[0049] The term "Fv" refers to the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a tightly, non-covalently associated heavy-chain variable domain and a light-chain variable domain. In this configuration, the three CDRs of each variable domain interact to define the 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 an Fv containing only three antigen-specific CDRs) can recognize and bind antigens, although with lower affinity than the entire binding site.

[0050] In some implementations, antibodies may include immunoglobulin molecules and the immunoactive portion of immunoglobulin molecules, i.e., molecules containing binding sites that specifically bind to 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 the constant domain, the light chains of antibodies (immunoglobulins) from any vertebrate species can be classified 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 classes based on the amino acid sequence of their heavy chain constant domains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Several of these can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, and IgG-4; and IgA-1 and IgA-2. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well-known.

[0052] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical except for the possible small number of naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody formulations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to specificity, monoclonal antibodies have the advantage of being synthesized from hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed 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 a 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 using a variety of methods, including but not limited to mouse hybridoma, phage display, recombinant DNA, direct molecular cloning of antibodies 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 chain and / or light chain is identical or homologous to the 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 the 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 (US 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 with a CDR derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived portion of the molecule derived from one (or more) human immunoglobulins. Additionally, the framework region support residues can 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)). Humanizing 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" refer to a biomolecule that 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 expressing it. Typically, purified polypeptides are prepared through at least one purification step. An "isolated" or "purified" antibody refers to an antibody that is substantially free of other antibodies with different antigen-binding specificities.

[0056] The term "immunogenicity" 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 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 similarly structured molecule that does not have binding activity). For instance, specific binding can be determined by competing with a target-like control molecule.

[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 specific 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.

[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 relative to the control, where KA or Ka refers to the association rate of the specific antibody-antigen interaction.

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

[0062] Example Example 1. Generation of anti-DLL3 antibody Rabbit antibodies immunity For immunization, New Zealand White rabbits were divided into two groups (see Tables 1A and 1A). Group 1 received a primary immunization with 100 μg of DLL3-His protein (catalog number DL3-H52H4) formulated with Freund's complete adjuvant, purchased from Acro. Then, the rabbits received four booster immunizations at 7-day intervals, including one 50 μg immunization and two 25 μg immunizations, alternating between Freund's incomplete adjuvant and Alum CpG2007. After the fourth immunization, the rabbits received three booster immunizations at 21-day intervals with 25 μg protein, alternating between Freund's incomplete adjuvant and Alum CpG2007. Twenty-eight days after the third booster immunization, another round of booster immunizations at 21-day intervals began, including three 25 μg booster immunizations and two 10 μg booster immunizations, alternating between Freund's incomplete adjuvant and Alum CpG2007. 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: a single 10 μg immunization followed by four 5 μg immunizations, with alternating adjuvants of Freund's incomplete adjuvant and Alum CpG2007. 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 (no blood was collected on days 98, 245, and 308), PBMCs were isolated, and stored in liquid nitrogen for subsequent sorting.

[0063] PBMC separation Blood diluted 1:1 with DPBS 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 tube was centrifuged at 2500 rpm for 30 minutes at room temperature without braking. The white PBMC layer was then carefully removed and the tube was transferred to a clean 50 mL centrifuge tube and diluted with DPBS to 50 mL. The tube was then centrifuged at 2500 rpm for 10 minutes at room temperature with braking to pellet the cells. 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. The tube was then centrifuged at 2500 rpm for 10 minutes at room temperature to pellet the cells again. The PBMCs were then resuspended 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 revived, the cells were stained in an antibody staining mixture containing specific rabbit B cell markers. B cells were enriched from the revived PBMCs by magnetically activated cell sorting (MACS) using a QuadroMACs magnetic column separator (Miltenyi Biotec, catalog number 130-090-976). After MACS separation, the cells were stained with live / dead cell efluor780 active dye (ebioscience, catalog number 65-0865-14), washed, resuspended in buffer, and maintained at 4°C until cells were sorted by fluorescence-activated cell sorting (FACS).

[0065] Before sorting B cells, appropriate compensation was performed using the sorting software and manual adjustments. The sorting gate was manually adjusted to obtain live IgG-positive rabbit B cells. 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 antibodies Before ELISA screening, B cell culture supernatant was harvested from each culture plate and stored at 4°C for screening. Remaining PBMCs in the culture plates were stored in RNA Later (Invitrogen, catalog number AM7021) at -80°C for subsequent RT-PCR amplification of the antibody variable region. 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 then 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 incubation with the primary antibody, the plate was washed again as described above. Then, HRP-conjugated anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, catalog number 111-035-046) diluted 1:15,000 (with DPBS containing 2% BSA) was added and incubated at 4°C for 30 minutes. The plate was washed, and the binding antibody was detected using TMB substrate (ThermoFisher Scientific, catalog number SS04). The plate was incubated at room temperature for 10 minutes, and the reaction was terminated using commercially available stop solution (ThermoFisher Scientific, catalog number SS04). The plate was read at 450 nm using a spectrophotometer. Based on the higher OD value compared to the negative control (secondary antibody only), DLL3-specific rabbit IgG antibody was detected.

[0067] The results of a direct sandwich ELISA used for screening and identifying anti-DLL3 antibodies are as follows: Figure 1 As shown. 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 binding DLL3-His protein were used for amplification of the antibody variable region to identify heavy and light chain sequences.

[0068] Example 2. Rabbit / human chimeric monoclonal antibody Amplification of antibody variable regions B cell clones selected by ELISA were thawed after storage at -80°C. Heavy and light chain variable sequences were amplified by multiplex RT-PCR using leader sequences and specific primers targeting rabbit IgG and rabbit κ sequences, as well as constant regions. In a secondary PCR, nested primers containing restriction enzyme 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. Sequencing-verified expression plasmids were transiently co-transfected into 293 HEK cells to generate rabbit / human chimeric antibodies.

[0069] Characterization of chimeric anti-DLL3 antibodies – Flow cytometry The binding of the supernatant containing the recombinant antibody to the surface-expressed DLL3 was screened by flow cytometry. Normally, DLL3 is located in the Golgi apparatus within cells. 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. ExpiCHO cells were transiently transfected with a eukaryotic expression vector encoding the human or cynomolgus monkey DLL3-EpCAM fusion construct (SEQ ID NO: 33-34). ExpiCHO cells transfected with unrelated DNA served as a negative control. Two days after transfection, cells were collected, stained with a cell tracking dye (ThermoFisher Scientific, catalog number C34565), and seeded at approximately 1.5 × 10^5 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 again, 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 bind to CHO cells transfected with human and cynomolgus monkey DLL3 but not to the negative control (see [link to relevant documentation]). Figure 2 ).

[0070] Characterization of chimeric anti-DLL3 antibodies – OCTET affinity The binding of the supernatant containing the recombinant antibody to DLL3 was verified using a ForteBio Octet Red 384 instrument via biolayer interferometry (also referred to as OCTET in the patent). 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 300 nM concentration using a 1:3 serial dilution. After establishing a baseline in kinetic buffer, the dissociation of DLL3 from the antibody was measured using biolayer interferometry. Curve fitting was performed using the manufacturer's software, and affinity was calculated. The biolayer interferometry affinity results of the disclosed antibody indicate that the chimeric antibody binds to recombinant human DLL3 with high affinity, with KD values ​​ranging from 0.1 to 1 nM. Figure 3 All antibodies bound to recombinant human and cynomolgus monkey DLL3.

[0071] Cross-species binding of DLL3 in mice and rats Referring to the method described in the foregoing embodiments, the cross-species binding of humanized anti-DLL3 clone 5018A1 GS to mouse and rat DLL3 was analyzed using OCTET. Figure 4 As shown, the humanized 5018A1 GS binds to recombinant mouse and rat DLL3 with high affinity using the biolayer interferometry method, with a KD value of less than 1 nM. However, after affinity modification, the OCTET response value and affinity decrease.

[0072] Example 3. Humanized anti-DLL3 antibody Humanization of variable regions in heavy and light chains The "Predict Humanizing Mutations" algorithm in Discovery Studio was used to humanize the variable regions of the antibody heavy and light chains selected as lead antibodies. The chosen humanization methods included Best Single Mutation (BSM), High-Frequency Residue Substitution (FRS), or Germline Transfer (GS). For 5029C10, computational extrapolation was also used to obtain the humanized sequence. Using Genewiz, the DNA sequences encoding the VH and VL regions of the humanized antibody were cloned into a mammalian expression vector containing the human IgG1 / κ constant region. Sequence analysis revealed the presence of an NG motif within the CDR-H3 of the heavy chain of clone 5018A1. To eliminate the risk of deamidation, a series of mutants targeting asparagine or glycine residues were generated, 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 engineered clones are shown in Table 2A, including ExpiCHO transfection titer and percentage of target protein after purification (% POI).

[0073] Antibody clusters Antibody epitopes were clustered using a ForteBio OCTET Red 384 instrument via a biolayer interference (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). Antibodies were clustered using the instrument software based on hierarchical clustering of response values ​​from the first 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] OCTET Affinity OCTET characterization was performed using humanized antibody supernatant, and 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 the 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 the manufacturer's software.

[0076] OCTET affinity and binding affinity of purified antibodies After protein A purification, the binding affinity and affinity of the disclosed humanized antibody to human DLL3 were characterized by OCTET using the method described in the previous embodiments. The purified disclosed antibodies SI-83M10 (also referred to in this text as 5018A1 GS VH-N99S) and SI-83M8 (also referred to in this text 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. High affinity detected by 5018A1-GS-N99S was not calculated because the value exceeded the limits of the software. However, further engineering to modify affinity reduced it to a detectable value of 0.5 nM. Mutations in both the heavy and light chains further reduced the affinity to 153 nM.

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

[0078] The binding of purified humanized antibodies to human cancer cells was characterized 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 the human DLL3-EpCAM fusion construct (SEQ ID NO: 33). Figure 6 Due to the low surface expression and intracellular localization of DLL3, surface binding and intracellular binding were analyzed. Figure 6(See the top and bottom images, respectively). As mentioned earlier, 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 for 15 minutes at 4°C in BD permeabilization wash buffer (BD, catalog number 554723). Cell pellet was then centrifuged, diluted to approximately 1.5 × 10⁵ cells / well, and stained with antibodies serially diluted from 10 μg / mL to 40 ng / mL at 4°C for 1 hour. After washing, cells 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, cells were washed, resuspended in FACS buffer, and binding was determined by flow cytometry using a BDFortessa flow cytometer. As expected, binding ( ) was detected on the surface of transiently transfected ExpiCHO cells. Figure 6 (See the image above).

[0080] Intracellular staining resulted in a >10-fold increase in binding ( Figure 6 (See figure below). Very weak surface staining was detected in 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. Figure 6 (B-6D). This is consistent with the low surface expression and high intracellular expression detected in DLL3-expressing cell lines. Further examination of the binding of reference clones SI-83C1 and SI-83C2 was conducted. Data indicate that the disclosed clones SI-83M8 and SI-83M10 bind to the neuroendocrine tumor-derived cell model.

[0081] Example 4. Comparative advantages of anti-DLL3 antibodies as therapeutic candidates When targeting tumors with heterogeneous antigen expression, antibody-dependent cytotoxicity (ADCC) against tumors with low-density antigen expression is often 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 activities. 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, with KD values ​​ranging from 0.2 to 4 nM. Affinity-modified antibodies derived from the parental clone 5018A1 are also disclosed. One of these variants has a low affinity 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 text) and tesculin-lovatotuzumab (Abbvie / StemCentRx, also referred to as SI-83C2 in this text), 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 (lovastatin) and SI-83C2 (talatumab) are shown in the cluster matrix table (see [link]). Figure 7 A clone 5257C8 was unidirectionally blocked by a clone 5029C10, indicating that there is partial overlap in their epitopes (in...). Figure 7 (Circled in the middle). None of the candidate antibodies blocked the binding of the reference antibody, nor were they blocked by the reference antibody, indicating that the epitopes that these antibodies bind to are different from those of the antibodies currently under investigation.

[0084] However, the clustering results of the biological layer interferometry method show that the disclosed clone binds to a unique epitope of DLL3 (see [link to original text]). Figure 7 (Clon 5018A1 in the middle).

[0085] Example 5. Antibody-dependent cell-mediated cytotoxicity The Fc-mediated ADCC activity of antibody-based therapeutic agents can be compared in vitro to determine whether the antibody has the potential to enhance the therapeutic mechanism of action. 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 properties or antibody internalization may affect ADCC activity

[17] .

[0086] To test this, human primary NK (natural killer) cells were enriched from whole blood through negative selection and then subjected to density gradient centrifugation to become “effective” cells. Cells used as target cells were transduced to express riboglobin (NR) and included Daudi (B lymphoblastoid 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 by using time-series fluorescence microscopy to assess the attenuation of the NR signal in target cells.

[0087] like Figure 8 As shown in Figure A, after 36 hours, CD20-specific rituximab detected ADCC activity in the Daudi-NR target cell line, demonstrating the effectiveness of this assay. As expected, no cytotoxic effect 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. Figure 8 B). This activity was significantly higher than lovatozumab, demonstrating that the latter had no killing effect on COR-L279 target cells. In transduced CHO cells, as expected, ADCC was not detected in CHO-NR. Figure 8 C). However, after 60 hours, both SI-83M10 and lovatozumab showed significant killing effects against CHO-NR-DLL3. The system controls, cetuximab and lovatozumab, showed negative results, as expected. Figure 8 D). In summary, the data indicate that SI-83M10 enhances ADCC activity in small cell lung cancer cell lines compared to lovatozumab.

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

[18] . After antibody binding, internalization facilitates the uptake and transport of the cytotoxic load to the lysosomes of cancer cells, 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. 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] Using time-series live-cell fluorescence microscopy, internalization and transport signals of SI-83M10 at a concentration of 10 nM can be easily detected. Figure 9 Signals were measured in red calibration units (RCU), automatically counted, and plotted over time. In the small cell lung cancer cell line COR-L279 expressing DLL3, SI-83M10 showed higher internalization efficiency and more signals transported to lysosomes (indicating cytotoxic load in the ADC case) compared to lovastatin. Figure 9 A). In SHP-77 cells, an epithelial cell line expressing DLL3 derived from non-encapsulated primary lung tumors, the internalization levels of lovatozumab were similar to those of SI-83M10 ( Figure 9 B). Finally, in the DLL3-negative gastric cancer cell line NUGC-4, no internalization of SI-83M10 or the control was observed (B). Figure 9 C).

[0091] Example 7. Sequence alignment of lead and reference antibodies To investigate the structural basis of their unique binding properties, the coding sequences of the published antibodies (SI-83M10, SI-83M8, 5257C8 BSM) and reference antibodies (see Table 3) were compared using Geneious Prime bioinformatics software according to the Kabat numbering rules. Figure 10 As shown. The heavy and light chain complementarity-determining regions (CDRs) of all antibodies were compared (in... Figure 10 (A, 10B are highlighted in the upper and lower diagrams). Figure 11 In sequences A, 11B, and 11C, the percentages of identity for CDR-only, frame region-only (FR), and complete variable region (CDR+FR) were calculated for comparison. In the aligned CDR sequences, SI-83M10 showed 13-46% identity with each reference antibody, while the identity among the reference antibodies was 25-45%. A notable exception 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. The broad range of sequence identity between the disclosed sequences and 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 of SI-83M10, SI-83M8, or 5257C8 BSM with a single reference antibody and the mean percentage of identity among the remaining reference antibodies. While sequence differences were observed among all antibodies, significant differences in the percentage of identity between reference antibodies and the published clones were observed only when compared with a few clinical antibodies (P < 0.05). For example, the percentage of identity of SI-83M10 with FZ-AD005 was significantly lower than the pooled percentage of identity for the listed reference antibodies (P < 0.0001 for CDR only). The same result was detected with SI-83M10 and Gocatamig (P = 0.013 for CDR only). The percentage of identity of SI-83M8 was significantly lower than the pooled percentage of identity for the listed reference antibodies compared to talatumab (P = 0.05 for CDR only). The 5257C8 BSM showed the highest sequence diversity among the reference antibody, with a significantly lower percentage of identity compared to FZ-AD005 (for CDR only, P=0.0093), Gensun (for CDR only, P=0.0331), and lovatozumab (for CDR only, P=0.0046). While not all differences detected in the amino acid sequences encoding the primary structure of the CDR were statistically significant, they formed the basis for various characteristics of the disclosed antibodies, including epitope clustering (…). Figure 7 ), and thus constitute the basis of biological activity.

[0093] References (each of these references is incorporated into this paper in its entirety through citation) 1.Oronsky, B., Reid, T. R., Oronsky, A., & Carter, C. A. (2017).What's New in SCLC? A Review.Neoplasia (New York, N.Y.), 19(10), 842–847.https: / / doi.org / 10.1016 / j.neo.2017.07.007 2.Gazdar, A. F., Bunn, P. A., & Minna, J. D. (2017).Small-cell lungcancer: what we know, what we need to know and the path forward.Naturereviews.Cancer, 17(12), 725–737.https: / / doi.org / 10.1038 / nrc.2017.87 3.Drapkin, B. J., & Rudin, C. M. (2021).Advances in Small-Cell LungCancer (SCLC) Translational Research.Cold Spring Harbor perspectives inmedicine, 11(4), a038240.https: / / doi.org / 10.1101 / cshperspect.a038240 4.Bray, F., Ferlay, J., Laversanne, M., Brewster, D. H., GombeMbalawa, C., Kohler, B., Piñeros, M., Steliarova-Foucher, E., Swaminathan,R., Antoni, S., Soerjomataram, I., & Forman, D. (2015).Cancer Incidence inFive Continents: Inclusion criteria, highlights from Volume X and the globalstatus 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 lungcancer: Tumor heterogeneity, diagnosis, therapy, and future directions.CA: acancer journal for clinicians, 10.3322 / caac.21785.Advance onlinepublication.https: / / doi.org / 10.3322 / caac.21785 6.Schwendenwein, A., Megyesfalvi, Z., Barany, N., Valko, Z., Bugyik,E., Lang, J., Ferencz, B., Paku, S., Lantos, A., Fillinger, J., Rezeli, M.,Marco-Varga, G., Bogos, K., Gal, Hoda, G. V., Reffy, F. MA,Klepetko, W., Hoetzenecker, K., Laszlo, V., & Dome, B. (2021).Molecularprofiles of small cell lung cancer subtypes: therapeuticimplications.Molecular therapy oncolytics, 20, 470–483.https: / / doi.org / 10.1016 / j.omto.2021.02.004 7.Saunders, LR, Bankovich, AJ, Anderson, WC, Aujay, MA,Bheddah, S., Black, K., Desai, R., Escarpe, PA, Hample, J., Laysang, A.,Liu, D., Lopez-Molina, J., Milton, M., Park, A., Pysz, B., Shalingerland, MA, B. Torgov, M., Williams, SA, Foord, O., … Dylla, SJ(2015).A DLL3-targeted antibody-drug conjugate eradicates high-gradepulmonary neuroendocrine tumor-initiating cells in vivo.Science translationalmedicine, 7(302), 302ra136.https: / / doi.org / 10.1126 / scitranslmed.aac9459 8.Kim, J. W., Ko, J. H., & Sage, J. (2022).DLL3 regulates Notchsignaling 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 ProcessingWith 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., MeazzaPrina, M., Cordani, N., Sala, E., & Canova, S. (2022).Harnessing DLL3inhibition: From old promises to new therapeutic horizons.Frontiers inmedicine, 9, 989405.https: / / doi.org / 10.3389 / fmed.2022.989405 11.Owen H.D., Giffin M.J., Bailis J.M., Damiette Smit M.A., CarboneD.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., Uchiyama K. (2021).Delta-like canonical Notch ligand 3as a potential therapeutic target in malignancies: A brief overview. CancerScience.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: AnOpen-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 immunotherapyon 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. Biomedicines, 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 F, 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 Table 1A. Immunization protocol for anti-DLL3 antibody generation - Group 1 Table 1B. Immunization regimens for anti-DLL3 antibody generation – Group 2 Table 2A. Characteristic profiles of engineered clones and controls targeting DLL3 Table 2B. Binding affinity of engineered clones targeting DLL3 and controls Table 2C. Binding affinity of engineered clones targeting DLL3 and controls Table 2D. Cell binding of engineered clones targeting DLL3 and controls * Transfected ExpiCHO cells (MFI at 1 μg / mL) Table 3. Summary of anti-DLL3 antibodies sequence list *The CDR region in the amino acid sequence is used Bold express Seq ID 1: 5018A1 light chain variable region nucleic acid sequence GATGTTGTGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGGATATTAGCGGTTGGTTGGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTCATCTACTGGGCATCCAATCTGGCA TCTGGGGTCTCATCGCGGTTCAAAGGCAGTAGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATTCTGCCACTTACTACTGTCAATCTACTTTTTATGGTACTAGTGATGTTGCTGCTTTCGGCGGAGGGACCGAGGTGGTGNTCAAA Seq ID 2: 5018A1 heavy chain variable region nucleic acid sequence CAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGCCTCTGGATTCGACTTCAATACCTACTACATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTTATGCTAGTGGTGGCACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGATGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGCCTATCCTGATAATGGTGATGGATTGGACATCTGGGGCCCAGGGACCCTCGTCACCGTCTCGAGC >Seq ID 3: Nucleotide sequence of the variable region of the light chain of 5029C10 GATGTTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTAGTTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTATCAGGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAGGCTATGATTCTAATAGTGTTGAGAATGCTTTCGGCGGAGGGACCGAGGTGGAGTTCAAA >Seq ID 4: Nucleotide sequence of the variable region of the heavy chain of 5029C10 CAGTCTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGAGGATCCCTGACACTCACCTGCACAGTCTCTGGAATCGACTTCAGTAGGAATGTAATTAATTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTGCTACTGCTGGTGACACATACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACACCCGAGGACACGGCCACCTATTTCTGTGCCGGAAAATATGGTGATACTTTTGATCTCTGGGGCCCGGGCACCCTGGTCACCGTCTCGAGC >Seq ID 5: Nucleotide sequence of the variable region of the light chain of 5257C8 GCCTATGATCTGACCCAGACTCCAGCCTCTGTGGAGGTAGATGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTGGTGGTAATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTATTCTGCATCCAAATTGGCAAGTGGGGTCCCATCGCGGTTCAGAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAACAGGCTTGGAGTTATAGTAATGTTGATAATACTTTCGGCGGAGGCACCGAGGTGGTGTTCAAA >Seq ID 6: Nucleotide sequence of the variable region of the heavy chain of 5257C8 CAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGTACAGTCTCTGGAATCGACCTCAGTAGTCACTATTTCAATTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGGATCGTTTATGCTAGTGGTAGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCTGGAGATCGGAGTGTTGCCTATTCAAACATCTGGGGCCAGGGGACCCTCGTCACCGTCTCGAGC >Seq ID 7: Nucleic acid sequence of the variable region of the light chain of 501A8-GS GACGTGGTGATGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTGACCATCACCTGCCAGGCCTCCGAGGACATCTCCGGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACTGGGCCTCCAACCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGTCCACCTTCTACGGCACCTCCGACGTGGCCGCCTTCGGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID 8: Nucleic acid sequence of the variable region of the heavy chain of 501A8-GS CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTTCGACTTCAACACCTACTACATGACCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCGTGATCTACGCCTCCGGCGGCACCTACTACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACACCATGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGCCTACCCCGACAACGGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 9: Nucleotide sequence of the variable region of the light chain of 5029C10-FRS GACGTGGTGATGACCCAGTCCCCCTCCTCCCTGTCCGCCTCCGTGGGCGACAGGGTGACCATCTCCTGCCAGGCCTCCCAGTCCATCTCCTCCTACCTGTCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACCAGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGGAGCCCGAGGACTTCGCCACCTACTACTGCCAGGGCTACGACTCCAACTCCGTGGAGAACGCCTTCGGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID 10: Nucleotide sequence of the variable region of the heavy chain of 5029C10-FRS CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCATCGACTTCTCCAGGAACGTGATCAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCATCATCGCCACCGCCGGCGACACCTACTACGCCAACTGGGCCAAGGGCAGGTTCACCATCTCCAAGACCTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCGGCAAGTACGGCGACACCTTCGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 11: Nucleic acid sequence of the variable region of the light chain of 5257C8-BSM GACTACCAGCTGACCCAGTCCCCCTCCTCCGTGGCCCTGACCGTGGGCCAGAGGGTGACCATCAACTGCCAGGCCTCCCAGTCCATCGGCGGCAACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGGCCCAAGCTGCTGATCTACTCCGCCTCCAAGCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGTCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTGGTCCTACTCCAACGTGGACAACACCTTCGGCGGCGGCACCAGGGTGGACATCAAG >Seq ID 12: Nucleic acid sequence of the variable region of the heavy chain of 5257C8-BSM CAGCAGCTGCAGGAGTCCGGCGGCAGGCTGATCAAGCCCGGCGAGCCCCTGAGGCTGACCTGCAAGACCTCCGGCATCGACCTGTCCTCCCACTACTTCAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCATCGTGTACGCCTCCGGCTCCACCTACTACGCCTCCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCACCAACACCGTGTTCCTGCAGATGAGGTCCCTGAGGTCCGAGGACACCGCCGTGTACTACTGCGCCGGCGACAGGTCCGTGGCCTACTCCAACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 13: Nucleotide sequence of the variable region of the heavy chain of 5018A1-GS VH-N99S CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTTCGACTTCAACACCTACTACATGACCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCGTGATCTACGCCTCCGGCGGCACCTACTACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACACCATGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGCCTACCCCGACAGCGGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCC >Seq ID 14: Nucleotide sequence of the variable region of the heavy chain of 5018A1-GS VH-N99S VH-Y33G CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTTCGACTTCAACACCTACGGCATGACCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCGTGATCTACGCCTCCGGCGGCACCTACTACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACACCATGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGCCTACCCCGACAGCGGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCC >Seq ID 15:Nucleic acid sequence of the variable region of the light chain of 5018A1-GS VL-Y93G GACGTGGTGATGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTGACCATCACCTGCCAGGCCTCCGAGGACATCTCCGGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACTGGGCCTCCAACCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGTCCACCTTCGGCGGCACCTCCGACGTGGCCGCCTTCGGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID 16:Amino acid sequence of the variable region of the light chain of 5018A1 DVVMTQTPSSVSAAVGGTVTIKC QASEDISGWLA WYQQKPGQPPKLLIY WASNLAS GVSSRFKGSRSGTEFTLTISDLECADSATYYC QSTFYGTSDVAA FGGGTEVVXK >Seq ID 17:Amino acid sequence of the variable region of the heavy chain of 5018A1 QSLEESGGRLVTPGTPLTLTCTASGFDFN TYYMT WVRQAPGKGLEWIG VIYASGGTYYATWAKG RFTISKTSTTMDLKITSPTTEDTATYFCAR AYPDNGDGLDI WGPGTLVTVSS >Seq ID 18: 5029C10 light chain variable region amino acid sequence DVVMTQTPASVEAAVGGTVTIKC QASQSISSYLS WYQQKPGQRPKLLIY QASTLAS GVPSRFKGSGSGTQFTLTISDLECADAATYYC QGYDSNSVENA FGGGTEVEFK >Seq ID 19: 5029C10 heavy chain variable region amino acid sequence QSQSVEESGGRLVTPGGSLTLTCTVSGIDFS RNVIN WVRQAPGKGLEWIG IIATAGDTYYANWAKG RFTISKTSSTTVDLKMTSLTPEDTATYFCAG KYGDTFDL WGPGTLVTVSS Seq ID 20: 5257C8 light chain variable region amino acid sequence AYDLTQTPASVEVDVGGTVTIKC QASQSIGGNLA WYQQKPGQRPKLLIY SASKLAS GVPSRFRGSGSGTEFTLTISDLECDDAATYYC QQAWSYSNVDNT FGGGTEVVFK >Seq ID 21: 5257C8 heavy chain variable region amino acid sequence QSVEESGGRLVTPGTPLTLTCTVSGIDLS SHYFN WVRQAPGKGLEWIG IVYASGSTYYASWAKG RFTISKTSTTTVDLKMTSLTTEDTATYFCAG DRSVAYSNI WGQGTLVTVSS >Seq ID 22: 5018A1-GS light chain variable region amino acid sequence DVVMTQSPSSVSASVGDRVTITC QASEDISGWLA WYQQKPGQAPKLLIY WASNLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QSTFYGTSDVAA FGGGTKVEIK >Seq ID 23: 5018A1-GS heavy chain variable region amino acid sequence QVQLVESGGGLVQPGGSLRLSCAASGFDFN TYYMT WVRQAPGKGLEWVG VIYASGGTYYATWAKG RFTISKSKNTMYLQMNSLRAEDTAVYYCAR AYPDNGDGLDI WGQGTLVTVSS >Seq ID 24: 5029C10-FRS light chain variable region amino acid sequence DVVMTQSPSSLSASVGDRVTISC QASQSISSYLS WYQQKPGQAPKLLIY QASTLAS GVPSRFSGSGSGTDFTLTISSLEPEDFATYYC QGYDSNSVENA FGGGTKVEIK >Seq ID 25: 5029C10-FRS heavy chain variable region amino acid sequence QVQLVESGGGLVKPGGSLRLSCAASGIDFS RNVIN WVRQAPGKGLEWVG IIATAGDTYYANWAKG RFTISKTSKNTVYLQMNSLRAEDTAVYYCAG KYGDTFDL WGQGTLVTVSS Seq ID 26: 5257C8-BSM light chain variable region amino acid sequence DYQLTQSPSSVALTVGQRVTINC QASQSIGGNLA WYQQKPGQRPKLLIY SASKLAS GVPSRFSGSGSGTDFTLTISSLQSEDFATYYC QQAWSYSNVDNT FGGGTRVDIK Seq ID 27: 5257C8-BSM heavy chain variable region amino acid sequence QQLQESGGRLIKPGEPLRLTCKTSGIDLS SHYFN WVRQAPGKGLEWIG IVYASGSTYYASWAKG RFTISKSTNTVFLQMRSLRSEDTAVYYCAG DRSVAYSNI WGQGTLVTVSS Seq ID 28: 5018A1-GS VH-N99S heavy chain variable region amino acid sequence QVQLVESGGGLVQPGGSLRLSCAASGFDFN TYYMTWVRQAPGKGLEWVG VIYASGGTYYATWAKG RFTISKSKNTMYLQMNSLRAEDTAVYYCAR AYPDSGDGLDI WGQGTLVTVSS >Seq ID 29: Amino acid sequence of the variable region of the heavy chain of 5018A1-GS VH-N99S VH-Y33G QVQLVESGGGLVQPGGSLRLSCAASGFDFN TYGMT WVRQAPGKGLEWVG VIYASGGTYYATWAKG RFTISKSKNTMYLQMNSLRAEDTAVYYCAR AYPDSGDGLDI WGQGTLVTVSS >Seq ID 30: Amino acid sequence of the variable region of the light chain of 5018A1-GS VL-Y93G DVVMTQSPSSVSASVGDRVTITC QASEDISGWLA WYQQKPGQAPKLLIY WASNLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QSTFGGTSDVAA FGGGTKVEIK >Seq ID 31: Amino acid sequence of the heavy chain of 5018A1-GS null Fc QVQLVESGGGLVQPGGSLRLSCAASGFDFN TYYMT WVRQAPGKGLEWVG VIYASGGTYYATWAKG RFTISKSKNTMYLQMNSLRAEDTAVYYCAR AYPDSGDGLDI WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 32: 5018A1-GS null Fc light chain amino acid sequence DVVMTQSPSSVSASVGDRVTITC QASEDISGWLA WYQQKPGQAPKLLIY WASNLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QSTFYGTSDVAA FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVCTKSFNRGEC Seq ID 33: Human DLL3 ECD nucleic acid sequence containing the EpCAM transmembrane domain and cytoplasmic domain. Seq ID 34: Cynomolgus monkey DLL3 ECD nucleic acid sequence containing EpCAM transmembrane and cytoplasmic domains. >Seq ID 35: Human IgG1 Amino Acid Sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 36: Human CK Amino Acid Sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 37: 5018A1-GS Heavy Chain Amino Acid Sequence QVQLVESGGGLVQPGGSLRLSCAASGFDFN TYYMT WVRQAPGKGLEWVG VIYASGGTYYATWAKG RFTISKSKNTMYLQMNSLRAEDTAVYYCAR AYPDNGDGLDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 38: Amino acid sequence of the light chain of 5018A1-GS DVVMTQSPSSVSASVGDRVTITC QASEDISGWLA WYQQKPGQAPKLLIY WASNLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QSTFYGTSDVAA FGGGTKVEIKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 39: Amino acid sequence of the heavy chain of 5029C10-FRS QVQLVESGGGLVKPGGSLRLSCAASGIDFS RNVIN WVRQAPGKGLEWVG IIATAGDTYYANWAKG RFTISKTSKNTVYLQMNSLRAEDTAVYYCAG KYGDTFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 40: Amino acid sequence of the light chain of 5029C10-FRS DVVMTQSPSSLSASVGDRVTISC QASQSISSYLS WYQQKPGQAPKLLIY QASTLAS GVPSRFSGSGSGTDFTLTISSLEPEDFATYYC QGYDSNSVENA FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 41: Amino acid sequence of the heavy chain of 5257C8-BSM QQLQESGGRLIKPGEPLRLTCKTSGIDLS SHYFN WVRQAPGKGLEWIG IVYASGSTYYASWAKG RFTISKSTNTVFLQMRSLRSEDTAVYYCAG DRSVAYSNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 42: Amino acid sequence of the light chain of 5257C8-BSM DYQLTQSPSSVALTVGQRVTINC QASQSIGGNLA WYQQKPGQRPKLLIY SASKLAS GVPSRFSGSGSGTDFTLTISSLQSEDFATYYC QQAWSYSNVDNT FGGGTRVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 43: Amino acid sequence of the VH-N99S heavy chain of 5018A1-GS QVQLVESGGGLVQPGGSLRLSCAASGFDFN TYYMT WVRQAPGKGLEWVG VIYASGGTYYATWAKG RFTISKSKNTMYLQMNSLRAEDTAVYYCAR AYPDSGDGLDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 44: Amino acid sequence of the light chain of 5018A1-GS VH-N99S DVVMTQSPSSVSASVGDRVTITC QASEDISGWLA WYQQKPGQAPKLLIY WASNLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QSTFYGTSDVAA FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 45: Amino acid sequence of the heavy chain of 5018A1-GS VH-N99S VH-Y33G QVQLVESGGGLVQPGGSLRLSCAASGFDFN TYGMT WVRQAPGKGLEWVG VIYASGGTYYATWAKG RFTISKSKNTMYLQMNSLRAEDTAVYYCAR AYPDSGDGLDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 46: Amino acid sequence of 5018A1-GS VH-N99S VL-Y93G light chain DVVMTQSPSSVSASVGDRVTITC QASEDISGWLA WYQQKPGQAPKLLIY WASNLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QSTFGGTSDVAA FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 47: Amino acid sequence of human IgG1 null APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAK >Seq ID 48: Amino acid sequence of human CK insert type RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVCTKSFNRGEC >Seq ID 49: Nucleic acid sequence of 5018A1 BSM heavy chain variable region CAGGTGCAGCTGCAGGAGTCCGGCGGCAGGCTGATCAAGCCCGGCGCCCCCCTGAGGCTGTCCTGCAAGGCCTCCGGCTTCGACTTCAACACCTACTACATGACCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCGTGATCTACGCCTCCGGCGGCACCTACTACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCACCAACACCATGTACCTGCAGATCAGGTCCCCCAGGTCCGAGGACACCGCCATCTACTACTGCGCCAGGGCCTACCCCGACAACGGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 50:5018A1 BSM heavy chain variable region amino acid sequence QVQLQESGGRLIKPGAPLRLSCKASGFDFN TYYMT WVRQAPGKGLEWIG VIYASGGTYYATWAK GRFTISKSTNTMYLQIRSPRSEDTAIYYCAR AYPDNGDGLD IWGQGTLVTVSS >Seq ID 51:5018A1 BSM heavy chain amino acid sequence QVQLQESGGRLIKPGAPLRLSCKASGFDFN TYYMTW VRQAPGKGLEWIG VIYASGGTYYATWAK GRFTISKSTNTMYLQIRSPRSEDTAIYYCAR AYPDNGDGLDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 52: Nucleic acid sequence of the variable region of the light chain of 5018A1 BSM GACGTGCAGATGACCCAGTCCCCCTCCTCCGTGTCCGCCACCGTGGGCCAGAGGGTGACCATCAAGTGCCAGGCCTCCGAGGACATCTCCGGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGCCCCCCAAGCTGCTGATCTACTGGGCCTCCAACCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGTGCGACGACTTCGCCACCTACTACTGCCAGTCCACCTTCTACGGCACCTCCGACGTGGCCGCCTTCGGCGGCGGCACCAAGGTGGACGTGAAG >Seq ID 53: Amino acid sequence of the variable region of the light chain of 5018A1 BSM DVQMTQSPSSVSATVGQRVTIKC QASEDISGWLA WYQQKPGKPPKLLIY WASNLAS GVPSRFSGSGSGTDFTLTISSLQCDDFATYYC QSTFYGTSDVAA FGGGTKVDVK >Seq ID 54: Amino acid sequence of the light chain of 5018A1 BSM DVQMTQSPSSVSATVGQRVTIKC QASEDISGWLAWYQQKPGKPPKLLIY WASNLAS GVPSRFSGSGSGTDFTLTISSLQCDDFATYYC QSTFYGTSDVAA FGGGTKVDVKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 55: 5018A1 FRS heavy chain variable region nucleic acid sequence CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTTCGACTTCAACACCTACTACATGACCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCGTGATCTACGCCTCCGGCGGCACCTACTACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACACCATGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGCCTACCCCGACAACGGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 56: 5018A1 FRS heavy chain variable region amino acid sequence QVQLVESGGGLVKPGGSLRLSCAASGFDFN TYYMT WVRQAPGKGLEWVG VIYASGGTYYATWAKG RFTISKSKNTMYLQMNSLRAEDTAVYYCAR AYPDNGDGLDI WGQGTLVTVSS >Seq ID 57: 5018A1 FRS heavy chain amino acid sequence QVQLVESGGGLVKPGGSLRLSCAASGFDFN TYYMT WVRQAPGKGLEWVG VIYASGGTYYATWAKG RFTISKSKNTMYLQMNSLRAEDTAVYYCAR AYPDNGDGLDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 58: Nucleic acid sequence of the variable region of the 5018A1 FRS light chain GACGTGGTGATGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTGACCATCTCCTGCCAGGCCTCCGAGGACATCTCCGGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACTGGGCCTCCAACCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGGAGCCCGAGGACTTCGCCACCTACTACTGCCAGTCCACCTTCTACGGCACCTCCGACGTGGCCGCCTTCGGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID 59: Amino acid sequence of the variable region of the 5018A1 FRS light chain DVVMTQSPSSVSASVGDRVTISC QASEDISGWLA WYQQKPGQAPKLLIY WASNLAS GVPSRFSGSGSGTDFTLTISSLEPEDFATYYC QSTFYGTSDVAA FGGGTKVEIK >Seq ID 60: Amino acid sequence of the 5018A1 FRS light chain DVVMTQSPSSVSASVGDRVTISC QASEDISGWLAWYQQKPGQAPKLLIY WASNLAS GVPSRFSGSGSGTDFTLTISSLEPEDFATYYC QSTFYGTSDVAA FGGGTKVEIKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 61:5029C10 BSM heavy chain variable region nucleic acid sequence CAGGTGCAGCTGGTGGAGTCCGGCGGCAGGCTGGTGAAGCCCGGCGGCTCCCTGAAGCTGACCTGCAAGACCTCCGGCATCGACTTCTCCAGGAACGTGATCAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCATCATCGCCACCGCCGGCGACACCTACTACGCCAACTGGGCCAAGGGCAGGTTCACCATCTCCAAGACCTCCACCAACACCGTGTTCCTGCAGATGAGGTCCCTGAAGTCCGAGGACACCGCCATCTACTACTGCGCCGGCAAGTACGGCGACACCTTCGACCTGTGGGGCCCCGGCACCCTGGTGACCGTGTCCTCC >Seq ID 62:5029C10 BSM heavy chain variable region amino acid sequence QVQLVESGGRLVKPGGSLKLTCKTSGIDFS RNVIN WVRQAPGKGLEWIG IIATAGDTYYANWAKG RFTISKTSTNTVFLQMRSLKSEDTAIYYCAG KYGDTFDL WGPGTLVTVSS >Seq ID 63:5029C10 BSM heavy chain amino acid sequence QVQLVESGGRLVKPGGSLKLTCKTSGIDFS RNVIN WVRQAPGKGLEWIG IIATAGDTYYANWAKG RFTISKTSTNTVFLQMRSLKSEDTAIYYCAG KYGDTFDLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 64: Nucleic acid sequence of the variable region of the light chain of 5029C10 BSM GACGTGCAGATGACCCAGTCCCCCTCCTCCCTGGCCGCCTCCGTGGGCCAGAGGGTGACCATCAACTGCCAGGCCTCCCAGTCCATCTCCTCCTACCTGTCCTGGTACCAGCAGAAGCCCGGCCAGAGGCCCAAGCTGCTGATCTACCAGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCAAGGGCTCCGGCTCCGGCACCCAGTTCACCCTGACCATCTCCTCCCTGCAGTGCGACGACTTCGCCACCTACTACTGCCAGGGCTACGACTCCAACTCCGTGGAGAACGCCTTCGGCGGCGGCACCAGGGTGGAGATCAAG >Seq ID 65: Amino acid sequence of the variable region of the light chain of 5029C10 BSM DVQMTQSPSSLAASVGQRVTINC QASQSISSYLS WYQQKPGQRPKLLIY QASTLAS GVPSRFKGSGSGTQFTLTISSLQCDDFATYYC QGYDSNSVENA FGGGTRVEIK >Seq ID 66: Amino acid sequence of the light chain of 5029C10 BSM DVQMTQSPSSLAASVGQRVTINC QASQSISSYLSWYQQKPGQRPKLLIY QASTLAS GVPSRFKGSGSGTQFTLTISSLQCDDFATYYC QGYDSNSVENA FGGGTRVEIKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 67: Nucleic acid sequence of the variable region of the heavy chain of 5029C10 GS CAGGTGCAGCTGCTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCATCGACTTCTCCAGGAACGTGATCAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCATCATCGCCACCGCCGGCGACACCTACTACGCCAACTGGGCCAAGGGCAGGTTCACCATCTCCAAGACCTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCGGCAAGTACGGCGACACCTTCGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 68: Amino acid sequence of the variable region of the heavy chain of 5029C10 GS QVQLLESGGGLVQPGGSLRLSCAASGIDFS RNVIN WVRQAPGKGLEWVG IIATAGDTYYANWAKG RFTISKTSKNTVYLQMNSLRAEDTAVYYCAG KYGDTFDL WGQGTLVTVSS >Seq ID 69: Amino acid sequence of the heavy chain of 5029C10 GS QVQLLESGGGLVQPGGSLRLSCAASGIDFS RNVIN WVRQAPGKGLEWVG IIATAGDTYYANWAKG RFTISKTSKNTVYLQMNSLRAEDTAVYYCAG KYGDTFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 70: Nucleotide sequence of the variable region of the 5029C10 GS light chain GACGTGGTGATGACCCAGTCCCCCTCCTCCCTGTCCGCCTCCGTGGGCGACAGGGTGACCATCACCTGCCAGGCCTCCCAGTCCATCTCCTCCTACCTGTCCTGGTACCAGCAGAAGCCCGGCCAGGTGCCCAAGCTGCTGATCTACCAGGCCTCCACCCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGGGCTACGACTCCAACTCCGTGGAGAACGCCTTCGGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID 71: Amino acid sequence of the variable region of the 5029C10 GS light chain DVVMTQSPSSLSASVGDRVTITC QASQSISSYLS WYQQKPGQVPKLLIY QASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QGYDSNSVENA FGGGTKVEIK >Seq ID 72: Amino acid sequence of the 5029C10 GS light chain DVVMTQSPSSLSASVGDRVTITC QASQSISSYLSWYQQKPGQVPKLLIY QASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QGYDSNSVENA FGGGTKVEIKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 73:5257C8 FRS heavy chain variable region nucleic acid sequence CAGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCATCGACCTGTCCTCCCACTACTTCAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCATCGTGTACGCCTCCGGCTCCACCTACTACGCCTCCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCGGCGACAGGTCCGTGGCCTACTCCAACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 74:5257C8 FRS heavy chain variable region amino acid sequence QQLVESGGGLVKPGGSLRLSCAASGIDLS SHYFN WVRQAPGKGLEWVG IVYASGSTYYASWAKG RFTISKSKNTVYLQMNSLRAEDTAVYYCAG DRSVAYSNI WGQGTLVTVSS >Seq ID 75:5257C8 FRS heavy chain amino acid sequence QQLVESGGGLVKPGGSLRLSCAASGIDLS SHYFN WVRQAPGKGLEWVG IVYASGSTYYASWAKG RFTISKSKNTVYLQMNSLRAEDTAVYYCAG DRSVAYSNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 76:5257C8 FRS light chain variable region nucleic acid sequence GACTACGTGCTGACCCAGTCCCCCTCCTCCGTGTCCGTGTCCGTGGGCGACAGGGTGACCATCTCCTGCCAGGCCTCCCAGTCCATCGGCGGCAACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACTCCGCCTCCAAGCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGGAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTGGTCCTACTCCAACGTGGACAACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID 77:5257C8 FRS light chain variable region amino acid sequence DYVLTQSPSSVSVSVGDRVTISC QASQSIGGNLA WYQQKPGQAPKLLIY SASKLAS GVPSRFSGSGSGTDFTLTISSLEPEDFATYYC QQAWSYSNVDNT FGGGTKVEIK >Seq ID 78:5257C8 FRS light chain amino acid sequence DYVLTQSPSSVSVSVGDRVTISC QASQSIGGNLAWYQQKPGQAPKLLIY SASKLAS GVPSRFSGSGSGTDFTLTISSLEPEDFATYYC QQAWSYSNVDNT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 79:5257C8 GS heavy chain variable region nucleic acid sequence CAGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCATCGACCTGTCCTCCCACTACTTCAACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCATCGTGTACGCCTCCGGCTCCACCTACTACGCCTCCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCGGCGACAGGTCCGTGGCCTACTCCAACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC >Seq ID 80:5257C8 GS heavy chain variable region amino acid sequence QQLVESGGGLVQPGGSLRLSCAASGIDLS SHYFN WVRQAPGKGLEWVG IVYASGSTYYASWAKG RFTISKSKNTVYLQMNSLRAEDTAVYYCAG DRSVAYSNI WGQGTLVTVSS >Seq ID 81:5257C8 GS heavy chain amino acid sequence QQLVESGGGLVQPGGSLRLSCAASGIDLS SHYFN WVRQAPGKGLEWVG IVYASGSTYYASWAKG RFTISKSKNTVYLQMNSLRAEDTAVYYCAG DRSVAYSNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 82: Nucleic acid sequence of the variable region of the light chain of 5257C8 GS GACTACCAGCTGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTGACCATCACCTGCCAGGCCTCCCAGTCCATCGGCGGCAACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACTCCGCCTCCAAGCTGGCCTCCGGCGTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTGGTCCTACTCCAACGTGGACAACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAG >Seq ID 83: Amino acid sequence of the variable region of the light chain of GS 5257C8 DYQLTQSPSSVSASVGDRVTITC QASQSIGGNLA WYQQKPGQAPKLLIY SASKLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAWSYSNVDNT FGGGTKVEIK >Seq ID 84: Amino acid sequence of the light chain of 5257C8 GS DYQLTQSPSSVSASVGDRVTITC QASQSIGGNLAWYQQKPGQAPKLLIY SASKLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAWSYSNVDNT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Seq ID 85: 5029C10 AP01 heavy chain variable region amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS RNVIN WVRQAPGKGLEWVG IIATAGDTYYANWAKG RFTISKDSSKNTVYLQMNSLRAEDTAVYYCAR KYGDTFDL WGQGTLVTVSS Seq ID 86: 5029C10 AP01 light chain variable region amino acid sequence DIQMTQSPSSLSASVGDRVTITC QASQSISSYLS WYQQKPGKAPKLLIY QASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYDSNSVENA FGQGTKVEIK Seq ID 87: 5029C10 OAS01 heavy chain variable region amino acid sequence QVHLVESGGGVVQPGKSLRLSCAASGIDFS RNVIN WVRQAPGKGLEWIG IIATAGDTYYANWAKG RFTISKDSSRNTVYLQMDSLRAEDTAMYYCAG KYGDTFDL WGQGTLVTVSS Seq ID 88: 5029C10 OAS01 light chain variable region amino acid sequence DVQMTQSPSTLSASVGDSVTITC QASQSISSYLS WYQQKPGKAPKLLIY QASTLAS GVPSRFSGSGSGTEFTLAISNLQPDDFATYYC QGYDSNSVENA FGQGTEVEIK Seq ID 89: 5029C10 OAS02 heavy chain variable region amino acid sequence EVQLVESGGGLVQPGRSLTVSCTTSGIDFS RNVIN WVRQAPGKGLEWIG IIATAGDTYYANWAKG RFTISKDSSKNTVYLQMNSLTNEDTAVYFCAG KYGDTFDL WGQGTLVTVSS Seq ID 90: 5029C10 OAS02 light chain variable region amino acid sequence DVQMTQSPSSLSASVGDRVTITC QASQSISSYLS WYQQKPGKRPKLLIY QASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QGYDSNSVENA FGGGTKVEIK Seq ID 91: 5029C10 OAS03 heavy chain variable region amino acid sequence EVQLVESGGGLVQPGGSLRLSCEASGIDFS RNVIN WVRQAPGKGLEWVG IIATAGDTYYANWAKG RFTISKTSKNTVYLQMNSLKTEDTAVYYCAR KYGDTFDL WGKGTTVTVSS Seq ID 92: 5029C10 OAS03 light chain variable region amino acid sequence DIQMTQSPSSLSASVGDRVTITC QASQSISSYLS WYQQKPGKAPKLLIY QASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QGYDSNSVENA FGGGTKVEIK >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 111:5029C10 CDR 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 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

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.

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