Methods of treating dLL3-expressing cancers

Abstract

The present disclosure provides methods of treating small cell lung cancer (SCLC) in a subject expressing a human delta-like ligand 3 (DLL3) protein. The methods comprise administering to the subject an antigen-binding molecule comprising at least a first binding domain that binds to human DLL3 wherein at least 25% of the SCLC cells express DLL3, or wherein at least 25% of the SCLC cells express DLL3 at an intensity equal to or greater than 2 +, as determined by the IHC assay.

Description

FIELD OF THE TECHNOLOGY

[0001] This disclosure relates to methods of treating cancers expressing DLL3, such as small cell lung cancer (SCLC). BACKGROUND OF THE TECHNOLOGY

[0002] Small cell lung cancer (SCLC) is an aggressive lung cancer with a poor prognosis and limited treatment options, accounting for approximately 13% of all newly diagnosed lung cancers, and more than 235,000 adults were diagnosed with SCLC in the United States in 2021. For decades, survival rates have remained low, with only 7% of SCLC patients surviving five years, largely due to a lack of new therapies to combat this form of lung cancer. Most patients present with extensive-stage disease, while approximately one-third of patients present with limited-stage disease, defined as a tumor present only on one side of the chest and suitable for a single radiation field. Diffuse, metastatic tumors with lymphom-like features are characteristic of SCLC. The first known diagnosis of an SCLC patient described SCLC as a lymphatic system disease, and it was not until 1926 that it was confirmed as a lung cancer, highlighting the unique nature of SCLC tumors compared to other solid tumors.

[0003] Patients typically respond well to current standards of care, including combinations of chemotherapy and thoracic radiotherapy (TRT), but chemoresistant disease always recurs quickly, for which there are no available treatment options. Recently, adding the anti-PD-L1 antibody atezolizumab to carboplatin and etoposide chemotherapy demonstrated an improvement in overall survival (OS) in the first-line setting, leading to the approval of this regimen by the US Food and Drug Administration (FDA) for first-line treatment of extensive-stage SCLC. Despite these treatment advances, the prognosis for recurrent refractory (RR) cases is very poor, with rapid disease progression and a short median survival of less than six months. In addition, SCLC patients have a high rate of complications, including hypertension, heart disease, diabetes, and paraneoplastic syndromes. These, combined with the typically advanced age of SCLC patients, affect the patient's ability to tolerate harsh chemotherapy regimens, further limiting treatment options.

[0004] Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand that is highly expressed in SCLC and other neuroendocrine tumors but is expressed at very low levels in normal tissues. In one study, evidence of DLL3 expression was shown by RNA sequencing (RNA-seq) in approximately 86% of SCLC tumors analyzed (Giffin et al., Clin. Cancer Res. [Clinical Cancer Research], 27(5):1526-1537 (2021). doi:10.1158 / 1078-0432.CCR-20-2845). In contrast, only a few normal cell types show expression of DLL3 (e.g., neurons, pancreatic islet cells, and pituitary cells), and such expression is mainly cytoplasmic. Recent studies have reported that DLL3 is also expressed in other tumor types of neuroendocrine origin, including melanoma, glioblastoma multiforme, neuroendocrine prostate cancer (NEPC), and large cell neuroendocrine lung tumors).

[0005] Therapies targeting DLL3 are being investigated for the treatment of cancers expressing DLL3. For example, rovalpituzumab tesirine (Rova-T) is an antibody-drug conjugate (ADC) that contains an antibody targeting DLL3 that is tethered to the cytotoxic agent pyrrolobenzodiazepine via a protease-cleavable linker. Although promising results were achieved in early clinical trials, the development of Rova-T was stopped after showing limited efficacy and a higher incidence of certain toxicities in phase 3 trials (Blackhall et al., J. Thoracic Oncology [Journal of Thoracic Oncology], 16(9):1547-1558 (2021); and Upetry et al., J. Thoracic Oncology [Journal of Thoracic Oncology], 16(9):1429-1433 (2021)). The therapeutic effect of an ADC is mediated by the cytotoxic / cytostatic agent conjugated to the antibody, and ADCs generally do not engage, recruit, and / or activate cells of the immune system. Thus, DLL3 remains a promising target for immunotherapeutic agents that engage and activate cytotoxic T cells, for example.

[0006] There remains a need for methods for identifying and treating patient populations that will benefit from immunotherapeutic agents targeting DLL3. SUMMARY OF THE DISCLOSURE

[0007] The present disclosure provides a method of treating small cell lung cancer (SCLC) in a subject, the method comprising administering to the subject a bispecific antigen-binding molecule that comprises at least a first binding domain that binds to human delta-like ligand 3 (DLL3), wherein at least 25% of SCLC cells express DLL3.

[0008] In some aspects of the method, at least 50% or at least 75% of SCLC cells express DLL3.

[0009] In some aspects of the method, immunohistochemistry (IHC) assays are used to determine DLL3 expression.

[0010] In some aspects of the method, formalin-fixed paraffin-embedded (FFPE) tissue specimens from a subject are used to determine DLL3 expression.

[0011] In some aspects of the method, after platinum-based therapy, SCLC progresses or recurs in a subject.

[0012] In some aspects of the method, after platinum-based therapy in combination with etoposide and optionally a PD-L1 inhibitor, SCLC progresses or recurs in a subject.

[0013] In some aspects of the method, the subject (i) has completed at most two cycles of platinum-based therapy in combination with etoposide and optionally a PD-L1 inhibitor, or (ii) has completed four to six cycles of platinum-based therapy in combination with etoposide and optionally a PD-L1 inhibitor and has not experienced disease progression.

[0014] In some aspects, the method further comprises administering to the subject a PD-L1 inhibitor and optionally a chemotherapeutic agent.

[0015] In some aspects of the method, the bispecific antigen-binding molecule comprises a second binding domain that binds to human CD3.

[0016] In some aspects of the method, the bispecific antigen-binding molecule is a protein.

[0017] In some aspects of the method, the bispecific antigen-binding molecule comprises an antibody, a single-chain variable fragment (scFv), a tandem single-chain variable fragment (scFv)2, a bispecific T cell engager molecule or a heteromultimer.

[0018] In some aspects of the method, the bispecific antigen-binding molecule comprises the amino acid sequences of SEQ ID NO:14 and SEQ ID NO:15.

[0019] In some aspects of the method, the bispecific antigen-binding molecule is tarlatamab.

[0020] In some aspects of the method, the bispecific antigen-binding molecule comprises a first heterodimer that binds to human DLL3 and a second heterodimer that binds to human CD3, wherein (a) the first heterodimer comprises a heavy chain (HC) having the amino acid sequence of SEQ ID NO: 17 and a light chain (LC) having the amino acid sequence of SEQ ID NO: 18; and (b) the second heterodimer comprises a heavy chain having the amino acid sequence of SEQ ID NO: 19 and a light chain having the amino acid sequence of SEQ ID NO: 20.

[0021] In some aspects, the method increases the progression-free survival (PFS) of a subject compared to a second SCLC subject in which less than 50% of SCLC cells express DLL3 and are treated with the same antigen-binding molecule.

[0022] In some aspects, the method increases one or more of the overall survival (OS), objective response rate (ORR), and / or disease control rate (DCR) and duration of response (DOR) compared to a second subject.

[0023] In some aspects, the method results in an objective response rate (ORR) in the subject that is greater than about 35%.

[0024] The present disclosure also provides a method of treating SCLC in a subject, the method comprising administering to the subject a bispecific antigen-binding molecule that comprises at least a first binding domain that binds to human DLL3, wherein the SCLC has a DLL3 expression level, wherein at least 25% of SCLC cells express DLL3 at an intensity equal to or greater than 2+, as determined by an IHC assay.

[0025] In some aspects of the method, the SCLC has a DLL3 expression level, wherein at least 50% or at least 75% of SCLC cells express DLL3 at an intensity equal to or greater than 2+, as determined by an IHC assay.

[0026] In some aspects of the method, at least 25% of SCLC cells express DLL3 at an intensity of 2+ or 3+, as determined by an IHC assay.

[0027] In some aspects of the method, a formalin-fixed paraffin-embedded (FFPE) tissue specimen from the subject is used to determine DLL3 expression.

[0028] In some aspects of the method, following platinum-based therapy, the SCLC progresses or recurs in the subject.

[0029] In some aspects of the method, following platinum-based therapy in combination with etoposide and optionally a PD-L1 inhibitor, the SCLC progresses or recurs in the subject.

[0030] In some aspects of the method, the subject (i) has completed at most two cycles of platinum-based treatment in combination with etoposide and optionally a PD-L1 inhibitor, or (ii) has completed four to six cycles of platinum-based treatment in combination with etoposide and optionally a PD-L1 inhibitor and has not experienced disease progression.

[0031] In some aspects, the method further comprises administering to the subject a PD-L1 inhibitor and optionally a chemotherapeutic agent.

[0032] In some aspects of the method, the bispecific antigen-binding molecule comprises a second binding domain that binds to human CD3.

[0033] In some aspects of the method, the bispecific antigen-binding molecule is a protein.

[0034] In some aspects of the method, the bispecific antigen-binding molecule comprises an antibody, a single-chain variable fragment (scFv), a tandem single-chain variable fragment (scFv)2, a bispecific T cell engager molecule or a heteromultimer.

[0035] In some aspects of the method, the bispecific antigen-binding molecule comprises SEQ ID NO:14 and SEQ ID NO:15.

[0036] In some aspects of the method, the bispecific antigen-binding molecule is talquetamab.

[0037] In some aspects of the method, the bispecific antigen-binding molecule comprises a first heterodimer that binds to human DLL3 and a second heterodimer that binds to human CD3, wherein (a) the first heterodimer comprises a heavy chain (HC) having an amino acid sequence containing SEQ ID NO:17 and a light chain (LC) having an amino acid sequence containing SEQ ID NO:18; and (b) the second heterodimer comprises a heavy chain having an amino acid sequence containing SEQ ID NO:19 and a light chain having an amino acid sequence containing SEQ ID NO:20.

[0038] In some aspects, the method increases the progression-free survival (PFS) of the subject compared to a second SCLC subject having a lower DLL3 expression level and treated with the same antigen-binding molecule.

[0039] In some aspects, the method increases one or more of OS, ORR, and / or DCR and DOR compared to a second subject.

[0040] In some aspects, the method results in an objective response rate (ORR) of the subject greater than about 35%.

[0041] In some aspects of the method, the cancer cells are live cancer cells.

[0042] In some aspects of the method, the bispecific antigen-binding molecule is talquetamab, and talquetamab is administered once every two weeks at a dose of 10 mg to 100 mg.

[0043] In some aspects of the method, talquetamab is administered once every two weeks at a dose of 10 mg.

[0044] In some aspects of the method, talquetamab is administered once every two weeks at a dose of 100 mg.

[0045] In some aspects, the method includes administering talquetamab once a week at a dose of 10 mg to 100 mg in weeks 1, 2, and 3, and then administering talquetamab once every two weeks.

[0046] In some aspects of the method, the bispecific antigen-binding molecule is talquetamab, and talquetamab is administered twice every three weeks at a dose of 10 mg to 100 mg.

[0047] In some aspects of the method, talquetamab is administered twice every three weeks at a dose of 10 mg, 30 mg, or 100 mg.

[0048] In some aspects of the method, talquetamab is administered on days 1 and 8 of a 21-day cycle.

[0049] In some aspects of the method, the bispecific antigen-binding molecule is talquetamab, and talquetamab is administered once every three weeks at a dose of 20 mg to 200 mg.

[0050] In some aspects of the method, talquetamab is administered once every three weeks at a dose of 20 mg to 100 mg.

[0051] In some aspects of the method, talquetamab is administered once every three weeks at a dose of 100 mg to 200 mg.

[0052] In some aspects of the method, talquetamab is administered at a dose of 20 mg, 60 mg, 100 mg, or 200 mg.

[0053] In some aspects of the method, talquetamab is administered on the first day of a 21-day cycle.

[0054] In some aspects, the method includes administering talquetamab once a week at a dose of 10 mg to 100 mg in weeks 1 and 2, and then administering talquetamab once every three weeks.

[0055] In some aspects of the method, the FFPE tissue specimen is prepared from a core needle biopsy tissue sample.

[0056] In some aspects of the method, the subject is a human. In some embodiments, the human has received at least one prior treatment for the cancer and has relapsed. In some embodiments, the at least one prior treatment is platinum-based chemotherapy and etoposide, and optionally an anti-PD-L1 antibody. In some embodiments, the human has not received prior systemic treatment for the cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1A Graph showing the best percent change in tumor burden (defined by the sum of the longest diameters (SLD) of all target lesions) relative to baseline for 94 patients with data cutoff at least 9 weeks after the first dose and for whom post-baseline tumor data were available. CR indicates complete response, PR indicates partial response, SD indicates stable disease, and NE indicates not evaluable. SD^ indicates that the patient had an initial response but no response was confirmed on subsequent scans, and PR** indicates that the patient had an initial PR and future confirmatory scans are still possible. One confirmed subject in cohort 30 lacked the sum of the diameters of the lesion measurements and was therefore not included in the graph. Escalating dosing (i.e., 1 mg lead-in dose) was used in these cohorts. Figure 1B Graph showing the time to response, duration of treatment, and patient status as of the data cutoff for taletrectinib doses according to all patients (N = 25) with confirmed responses.

[0058] Figure 2A Kaplan-Meier curve showing the progression-free survival of patients (N = 107) with data cutoff at least 9 weeks after the first dose. Figure 2B Kaplan-Meier curve showing the overall survival of patients (N = 107) with data cutoff at least 9 weeks after the first dose.

[0059] Figure 3 Is an ROC curve reflecting the true positive rate (TPR) and false positive rate (FPR) of objective response enrichment when retrospectively examining DLL3 expression, as assessed using the Ventana SP347 assay, for selection of FIH subjects (1 - 100 mg cohort). The ROC analysis included 77 subjects enrolled in the 1 - 100 mg cohort for whom pre-treatment DLL3 expression readings were available.

[0060] Figure 4 Graph showing the objective response rate (ORR) and disease control rate (DCR) versus the percentage of tumor cells expressing DLL3.

[0061] Figure 5AA graph showing the relationship between total DLL3 expression and tumor reduction in the FIH cohort receiving talazoparib monotherapy at the target dose in 1 step as described in Example 1. Figure 5B A graph showing the relationship between tumors with 3+ DLL3 expression intensity and tumor reduction. BOR represents Best Overall Response, CR represents Complete Response, PR represents Partial Response, PD represents Progressive Disease, SD represents Stable Disease, NE represents Not Evaluable, and NA represents "Not Available".

[0062] Figures 6A-6D A graph showing that peak cytokine levels (6A: IL-6; 6B: IL-8; 6C: IL-10; 6D: TNF-α) tend to be higher in patients with CRS compared to patients without CRS. Biomarker evaluable patients (N = 86); patients with any grade of CRS in Cycle 1 (n = 45); patients without CRS (n = 40). C1, Cycle 1; CRS, Cytokine Release Syndrome; G, Grade.

[0063] Figure 7 A graph showing the longitudinal analysis of IL-10 expression in patients from the Phase I study described in Example 1. IL-10 showed a significant elevation above the reference normal range and was higher in patients with CRS. Adjusted P value for JT trend test = 0.049 (significant at 95% confidence); adjusted P value for KW association test = 0.096 (significant at 90% confidence; not significant at 95% confidence). The yellow dashed line indicates the reference normal range.

[0064] Figure 8A and Figure 8B A graph showing the analysis of IFN-γ expression in patients from the Phase I study described in Example 1. IFN-γ induction was above the physiological range; the induction was similar between patients with and without Cycle 1 CRS. Adjusted P value for JT trend test = 0.234 (not significant at 95% confidence); adjusted P value for KW association test = 0.317 (not significant at 90% or 95% confidence). The yellow dashed line indicates the reference normal range.

[0065] Figure 9 is a schematic diagram of the clinical study described in Example 2.

[0066] Figure 10 A waterfall-lane graph showing the efficacy of talazoparib in DLL3+ NEPC tumors as described in Example 4. Detailed Description

[0067] This disclosure is at least in part based on the discovery that DLL3 is expressed by certain types of cancer. It was identified as a tumor-associated antigen and a target for T cell-based therapies by analyzing differential expression of DLL3 in small cell lung cancer (SCLC) tumors and a large panel of normal tissues (Giffin et al., J Thorac Oncol. [Journal of Thoracic Oncology], 13(10):S971 (2018)). DLL3 overexpression has been observed on the surface of SCLC and large cell neuroendocrine carcinoma (LCNEC) (Rudin et al., Lancet Oncol. [The Lancet Oncology] 2017; 18:42-51; and Saunders et al., SciTranslMed. [Science Translational Medicine], 2015; 7:302ra136). DLL3 expression has also been observed in cells of other cancers, including pancreatic cancer, gastrointestinal neuroendocrine cancer, small cell bladder cancer, neuroendocrine prostate cancer, isocitrate dehydrogenase mutant glioma, gynecologic cancers, and Merkel cell carcinoma (Matsuo et al., Cancer Science [Cancer Science], 112:2984-2992 (2021)). Thus, DLL3 is a potential therapeutic target for multiple cancers.

[0068] This disclosure provides methods of treating cancers (such as small cell lung cancer (SCLC)) that express DLL3 in a subject (e.g., a human subject), the methods comprising administering to the subject a bispecific antigen-binding molecule that comprises at least a first binding domain that binds human DLL3. Administration of the bispecific antigen-binding molecules described herein to SCLC cancer cells that express a certain level of DLL3 is advantageously associated with improved treatment outcomes (such as increased progression-free survival (PFS) and objective response rate (ORR)) compared to SCLC cancer cells with lower or no DLL3 expression treated with the same bispecific antigen-binding molecule.

[0069] Definition

[0070] To facilitate understanding of the technology, several terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0071] As used herein, the terms "biomarker", "marker", or "biological marker" refer to an analyte (e.g., nucleic acid, DNA, RNA, peptide, protein, or metabolite) that can be objectively measured and evaluated as an indicator of a biological process or condition. In some aspects, an analyte is differentially detectable if it can be quantitatively or qualitatively distinguished in tumor cells as compared to a control (e.g., a healthy subject or a subject with SCLC that does not express DLL3).

[0072] As used herein, the term "antigen-binding molecule" refers to a molecule or compound that specifically binds an antigen. In some embodiments, the antigen-binding molecule is a protein. As used herein, the term "antigen-binding protein" refers to a protein molecule that specifically binds an antigen. For example, an antigen-binding protein can comprise an antibody or an antigen-binding fragment thereof. An antigen-binding protein typically comprises the variable heavy chain (VH) and / or variable light chain (VL) of an antibody, or a domain derived therefrom. In some embodiments, the antigen-binding protein comprises the minimal structural requirements of an antibody that permit immunospecific target binding. Such minimal requirements can be defined, for example, by the presence of at least three light chain complementarity-determining regions (CDRs) (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region), and desirably all six CDRs. The position (and order) of these CDRs in the antigen-binding protein is within the knowledge of one of ordinary skill in the art.

[0073] In certain aspects, the antigen-binding proteins of the present disclosure are "bispecific", meaning that the antigen-binding protein is capable of specifically binding two different antigens. In another aspect, the antigen-binding proteins of the present disclosure can be "trispecific", meaning that the antigen-binding protein is capable of specifically binding three different antigens. In another aspect, the antigen-binding proteins of the present disclosure can be "tetraspecific", meaning that the antigen-binding protein is capable of specifically binding four different antigens.

[0074] As used herein, the term "antibody" refers to a whole antibody molecule or a fragment thereof (e.g., fragments such as scFv, Fab, Fab', and F(ab')2), unless otherwise specified; the antibody can be a polyclonal or monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, etc. In a native whole antibody, the heavy chain comprises the variable region VH and three constant regions CH1, CH2, and CH3. The VH domain is located at the amino terminus of the heavy chain, and the CH3 domain is located at the carboxyl terminus. In a native whole antibody, the light chain comprises the variable region VL and the constant region CL. The variable region of the light chain is located at the amino terminus of the light chain. In a native whole antibody, the variable regions of each light chain / heavy chain pair typically form an antigen-binding site. The constant regions typically are responsible for effector functions.

[0075] In a human antibody, CH1 refers to the region having an amino acid sequence at positions 118 to 215 of the EU index or EU numbering system, which is based on the sequence numbering of the first human IgG1 sequenced (i.e., “EU antibody”) (Edelman et al., Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 63(1):78-85 (1969)). A highly flexible amino acid region called the “hinge region” exists between CH1 and CH2. CH2 represents the region having an amino acid sequence at positions 231 to 340 of the EU index, and CH3 represents the region having an amino acid sequence at positions 341 to 446 of the EU index.

[0076] “CL” represents the constant region of the light chain. In the case of the κ chain of a human antibody, CL represents the region having an amino acid sequence at positions 108 to 214 of the EU index. In the λ chain, CL represents the region having an amino acid sequence at positions 108 to 215.

[0077] In a native antibody, the variable regions typically exhibit the same general structure, in which relatively conserved framework regions (FRs) are connected by three hypervariable regions (also called complementarity-determining regions (CDRs)). The CDRs from each pair of the two chains are typically aligned by the framework regions, which can enable binding to a specific epitope. From the N-terminus to the C-terminus, the light chain variable region and the heavy chain variable region typically each contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs on the heavy chain are called H1, H2, and H3, while the CDRs on the light chain are called L1, L2, and L3. Typically, CDR3 is the greatest source of molecular diversity within the antigen-binding site. The assignment of amino acids for each domain is typically according to the following definitions: Kabat et al. (1991) Sequences of Proteins of Immunological Interest (National Institutes of Health [National Institutes of Health of the United States], Publication No. 91-3242, Volumes 1-3, Bethesda, Maryland); Chothia, C., and Lesk, A.M. (1987) J. Mol. Biol. [Journal of Molecular Biology], 196:901-917; or Chothia C. et al., Nature [Nature], 342:878-883 (1989). In some embodiments, the CDRs of an antigen-binding protein are defined according to the Kabat or Chothia definitions. In the present application, unless otherwise specified, the term “CDR” refers to the CDRs from the light chain or the heavy chain.

[0078] An antibody can comprise any constant region known in the art. Human light chains are classified as kappa light chains and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. Embodiments of the present disclosure include all such antibody classes or isotypes. The light chain constant region can be, for example, a kappa-type or lambda-type light chain constant region, such as a human kappa-type or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-type, delta-type, epsilon-type, gamma-type, or mu-type heavy chain constant region, such as a human alpha-type, delta-type, epsilon-type, gamma-type, or mu-type heavy chain constant region. Thus, in an exemplary embodiment, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the antibody is of isotype IgG (e.g., IgG1).

[0079] The antibody can be a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B lymphocytes directed against a single epitope on an antigen. Monoclonal antibodies are typically produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol., 5:511-519 (1976). Monoclonal antibodies can also be produced using recombinant DNA methods (see, e.g., U.S. Patent 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol., 222:581-597 (1991)), or produced by transgenic mice carrying a fully human immunoglobulin system (see, e.g., XENOMOUSE TM mice, Green et al. (1994) Nature Genetics 7:13-21, US2003-0070185, WO 96 / 34096, and WO 96 / 33735). In contrast, a "polyclonal" antibody is an antibody secreted by different B cell lineages in an animal. A polyclonal antibody is a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0080] As used herein, the term "chimeric antibody" refers to an antibody that contains domains from two or more different antibodies. A chimeric antibody can, for example, contain a constant domain from one species and a variable domain from a second species, or more generally, can contain segments of amino acid sequences from at least two species. A chimeric antibody can also contain domains from two or more different antibodies within the same species. The term "humanized," when used in reference to an antibody, refers to an antibody that has at least the CDR regions from a non-human source that have been engineered to have a structure and immunological function more similar to that of a true human antibody. For example, humanization can involve grafting the CDRs from a non-human antibody (such as a murine antibody) into a human antibody. Humanization can also involve selected amino acid substitutions to make the non-human sequence more similar to a human sequence.

[0081] Antibodies can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce an F(ab’)2 fragment and a pFc’ fragment. In an exemplary aspect, the bispecific antigen-binding proteins of the present disclosure comprise antigen-binding antibody fragments. As used herein, the term "antigen-binding antibody fragment" refers to the portion of an antibody molecule that is capable of binding to the antigen of the antibody, also referred to as the "antigen-binding fragment" or "antigen-binding portion." In some embodiments, the antigen-binding antibody fragment is a Fab fragment or an F(ab')2 fragment.

[0082] Antibody architectures have been used to build an increasing number of alternative forms that span a molecular weight range of at least about 12 - 150 kDa and have a valence (n) range from monomer (n = 1), dimer (n = 2), and trimer (n = 3) to tetramer (n = 4) and potentially higher; such alternative forms are referred to herein as "antibody protein products". Antibody protein products include those based on the complete antibody structure and those that mimic antibody fragments retaining full antigen-binding capacity, such as single-chain variable fragments (scFv), Fab, and VHH / VH (discussed below). The smallest antigen-binding antibody fragment that retains its full antigen-binding site is the Fv fragment, which consists entirely of variable (V) regions. The V regions are linked to the scFv (single-chain variable fragment) fragment using a soluble flexible amino acid peptide linker to stabilize the molecule, or a constant (C) domain is added to the V region to produce the Fab fragment. Both scFv and Fab fragments can be readily produced in host cells, such as prokaryotic host cells. Other antibody protein products include tandem single-chain variable fragments (scFv)2, disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and dimeric and multimeric antibody forms, such as bispecific antibodies, trispecific antibodies, and tetraspecific antibodies, or different forms of miniantibodies (miniAb) consisting of scFv linked to oligomeric domains. Peptibodies or peptide-Fc fusions are another type of antibody protein product. The structure of a peptibody consists of a bioactive peptide grafted onto an Fc domain. Peptibodies are well described in the art (see, for example, Shimamoto et al., mAbs [Monoclonal Antibodies] 4(5):586 - 591 (2012)).

[0083] The bispecific antigen-binding molecules of the present disclosure can comprise any of the above-described antibody protein products. In an exemplary aspect, the bispecific antigen-binding proteins of the present disclosure comprise scFv, (scFv)2, Fab, VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibodies, multimeric antibodies (such as bispecific antibodies, trispecific antibodies, tetraspecific antibodies), miniantibodies, peptibodies of camelid heavy-chain antibodies VHH / VH, scFv-single domain mAb, multimeric proteins, sdAb, bispecific antibodies; trispecific antibodies; tetraspecific antibodies; bispecific or trispecific antibodies, BsIgG, additional IgG, BsAb fragments, or bispecific fusion proteins. In certain embodiments, the bispecific antigen-binding protein comprises (scFv)2, scFab, scFv-single domain mAb, or multimeric protein. In certain embodiments, the bispecific antigen-binding protein comprises (scFv)2 or multimeric protein.

[0084] As used herein, an antigen-binding protein "specifically binds to" an antigen when its binding affinity for the target antigen is significantly higher than its affinity for other unrelated proteins under similar binding assay conditions and can thus be distinguished. An antigen-binding protein that specifically binds an antigen may have an equilibrium dissociation constant (K -6 ) of ≤ 1 x 10 D M. In exemplary aspects, the K D of the bispecific antigen-binding proteins provided herein is in the micromolar, nanomolar, picomolar, or femtomolar range. When K D ≤ 3 x 10 -8 M, the antigen-binding protein specifically binds the antigen with "high affinity". In some embodiments, the bispecific antigen-binding proteins of the present disclosure bind to one or more target antigens with a K D ≤ 100 nM (e.g., 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, or a range defined by any two of the foregoing values). In other embodiments, the bispecific antigen-binding proteins of the present disclosure bind to one or more target antigens with a K D of about 10 nM - 30 nM (e.g., about 15 nM, 20 nM, or 25 nM).

[0085] Multiple techniques can be used to determine affinity, an example of which is enzyme-linked immunosorbent assay (ELISA). In various embodiments, affinity is determined by surface plasmon resonance assays (e.g., assays based on ). Using this method, the association rate constant (k a , expressed in M -1 s -1 ) and the dissociation rate constant (k d , expressed in s -1 ) can be measured. The equilibrium dissociation constant (K d , expressed in M) can then be calculated from the ratio of the kinetic rate constants (k a / k D ). In some embodiments, affinity can be determined by kinetic methods, such as kinetic exclusion assay (KinExA) (as described in Rathanaswami et al., Analytical Biochemistry, 373:52 - 60 (2008)). Using a KinExA assay, the equilibrium dissociation constant (K D , expressed in M) and the association rate constant (ka, expressed in M -1 s -1 ) can be measured. The dissociation rate constant (k -1 , expressed in s d ) can be calculated from these values (KD x k a )。In other embodiments, affinity is determined by an equilibrium / solution method. In certain embodiments, affinity is determined using flow cytometry in a cell-bound assay. In certain embodiments of the present disclosure, the bispecific antigen-binding protein binds specifically to one or more target antigens expressed by mammalian cells (e.g., CHO, HEK 293, Jurkat), as determined by kinetic exclusion assay (Rathanaswami et al., supra). In some embodiments, the bispecific antigen-binding proteins described herein exhibit desired characteristics, e.g., a binding affinity for one or more target antigens of about 10 -8 M) or lower K D 、10 nM (1.0 x 10 -8 M) or lower K D 、1 nM (1.0 x 10 -9 M) or lower K D 、500 pM (5.0 x 10 -10 M) or lower K D 、200 pM (2.0 x 10 -10 M) or lower K D 、150 pM (1.50 x 10 -10 M) or lower K D 、125 pM (1.25 x 10 -10 M) or lower K D 、105 pM (1.05 x 10 -10 M) or lower K D 、50 pM (5.0 x 10 -11 M) or lower K D 、20 pM (2.0 x 10 -11 M) or lower K D and as determined by kinetic exclusion assay (Rathanaswami et al., supra). In some embodiments, the bispecific antigen-binding proteins described herein exhibit desired characteristics, e.g., a binding affinity for one or more target antigens of about 10 d 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 s -1 or lower (lower values indicate higher binding affinity), and / or an off-rate of about 10 D 、10 -9 、10 -10 、10 -11 、10 -12 、10 -13, 10 -14 , 10 -15 , 10 -16 M or lower binding affinity for one or more target antigens (a lower value indicates a higher binding affinity).

[0086] In certain embodiments of the present disclosure, the bispecific antigen-binding protein can be multivalent. The valency of the binding protein indicates the number of individual antigen-binding domains within the binding protein. In some embodiments, the bispecific antigen-binding protein can be multivalent. For example, in certain embodiments, the bispecific antigen-binding protein can be tetravalent, comprising four antigen-binding domains: two antigen-binding domains that bind to a first target antigen and two antigen-binding domains that bind to a second target antigen. A tetra-specific antigen-binding protein is tetravalent and comprises four antigen-binding domains: an antigen-binding domain that binds to a first target antigen, an antigen-binding domain that binds to a second target antigen, an antigen-binding domain that binds to a third target antigen, and an antigen-binding domain that binds to a fourth target antigen.

[0087] As used herein, the term "antigen-binding domain" can be used interchangeably with "binding domain" and refers to the region of an antigen-binding protein that contains the amino acid residues that interact with an antigen and confer specificity and affinity of the antigen-binding protein for the antigen. In some embodiments, the binding domain can be derived from the natural ligand of one or more target antigens. As used herein, the term "one or more target antigens" refers to the first target antigen and / or the second target antigen of a bispecific antigen-binding molecule, and also refers to the first target antigen, the second target antigen, the third target antigen, and / or the fourth target antigen of a tetra-specific molecule.

[0088] As used herein, the term "immunoglobulin domain" refers to a peptide that contains an amino acid sequence similar to that of an immunoglobulin and contains approximately 100 amino acid residues (including at least two cysteine residues). Examples of immunoglobulin domains include VH, CH1, CH2, and CH3 of the immunoglobulin heavy chain, and VL and CL of the immunoglobulin light chain. Additionally, immunoglobulin domains are found in proteins other than immunoglobulins. Examples of immunoglobulin domains in proteins other than immunoglobulins include the immunoglobulin domains contained in proteins belonging to the immunoglobulin superfamily (such as the major histocompatibility complex (MHC), CD1, B7, the T cell receptor (TCR), etc.). Any immunoglobulin domain can be used as the immunoglobulin domain of the bispecific antigen-binding protein described herein.

[0089] A binding domain that specifically binds to one or more target antigens can be derived from a) known antibodies to these antigens, or b) new antibodies or antibody fragments obtained by de novo immunization methods using the antigen protein or fragments thereof, by phage display or other conventional methods. The antibodies from which the binding domains of the bispecific antigen-binding proteins are derived can be monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies or humanized antibodies. In certain embodiments, the antibody from which the binding domain is derived is a monoclonal antibody. In these and other embodiments, the antibody is a human antibody or a humanized antibody and can be of the IgG1 type, IgG2 type, IgG3 type or IgG4 type. For example, the binding domain can be obtained from or based on an IgG1 monoclonal antibody.

[0090] As used herein, the term "multimeric protein" refers to a protein that contains more than one separate polypeptide chain or protein chain, which associate with each other to form a single protein in vitro or in vivo. A multimeric protein can contain more than one of the same type of polypeptide to form a "homomultimer". Alternatively, a multimeric protein can also be composed of more than one polypeptide having different sequences to form a "heteromultimer". Thus, a "heteromultimer" is a molecule that contains at least a first polypeptide and a second polypeptide, wherein the second polypeptide differs from the first polypeptide by at least one amino acid residue in amino acid sequence. A heteromultimer can contain a "heterodimer" formed by the first and second polypeptides or can form a higher-order tertiary structure in which there are more than two polypeptides.

[0091] As used herein, the terms "stability" and "stable" are defined as maintaining the chemical or physical integrity and / or biological activity of a bispecific antigen-binding polypeptide or protein over a period of time. A stable antigen-binding polypeptide or protein includes preventing or delaying the degradation or degeneration of the antigen-binding polypeptide or protein from its biologically and / or therapeutically active form into an inactive form. Instability can result from events such as aggregation, denaturation, fragmentation or chemical modification (such as oxidation, crosslinking, deamidation) and reactions with other components specific to the composition containing the antigen-binding polypeptide or protein.

[0092] The stability of an antigen-binding protein or polypeptide can be characterized using methods known in the art, including but not limited to measuring biological activity (such as antigen-binding activity) using immunoassay techniques (such as ELISA), or other techniques for determining the purity or physical / chemical changes of the antigen-binding protein or polypeptide, such as size exclusion chromatography, capillary gel electrophoresis, circular dichroism or mass spectrometry. Stability is determined by comparing the measurements obtained via these types of characterization methods at an initial time point (such as at the time of formulation or preparation of the composition (i.e., a suspension or dispersion, as appropriate)) with the measurements obtained at a later time point (i.e., after storage in a given environment or conditions).

[0093] As used herein, the term "CD3 receptor complex" refers to a protein complex composed of four chains. In mammals, the complex contains the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains. These chains associate with the T cell receptor (TCR) and the so-called ζ (zeta) chain to form the T cell receptor CD3 complex and generate activation signals in T lymphocytes. The CD3γ (gamma), CD3δ (delta), and CD3ε (epsilon) chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. The intracellular tails of the CD3 molecules contain a single conserved motif essential for the signaling capacity of the TCR, called the immunoreceptor tyrosine-based activation motif or simply ITAM. The CD3ε molecule is a polypeptide that is encoded in humans by the CD3E gene located on chromosome 11. The most preferred epitopes of CD3ε include within amino acid residues 1 - 27 of the extracellular domain of human CD3ε.

[0094] The term "treatment" includes, for example, ameliorating or reducing the severity of a disease, or shortening the duration of a disease. In addition, the term "treatment" and words associated therewith do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment that are considered by those of ordinary skill in the art to have potential benefit or therapeutic effect. In this regard, the cancer treatment methods of the present disclosure can provide any amount or any level of treatment. In addition, the treatment provided by the methods of the present disclosure can include treating one or more conditions or symptoms or signs of the cancer being treated.

[0095] The efficacy of treatment can be monitored by regularly evaluating the patient being treated. For repeated administrations over several days or longer, depending on the condition, treatment can be repeated until the desired containment of the disease symptoms occurs. However, other dosing regimens may be useful and are within the scope of the present disclosure.

[0096] When "about" or "approximately" is used in connection with a measurable numerical variable, it refers to the indicated value of the variable and all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval of the mean) or ±10% of the indicated value, whichever is greater. Numerical ranges include the numbers defining the range.

[0097] The present disclosure is not limited to the specific amino acid sequences described herein. In some embodiments, the present disclosure provides amino acid sequences that are at least about 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to any of the amino acid sequences identified herein. Amino acid or nucleic acid sequence “identity” can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percentage of identity is the number of nucleotide or amino acid residues that are identical (i.e., have identity) between the sequence of interest and the reference sequence divided by the length of the longest sequence (i.e., the length of the sequence of interest or the reference sequence, whichever is longer). A number of mathematical algorithms for obtaining the best alignment between two or more sequences and calculating their identity are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL Omega, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.13, BL2SEQ and their successors), and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searching). Sequence alignment algorithms are also disclosed, for example, in Altschul et al., J. Mol. Biol., 275(3):403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009); Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 27(7):951-960 (2005); Altschul et al., Nucleic Acids Res., 25(17):3389-3402 (1997); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge, UK (1997).

[0098] Bispecific antigen-binding molecule

[0099] As discussed above, the antigen-binding molecules provided herein are "bispecific" in that they bind two different antigens or epitopes. In some embodiments, the bispecific antigen-binding molecule is an antigen-binding protein that comprises at least a first binding domain that binds human DLL3. In a preferred embodiment, the bispecific antigen-binding molecule is an antigen-binding protein that comprises at least a first binding domain that binds human DLL3 and a second binding domain that binds human CD3 (a subunit of the T cell receptor complex on T cells as described above).

[0100] Delta-like ligand 3 (DLL3) is an atypical Notch ligand that is expressed primarily during embryonic development (where it plays a role in somitogenesis). DLL3 accumulates in the Golgi apparatus of normal tissues (Geffers et al., J Cell Biol. [Journal of Cell Biology], 178:465-476 (2007)). The human DLL3 protein comprises several extracellular domains: a signal peptide, an N-terminus, DSL, EGF1, EGF2, EGF3, EGF4, EGF5, EGF6, and a membrane-proximal domain. Exemplary amino acid sequences of human DLL3 include, for example, UniProt Q9NYJ7 and NCBI reference sequence: NP_058637.1.

[0101] The first binding domain can bind any suitable region or epitope of human DLL3. In some embodiments, the first binding domain binds an epitope of human DLL3 that is contained within the EGF3 and EGF4 domains, such as an epitope within the amino acid sequence of SEQ ID NO:1. For example, the first binding domain can bind an epitope of human DLL3 within the EGF3 domain that is contained within the amino acid sequence of SEQ ID NO:2. In other embodiments, the first binding domain of the bispecific antigen-binding molecule binds an epitope of human DLL3 that is contained within the EGF5 domain (SEQ ID NO:3) and / or the EGF6 domain (SEQ ID NO:4). An example of an agent that targets DLL3 is a bispecific T cell-engaging antigen-binding polypeptide that binds DLL3 and CD3, such as molecule. The molecule is a recombinant protein composed of two flexibly linked binding domains, each domain being derived from an antibody. One binding domain of the molecule is specific for a tumor-associated surface antigen, such as DLL3; the second binding domain is specific for CD3. By its particular design, The antibody construct is uniquely suited to transiently link T cells to target cells and, concomitantly, potently activate the innate cytolytic potential of T cells against the target cells. See, e.g., WO 99 / 54440, WO 2005 / 040220, and WO 2008 / 119567.

[0102] In some embodiments, the bispecific antigen-binding protein may comprise two tandem scFv amino acid sequences (scFv)2: one that binds to human DLL3 and another that binds to human CD3. In this regard, for example, the amino acid sequence of the first scFv comprises a heavy-chain variable region (VH) and a light-chain variable region (VL) that bind to human DLL3, while the amino acid sequence of the second scFv comprises a VH and a VL that bind to human CD3. In some embodiments, the VH and VL are linked by a linker to form a single-chain Fv (scFv). In some embodiments, the linker is a peptide linker comprising a sequence selected from any one of SEQ ID NOs: 5-13. In some embodiments, the linker is a GS linker, such as Gly-Gly-Gly-Gly-Ser (G4S, SEQ ID NO: 6) or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer of 1 or greater (e.g., 2 or 3) (e.g., SEQ ID NO: 12 or 13).

[0103] In certain embodiments, the anti-DLL3 antigen-binding protein described herein comprises a first binding domain that binds to DLL3 (preferably human DLL3) and comprises the amino acid sequence of SEQ ID NO: 14, and a second binding domain that binds to CD3 (preferably human CD3) and comprises the amino acids of SEQ ID NO: 15. In certain embodiments, the anti-DLL3 antigen-binding protein described herein comprises the amino acid sequence of SEQ ID NO: 16.

[0104] In various embodiments, the bispecific antigen-binding protein is talquetamab (International Nonproprietary Name (INN): proposed INN: List 123, WHO Drug Information 34(2):395-397 (2020)), also known as AMG 757, which is a half-life extended Molecule. The activity of taletrectinib requires simultaneous binding to both the target cell (DLL3+ cell) and the T cell. The pharmacological effect of taletrectinib is mediated by specifically redirecting previously primed cytotoxic CD8+ or CD4+ T lymphocytes to kill DLL3+ cells. Taletrectinib demonstrated antitumor activity in phase 1 studies in SCLC patients, and clinical evaluations are ongoing (see, e.g., ClinicalTrials.gov identifier: NCT03319940 and Owonikoko et al., Journal of Clinical Oncology 39:15_suppl, 8510-8510 (2021)). For example, WO 2017 / 021349 and WO 2021 / 092134 describe taletrectinib in detail.

[0105] In some embodiments, the bispecific antigen-binding protein can be a heteromultimer. Exemplary heteromultimers include, but are not limited to, heterodimeric antibodies (which may be used interchangeably with "heteroimmunoglobulin" or "heteroIg" herein), which are antibodies comprising two different light chains and two different heavy chains. In some embodiments, the heteromultimer encompassed by this disclosure comprises a first heterodimer that binds to human DLL3 and a second heterodimer that binds to human CD3.

[0106] In some embodiments, the first heterodimer comprises a heavy chain (HC) having an amino acid sequence containing SEQ ID NO:17 or SEQ ID NO:21, and a light chain (LC) having an amino acid sequence containing SEQ ID NO:18 or SEQ ID NO:22; and the second heterodimer comprises a heavy chain having an amino acid sequence containing SEQ ID NO:19 or SEQ ID NO:24, and a light chain having an amino acid sequence containing SEQ ID NO:20. For example, the first heterodimer may comprise a heavy chain having an amino acid sequence containing SEQ ID NO:17 and a light chain having an amino acid sequence containing SEQ ID NO:18. Alternatively, the first heterodimer may comprise a heavy chain having an amino acid sequence containing SEQ ID NO:21 and a light chain having an amino acid sequence containing SEQ ID NO:22. In some embodiments, for example, the second heterodimer may comprise a heavy chain having an amino acid sequence containing SEQ ID NO:19 and a light chain having an amino acid sequence containing SEQ ID NO:20. In other embodiments, for example, the second heterodimer may comprise a heavy chain having an amino acid sequence containing SEQ ID NO:24 and a light chain having an amino acid sequence containing SEQ ID NO:20. In some embodiments, the first heterodimer may comprise a heavy chain having an amino acid sequence containing SEQ ID NO:23. In additional embodiments, the second heterodimer may comprise a heavy chain having an amino acid sequence containing SEQ ID NO:25.

[0107] Accordingly, exemplary heteromultimers provided herein may include a first heterodimer comprising a heavy chain amino acid sequence of SEQ ID NO:17 and a light chain amino acid sequence of SEQ ID NO:18; and a second heterodimer comprising a heavy chain amino acid sequence of SEQ ID NO:19 and a light chain amino acid sequence of SEQ ID NO:20. In other embodiments, exemplary heteromultimers provided herein may include a first heterodimer comprising a heavy chain amino acid sequence of SEQ ID NO:17 and a light chain amino acid sequence of SEQ ID NO:18; and a second heterodimer comprising a heavy chain amino acid sequence of SEQ ID NO:24 and a light chain amino acid sequence of SEQ ID NO:20. In some embodiments, exemplary heteromultimers provided herein include a first heterodimer comprising a heavy chain amino acid sequence of SEQ ID NO:21 and a light chain amino acid sequence of SEQ ID NO:22; and a second heterodimer comprising a heavy chain amino acid sequence of SEQ ID NO:19 and a light chain amino acid sequence of SEQ ID NO:20. In other embodiments, exemplary heteromultimers provided herein include a first heterodimer comprising a heavy chain amino acid sequence of SEQ ID NO:21 and a light chain amino acid sequence of SEQ ID NO:22; and a second heterodimer comprising a heavy chain amino acid sequence of SEQ ID NO:24 and a light chain amino acid sequence of SEQ ID NO:20.

[0108] Other exemplary antigen-binding proteins that bind DLL3 and CD3 and are included in this disclosure include multispecific binding proteins, e.g., as described in Hipp et al., Clin Cancer Res 2020;26:5258–68; WO2019 / 234220; and WO 2020 / 069028. In certain embodiments, the bispecific antigen-binding protein is a protein comprising: (a) a first domain that is a single-chain variable fragment (scFv) that specifically binds to human CD3; (b) a second domain that is a single-domain antibody that specifically binds to human serum albumin; and (c) a third domain that is a single-domain antibody that specifically binds to the DLL3 protein. In certain embodiments, the bispecific antigen-binding protein comprises or consists of the amino acid sequence of SEQ ID NO:26 or 27. In certain embodiments, the bispecific antigen-binding protein is a protein comprising: (a) a first antigen-binding domain that specifically binds to human DLL3; (b) a second antigen-binding domain that specifically binds to human CD3, and (c) a first and a second Fc domain, wherein the first Fc domain is covalently linked to the first antigen-binding domain, and the second Fc domain is covalently linked to the second antigen-binding domain. In certain embodiments, the first binding domain specifically binds to the membrane-proximal region of human DLL3 (e.g., SEQ ID NO:28). In certain embodiments, the first binding domain comprises, from its N-terminus to its C-terminus, a first light-chain variable domain, a first light-chain constant domain, a first peptide linker, a first heavy-chain variable domain, and a first heavy-chain constant CH1 domain; and the second binding domain comprises, from its N-terminus to its C-terminus, a second light-chain variable domain, a second light-chain constant domain, a second peptide linker, a second heavy-chain variable domain, and a second heavy-chain constant CH1 domain.

[0109] In some embodiments, the antigen-binding molecule comprises an immunoglobulin constant region. As used herein, the term “constant region” refers to all domains of an antibody other than the variable regions. The constant regions do not directly participate in antigen binding but exhibit various effector functions. As described above, antibodies are classified into specific isotypes (IgA, IgD, IgE, IgG, and IgM) and subtypes (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) depending on the amino acid sequence of their heavy-chain constant regions. The light-chain constant region can be, for example, the κ or λ light-chain constant region found in all five antibody isotypes, e.g., the human κ or λ light-chain constant region. In some embodiments, the antigen-binding proteins included in this disclosure are of the IgG1 or IgG4 isotype.

[0110] DLL3 expression

[0111] The present disclosure provides methods for treating cancers that express DLL3 in a subject (e.g., small cell lung cancer (SCLC)). For any of the methods of treating SCLC described herein, SCLC cancer cells desirably express DLL3 on the cell surface. In other embodiments, SCLC cancer cells express DLL3 on the cell surface and in the cytoplasm. In some aspects, cell surface expression and intercellular expression of the DLL3 protein can be determined by immunohistochemistry (IHC) or positron emission tomography (PET). Any suitable IHC assay for determining DLL3 protein expression can be used in conjunction with the present disclosure. Desirably, the IHC assay is approved by a regulatory agency such as the US Food and Drug Administration (FDA) or an agency with CE-IVD registration authority. DLL3-specific IHC assays and their components are known in the art and are commercially available from a variety of sources. For example, one can use the DLL3 (SP347) assay (Roche Diagnostics GmbH, Mannheim, Germany) to detect DLL3 expression in cancer cells or tumor cells. Other anti-DLL3 antibodies that can be used to detect DLL3 expression in an IHC assay include, but are not limited to, NBP2-24669 (Novus Biologicals, Littleton, CO); PA5-26336 (Thermo Fisher Scientific, Waltham, MA); and ab229902 (Abcam, Cambridge, MA).

[0112] In other embodiments, DLL3 gene expression in SCLC can be assayed. Methods for detecting and quantifying gene expression (e.g., mRNA levels) are known in the art and can be used in the context of the present disclosure. These methods include, for example, flow cytometry-based methods, polymerase chain reaction (PCR) analysis, sequencing analysis (e.g., RNA sequencing), electrophoretic analysis, restriction fragment length polymorphism (RFLP) analysis, Northern blot analysis, quantitative PCR, reverse transcriptase-PCR analysis (RT-PCR), and the like.

[0113] In some embodiments, at least 5% (e.g., 5%, 10%, or 20%) of the cancer cells can be positive for DLL3, as determined by IHC. The amount of DLL3-positive SCLC cells in a sample obtained from a subject can be represented by a tumor proportion score (TPS). The "tumor proportion score" is the percentage of viable cancer cells or tumor cells showing partial or complete membranous and cytoplasmic staining for DLL3. In some embodiments, at least 25% (e.g., 30%, 35%, 40%, or 45%) of the SCLC cells in a subject or in an SCLC sample obtained from a subject express DLL3 (25% TPS). In certain embodiments, at least 50% (e.g., 55%, 60%, 65%, 70%, 80%, 90%, 99%, or 100%) of the SCLC cells in a subject or in an SCLC sample obtained from a subject express DLL3 (50% TPS). For example, at least 75% (e.g., 85%, 95%, or 100%) of the SCLC cells in a subject or in an SCLC sample obtained from a subject express DLL3 (75% TPS). Viable cancer cells or tumor cells are those cancer cells or tumor cells with normal morphology and intact nuclei (e.g., without nuclear blebbing), and are subject to qualitative assessment routinely performed by a person of ordinary skill in the art (e.g., a pathologist). In certain embodiments, viable cancer cells or tumor cells are determined by a pathologist.

[0114] For example, in embodiments using the DLL3 (SP347) assay (Roche Diagnostics, GmbH, Mannheim, Germany), the following DLL3 scoring algorithm can be employed:

[0115] Positive status of DLL3 (SP347) at 25% cut-off: ≥25% of viable tumor cells exhibit moderate to strong membranous and / or cytoplasmic staining using any objective lens.

[0116] Positive status of DLL3 (SP347) at 75% cut-off: ≥75% of viable tumor cells exhibit moderate to strong membranous and / or cytoplasmic staining using any objective lens.

[0117] Some considerations of the above scoring algorithm include, for example, (i) there is no limitation on the magnification used; (ii) at least 100 viable tumor cells with well-preserved morphology are required; and (iii) partial / incomplete cell (cellular or membranous) staining is considered, and the highest intensity is considered in the evaluation.

[0118] The DLL3 expression level by IHC can also be determined by giving an IHC score based on the DLL3 positive signal intensity to the tumor sample. In some embodiments, the tumor scoring is based on the method described in Huang et al., Arch Pathol Lab Med [Archives of Pathology & Laboratory Medicine], 143(11):1373 - 1379 (2019). For example, to evaluate DLL3 staining, a combined H score of the membrane and cytoplasm can be collected at any magnification. The H score can be calculated using the following formula: 1x (percentage of cells stained with intensity 1) + 2x (percentage of cells stained with intensity 2) + 3x (percentage of cells stained with intensity 3), and typically ranges from 0 - 300. Surprisingly, combining cytoplasmic DLL3 staining with membrane DLL3 staining does not reduce the predictive / correlative value of DLL3 expression for treatment outcome, and in fact results in a more effective analysis. Negative or weak staining can be confirmed at a magnification of at least 20x. DLL3 positivity can be defined as ≥1% stained tumor cells. Hematoxylin and eosin (H&E) staining can be performed using conventional methods, and H&E stained slides can be used to evaluate tumor content and tissue quality assessment. In some embodiments, at least 100 live tumor cells (e.g., 120, 130, 140, 150, 200, 300, 400, 500 or more live tumor cells) are used for DLL3 assessment. As discussed above, a specific percentage of tumor cell staining in the tumor tissue can be scored. The total intensity of DLL3 staining of tumor cells in the tumor tissue can also be scored, ranging from 0 to 3, in increments of 1. For example, strong cytoplasmic and / or membrane DLL3 staining of tumor cells can be scored as "3" or "3+", moderate staining can be scored as "2" or "2+", and weak staining can be scored as "1" or "1+". No DLL3 positive staining can be given a score of "0". In some embodiments, an IHC intensity score of 2, 2+, 3 or 3+ (e.g., 2+, 3+ or 2+ and 3+) indicates a high level of DLL3 expression or DLL3 overexpression. In other embodiments, 3+ staining of tumor cells by IHC indicates a high level of DLL3 expression or DLL3 overexpression. In some embodiments, the IHC intensity score (e.g., 1+, 2+ or 3+) is determined by a pathologist.

[0119] Accordingly, the present disclosure also provides a method of treating SCLC in a subject, the method comprising administering to the subject a bispecific antigen-binding molecule that comprises at least a first binding domain that binds to human DLL3, wherein at least 25% (e.g., 30%, 35%, 40%, 45%) of SCLC cells express DLL3, e.g., as determined by an IHC assay. In certain embodiments, at least 50% (e.g., 55%, 60%, 65%, 70%, 80%, 90%, 99% or 100%) of SCLC cells express DLL3. For example, at least 75% (e.g., 85%, 95% or 100%) of SCLC cells express DLL3. In certain embodiments, the present disclosure provides a method of treating SCLC in a subject, the method comprising administering to the subject a bispecific antigen-binding molecule that comprises at least a first binding domain that binds to human DLL3, wherein the SCLC has a DLL3 expression level, wherein at least 25% (e.g., 30%, 40%, 50%, 60%, 70% or more) of SCLC cells express DLL3 at an intensity equal to or greater than 2+ (e.g., 2+, 3+ or 2+ and 3+), as determined by an IHC assay. For example, in some embodiments, the SCLC has a DLL3 expression level, wherein at least 75% (e.g., 80%, 85%, 90%, 95%, 99% or 100%) of SCLC cells express DLL3 at an intensity equal to or greater than 2+ (e.g., 2+, 3+ or 2+ and 3+), as determined by an IHC assay.

[0120] In the methods according to the invention, the intensity of one or more IHC signals detected in a sample can be compared to a predetermined reference value, e.g., a predetermined threshold. The term "predetermined reference value" refers to a measured value or defined amount of analyte used to evaluate the result of an assay. For example, the predetermined reference value can be a DLL3 positive signal corresponding to a known amount of DLL3 protein. The terms "predetermined threshold" and "predetermined cut-off value" are used interchangeably herein and refer to a measured value or defined amount of analyte used to evaluate DLL3 expression in a tumor sample of a subject by comparing the result of the assay to the predetermined threshold or cut-off value. While the present disclosure may provide exemplary predetermined threshold levels, it is well known that the cut-off value can vary depending on the nature of the assay (e.g., the antibody used, etc.). In addition, it is well within the scope of one of ordinary skill in the art to apply the disclosures herein to other samples to obtain assay-specific references or cut-off values for such assays based on the present disclosure. Without being bound by theory, using paired nested cut-off values (e.g., 25% TPS or 75% TPS) to identify cancers expressing DLL3 ensures inclusion of both moderately and highly expressing tumor cells while also allowing analysis of strong DLL3 expression.

[0121] Subjects and Samples

[0122] In various instances of the disclosed methods, the subject is a human subject. In an exemplary instance, the human subject has small cell lung cancer (SCLC), optionally histologically or cytologically confirmed SCLC. In various aspects, the human is male or female with SCLC and / or is 18 years of age or older. In an exemplary aspect, the human subject has been treated with platinum-based chemotherapy. In an exemplary aspect, the human subject has recurrent refractory (RR) SCLC, which optionally progresses or recurs after at least one platinum-based chemotherapy with or without a PD-L1 inhibitor. In an exemplary aspect, the human subject has extensive stage small cell lung cancer (ES-SCLC), optionally histologically or cytologically confirmed ES-SCLC. In an exemplary aspect, the human subject has limited SCLC that optionally progresses or recurs after at least one platinum-based chemotherapy. In an exemplary aspect, the human subject has ES-SCLC and has not received prior systemic treatment for ES-SCLC. In an exemplary instance, the human subject has an Eastern Cooperative Oncology Group (ECOG) performance status of 0-1 (Oken et al., Am J Clin Oncol [Journal of the American Society of Clinical Oncology] 5:649-655 (1982)). In various aspects, the human subject has one or more treated brain metastases. In various aspects, the platinum-based chemotherapy includes carboplatin or cisplatin or platinum-irinotecan.

[0123] The DLL3 expression analysis described herein is ideally performed on tumor or cancer cell samples obtained from a subject diagnosed with cancer. As used herein, the term "sample" or "biological sample" refers to a sample of a biological fluid, tissue, or cell obtained from a subject in a healthy and / or pathological state. A variety of cell types, tissues, or body fluids can be used as samples. These cell types, tissues, and body fluids can include tissue sections, such as biopsy and autopsy samples, frozen sections for histological purposes, blood (such as whole blood), plasma, serum, red blood cells, platelets, bronchoalveolar lavage fluid, interstitial fluid, cerebrospinal fluid, and the like. Tissue or cell samples can be provided by removing cells from a human body, but can also be accomplished by using previously isolated cells (e.g., isolated by another person at another time and / or for other purposes). Archived tissues can also be used, such as those with a history of treatment or outcome. In some aspects, the sample includes a formalin-fixed paraffin-embedded (FFPE) cytology or tissue specimen from a subject. Exemplary cytology or tissue specimens include core needle biopsy (CNB), fine needle aspiration (FNA), endobronchial ultrasound-guided biopsy (EBUS). A cytology or tissue specimen is eligible for testing as long as it is fixed in formalin and prepared as an FFPE (e.g., FFPE of a fresh core needle biopsy) specimen.

[0124] In an exemplary aspect, the cancer is histologically or cytologically confirmed SCLC. Optionally, SCLC can be measured by modified Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, where measurable lesions include (a) non-nodular lesions with clear boundaries that can be accurately and continuously measured in one dimension in the axial plane (longest diameter ≥ 10 mm, measured by magnetic resonance imaging / computed tomography (MRI / CT), scan slice thickness ≤ 5 mm) and / or (b) nodular lesions with a longest diameter perpendicular to the long axis (short axis) ≥ 15 mm according to MRI / CT, and / or excluding simple cysts, pleural / pericardial effusions, and ascites.

[0125] The cancer may be a neuroendocrine carcinoma other than SCLC. Neuroendocrine carcinoma or neoplasm (NEC or NEN) is a relatively rare and heterogeneous type of tumor, accounting for approximately 2% of all malignancies, with a prevalence in the United States of <200,000 (Oronsky et al., Neoplasia, 19(12):991-1002 (2017)). The term "neuroendocrine" applies to widely distributed cells that have properties similar to nerve cells (such as the presence of dense core granules (DCGs4), which are similar to the DCGs present in serotonergic neurons (which store monoamines)) and "endocrine" properties (such as the synthesis and secretion of these monoamines). The neuroendocrine (NE) system includes endocrine glands such as the pituitary, parathyroid, and NE adrenal glands, as well as endocrine islet tissue embedded within glandular tissue (thyroid or pancreas) and dispersed cells within the exocrine parenchyma, such as endocrine cells of the digestive and respiratory tracts, which belong to the so-called diffuse endocrine system. Most neuroendocrine tumors occur in the lung, appendix, small intestine, rectum, and pancreas. Neuroendocrine carcinomas include, but are not limited to, small cell lung cancer (SCLC), neuroendocrine prostate cancer (NEPC), and neuroblastoma. In some embodiments, the tumor or cancer is a lung cancer such as small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), glioma, glioblastoma, melanoma, prostate cancer such as neuroendocrine prostate cancer, neuroendocrine pancreatic cancer, hepatoblastoma, large cell lung neuroendocrine carcinoma, pancreatic neuroendocrine carcinoma, bladder neuroendocrine carcinoma, gastric neuroendocrine carcinoma, adrenocortical carcinoma, Merkel cell carcinoma, neuroblastoma, head and neck carcinoid or neuroendocrine carcinoma, head and neck paraganglioma, or cervical small cell neuroendocrine carcinoma. In some embodiments, the tumor or cancer is prostate cancer (e.g., neuroendocrine prostate cancer) or lung cancer (e.g., small cell lung cancer).

[0126] Administration regimen

[0127] Disclosed herein are methods of treating cancer (e.g., SCLC) in which at least 25% of the cancer cells express DLL3 or in which at least 25% of the cancer cells express DLL3 at an intensity equal to or greater than 2+, comprising administering to a subject in need thereof a bispecific antigen-binding molecule, such as an anti-DLL3 antigen-binding molecule described herein. In some aspects, the anti-DLL3 antigen-binding molecule is administered to the subject once every two weeks at a dose of about 10 mg to about 100 mg, about 30 mg to about 100 mg, about 10 mg, or about 100 mg. In other embodiments, the anti-DLL3 antigen-binding molecule is administered to the subject twice every three weeks at a dose of about 10 mg to about 100 mg, about 30 mg to about 100 mg, about 10 mg, or about 100 mg. In other embodiments, the anti-DLL3 antigen-binding molecule is administered to the subject once every three weeks at a dose of about 20 mg to about 200 mg, about 30 mg to about 100 mg, about 20 mg, about 60 mg, or about 200 mg. As discussed above, in certain embodiments, the DLL3-positive cancer is small cell lung cancer (SCLC). In certain embodiments, the SCLC is limited SCLC, recurrent / refractory SCLC (RR SCLC), or extensive disease SCLC (ED SCLC). In certain embodiments, the subject is a human having SCLC (e.g., RR SCLC or ED SCLC).

[0128] Exemplary dosing regimens include administering the anti-DLL3 antigen-binding molecule to the subject once a week for three weeks (i.e., weeks 1, 2, and 3), and then administering the anti-DLL3 antigen-binding molecule to the subject once every two weeks. Another exemplary dosing regimen includes administering the anti-DLL3 antigen-binding molecule to the subject once a week for two weeks (i.e., weeks 1 and 2), and then administering the anti-DLL3 antigen-binding molecule to the subject once every three weeks. The anti-DLL3 antigen-binding molecule can be administered to the subject at a dose of about 10 mg to about 100 mg, about 30 mg to about 100 mg, about 10 mg, or about 100 mg.

[0129] In certain embodiments, the anti-DLL3 antigen-binding molecule is administered on day 1 and day 15 of a 28-day cycle. In other embodiments, the anti-DLL3 antigen-binding molecule is administered on day 1 of a 21-day cycle, or on day 1 and day 8 of a 21-day cycle.

[0130] The anti-DLL3 antigen-binding molecule can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intralung, intranasal, and / or intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the anti-DLL3 antigen-binding molecule is administered once every two weeks, once every three weeks, or twice every three weeks by intravenous (IV) infusion (e.g., short intravenous infusion (about 60 minutes)).

[0131] Due to the mechanism of action of bispecific antigen-binding molecules (e.g., anti-DLL3 antigen-binding molecules, such as those described herein), during the initiation of treatment therewith, a subject may have an increased risk of cytokine release syndrome (CRS), and a stepwise dosing method can be implemented. Thus, in certain embodiments, the bispecific antigen-binding molecule (e.g., anti-DLL3 antigen-binding molecule) is administered using a stepwise dosing method during the initiation of the pharmaceutical treatment (e.g., the first cycle of treatment), after which the anti-DLL3 antigen-binding molecule is administered according to the bi-weekly, twice every three weeks, or once every three weeks regimens described above. For example, the anti-DLL3 antigen-binding molecule can be administered in 21-day or 28-day cycles according to the following stepwise dosing protocol during the initiation of treatment (cycle 1), such as those described above: a first step dose or priming dose on day 1, a step dose equal to the target dose on day 8, and the target dose on day 15. Exemplary priming doses of the anti-DLL3 antigen-binding molecule include 1 mg. Exemplary target doses of the anti-DLL3 antigen-binding molecule include doses in the range of 10 mg to 100 mg (e.g., 10 mg or 100 mg) or doses in the range of 20 mg to 200 mg (e.g., 20 mg, 60 mg, or 200 mg).

[0132] Combination therapy

[0133] In some embodiments of the methods disclosed herein, the bispecific antigen-binding molecule (e.g., anti-DLL3 antigen-binding protein) described above can be administered alone (i.e., as a "monotherapy") to treat cancer (e.g., SCLC) in which at least 25% of the cancer cells express DLL3 or in which at least 25% of the cancer cells express DLL3 at an intensity equal to or greater than 2+, or in combination with at least one additional therapeutic agent to achieve a desired biological effect in a subject (i.e., as part of a "combination therapy"). In various embodiments disclosed herein, "combination therapy" or "in combination with" refers to administering to a subject (e.g., a human) having cancer that expresses DLL3 one therapeutic modality (e.g., an anti-DLL3 antigen-binding protein) in addition to another therapeutic modality (e.g., an anti-PD-L1 antibody and optionally one or more chemotherapeutic agents). In a combination therapy involving an anti-DLL3 antigen-binding protein and an anti-PD-L1 antibody (described below), one therapeutic modality can be administered before, during, or after the administration of the other therapeutic modality to the subject. However, such combination therapy does not include a situation in which 28 days or more have elapsed between the end of the administration of one therapeutic modality and the start of the administration of the other therapeutic modality.

[0134] In some embodiments, the anti-DLL3 antigen-binding molecule is administered in combination with one or more chemotherapeutic agents. A "chemotherapeutic agent" is also referred to as an anti-tumor agent and includes compounds that can be used to treat cancer. Chemotherapeutic agents can be classified according to their mechanism of action and further divided into subgroups within each category. Exemplary categories of chemotherapeutic agents include alkylating agents, antimetabolites, topoisomerase inhibitors, anti-tumor antibiotics, mitotic inhibitors, and protein kinase inhibitors. Alkylating agents include subgroups such as oxazaphosphorine, nitrogen mustard, imidazotetrazine, nitrosourea, alkyl sulfonate, hydrazine, and platinum-based agents. Platinum-based agents include cisplatin, carboplatin, and oxaliplatin. Topoisomerase inhibitors include topoisomerase I inhibitors and topoisomerase II inhibitors. Mitotic inhibitors include vinca alkaloids, taxanes, and non-taxane microtubule inhibitors. Anti-tumor antibiotics include bleomycin, actinomycin D (dactinomycin), and mitomycin.

[0135] In certain embodiments, the chemotherapeutic agent useful in the methods disclosed herein is an alkylating agent. In an exemplary embodiment, the alkylating agent can be a platinum-based agent such as cisplatin, carboplatin, or oxaliplatin. In certain embodiments, the alkylating agent is lurbinectedin (ZEPZELCA TM ). In other embodiments, the chemotherapeutic agent can be a topoisomerase inhibitor such as a topoisomerase II inhibitor (e.g., etoposide). In certain embodiments, the chemotherapeutic agent useful in the methods disclosed herein includes a platinum-based agent (cisplatin, carboplatin, or oxaliplatin), a topoisomerase II inhibitor (etoposide), or a combination of a platinum-based agent and a topoisomerase II inhibitor.

[0136] In some aspects of the disclosed methods, the anti-DLL3 antigen-binding molecule is administered in combination with an antagonist of the PD-1 / PD-L1 signaling pathway. Programmed cell death protein 1 (PD-1), also known as CD279, SLEB2, and hSLE1, is a transmembrane protein expressed on activated T cells, natural killer (NK) cells, B lymphocytes, macrophages, dendritic cells (DCs), and monocytes. Notably, PD-1 is highly expressed on tumor-specific T cells (Han et al., Am J Cancer Res [American Journal of Cancer Research] 10(3):727-742 (2020)). PD-1 binds to members of the B7 protein family, PD-1 ligand 1 (PD-L1; also known as CD279 and B7-H1), and PD-1 ligand 2 (also known as PD-L2, CD273, and B7-DC). PD-L1 is constitutively expressed on T cells and B cells, macrophages, and dendritic cells, while PD-L2 expression is generally restricted to activated DCs and macrophages (Xing et al., Oncoimmunology [Cancer Immunology] 7(3):e1356144 (2017) (doi:10.1080 / 2162402X.2017.1356144)). PD-1 inhibits adaptive and innate immune responses. The PD-1 / PD-L1 axis is associated with the inhibition of T cell immune responses in cancer. Antagonists of this pathway have been clinically demonstrated in many solid tumor indications. PD-1 inhibitors (e.g., nivolumab, pembrolizumab, and cemiplimab) and PD-L1 inhibitors (e.g., atezolizumab, avelumab, and durvalumab) target the PD-1 / PD-L1 pathway, and each has been approved by the US Food and Drug Administration (FDA) for the treatment of various cancers. In various embodiments, an agent that targets PD-L1 (e.g., a PD-L1 blocker) can be used in the methods disclosed herein to treat cancers expressing DLL3. Exemplary agents that target PD-L1 include anti-PD-L1 antibodies, such as atezolizumab, avelumab, and durvalumab.

[0137] In certain embodiments, the anti-PD-L1 antibody is atezolizumab (International Nonproprietary Name (INN), WHO Drug Information, Volume 29, Issue 3, 2015, Recommended INNs: List 74). Atezolizumab is a humanized PD-L1 blocking antibody. It is an immunoglobulin G1-κ, anti-[Homo sapiens CD274 (programmed death ligand 1, PDL1, PD-L1, B7 homolog 1, B7H1)], humanized monoclonal antibody; γ1 heavy chain (1-448) [humanized VH (Homo sapiens IGHV3-23*04 (86.70%)-(IGHD)-IGHJ4*01) [8.8.11] (1-118)-Homo sapiens IGHG1*03 (CH1 R120>K(215)(119-216), hinge (217-231), CH2 N84.4>A(298)(232-341), CH3 (342-446), CHS (447-448)) (119-448)], (221-214')-disulfide with κ light chain (1'-214') [humanized V-κ (Homo sapiens IGKV1-5*01 (87.90%)-IGKJ1*01) [6.3.9] (1'-107')-Homo sapiens IGKC*01 (108'-214')]; dimer (227-227”:230-230”)-bis-disulfide. Atezolizumab is commercially available, for example, it is available as on the market.

[0138] In certain embodiments, the anti-PD-L1 antibody is avelumab (International Nonproprietary Name (INN), WHO Drug Information, Volume 30, Issue 1, 2016, Recommended INNs: List 75). Avelumab is a PD-L1 blocking monoclonal antibody produced in CHO cells. It is an immunoglobulin G1-λ1, anti-[Homo sapiens CD274 (programmed death ligand 1, PDL1, PD-L1, B7 homolog 1, B7H1)], Homo sapiens monoclonal antibody; γ1 heavy chain (1-450) [Homo sapiens VH (IGHV3-23*01 (90.80%)-(IGHD)-IGHJ4*01)[8.8.13](1-120)-IGHG1*01, Gm17,1 (CH1(121-218), hinge(219-233), CH2(234-343), CH3(344-448), CHS(449-450)(121-450)], (223-215')-disulfide bond with λ1 light chain (1'-216') [Homo sapiens V-λ (IGLV2-14*01 (99.00%)-IGLJ1*01)[9.3.10](1'-110')-IGLC1*02(111'-216')]; dimer (229-229”:232-232”)-bis-disulfide bond. Avelumab is commercially available, for example, it is available under the market.

[0139] In certain embodiments, the anti-PD-L1 antibody is durvalumab (International Nonproprietary Name (INN), WHO Drug Information, Volume 29, Number 3, 2015, Recommended INNs: List 74). Durvalumab is a PD-L1 blocking monoclonal antibody produced in CHO cells. It is an immunoglobulin G1-κ, anti-[Homo sapiens CD274 (programmed death ligand 1, PDL1, PD-L1, B7 homolog 1, B7H1)], Homo sapiens monoclonal antibody; γ1 heavy chain (1-451) [Homo sapiens VH (IGHV3-7*01 (99.00%)-(IGHD)-IGHJ4*01) [8.8.14] (1-121) - IGHG1*03 (CH1 (122-219), hinge (220-234), CH2 (235-344) L1.3>F (238), L1.2>E (239), P116>S (335), CH3 (345-449), CHS (450-451)) (122-451)], (224-215’)-disulfide with κ light chain (1’-215’) [Homo sapiens V-κ (IGKV3-20*01 (96.90%)-IGKJ1*01) [7.3.9] (1’-108’)-IGKC*01 (109’-215’)]; dimer (230-230”:233-233”)-bis-disulfide. Durvalumab is commercially available, for example, it is available as on the market.

[0140] In one aspect, the present disclosure provides a method of treating a cancer expressing DLL3 (e.g., SCLC) in which at least 25% of the cancer cells express DLL3 or in which at least 25% of the cancer cells express DLL3 at an intensity equal to or greater than 2+, the method comprising administering to a subject in need thereof an anti-DLL3 antigen-binding molecule, an anti-PD-L1 antibody, and optionally one or more chemotherapeutic agents, wherein the anti-DLL3 antigen-binding molecule is administered once every two weeks at a dose of about 10 mg to about 100 mg. In certain embodiments, the anti-DLL3 antigen-binding molecule is administered once every two weeks at a dose of about 10 mg, about 30 mg, about 50 mg, or about 100 mg. In certain embodiments, the anti-DLL3 antigen-binding molecule is administered on Day 1 and Day 15 of a 28-day cycle.

[0141] In one aspect, the present disclosure provides a method of treating a cancer expressing DLL3 (e.g., SCLC) in which at least 25% of the cancer cells express DLL3 or in which at least 25% of the cancer cells express DLL3 at an intensity equal to or greater than 2+, the method comprising administering to a subject in need thereof an anti-DLL3 antigen-binding molecule, an anti-PD-L1 antibody, and optionally one or more chemotherapeutic agents, wherein the anti-DLL3 antigen-binding molecule is administered at a dose of about 10 mg to about 100 mg twice every three weeks, or at a dose of about 20 mg to about 200 mg once every three weeks. In certain embodiments, the anti-DLL3 antigen-binding molecule is administered at a dose of about 10 mg, about 30 mg, about 50 mg, or about 100 mg twice every three weeks, e.g., on day 1 and day 8 of a 21-day cycle. In certain embodiments, the anti-DLL3 antigen-binding molecule is administered at a dose of about 20 mg to about 100 mg (e.g., about 20 mg, about 60 mg, or about 100 mg) once every three weeks, e.g., on day 1 of a 21-day cycle. In certain embodiments, the anti-DLL3 antigen-binding molecule is administered at a dose of about 100 mg to about 200 mg (e.g., about 120 mg or about 200 mg) once every three weeks, e.g., on day 1 of a 21-day cycle.

[0142] In various embodiments in which the anti-DLL3 antigen-binding molecule is administered together with the anti-PD-L1 antibody and optionally one or more chemotherapeutic agents, the anti-DLL3 antigen-binding molecule can be administered according to a stepwise dosing regimen during the start of treatment (e.g., cycle 1) to minimize potential side effects (e.g., CRS) associated with the anti-DLL3 antigen-binding molecule, as described herein. Thus, in certain embodiments, the anti-DLL3 antigen-binding molecule is administered in a 21-day cycle according to the following regimen during cycle 1 of treatment: a first dose of about 0 mg or about 1 mg on day 1, a second dose of from about 1 mg to about 100 mg on day 8, and a third dose of from about 10 mg to about 200 mg on day 15. In certain embodiments, the anti-DLL3 antigen-binding molecule is administered during cycle 1 according to the following regimen: a first dose of about 1 mg on day 1, a second dose of from about 10 mg to about 100 mg on day 8, and a third dose of from about 10 mg to about 100 mg on day 15. In certain embodiments, the anti-DLL3 antigen-binding molecule is administered during cycle 1 according to the following regimen: a first dose of about 1 mg on day 1, a second dose of from about 10 mg to about 100 mg on day 8, and a third dose of from about 20 mg to about 200 mg on day 15. Each cycle 1 regimen can be used prior to the bi-weekly, twice every three weeks, or once every three weeks regimen of the anti-DLL3 antigen-binding molecule described above.

[0143] In certain embodiments, a method of treating a cancer that expresses DLL3 (such as SCLC) is disclosed herein, wherein at least 25% of the cancer cells express DLL3 or wherein at least 25% of the cancer cells express DLL3 at an intensity equal to or greater than 2+, the method comprising administering to a subject in need thereof an anti-DLL3 antigen-binding molecule, an anti-PD-L1 antibody, and optionally one or more chemotherapeutic agents, wherein the anti-DLL3 antigen-binding molecule is administered according to the following schedule: a) Cycle 1 (21 days): a first dose of about 1 mg on day 1, a second dose on day 8, and a third dose on day 15, b) Cycles 2 and 3 (each cycle 21 days): a fourth dose on day 1 and a fifth dose on day 8 of each cycle, and c) at the start of Cycle 4 and one or more subsequent doses every two weeks in 28-day cycles thereafter, wherein the second dose, the third dose, the fourth dose, the fifth dose, and the one or more subsequent doses are the same and are each from about 10 mg to about 100 mg (e.g., about 10 mg, about 30 mg, or about 100 mg).

[0144] In certain embodiments, a method of treating a cancer that expresses DLL3 (such as SCLC) is disclosed herein, wherein at least 25% of the cancer cells express DLL3 or wherein at least 25% of the cancer cells express DLL3 at an intensity equal to or greater than 2+, the method comprising administering to a subject in need thereof an anti-DLL3 antigen-binding molecule, an anti-PD-L1 antibody, and optionally one or more chemotherapeutic agents, wherein the anti-DLL3 antigen-binding molecule is administered in 21-day cycles according to the following schedule: a) Cycle 1: a first dose of about 1 mg on day 1, a second dose on day 8, and a third dose on day 15, b) Cycles 2 and 3: a fourth dose on day 1 and a fifth dose on day 8 of each cycle, and c) at the start of Cycle 4 and one or more subsequent doses every three weeks thereafter, wherein the second dose, the third dose, the fourth dose, and the fifth dose are the same and are each from about 10 mg to about 100 mg (e.g., about 10 mg, about 30 mg, or about 100 mg), and wherein the one or more subsequent doses are the same and are each from about 20 mg to about 200 mg (e.g., about 20 mg, about 60 mg, or about 200 mg).

[0145] In certain embodiments, a method of treating DLL3-positive cancer (e.g., SCLC) is disclosed herein, wherein at least 25% of the cancer cells express DLL3 or at least 25% of the cancer cells express DLL3 at an intensity equal to or greater than 2+, the method comprising administering to a subject in need thereof an anti-DLL3 antigen-binding molecule, an anti-PD-L1 antibody, and optionally one or more chemotherapeutic agents, wherein the anti-DLL3 antigen-binding molecule is administered in 21-day cycles according to the following schedule: a) Cycle 1: a first dose of about 1 mg on day 1, a second dose of from about 10 mg to about 100 mg on day 8, a third dose on day 15, b) Cycles 2 and 3: a fourth dose on day 1 of each cycle, and c) at the start of Cycle 4 and one or more subsequent doses every three weeks thereafter, wherein the third dose, the fourth dose, and the one or more subsequent doses are the same and are each from about 20 mg to about 200 mg (e.g., about 20 mg, about 60 mg, or about 200 mg).

[0146] In various embodiments in which the anti-DLL3 antigen-binding molecule is administered in combination with an anti-PD-L1 antibody and optionally one or more chemotherapeutic agents, the anti-PD-L1 antibody is a PD-L1 blocking antibody. Examples of such anti-PD-L1 antibodies include atezolizumab, durvalumab, and avelumab. In certain embodiments, the anti-PD-L1 antibody is atezolizumab or durvalumab. In certain embodiments, the anti-PD-L1 antibody is durvalumab. When used in the methods disclosed herein, the dose and schedule of the anti-PD-L1 antibody are the same as those approved by a regulatory agency (e.g., the FDA). For example, atezolizumab can be administered at a dose of about 840 mg every 2 weeks, or about 1200 mg every 3 weeks, or about 1680 mg every 4 weeks. For example, durvalumab can be administered at a dose of about 10 mg / kg every 2 weeks, or about 1500 mg every 3 weeks, or about 1500 mg every 4 weeks.

[0147] In various embodiments in which the anti-DLL3 antigen-binding molecule is administered in combination with an anti-PD-L1 antibody and optionally one or more chemotherapeutic agents, the one or more chemotherapeutic agents include alkylating agents, topoisomerase inhibitors, or a combination thereof. In certain embodiments, the one or more chemotherapeutic agents include platinum-based agents (e.g., cisplatin, carboplatin, or oxaliplatin), topoisomerase II inhibitors (e.g., etoposide), or a combination thereof. In certain embodiments, the one or more chemotherapeutic agents comprise cisplatin or carboplatin and etoposide. In certain embodiments, the one or more chemotherapeutic agents is etoposide. In various embodiments, the one or more chemotherapeutic agents are administered according to the dose and / or schedule approved by a regulatory agency (e.g., the FDA). For example, in various embodiments, carboplatin is administered at a dose sufficient to achieve an AUC = 5 mg / ml / min, and etoposide is administered at 100 mg / m2 Administration of a dose.

[0148] In various embodiments, the anti-DLL3 antigen-binding molecule, the anti-PD-L1 antibody, and optionally one or more chemotherapeutic agents are each administered by intravenous (IV) infusion. In certain embodiments, when administered on the same day, the anti-DLL3 antigen-binding molecule is administered after the anti-DP-L1 antibody and one or more chemotherapeutic agents.

[0149] In certain embodiments, provided herein is a method of treating a cancer expressing DLL3 (e.g., SCLC) in which at least 25% of the cancer cells express DLL3 or in which at least 25% of the cancer cells express DLL3 at an intensity equal to or greater than 2+, the method comprising administering to a subject in need thereof an anti-DLL3 antigen-binding molecule and an alkylating agent, wherein the anti-DLL3 antigen-binding molecule is administered once every three weeks at a dose of about 10 mg to about 200 mg. In certain embodiments, the anti-DLL3 antigen-binding molecule is administered once every three weeks at a dose of about 10 mg to about 100 mg (e.g., 10 mg, 20 mg, 60 mg, or 100 mg). In certain embodiments, the anti-DLL3 antigen-binding molecule is administered once every three weeks at a dose of about 20 mg to about 200 mg (e.g., 20 mg, 60 mg, 100 mg, or 200 mg). In certain embodiments, the anti-DLL3 antigen-binding molecule is administered on day 1 of a 21-day cycle. In various embodiments, the alkylating agent is lurbinectedin.

[0150] In certain embodiments, the method comprises administering to a subject in need an anti-DLL3 antigen-binding molecule and an alkylating agent, wherein the anti-DLL3 antigen-binding molecule is administered to the subject according to the following 21-day cycle: a first dose of 0 mg or about 1 mg on day 1, a second dose of from about 10 mg to about 100 mg on day 8, a third dose of from about 10 mg to about 200 mg on day 15, and one or more subsequent doses of from about 10 mg to about 200 mg starting on day 22 and every three weeks thereafter. In certain embodiments, the first dose is 1 mg, the second dose is from 10 mg to 100 mg (e.g., 10 mg, 20 mg, 60 mg, or 100 mg), the third dose is from 10 mg to 200 mg (e.g., 10 mg, 20 mg, 60 mg, 100 mg, or 200 mg), and the one or more subsequent doses are the same and the same as the third dose (e.g., 10 mg, 20 mg, 60 mg, 100 mg, or 200 mg). In certain embodiments, the method comprises administering only the alkylating agent in cycles 1 and 2, and administering the alkylating agent and the anti-DLL3 antigen-binding molecule in cycle 3 and thereafter. In various embodiments, the alkylating agent is lurbinectedin. In various embodiments, lurbinectedin can be administered according to a dose and / or regimen approved by a regulatory agency (e.g., the FDA), e.g., at a dose of about 3.2 mg / m 2 , 2.6 mg / m 2 or 2 mg / m 2 administered once every three weeks.

[0151] In various embodiments, the anti-DLL3 antigen-binding molecule and the alkylating agent (e.g., lurbinectedin) are each administered by intravenous (IV) infusion. In certain embodiments, when administered on the same day, the anti-DLL3 antigen-binding molecule is administered after the alkylating agent.

[0152] Other cancer treatments that can be administered in combination with the antigen-binding molecules described herein (optionally in combination with one or more chemotherapeutic agents and / or anti-PD-L1 antibodies) include, but are not limited to, surgery, radiation therapy, targeted therapy, immunotherapy, hormone therapy, and stem cell transplantation. Exemplary targeted cancer therapies include, but are not limited to, protein kinase inhibitors (e.g., BCR-ABL and c-KIT tyrosine kinase inhibitors, EGFR tyrosine kinase inhibitors, ALK tyrosine kinase inhibitors, V600E mutant-BRAF oncogene inhibitors, MEK inhibitors, Bruton kinase inhibitors, Janus kinase inhibitors, and CDK inhibitors).

[0153] Additional therapeutic agents

[0154] In some embodiments, the methods disclosed herein further comprise using one or more additional therapeutic agents to prevent, reduce, or mitigate the risk of any side effects associated with administering a bispecific antigen-binding molecule (e.g., an anti-DLL3 antigen-binding protein) alone or in combination with an anti-PD-L1 antibody and / or a chemotherapeutic agent. The major side effect that may be associated with the use of an anti-DLL3 antigen-binding protein is cytokine release syndrome (CRS). Additional therapeutic agents that can be used to prevent, reduce, or mitigate the risk of CRS include, but are not limited to, corticosteroids (e.g., dexamethasone), fluids (e.g., saline), and anti-IL6 antibodies (e.g., tocilizumab or siltuximab). Dexamethasone can be administered intravenously (IV) prior to all cycle 1 doses (including all ramp doses) of the anti-DLL3 antigen-binding protein (e.g., AMG 757), saline (e.g., 1 liter) can be administered intravenously (IV) after all doses of the anti-DLL3 antigen-binding protein (e.g., AMG 757) in cycle 1, and an anti-IL6 antibody (e.g., tocilizumab or siltuximab) can be administered as needed (e.g., if the subject is unresponsive to intravenous (IV) fluids). Additional corticosteroid prophylaxis can be achieved with oral dexamethasone as needed. Exemplary doses of dexamethasone include 8 mg / dose (maximum 24 mg / day). Exemplary doses of tocilizumab include 8 mg / kg (not to exceed 800 mg). Symptoms of CRS include fever, nausea, fatigue, headache, myalgia, and asthenia, and therapeutic agents that can be used to treat these symptoms (e.g., acetaminophen for treating fever) can also be used. In certain embodiments, additional therapeutic agents that can also be used to reduce or mitigate side effects associated with anti-DLL3 antigen-binding protein therapy include granulocyte colony-stimulating factor (e.g., filgrastim or pegfilgrastim).

[0155] Accordingly, in certain embodiments, the methods disclosed herein further comprise administering one or more additional therapeutic agents selected from corticosteroids (e.g., prednisone, hydrocortisone, and dexamethasone), fluids (saline), and anti-IL6 antibodies (e.g., tocilizumab or siltuximab). In certain embodiments, these methods further comprise administering one or more additional therapeutic agents selected from corticosteroids (e.g., dexamethasone), fluids (saline), and tocilizumab or siltuximab. In certain embodiments, one or more of the corticosteroid, fluid, and tocilizumab are administered during cycle 1 in which the anti-DLL3 antigen-binding protein (e.g., AMG 757) is administered.

[0156] Therapeutic response

[0157] The efficacy of the disclosed methods for treating cancer (e.g., SCLC) can be evaluated by a variety of clinical outcomes, endpoints, and / or metrics. In some embodiments, the outcome of a subject treated according to the disclosed methods can be compared to a second SCLC subject with a lower DLL3 expression level treated with the same bispecific antigen-binding molecule. In this regard, evaluable clinical outcomes include, but are not limited to, progression-free survival (PFS), overall survival (OS), objective response rate (ORR), disease control rate (DCR), duration of response (DOR). As used herein, the term "progression-free survival (PFS)" refers to the time from randomization until the first evidence of disease progression or death. As used herein, the term "overall survival (OS)" refers to the time from randomization to death. As used herein, the term "objective response rate (ORR)" is a measure of how a particular treatment affects tumor burden in patients with a history of solid tumors and refers to the proportion of patients who have a partial or complete response to the therapy. As used herein, the term "duration of response (DoR)" refers to the time from randomization to disease progression or death in patients who achieve a complete or partial response. As used herein, the term "disease control rate (DCR)" describes the percentage of patients with advanced cancer in whom a therapeutic intervention results in a complete response, partial response, or stable disease. In some aspects, compared to a second SCLC subject with a lower DLL3 expression level treated with the same antigen-binding molecule, the disclosed methods are expected to increase one or more of progression-free survival (PFS), overall survival (OS), objective response rate (ORR), and / or disease control rate (DCR) and duration of response (DOR). Clinical endpoints regarding cancer therapies are further described, for example, in Delgado A. and Guddati, A.K. et al., Am J Cancer Res [American Journal of Cancer Research] 2021; 11(4):1121-1131.

[0158] As discussed above, administration of the antigen-binding molecules described herein to cancers expressing DLL3 (e.g., SCLC) where the cancer cells express a particular level of DLL3 is advantageously associated with improved treatment outcomes, particularly as compared to standard of care (SOC). It should be understood that the overall response rate (ORR) and median progression-free survival (PFS) for SCLC patients receiving SOC second-line treatment are approximately 51% and 4.6 months, respectively. (Simos et al., Clin Lung Cancer, 15:110-118 (2014)). For recurrent SCLC patients receiving SOC third-line chemotherapy, the ORR is approximately 18% and the median PFS is approximately 2.0 months (Gong, J. and R. Salgia, J Oncol Practice, 14(6):359-366 (2018)). Thus, in some embodiments, the methods described herein are expected to increase ORR, PFS, and / or OS as compared to standard of care. For example, when the anti-DLL3 antigen-binding agent is used as first-line treatment for SCLC, the disclosed methods are expected to produce an ORR of at least about 65% (e.g., about 65% to about 70%), a median PFS greater than about 5 months (e.g., 7 months or more), and a median OS of at least about 13 months (e.g., about 13 to 15 months or about 16 to 18 months). When the anti-DLL3 antigen-binding agent is used as second-line treatment for SCLC, the disclosed methods are expected to produce an ORR of at least about 35%, a median PFS greater than about 7 months (e.g., 8 months or more), and a median OS of at least about 11 months (e.g., 11.5 months or more). When the anti-DLL3 antigen-binding agent is administered as third-line treatment for SCLC, the method is expected to produce an ORR of at least about 20% (e.g., about 24% or more), a median PFS greater than about 6 months, and a median OS of at least about 7.5 months (e.g., 8 months or more).

[0159] Ideally, the treatment provided by the methods disclosed herein slows the progression of cancer. For example, these methods can treat cancer by enhancing T cell activity or the immune response against cancer, reducing tumor or cancer growth, reducing tumor cell metastasis, increasing cell death of tumors or cancer cells, etc. In exemplary aspects, these methods can delay cancer recurrence by at least about 30 days, two months, four months, six months, one year, two years, four years, or longer. In exemplary aspects, the treatment can encompass increasing the survival period of the subject. In various aspects, the treatment provided by the methods disclosed herein includes a therapeutic response according to the Response Evaluation Criteria in Solid Tumors (RECIST) or other similar criteria. RECIST is a set of criteria established jointly by the National Cancer Institute of the United States, the National Cancer Institute of Canada Clinical Trials Group, and the European Organisation for Research and Treatment of Cancer for assessing the progression, stabilization, or responsiveness of tumors and / or cancer cells. RECIST defines tumor size as the sum of the longest diameters (SLD) of target lesions and classifies patients as complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). CR and PR indicate no new tumor lesions, a 100% shrinkage rate and a shrinkage rate greater than 30%, respectively; PD indicates an increase in tumor size by 20% from the smallest size observed up to that point, or the appearance of new lesions.

[0160] Article

[0161] This disclosure provides articles comprising: (a) a container comprising a bispecific antigen-binding molecule (e.g., an anti-DLL3 antigen-binding molecule such as AMG 757); and (b) a package insert with instructions for treating a subject with cancer expressing DLL3 (or treating SCLC) by administering an anti-DLL3 antigen-binding molecule (e.g., AMG 757), wherein the instructions specify that at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 or at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 with an intensity of at least 2+, and the anti-DLL3 antigen-binding molecule is administered by intravenous (IV) infusion at a dose of about 10 mg to about 100 mg once every two weeks. In some embodiments, the instructions specify that at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 or at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 with an intensity of at least 2+, and the anti-DLL3 antigen-binding molecule is administered to the subject at a dose of about 10 mg to about 100 mg (e.g., 10 mg, 30 mg, or 100 mg) twice every three weeks, e.g., on day 1 and day 8 of a 21-day cycle. In certain embodiments, the instructions specify that at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 or at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 with an intensity of at least 2+, and the anti-DLL3 antigen-binding molecule is administered to the subject at a dose of about 20 mg to about 200 mg (e.g., 20 mg, 60 mg, 100 mg, 160 mg, or 200 mg) once every three weeks, e.g., on day 1 of a 21-day cycle. In certain embodiments, the instructions further specify that in the first cycle of administering the anti-DLL3 antigen-binding molecule to the subject, the anti-DLL3 antigen-binding molecule is administered by an extended intravenous infusion lasting from 2 days to 7 days (e.g., lasting 3 days). In certain embodiments, the instructions further specify that the anti-DLL3 antigen-binding molecule is administered according to a stepwise dosing regimen in cycle 1 (21-day cycle): 1 mg on day 1, a second dose on day 8, and a third dose on day 15, wherein the second dose and the third dose are the same and each is from about 10 mg to about 100 mg.

[0162] In certain embodiments, the article comprises: (a) a container comprising a bispecific antigen-binding molecule (e.g., an anti-DLL3 antigen-binding molecule such as AMG 757); and (b) a package insert with instructions for treating a subject with cancer expressing DLL3 (or treating SCLC) by administering a combination of an anti-DLL3 antigen-binding molecule (e.g., AMG 757) and an anti-PD-L1 antibody (e.g., atezolizumab or durvalumab), wherein the instructions specify that at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 or at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 with an intensity of at least 2+, and the anti-DLL3 antigen-binding molecule is administered to the subject once every three weeks at a dose of about 20 mg to about 200 mg (e.g., 20 mg, 60 mg, 100 mg, 160 mg, or 200 mg), such as on day 1 of a 21-day cycle. In certain embodiments, the instructions specify that at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 or at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 with an intensity of at least 2+, and the anti-DLL3 antigen-binding molecule is administered to the subject twice every three weeks at a dose of about 10 mg to about 100 mg, such as on day 1 and day 8 of a 21-day cycle. In certain embodiments, the instructions specify that at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 or at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 with an intensity of at least 2+, and the anti-DLL3 antigen-binding molecule is administered to the subject once every two weeks at a dose of about 10 mg to about 100 mg, such as on day 1 and day 15 of a 28-day cycle. In certain embodiments, the instructions further specify administering to the subject a combination of one or more chemotherapeutic agents (e.g., carboplatin or cisplatin and / or etoposide) with the anti-DLL3 and anti-PD-L1 agents.

[0163] In certain embodiments, the article comprises: (a) a container comprising a bispecific antigen-binding molecule (e.g., an anti-DLL3 antigen-binding molecule such as AMG 757); and (b) a package insert with instructions for treating a cancer expressing DLL3 (or treating SCLC) in a subject by administering a combination of an anti-DLL3 antigen-binding molecule (e.g., AMG 757) and an alkylating agent (e.g., lurbinectedin), wherein the instructions specify that at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 or at least 25% (e.g., 30%, 50%, 60%, 70%, or 75%) of the cancer cells express DLL3 at an intensity of at least 2+, and the anti-DLL3 antigen-binding molecule is administered to the subject at a dose of about 20 mg to about 200 mg (e.g., 20 mg, 60 mg, 100 mg, 160 mg, or 200 mg) once every three weeks, e.g., on day 1 of a 21-day cycle.

[0164] In some embodiments, the package insert further includes instructions for detecting and measuring human DLL3 protein expression in tumor cells from a subject. For example, the package insert desirably includes instructions for measuring DLL3 expression by immunohistochemistry (IHC) assays, such as those described herein. In certain embodiments, the IHC assay is approved by a regulatory agency, such as the US Food and Drug Administration (FDA) or an agency with CE-IVD registration authority. Additionally, the package insert may include instructions for scoring the intensity of DLL3 expression measured by IHC to select subjects suitable for treatment and an appropriate dose of an anti-DLL3 antigen-binding molecule to administer to the selected subjects. In various embodiments, the package insert may further instruct to hospitalize the subject and monitor for up to about 48 hours (e.g., about 24 hours, 12 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour) after administration of the anti-DLL3 antigen-binding molecule (e.g., AMG 757) in Cycle 1 and / or Cycle 2. In various embodiments, the package insert may further instruct to measure or test one or more cytokines of the subject, such as IL-6, IL-8, IL-10, TNF-α, and IFN-γ (e.g., the levels of one or more cytokines in the subject's blood or serum) after administration of the anti-DLL3 antigen-binding molecule (e.g., AMG 757) in Cycle 1, and to hospitalize the subject and monitor for up to about 48 hours (e.g., about 24 hours, 12 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour) if any level of any cytokine is above the normal reference level. For example, the package insert may instruct to measure or test the subject's IL-10 after administration of the anti-DLL3 antigen-binding molecule (e.g., AMG 757) in Cycle 1, and to hospitalize the subject and monitor for up to about 48 hours (e.g., about 24 hours, 12 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour) if the level of IL-10 is above the normal reference level. Cytokines can be measured or detected using methods known in the art.

[0165] In various embodiments, the container contains a bispecific antigen-binding molecule (e.g., an anti-DLL3 antigen-binding molecule (e.g., AMG 757)) in an amount of about 1 mg, 5 mg, 10 mg, or 25 mg. For example, the anti-DLL3 antigen-binding molecule (e.g., AMG 757) is provided as a sterile, single-use, preservative-free lyophilized drug product, and each container (e.g., vial) contains 1 mg, 5 mg, 10 mg, or 25 mg of the anti-DLL3 antigen-binding molecule. In various embodiments, the instructions specify reconstituting the lyophilized drug product with water for infusion. In various embodiments, the instructions specify that the subject is a human (e.g., a human with SCLC).

[0166] The following examples further illustrate the present invention, but should of course not be construed as limiting its scope in any way.

[0167] Example 1

[0168] This example describes a Phase 1 clinical study evaluating talquetamab in patients with recurrent / refractory small cell lung cancer.

[0169] Small cell lung cancer (SCLC) is an aggressive subtype of lung cancer, with neuroendocrine differentiation diagnosed in more than 150,000 people worldwide each year. From 2001 - 2016, the 2-year survival rate for patients with SCLC in the United States was 11% - 17%. Adding the immune checkpoint inhibitors atezolizumab or durvalumab to platinum and etoposide chemotherapy, followed by maintenance therapy with checkpoint inhibitors alone as first-line treatment for SCLC, reduced the risk of death by approximately 30% in a subset of patients with extensive-stage SCLC (ES-SCLC) and resulted in a durable but modest increase in survival. For most patients with relapsed SCLC, the available treatments are limited. Topotecan is the most widely used second-line agent globally, with limited efficacy and poor safety. In 2020, lurbinectedin became the first drug approved by the FDA for second-line treatment in over 20 years and received conditional approval based on an objective response rate (ORR) of 35%; however, a randomized study failed to demonstrate an OS benefit. No agent has been specifically approved for third-line treatment of recurrent SCLC.

[0170] The Notch signaling pathway is a regulator of neuroendocrine differentiation in SCLC. As discussed herein, the inhibitory Notch ligand delta-like ligand 3 (DLL3) is aberrantly expressed on the surface of up to 85% of SCLC cells and is minimally expressed in normal tissues, making it an attractive therapeutic target. In vitro SCLC models have demonstrated the role of DLL3 in promoting tumor growth, migration, and invasion. The antibody-drug conjugate (ADC) tesirine-lovastatin targeting DLL3 has shown clinical anti-tumor activity in patients with SCLC.

[0171] As discussed herein, talquetamab is a half-life extended bispecific T cell engager (HLE ) molecule that binds DLL3 on cancer cells and CD3 on T cells, resulting in T cell-mediated tumor lysis. Talquetamab promotes tumor regression in preclinical models of SCLC. Talquetamab is the first DLL3-targeted immunotherapy to be clinically evaluated in SCLC.

[0172] The Phase 1 trial evaluated the safety, pharmacokinetics, and preliminary efficacy of talquetamab in patients with SCLC.

[0173] Patients and Methods

[0174] Study design and participants

[0175] DeLLphi-300 is a phase 1, multinational, open-label, dose-escalation study evaluating talazoparib monotherapy. The results reported herein pertain to the monotherapy regimen and include dose-escalation and expansion cohorts. Eligible patients were 18 years of age or older, had histologically or cytologically confirmed SCLC that had progressed or recurred after at least 1 prior platinum-based regimen, and, if standard of care, were receiving a PD-L1 inhibitor in addition to chemotherapy. Included patients needed to have an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or lower and at least 2 measurable lesions as defined by the modified Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Key exclusion criteria were untreated active brain metastases and severe or recurrent immune-mediated adverse events or infusion-related reactions during prior immunotherapy.

[0176] The protocol and amendments were approved by the institutional review board or ethics committee of each participating institution. The trial was conducted in accordance with the guidelines of the International Council for Harmonisation Good Clinical Practice and the principles of the Declaration of Helsinki. All patients provided written informed consent.

[0177] Procedures

[0178] The planned talazoparib dose levels were 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 mg, administered intravenously (IV) by infusion every 2 weeks (Q2W). Because adverse events (AEs) were expected to be low, the first 4 dose levels were planned as single-patient cohorts. Talazoparib administration continued until disease progression, unacceptable side effects occurred, or informed consent was withdrawn.

[0179] Outcomes

[0180] The primary endpoints were safety, including dose-limiting toxicity (DLT; defined as talquetamab-related toxicity within 28 days after the first dose and meeting the protocol criteria), adverse events (AEs) during treatment (treatment-emergent AEs [TEAEs]), and TEAEs potentially related to talquetamab according to the investigator review (treatment-related adverse events [TRAEs]). The secondary endpoints included pharmacokinetics, antitumor activity of objective response according to the investigator assessment using the modified RECIST 1.1 (which applies multiple aspects of immune-related response criteria, including confirmation of disease progression to RECIST 1.1), duration of response (DOR), time to response (TTR), progression-free survival (PFS), and overall survival (OS).

[0181] Statistical Analysis

[0182] The analysis included patients enrolled in the dose-escalation and expansion cohorts. The data cutoff date was July 19, 2022. A two-parameter Bayesian logistic regression model (BLRM) guided dose exploration. Safety data were continuously reviewed. In the dose-level review meetings (DLRMs), the sponsor consulted with the site investigators to review the dose levels recommended by the BLRM and all available cumulative data by cohort before making dose-escalation decisions. AEs and DLTs observed in all subjects were continuously evaluated and fully incorporated into all DLRMs. Based on the overall benefit-risk profile of 100 mg, it was decided to further evaluate it as an expansion dose. Descriptive statistics were provided for selected demographic, safety, pharmacokinetic (PK), pharmacodynamic, and biomarker data. Exploratory analyses were conducted to evaluate the relationship between DLL3 baseline expression and clinical benefit and are further detailed below. Kaplan-Meier estimates were used to determine the median and percentiles of the time to event endpoints, with confidence intervals (CIs) calculated using the Brookmeyer and Crowley methods.

[0183] Additional Notes on Endpoints

[0184] The maximum tolerated dose (MTD) was the highest dose determined by the investigator and the study team to be safe, taking into account the Bayesian logistic regression model (BLRM).

[0185] Adverse events were graded using the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. CRS events were graded using the Lee criteria.

[0186] In addition, cytokine release syndrome (CRS), neutropenia, and neurological events were considered events of interest in this study and were monitored using the Amgen MedDRA Query (AMQN) search method. All events were coded using version 24.1 of MedDRA. The AMQN search for cytokine release syndrome included cytokine abnormalities, cytokine release syndrome, cytokine storm, and cytokine assays. CRS events were graded using the criteria of Lee et al. (2014). Neutropenia was based on the AMQN search and was graded using version 4.0 of the Common Terminology Criteria for Adverse Events (CTCAE). Neurological events were based on the AMQN search for "central neuropsychiatric events due to direct neurotoxicity" and were graded using version 4.0 of CTCAE.

[0187] The efficacy data disclosed herein are based on local investigator assessments. A patient was defined as efficacy evaluable if the data cutoff was at least 9 weeks after the first dose to allow time for assessment.

[0188] An exploratory analysis of T cells and peripheral cytokines was performed on sequentially collected blood samples.

[0189] For immunogenicity assessment, blood samples were collected from patients receiving talquetamab at study day 1 (before dosing) and at multiple time points during the study to detect anti-talquetamab binding antibodies using a validated electrochemiluminescent bridging immunoassay.

[0190] Assessment of DLL3 expression

[0191] Subjects enrolled in the study provided fresh biopsies or archived formalin-fixed and paraffin-embedded (FFPE) tissue biopsies for retrospective DLL3 analysis by immunohistochemistry. Of the 107 subjects in the safety / efficacy population, 90 (84%) had evaluable tumor DLL3 expression. DLL3 expression in tumor cells was detected by using an anti-DLL3 antibody (clone SP347, Ventana, Tucson, AZ). Briefly, FFPE SCLC tissue blocks were sectioned at 4 μm or 5 μm thickness on charged slides and stained on the BenchMark ULTRA using the UOptiView DAB IHC Detection Kit (Ventana) for visualization. Rabbit monoclonal antibody was used as a negative marker control. Acceptable background and signal / noise staining of the negative marker control were evaluated for each specimen. To evaluate DLL3 staining, combined H scores of the membrane / cytoplasm were collected at any magnification. Negative or weak staining was confirmed at a magnification of at least 20×. Interpretation of DLL3 expression was performed by a qualified pathologist. DLL3 positivity was defined as ≥1% stained tumor cells. Hematoxylin and eosin (H&E) staining was performed on a Sakura TissueTek Prisma instrument. H&E-stained slides were used for assessment of tumor content and tissue quality assessment. Biomarker assessment required at least 100 viable tumor cells.

[0192] Tumor cell staining was scored for a specific percentage of tumor cells. The total intensity of tumor cell staining was also evaluated on the DLL3 (SP347)-stained slides, ranging from 0 to 3 in increments of 1, as described by Huang et al., Arch Pathol Lab Med [Archives of Pathology & Laboratory Medicine], 143(11):1373-1379 (2019). Strong cytoplasmic and / or membrane staining of tumor cells was scored as "3" or "3+", moderate staining as "2" or "2+", and weak staining as "1" or "1+". No staining was given a score of "0".

[0193] ROC analysis

[0194] Receiver operating characteristic (ROC) analysis included 77 subjects enrolled in the 1-100 mg dose cohorts who had pre-treatment DLL3 expression readings available. Total DLL3 expression (percent positive tumor cells for DLL3, 0%-100%) at 10x magnification using the SP347 assay described above and exploratory scores were retrospectively used to examine the impact of patient selection on enrichment of clinical benefit (confirmed OR). True positive rate (TPR, y-axis) and false positive rate (FPR, x-axis) were calculated across the full range of DLL3 expression. For each threshold considered, true positives were defined as high DLL3 responders, false positives were defined as high DLL3 non-responders, true negatives were defined as low DLL3 non-responders, and false negatives were defined as low DLL3 responders.

[0195] Immune cell and cytokine analysis

[0196] Whole blood samples drawn into EDTA tubes were collected according to the assessment plan specified in the study protocol. Whole blood samples were stained using a panel of fluorescently labeled antibodies, CD4 BV510 (clone SK3, BD Biosciences), CD8 BV605 (clone SK1, BD Biosciences), CD3 Alexa Fluor 700 (clone SK7, BioLegend), and CD279 (PD-1) BB515 (clone EH12.1, BD Biosciences), using a combination validated by flow cytometry. Data were collected centrally by Q2 Solutions Laboratories Europe on a BD FACSCanto flow cytometer. To assess cytokine production, serum samples were collected and IFNγ levels were evaluated using a Meso Scale Discovery (MSD) V-plex proinflammatory panel 1. Assays were performed according to the manufacturer's instructions. Briefly, samples were diluted 1:2 with diluent 2 (MSD). Diluted samples and standards were added in duplicate to 96-well plates that had capture antibodies pre-coated independently at 10 defined spots, and incubated for 2 hours at ambient temperature. Plates were washed three times with wash buffer, and detection antibody mixture was added to each well and the plates were incubated for 2 hours at ambient temperature. Plates were washed three times with wash buffer, and 2x read buffer T (MSD) was added to each well, and read on an MSD plate reader. Concentrations were interpolated from a standard curve in the established range of 2.61 - 542,720 pg / mL.

[0197] Characterization of cytokine release syndrome (CRS)

[0198] Perform an analysis to explore the correlation between cytokine levels within 24 hours after the initial dose of talquetamab and the occurrence of CRS in Cycle 1. Included in this analysis were the cohort that received 1 mg as the initial dose of talquetamab in Cycle 1 and subsequent doses of 1 mg to 100 mg.

[0199] Serum was drawn at time points up to 24 hours for cytokine analysis. The incidence, onset time, severity, management, and recurrence of CRS were evaluated. In Cycle 1, serum peak levels and rate of increase within 24 hours after the initial dose of talquetamab were evaluated for a panel of soluble factors in patients with CRS versus those without CRS. Patients from the 1 mg initial dosing cohort were included.

[0200] The Kruskal–Wallis (KW) rank test with false discovery rate correction was used to identify whether analyte values from the CRS and no-CRS categories were drawn from different distributions. The Jonckheere–Terpstra (JT) trend test with false discovery rate correction was used to determine the trend of increasing analyte values from the no-CRS to the CRS group. Univariate logistic regression was used to determine whether the growth rate and peak of each analyte could predict the occurrence of CRS in Cycle 1 after dosing.

[0201] Results

[0202] Patients

[0203] As of July 19, 2022, 107 patients received talquetamab in the dose-escalation (0.003 mg to 100 mg; n = 73) and expansion (100 mg; n = 34) cohorts (Figure 1). Due to the observation of cytokine release syndrome (CRS) in previous cohorts, starting from the 3 mg cohort, a stepwise dosing (using 1 mg as the lead-in dose, followed by the target dose on Day 8, Day 15, and Q2W thereafter) was used. Cytokine release syndrome (CRS) is an expected risk of talquetamab based on its mechanism of action (MOA).

[0204] Baseline characteristics are summarized in Table 1. The median age was 63 years (range 32 to 80 years). The ECOG performance status was 0 to 1 in 99% of patients. More than 70% of patients had received ≥2 lines of prior therapy, 25% of patients were platinum refractory, and 50% of patients had received prior PD-1 / PD-L1 inhibitors.

[0205] The median follow-up period was 8.7 months (range, 0.2 to 31.8 months). Ninety-two patients (86%) discontinued treatment, most commonly due to disease progression (n = 77 [72%]). At data cutoff, 47 patients (43.9%) had their study terminated due to death. The median number of treatment cycles initiated was 3 (interquartile range [IQR]: 1, 8), and the median dose of taletrectinib received was 6 (IQR: 3, 16).

[0206] Table 1. Patient demographics and baseline characteristics

[0207]

[0208]

[0209] *The disease stage of 1 patient was unknown at baseline.

[0210] ECOG, Eastern Cooperative Oncology Group; IQR, interquartile range; PD-1, programmed cell death protein 1; PD-L1, programmed death ligand 1.

[0211] As described above, retrospective DLL3 immunohistochemical analysis was performed on fresh or archived biopsy specimens. DLL3 was expressed in 85 of 90 (94%) evaluable patients (≥1%); the median H score was 186 (range, 0 to 300), and the median tumor cell positivity rate was 95% (range, 0 to 100%).

[0212] Safety and tolerability

[0213] DLT occurred in 6 patients, including pulmonary inflammation (n = 1 [last prior dose, 0.3 mg]), elevated alanine aminotransferase (n = 1 [1 mg]), CRS (n = 1 [1 mg]), encephalopathy (n = 1 [10 mg]), chills, fever, and neutropenia (n = 1 each [100 mg]). The maximum tolerated dose (MTD) was not reached; the highest dose (100 mg) was evaluated in the expansion cohort. Four patients (3.7%) discontinued talquetamab due to AEs such as encephalopathy (n = 1), immune effector cell-associated neurotoxicity (ICANS) (n = 1), and pulmonary inflammation (n = 2), all of which were treatment-related. A single G5 pulmonary inflammation event was recorded in a 70-year-old elderly male with a history of prior carboplatin / etoposide chemotherapy, chronic obstructive pulmonary disease, and radiotherapy for lung nodules and pleural nodules. The event onset was on day 18 of cycle 1, 3 days after the second talquetamab treatment (both doses 0.3 mg), and was confused with clinically significant disease progression when pulmonary inflammation required urgent palliative radiotherapy for a soft tissue mass causing spinal cord compression in the lung and thoracic spine. The investigators attributed the cause of death to disease progression and pulmonary inflammation. One additional G3 and three additional G2 pulmonary inflammation TEAEs were observed (overall incidence of pulmonary inflammation 5 / 107 [4.7%]). Among patients with G2 pulmonary inflammation, 1 patient discontinued treatment due to neurotoxicity (non-pulmonary inflammation), 1 patient's pulmonary inflammation resolved before discontinuation due to PD, and 1 patient resumed treatment without dose modification.

[0214] Any-cause / grade TEAEs occurred in 107 patients (100%). The most common were CRS (56 patients [52.3%]), fever (43 [40.2%]), constipation (33 [30.8%]), and fatigue (32 [29.9%]). ≥Grade 3 AEs occurred in 61 patients (57.0%), the most common being neutropenia (8.4%), lymphocytopenia (6.5%), and hypertension (5.6%). Serious adverse events (SAEs) occurred in 55 patients (51.4%). TEAEs led to dose reduction in 9 patients (8.4%), of which 4 (3.7%) had CRS-related reduction. Dose interruptions occurred in 20 patients (18.7%), most commonly due to neutropenia and neutropenia count decrease. Any-grade and ≥grade 3 TRAEs occurred in 97 (90.7%) and 33 (30.8%) patients, respectively.

[0215] Based on the use of talquetamab, other BiTE TMPreclinical, clinical, and mechanistic data on the molecule and other T cell-related therapies monitored CRS, neutropenia, and neurological events as events of interest. An Amgen MedDRA Query Narrowing (AMQN) search was performed to supplement standard system organ class single preferred term safety reports (as defined above and summarized in Table 2). Measures to mitigate the likelihood of CRS included prophylactic corticosteroids (cycle 1 only) and intravenous (IV) fluid hydration in some patients. Fifteen patients (14.0%) reported ≥ grade 2 on-treatment CRS, and 1 patient (0.9%) reported grade 3 CRS; no grade 4 or 5 CRS was reported. For CRS of any grade (n = 56), the median time to first onset was 2 days (range 1 day to 30 days) after first dose, based on the recorded date; more precise time-based reporting was implemented to better characterize CRS, where in a subset of patients with available hourly data (n = 47), the median time to onset was 17.5 hours. CRS was transient (median duration 3 days [IQR: 2 days to 4 days]) and resolved in all cases. Eight patients (7.5%) received tocilizumab for CRS. CRS was mainly confined to cycle 1. A total of 5 patients (4.7%) had CRS in cycle 2; 4 of these patients also had CRS in cycle 1, while 1 patient experienced CRS for the first time in cycle 2 or later. Seventy-five patients (70.1%) had any grade on-treatment neurological AEs, and most neurological AEs were grade 1; the most common were dysgeusia (29.0%), headache (19.6%), and dizziness (10.3%). Twelve patients (11.2%) had ≥ grade 3 on-treatment neurological events, including confusional state (4.7%), delirium (1.9%), and encephalopathy (1.9%). One subject had a grade 4 neurological event (confusion), and no patient had a grade 5 neurological event. All ≥ grade 3 neurological AEs resolved, with 1 subject discontinuing talquetamab due to G3 encephalopathy and the other 2 subjects continuing treatment at a reduced dose. G2 ICANS was another neurological reason for discontinuation in 1 subject. The first onset of neurological events of any grade occurred mostly within the first 30 days of treatment (median 9 days [IQR, 2 days to 29 days]), with a median duration of 5 days (IQR, 2 days to 15 days). Eleven patients (10.3%) had ≥ grade 3 neutropenia. The first onset of neutropenia of any grade occurred at a median time of 30 days (IQR, 21 days to 31 days) after first talquetamab administration, and the median duration was 7 days (IQR, 4 to 13); overall, 10 patients (9.3%) received G-CSF. Febrile neutropenia occurred in 1 patient, but it was considered unrelated to treatment.

[0216] Table 2. Adverse events occurring during treatment (AMQ of preferred terms and selected terms)

[0217]

[0218]

[0219] * Incidence based on a single preferred term. Coded using MedDRA version: 25.0. Adverse events were graded using CTCAE version 4.0, and CRS events were graded using the criteria of Lee et al. (2014).

[0220] Narrow - scope search based on AMQ, including cytokine abnormalities, cytokine release syndrome, cytokine storm, cytokine detection. Neutropenia was based on a narrow - scope search of AMQ. Neurological events were based on a narrow - scope search of the AMQ of "central neuropsychiatric events caused by direct neurotoxicity". Coded using MedDRA version: 25.0. CRS events were graded using the criteria of Lee et al. (2014). Neutropenia and neurological events were graded using CTCAE version 4.0.

[0221] AMQ, Amgen MedDRA Query.

[0222] Efficacy

[0223] The confirmed ORR was 23.4% (95% confidence interval [CI]: 15.7, 32.5), including 2 complete responses and 23 partial responses (Table 3).

[0224] Table 3. Tumor responses induced by talquetamab according to investigator assessment

[0225]

[0226] * The interim efficacy analysis set was a subset of the safety analysis set. The interim efficacy analysis set included patients with a data cut - off date of at least 9 weeks after the first dose administration.

[0227] This included 32 patients who had PD on post - baseline scans but without further confirmatory scans (unconfirmed PD according to revised RECIST 1.1).

[0228] Reasons for not performing imaging assessments included withdrawal of informed consent (n = 5), death (n = 2), clinical PD (n = 1), and initiation of a new anticancer therapy (n = 1).

[0229] Figure 1A The sum of diameters relative to baseline of patients (n = 94) with evaluable post-baseline assessment results is shown as the best percentage change relative to baseline. The disease control rate was 51.4% (95% CI: 41.5, 61.2). Responses were visible starting from the 0.3 mg dose, and higher response rates were generally observed at 3 mg and higher doses. Tumor shrinkage of at least 30% in target lesions was observed at post-baseline assessment in 39 patients (36.4%). Among confirmed responders, the median TTR was 1.8 months (range 1.2 to 7.4), and the median DOR was 12.3 months (95% CI: 6.6, 14.9)( Figure 1B ). The longest duration of response was 14.9 months, and 11 patients (44% of responders) had ongoing responses at data cutoff. Separately, the median PFS was 3.7 months (95% CI: 2.1, 5.4), and the median OS was 13.2 months (95% CI: 10.5, NE) (Figure 2). A total of 28 patients (26.2%) continued with subsequent anti-cancer therapy after talquetamab.

[0230] Analyses were performed to investigate the relationship between DLL3 expression and clinical benefit of talquetamab. Figure 3 Showed enhanced sensitivity and specificity for responders (using confirmed OR) when total DLL3 expression for patient selection was considered retrospectively. Figure 4 Showed that DLL3 expression levels were associated with improved ORR and DCR. Figure 5A and 5B Showed the correlation between DLL3 expression and tumor reduction. Clinical benefit was observed within the threshold range.

[0231] Clinical Pharmacokinetics

[0232] As of April 15, 2022, preliminary pharmacokinetic data for 101 patients were obtained from the dose escalation and expansion cohorts. Briefly, talquetamab showed an approximately dose-proportional increase in serum exposure. Serum talquetamab exposure reached an approximately steady state within 4 weeks after the start of the every-other-week target regimen, with minimal accumulation. Within the target dose range evaluated, the mean (±SD) terminal elimination half-life estimated at steady state was approximately 5.7 (±2.2) days, which is consistent with the expected extended half-life of the HLE platform relative to the non-HLE BiTE TM molecule.

[0233] Immunogenicity

[0234] In patients with available samples, 10 out of 97 patients (10.3%) developed anti-talarotamab antibodies after talarotamab administration. Two out of 99 patients (2.0%) had pre-existing antibodies at baseline. In these patients, anti-drug antibodies (ADA) had no significant effect on talarotamab exposure or on the safety profile.

[0235] Pharmacodynamics

[0236] The pharmacodynamic response after the first dose of talarotamab infusion was characterized by an initial T-cell redistribution, T-cell activation, and a transient increase in IFN-γ. For the escalated dose cohort, the pharmacodynamic response was maximal after the initial administration of the 1 mg escalated dose and did not exceed this result when administered at the target dose.

[0237] Clinical CRS summary

[0238] Most CRS were grade 1 (39%), occurred in cycle 1, and were reversible in all patients (see Table 4). CRS was clinically manageable.

[0239] Table 4

[0240]

[0241] To mitigate the risk of CRS, ≥1 of the following prophylactic measures could be used during cycle 1: additional corticosteroid prophylaxis with

[0242] oral dexamethasone, administration of tocilizumab, etanercept, or

[0243] acetaminophen.

[0244] *CRS includes cytokine abnormalities, cytokine release syndrome, cytokine storm, cytokine

[0245] testing.

[0246] Percentages are based on the total number of patients with any grade of CRS.

[0247] Onset time of patients (n = 47) with data on both date and time of onset.

[0248] Data cut-off: June 15, 2022.

[0249] CRS, cytokine release syndrome.

[0250] Cytokine and CRS analysis

[0251] In biomarker-evaluable patients, the ratios of peak levels within 24 hours to baseline levels of IL-6, IL-8, IL-10, and TNF-α tended to be higher in patients with CRS in cycle 1 compared with patients without CRS ( Figures 6A through 6D ). IL-10 showed an elevation significantly higher than the reference normal range and was higher in patients with CRS ( Figure 7 ). Since the effector cytokine IFN-γ had a strong induction in preclinical trials, it was investigated (see, for example, Giffin MJ et al. Clin Cancer Res. [Clinical Cancer Research] 2021;27:1526 - 1537). As expected from the mechanism of action of talquetamab, IFN-γ induction was above the physiological range; the induction was similar between patients with cycle 1 CRS and patients without cycle 1 CRS ( Figure 8A and Figure 8B ).

[0252] Discussion

[0253] With the extended dose of 100 mg, talquetamab demonstrated a manageable safety profile across a wide dose range and was associated with encouraging response rates in a heavily pretreated population of SCLC patients. The confirmed responses were durable and appeared promising for OS. Among all doses (N = 107), only 4 patients (3.7%) discontinued talquetamab, and 9 patients had dose reductions due to AEs. The MTD was not reached; the highest dose (100 mg) was further evaluated in the dose expansion cohort.

[0254] Based on the MOA of talquetamab, CRS was expected to occur. Although CRS was the most common TEAE observed in this study (56% of patients), it was generally low-grade, transient, and typically occurred in the first cycle. CRS was usually reversible and managed with steroids, intravenous (IV) fluids, and antipyretics, and tocilizumab was used to treat CRS in 8 of 107 patients (7.5%) receiving talquetamab. Neutropenia was a risk associated with talquetamab observed in this study and was unexpected based on preclinical data; the mechanism was unknown. Therefore, the study protocol was updated for specific monitoring and management. Further evaluation of neutropenia will be relevant to trials combining talquetamab with other myelosuppressive therapies. Neurological assessments were performed as part of frequent clinical evaluations because of the known association with immunoeffector cell therapies to assess CRS and / or neurological AEs in study patients. Although 12 patients (11.2%) had ≥ grade 3 neurological AEs, most neurological AEs were mild and self-limiting and did not require treatment discontinuation or dose reduction. Talquetamab was discontinued in 2 patients because of neurological AEs (encephalopathy, ICANS). Careful evaluation of neurological AEs is ongoing to better characterize these events and identify risk factors or interventions that can specifically improve management.

[0255] There are few approved therapies for post - first - line SCLC. A phase 2 study of lurbinectedin in second - line SCLC found an ORR of 35% and a median DOR of 5.3 months. In a randomized study of topotecan versus combination chemotherapy in recurrent SCLC, the ORR of topotecan was 24% and the median DOR was 3.3 months. The previous conditional approvals by the US FDA for nivolumab and pembrolizumab for third - line or later SCLC were based on response rates of 12% and 19% respectively, with durable responses of ≥12 months observed in >60% of the responding patients. These approvals were subsequently withdrawn due to lack of demonstrated survival benefit. The ORR of talazoparib was 23% and the median DOR was 12.3 months, which was favorable compared to other therapies, especially considering that over 70% of the patients had received at least 2 prior lines of therapy. Half of the patients (50%) in this study had received prior PD - 1 / PD - L1 therapy, which represents current practice in first - line SCLC. Although a median PFS of 3.7 months was observed with talazoparib, the median OS (13.2 months) was relatively high and superior to the previously reported median OS with lurbinectedin (9.3 months) or OS with topotecan (approx. 6 months), but the value of the comparison was limited by differences in study design and patient population. The promising OS benefit may reflect the long - term durability of responses observed so far in those patients who responded to talazoparib, but further follow - up in larger - scale randomized studies is needed. An alternative explanation for the relatively long OS and short PFS could be that the OS benefit stems from post - talazoparib treatment, but this is unlikely to be the main factor as only 26.2% of the patients in this heavily pretreated cohort received such treatment. Efforts are underway to identify clinical, demographic, and biological factors (e.g., prior therapy, DLL3 expression) that can predict response and / or toxicity. Increased DLL3 expression seems to tend towards a higher degree of clinical benefit.

[0256] The results of this example demonstrated the promising activity of talazoparib in patients with a high unmet medical need and have led to several ongoing studies of talazoparib as a single therapy for SCLC and other neuroendocrine cancers.

[0257] Example 2

[0258] This example describes a phase 2 study that evaluated the efficacy, safety, tolerability, and pharmacokinetics of AMG 757 / talazoparib in subjects with recurrent / refractory small - cell lung cancer after two or more prior lines of therapy.

[0259] Study design

[0260] The DELLphi-301 study (20200491) is a Phase 2, open-label, global study of AMG 757 / talquetamab in subjects with recurrent SCLC who have progressed or relapsed after 1 platinum-based regimen (with or without checkpoint inhibitors) and at least 1 additional line of therapy (re-treatment with a platinum-based regimen is considered second-line therapy).

[0261] The study was conducted in 3 parts. Part 1 was a dose comparison in which approximately 180 patients were randomized in a 1:1 ratio to receive 10 mg or 100 mg talquetamab (as a 60-minute intravenous infusion). A pre-specified interim analysis was used to select the doses for Parts 2 and 3. Part 2 was dose expansion in which a total of approximately 100 patients were enrolled at the selected dose (combined Parts 1 and 2). Part 3 was a sub-study conducted after enrollment in Part 2 was completed to evaluate the safety of talquetamab with reduced inpatient monitoring (from 48 hours to 24 hours) during Cycle 1.

[0262] The primary efficacy endpoint was objective response (complete or partial response) confirmed by blinded independent central review (BICR) according to RECIST 1.1. Secondary endpoints included DoR, disease control rate, duration of disease control, progression-free survival (PFS), overall survival (OS), incidence of treatment-emergent adverse events (TEAE), talquetamab serum concentration, and incidence of anti-talquetamab antibody formation. Exploratory endpoints included cytokine levels, target expression in tumor tissue, immune-related biomarkers, and change from baseline in health-related quality of life. DLL3 immunohistochemistry was performed retrospectively on formalin-fixed paraffin-embedded tissue using the SP347 antibody at Roche Tissue Diagnostics (Tucson, USA). The study objectives and endpoints are shown in Table 5. The study protocol is shown in Figure 9.

[0263] Table 5

[0264]

[0265]

[0266]

[0267] Treatment

[0268] For all 3 parts, talazoparib was initiated at a starting dose of 1 mg on Cycle 1 Day 1 (C1D1), then at 10 mg or 100 mg on C1D8 and C1D15, and then every 2 weeks thereafter (28-day cycle). Patients were treated until disease progression. Imaging assessments were planned every 6 weeks for the first year and then every 12 weeks thereafter. If the criteria specified in the protocol were met, the investigator could, at their discretion, allow treatment beyond radiographic disease progression for patients with continued clinical benefit. Safety follow-up occurred 6 weeks after the last dose of talazoparib, and long-term follow-up occurred every 3 months for 1 year after the last dose of talazoparib or 5 years after the first patient was enrolled, whichever occurred first.

[0269] Statistical Considerations

[0270] Based on published literature, a 15% objective response rate (ORR) was pre-specified in the protocol as a historical control benchmark (Ready et al., J Thorac Oncol [Journal of Thoracic Oncology], 14:237-44 (2019); and Chung et al., J Thorac Oncol [Journal of Thoracic Oncology], 15:618-27 (2020)). Assuming an ORR of 30%, a sample size of 100 patients at the target dose from Parts 1 and 2 was selected to provide approximately a 92% probability that the lower limit of the 97.5% confidence interval (CI) for the ORR would exceed 15%. The 97.5% CI was selected as the primary endpoint in a pre-specified interim analysis to adjust for multiplicity in dose selection. The safety analysis set consisted of all patients (Parts 1-3) who received at least one dose of talazoparib. The efficacy analysis set included all patients in Parts 1 and 2 who received at least one dose of talazoparib and had at least one measurable lesion at baseline according to BICR. Patients in Part 3 were not included in the efficacy analysis set because the data were not yet mature.

[0271] Confidence intervals for proportions were calculated using the Clopper-Pearson method. Event endpoint times were estimated using the Kaplan-Meier method.

[0272] Summary of Subject Eligibility Criteria

[0273] Male and female subjects (≥ 18 years of age [or the legal age of majority in the country]) with histologically or cytologically confirmed recurrent / refractory SCLC who have progressed or relapsed after 1 platinum-based regimen and at least 1 other prior line of treatment (Note: [1] Retreatment with a platinum-based regimen is considered second-line treatment; [2] Use of checkpoint inhibitors / anti-programmed death ligand 1 [PD-L1] as maintenance treatment after a platinum-based regimen is considered 1 line of treatment; [3] In countries where the standard of care first-line systemic treatment includes a combination of platinum-containing chemotherapy and a PD-L1 inhibitor, subjects are required to have failed the PD-L1 inhibitor as part of their first-line systemic treatment or to be ineligible for PD-L1 inhibitor treatment).

[0274] Once consent is obtained for the study, subjects provide their medical history and undergo screening safety tests to confirm that all eligibility requirements for the study are met. Subjects must have measurable lesions as defined by RECIST 1.1 within 21 days prior to the first dose of talazoparib and must have adequate organ function.

[0275] Study product

[0276] The dosing and administration of talazoparib are summarized in Table 6 as follows.

[0277] Table 6

[0278]

[0279]

[0280] Before medication

[0281] Dexamethasone 8 mg IV (or other corticosteroid of equivalent dose) is administered only within 1 hour before the infusion of talazoparib on Day 1 and Day 8 of Cycle 1.

[0282] Intravenous (IV) fluid replacement (1 L of normal saline) is administered immediately after all doses of talazoparib in Cycle 1 for prophylaxis.

[0283] Biomarker assessment during the study

[0284] Blood samples: Peripheral blood, serum, and plasma samples are collected for the evaluation of exploratory biomarkers, which may include but are not limited to cytokines, DNA, RNA analysis, and circulating tumor cell counts. These samples can be used to help further understand the subject's disease and its response to treatment.

[0285] Flow cytometry whole blood samples: Whole blood samples are collected for immunophenotyping flow cytometry to identify changes in peripheral blood cell subsets and activation status.

[0286] Tumor tissue before archiving and / or fresh administration: Evaluate the baseline DLL3 expression and other exploratory biomarkers that may predict response in tumor tissue before fresh administration or archived tumor tissue by IHC.

[0287] Subjects must provide an archived tumor tissue sample collected after the last cancer treatment (formalin-fixed, paraffin-embedded [FFPE] sample collected within 2 years) or be willing to undergo a pre-treatment tumor biopsy. With the consent of the investigator and medical monitor, subjects who do not have archived tumor tissue available after the last cancer treatment or are unable to undergo a pre-treatment tumor biopsy due to extenuating circumstances (e.g., cannot be performed safely or determined by the investigator to be infeasible) can be enrolled without a tumor biopsy.

[0288] Submit the corresponding pathology report for any biopsy provided for the study. Tumor biopsies are optional at the time of the first radiological assessment and after the end of treatment.

[0289] Results

[0290] Patients

[0291] Between December 2021 and May 2023, a total of 222 patients were enrolled at 56 sites in 17 countries. In Part 1, 176 patients were randomly assigned to receive talquetamab at 10 mg (n = 88) or 100 mg (n = 88). Based on the results of a pre-specified interim analysis, a 10 mg dose was selected for Part 2 (dose expansion; n = 12) and Part 3 (shortened inpatient monitoring period; n = 34). At the data cut-off (June 27, 2023), the median treatment duration was 5.1 months (range 0.0 - 15.2) in the 10 mg group and 3.7 months (range 0.0 - 15.2) in the 100 mg group. The baseline demographics and clinical characteristics were comparable between the two dose groups, although the proportion of patients with brain metastases was higher at baseline in the 100 mg group.

[0292] Efficacy

[0293] A total of 185 patients in Part 1 and Part 2 received talazoparib, had at least 1 measurable lesion at baseline, and were included in the efficacy analysis; the median follow-up time was 10.6 months (95% CI, 9.7 - 11.3). The ORR by BICR was 40.4% (97.5% CI, 29.4 - 52.2) in the 10 mg group and 31.4% (97.5% CI, 20.6, 43.8) in the 100 mg group. The ORR was consistent across predefined subgroups, including the presence of brain or liver metastases, sensitivity to platinum-based chemotherapy in first-line treatment, and prior PD-(L)1 inhibitor treatment. The disease control rate was 70.7% (95% CI, 60.7, 79.4) in the 10 mg group and 62.8% (95% CI, 51.7, 73.0) in the 100 mg group. Most responses (61 / 67, 91.0%) occurred at the first planned assessment 6 ± 1 weeks after the start of talazoparib. The median DoR was not reached in either group (10 mg: 95% CI, 5.9 months - not evaluable; 100 mg: 95% CI, 6.6 months - not evaluable). Among all 67 responders in the 10 mg and 100 mg groups, 39 (58.2%) had a response duration of at least 6 months and 37 (55.2%) had a continuing response at the data cutoff. The investigator's responses were consistent with the central review.

[0294] The median PFS (mPFS) was 4.9 months (95% CI, 3.0 - 6.7) in the 10 mg group and 3.9 months (95% CI, 2.6 - 4.4) in the 100 mg group. At 6 months and 9 months, the Kaplan-Meier estimates of PFS were 40.8% (95% CI, 30.6 - 50.7) and 28.5% (95% CI, 19.2 - 38.6) in the 10 mg group, and 33.2% (95% CI, 23.0 - 43.8) and 25.5% (95% CI, 16.1 - 35.9) in the 100 mg group, respectively. The median overall survival (mOS) was 14.3 months (95% CI, 10.8 - not estimable) in the 10 mg group and not reached in the 100 mg group (95% CI, 12.4 - not estimable). At 6 months and 9 months, the Kaplan-Meier estimates of overall survival were 73.4% (95% CI, 63.2 - 81.2) and 68.0% (57.1 - 76.6) in the 10 mg group, and 71.4% (95% CI, 60.1 - 80.0) and 65.5% (95% CI, 53.8 - 75.0) in the 100 mg group, respectively. At the last follow-up, 57.6% (57 / 99) in the 10 mg group and 51.7% (45 / 87) in the 100 mg group were still alive, and the OS data were not yet mature.

[0295] Immunohistochemical analysis showed that DLL3 expression was detected in 96.2% (150 / 156) of patient samples. The analysis of ORR and DoR according to the DLL3 expression level evaluated by BICR is shown in Table 7.

[0296] Table 7.

[0297]

[0298]

[0299] Safety

[0300] The most common treatment-emergent adverse events (TEAEs) were cytokine release syndrome (CRS) (55.0%), decreased appetite (34.5%), fever (34.1%), constipation (26.4%), and anemia (25.9%). Grade 3 or higher TEAEs occurred in 59.4% of patients in the 10 mg group and 64.4% of patients in the 100 mg group. Grade 3 or higher treatment-related adverse events (TRAEs) occurred in 25.6% of patients in the 10 mg group and 33.3% of patients in the 100 mg group. TRAEs led to dose interruption and / or reduction in 12.8% (10 mg) and 28.7% (100 mg) of patients, and discontinuation in 3.0% (10 mg) and 3.4% (100 mg) of patients. There was one case (0.8%) of grade 5 TRAE due to respiratory failure in the 10 mg group.

[0301] CRS was reported in 51.1% (68 / 133) of patients in the 10 mg group and 60.9% (53 / 87) of patients in the 100 mg group, mainly grade 1 (10 mg: 40 / 133 [30.1%], 100 mg: 28 / 87 [32.2%]) or grade 2 (10 mg: 27 / 133 [20.3%], 100 mg: 20 / 87 [23.0%]), and was mainly limited to the first two doses (day 1 of cycle 1 [C1D1] and day 8 of cycle 1 [C1D8]). Grade 3 CRS occurred in 0.8% (1 / 133) of patients in the 10 mg group and 5.7% (5 / 87) of patients in the 100 mg group. Among patients who experienced CRS, the most common symptoms were fever (96.7%), hypotension (19.8%) and hypoxia (17.4%). The median onset time of CRS after the last talquetamab dose was 13.1 hours (Q1, Q3: 7.8, 27.4). The median duration of CRS was 4 days (Q1, Q3: 2, 6) and was usually controllable with supportive care including the use of paracetamol, intravenous (IV) fluids and / or corticosteroids. In a few cases, additional interventions included tocilizumab (10 mg: 7 / 133 [5.3%]; 100 mg: 9 / 87 [10.3%]), oxygen supplementation (10 mg: 11 / 133 [8.3%]; 100 mg: 8 / 87 [9.2%]) and / or vasopressor support (10 mg: 1 / 133 [0.8%]; 100 mg: 1 / 87 [1.1%]). Compared with the 10 mg group, CRS led to a higher frequency of dose interruption and / or dose reduction in the 100 mg group (8 / 87 [9.2%] vs 4 / 133 [3.0%]). Almost all cases of CRS (98.4%) resolved.

[0302] Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) was graded according to the 2019 consensus of the American Society for Transplantation and Cellular Therapy (ASTCT) (Lee et al., Journal of the American Society for Blood and Marrow Transplantation, 25:625-38 (2019)). Analysis of these events included ICANS terminology and possible associated neurological adverse events. ICANS including associated neurological events occurred in 11 patients (8.3%) in the 10 mg group and 24 patients (27.6%) in the 100 mg group. Grade ≥3 events were not observed in the 10 mg group (0%; 0 / 133) and were observed in 4 patients (4.6%) in the 100 mg group. ICANS including associated neurological events mainly occurred in Cycle 1, with a median onset time of 5 days. The most common manifestations were immune effector cell-associated toxicity syndrome (10 mg: 6 / 11 patients, 100 mg: 9 / 24 patients), myasthenia (10 mg: 4 / 11 patients, 100 mg: 6 / 24 patients), aphasia (10 mg: 1 / 11 patients, 100 mg: 2 / 24 patients), and cognitive impairment (100 mg: 3 / 24 patients). ICANS including associated neurological events led to dose interruption and / or dose reduction in 1 patient (0.8%) in the 10 mg group and 5 patients (5.7%) in the 100 mg group, and treatment interruption in 1 patient in each group. The median resolution time was 6.5 days (95% CI: 4.0, 17.0).

[0303] Neutropenia was observed in 17.3% of patients in the 10 mg group and 16.1% of patients in the 100 mg group. Grade 3 febrile neutropenia was observed in 1 patient in each dose group. Neutropenia or febrile neutropenia did not lead to treatment interruption in any patient.

[0304] Similar safety was observed in patients who received 24-hour or 48-hour inpatient monitoring after talquetamab infusion in Cycle 1.

[0305] Pharmacokinetics

[0306] Within the evaluated dose range, talquetamab demonstrated an approximate dose-proportional increase in serum exposure. Steady-state serum talquetamab exposure was reached on Day 15 of Cycle 2. After the 10 mg and 100 mg Q2W regimens, the mean (SD) C of talquetamab at steady state (before dosing on Day 15 of Cycle 2) 谷The values were 0.51 (0.22) μg / mL and 6.8 (3.4) μg / mL, respectively. The results were consistent with those observed in the Phase 1 study described in Example 1 and Paz-Ares et al., J Clin Oncol [Journal of Clinical Oncology], 41(16):2893-2903 (2023), and supported the Q2W dosing interval.

[0307] Immunogenicity

[0308] Among 199 patients with at least one post-baseline immunogenicity assessment reportable, 4 patients (4 / 119; 3.4%) in the 10 mg group and 3 patients (3 / 80; 3.8%) in the 100 mg group developed treatment-emergent anti-talazoparib-binding antibodies. None of these patients developed anti-talazoparib neutralizing antibodies. The presence of anti-talazoparib antibodies did not appear to affect drug exposure, efficacy, or safety.

[0309] Example 3

[0310] This example describes a Phase 3 study comparing talazoparib with standard-of-care (SOC) chemotherapy in recurrent small cell lung cancer (SCLC).

[0311] An open-label, randomized, multi-center, Phase 3 study is described below that will evaluate the efficacy and safety of talazoparib compared to SOC therapy in subjects with small cell lung cancer (SCLC) who have progressed after prior first-line platinum-containing therapy.

[0312] Study Design

[0313] The study included a pre-screening period, a 21-day screening period, a treatment period, a safety follow-up (SFU) visit, and a long-term follow-up (LTFU) period.

[0314] At the time of enrollment, an evaluable archived tissue sample (formalin-fixed paraffin-embedded [FFPE] block collected within 5 years or sections cut within 30 days before signing the relevant informed consent form) or a new core needle biopsy for central evaluation was required, and the subject had to consent to tumor tissue collection and testing. Tumor material collection could be performed at any time after the initial diagnosis, including before the 21-day screening window, provided that the pre-screening informed consent form was completed. To be eligible for the study, tumor tissue evaluability had to be confirmed before randomization.

[0315] Subjects were randomly assigned at a 1:1 allocation ratio to receive talazoparib or the approved SOC treatment (lurbinectedin or topotecan in the United States (US), Canada, Australia, Singapore, South Korea; amrubicin in Japan; topotecan in all countries except Japan).

[0316] Randomization will be stratified as follows:

[0317] ·Prior anti-PD-(L)1 exposure (yes or no)

[0318] ·Chemotherapy-free interval (≥180 days; <180 to ≥90 days; <90 days)

[0319] ·Presence of brain metastases (yes or no)

[0320] Approximately 350 subjects per group, with a total of approximately 700 subjects enrolled. The primary efficacy endpoint included OS, and the secondary efficacy endpoints included PFS.

[0321] Study population

[0322] Inclusion criteria

[0323] Subjects were eligible for inclusion in the study only if all of the following criteria applied: prior to the start of any study-specific activity / procedure, the subject had provided informed consent; age ≥18 years (or the national legal age of majority, whichever is greater) at the time of signing the informed consent; histologically or cytologically confirmed recurrent / refractory SCLC; the subject had progressed or relapsed after 1 platinum-based regimen (the first documented disease progression must have occurred during or after first-line platinum-based systemic chemotherapy for ES or LS disease; patients who received treatment for LS disease and relapsed were eligible; patients who received adjuvant platinum-etoposide after resection of SCLC and relapsed were eligible; in countries where the SOC first-line systemic treatment for ES disease included a combination of platinum-based chemotherapy and a PD-L1 inhibitor, patients were required to have failed the PD-(L)1 inhibitor as part of their first-line systemic treatment or to be ineligible for PD-(L)1 inhibitor treatment); an evaluable tumor sample was provided for central testing (the tumor sample had to be archived (FFPE blocks collected within 5 years or sections cut within 30 days prior to signing the relevant informed consent) or fresh core needle biopsy); measurable disease as defined by RECIST 1.1 within a 21-day screening period (if completed within the 21-day screening period, screening scans performed as the standard of care and prior to the informed consent could be used to confirm subject eligibility, provided informed consent for the use of these scans was obtained prior to any data transfer); Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; minimum life expectancy of 12 weeks.

[0324] Subjects must have had adequate organ function, defined as follows: blood function (absolute neutrophil count ≥1.5×10 9 / L; platelet count ≥100×10 9 / L; Hemoglobin > 9 g / dL (90 g / L)); Coagulation function (Prothrombin time (PT) / International Normalized Ratio (INR) and Partial Thromboplastin Time (PTT) or Activated Partial Thromboplastin Time (APTT) ≤ 1.5 × institutional normal upper limit (ULN). Subjects receiving chronic anticoagulation therapy who do not meet the above criteria are eligible for enrollment.); Renal function (Estimated glomerular filtration rate (eGFR) > 30 mL / min / 1.73 m 2 2); Liver function (Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) < 3 × ULN (or for subjects with liver involvement, < 5 × ULN); Total bilirubin < 1.5 × ULN (or for subjects with liver involvement, < 2 × ULN); For subjects who may receive lurbinectedin as SOC, total bilirubin < 1.5 × ULN and any AST); Pulmonary function (No clinically significant pleural effusion. Use of indwelling pleural catheter (e.g., PleurX) to manage pleural effusion is permitted; Baseline oxygen saturation > 90% in room air); and Cardiac function (Cardiac ejection fraction ≥ 50%, no clinically significant pericardial effusion determined by echocardiogram (ECHO) or multiple gated acquisition (MUGA) scan, and no clinically significant electrocardiogram (ECG) findings).

[0325] Exclusion Criteria

[0326] Subjects will be excluded from the study if any of the following criteria apply.

[0327] Related diseases: Untreated or symptomatic central nervous system (CNS) metastases or leptomeningeal disease (Subjects with asymptomatic CNS metastases are eligible if clinically stable for at least 4 weeks and do not require intervention (including use of corticosteroids); Subjects with treated brain metastases are eligible if the following criteria are met: Definitive treatment was completed at least 2 weeks before the first dose of study treatment (stereotactic radiosurgery was completed at least 7 days before the first dose of study treatment), any CNS disease is clinically stable, the subject has discontinued corticosteroids due to CNS disease (unless corticosteroids are prescribed for reasons unrelated to CNS disease), and the subject has discontinued or is taking a stable dose of antiepileptic drugs at least 7 days before the first dose of study treatment); History of prior immune checkpoint inhibitors, resulting in: Any severe or life-threatening immune-mediated adverse event, history of immune-mediated encephalitis or other immune-mediated CNS events (any grade), ≥ grade 2 immune-mediated recurrent pneumonia, infusion-related reactions that led to permanent discontinuation of immunotherapy agents (Exception: Subjects with a history of immune checkpoint inhibitor-induced endocrinopathy and clinically stable on alternative therapy).

[0328] Other medical conditions: Active autoimmune disease requiring systemic treatment (except alternative therapies) within the past 2 years or any other disease requiring immunosuppressive therapy at the time of the study; Active infection requiring systemic treatment or any uncontrolled infection. Uncomplicated urinary tract infection (UTI) and uncomplicated bacterial pharyngitis are permitted if responsive to active treatment. Subjects who require oral antibiotics and have had no fever for >24 hours, no leukocytosis, and no signs of any clinical infection are eligible; History of solid organ transplantation; History of other malignancies within the past 2 years (Exception: Low-risk malignancies treated with curative intent and with no known active disease ≥1 year prior to enrollment and with a low risk of recurrence as determined by the investigator; Non-melanoma skin cancer or lentigo maligna melanoma with no evidence of disease, fully treated; Cervical carcinoma in situ with no evidence of disease, fully treated; Ductal carcinoma in situ of the breast with no evidence of disease, fully treated; Prostatic intraepithelial neoplasia with no evidence of prostate cancer; Papillary non-invasive urothelial carcinoma or carcinoma in situ, fully treated); Myocardial infarction and / or symptomatic congestive heart failure (New York Heart Association > Class II) within 12 months prior to the first dose of study treatment; History of arterial thrombosis (e.g., stroke or transient ischemic attack) within 12 months prior to the first dose of study treatment; History of hypophysitis or pituitary dysfunction (any grade); Known human immunodeficiency virus (HIV) infection, hepatitis C infection (hepatitis C subjects permitted who have achieved a sustained virologic response after antiviral treatment), or hepatitis B infection (subjects with hepatitis B surface antigen (HBsAg) or core antibody permitted who have achieved a sustained virologic response through antiviral treatment, but who are required to be monitored regularly for reactivation during study treatment); Systemic corticosteroid treatment or any other form of immunosuppressive treatment within 7 days prior to the first dose of study treatment (permitted are prophylactic dexamethasone as required by the protocol and any antiemetic therapy, low-dose corticosteroids (prednisone ≤10 mg / day or equivalent permitted during the trial)); Evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (subjects are eligible if they have no acute symptoms of coronavirus disease 2019 (COVID-19) within 14 days prior to the first dose of study treatment (counting from the day an asymptomatic subject tests positive)); Evidence of interstitial lung disease or active non-infectious pneumonia.

[0329] Prior / Concomitant Therapy: Prior therapy with talazoparib or any SOC chemotherapy as part of this trial; prior therapy with any selective inhibitor of the DLL3 pathway; the subject has previously received more than one SCLC systemic treatment regimen (anti-PD-(L)1 maintenance therapy after platinum / etoposide / anti-PD-(L)1 therapy is considered one regimen); prior anticancer treatment within 21 days before the first dose of study treatment (Exception: Subjects who received conventional chemotherapy are eligible if at least 14 days have passed since the first dose of study treatment and if all treatment-related toxicities have resolved to Grade ≤1, or the level specified in the eligibility criteria, except for alopecia or toxicities considered irreversible (defined as present and stable >30 days) that are not otherwise described in the exclusion criteria; prior palliative radiotherapy must be completed at least 7 days before the first dose of study treatment); receiving anticancer treatment such as chemotherapy, immunotherapy, or targeted therapy (patients receiving adjuvant hormonal therapy for resected breast cancer may be considered (also refers to exclusions related to a history of other malignancies)); any herbal or prescription / non-prescription drug (including but not limited to cyclosporine, clarithromycin, itraconazole, or ketoconazole) known to inhibit membrane transporter P-gp and / or breast cancer resistance protein (BCRP) within 7 days before the first dose of study treatment; any herbal or prescription / non-prescription drug (including but not limited to clarithromycin, itraconazole, ketoconazole) known to be a moderate or strong inhibitor of cytochrome P450 3A (CYP3A) enzyme within 7 days before the first dose of study treatment; any herbal or prescription / non-prescription drug (including but not limited to efavirenz, phenobarbital, phenytoin, rifampin, St. John's wort) known to be a moderate or strong inducer of CYP3A enzyme within 28 days before the first dose of study treatment; subjects who have reached the maximum tolerated dose of prior cardiotoxic drugs (e.g., other anthracyclines) (total dose of daunorubicin hydrochloride is 25 mg / kg body weight, total dose of doxorubicin hydrochloride is 500 mg / m 2 body surface area, total dose of epirubicin hydrochloride is 900 mg / m 2 body surface area, total dose of pirarubicin hydrochloride is 950 mg / m 2 body surface area, etc.); major surgery within 28 days before the first dose of study treatment; live virus therapy, including administration of live attenuated vaccines, within 4 weeks before the first dose of study treatment (inactive vaccine administration (e.g., non-live or non-replicating agents), including COVID-19 vaccines, is not permitted within 3 days after the first dose of talazoparib (Note: Vaccination prophylaxis for SOC treatment is based on regional prescribing information)).

[0330] Prior / Current Clinical Study Experience: Currently receiving treatment in another investigational device or drug study, or less than 30 days have elapsed since the end of treatment in another investigational device or one or more drug studies. Exclude other concurrent investigational procedures for participation in this study.

[0331] Other exclusions: Female subjects of childbearing potential who are unwilling to use the contraceptive methods specified in the protocol during the treatment period and for an additional 72 days after the last dose of talazoparib (Note: The contraceptive requirements for SOC therapy are based on regional prescribing information); Female subjects who are breastfeeding or plan to breastfeed during the study period up to 72 days after the last dose of talazoparib (Note: The breastfeeding restrictions for SOC therapy are based on regional prescribing information); Female subjects who plan to become pregnant or donate eggs during the study period up to 72 days after the last dose of talazoparib (Note: The contraceptive requirements for SOC therapy are based on regional prescribing information); Female subjects of childbearing potential who are found to have a positive pregnancy test by serum pregnancy test at screening; Male subjects with female partners of childbearing potential who are unwilling to practice sexual abstinence (avoid heterosexual intercourse) or use contraception during the treatment period and for an additional 132 days after the last dose of talazoparib; Male subjects with pregnant partners who are unwilling to practice abstinence or use a condom during the treatment period and for an additional 132 days after the last dose of talazoparib; Male subjects who are unwilling to refrain from donating sperm during the treatment period and for an additional 132 days after the last dose of talazoparib; Subjects with known sensitivity to any product or component to be administered or potentially administered during dosing; To the knowledge of the subject and the investigator, the subject may be unable to complete all study visits or procedures required by the protocol and / or comply with all required study procedures (e.g., clinical outcome assessments); History or evidence of any other clinically significant obstacle, condition, or disease (other than those outlined above) that, in the opinion of the investigator or medical monitor (if consulted), would pose a risk to the safety of the subject or interfere with the study evaluation, procedures, or completion.

[0332] Standard of care treatment regimen

[0333] Lurbinectedin will be administered at 3.2 mg / m 2 on Day 1 of each 21-day cycle according to the approved protocol. Lurbinectedin will be administered as an intravenous (IV) infusion over 60 minutes. Lurbinectedin is provided as a sterile, preservative-free, white to off-white lyophilized powder, containing 4 mg in a single-dose clear glass vial. Lurbinectedin is intended to be reconstituted with Sterile Water for Injection USP. The lyophilized formulation contains sucrose, lactic acid, and sodium hydroxide.

[0334] Topotecan will be administered at 1.5 mg / m 2 IV on Days 1 to 5 of each 21-day cycle or 2.3 mg / m 2Administered according to the approved regimen orally per day. Topotecan injection will be administered by intravenous (IV) infusion over 30 minutes. Topotecan capsules should be swallowed whole. Topotecan injection is provided as a sterile, pyrogen-free, clear, light yellow to green solution, packaged in a single-dose vial containing a free base concentration of 4 mg / 4 mL (1 mg / mL). Each mL contains topotecan hydrochloride (equivalent to 1 mg of topotecan free base), mannitol, USP, and tartaric acid, NF. Hydrochloric acid and sodium hydroxide may also be included to adjust the pH. Topotecan capsules are provided in the form of 0.25 mg opaque white to yellowish-white capsules imprinted with HYCAMTIN and 0.25 mg or 1 mg opaque pink capsules imprinted with HYCAMTIN and 1 mg. The capsules contain topotecan hydrochloride (the content is expressed as topotecan free base), and the excipients are gelatin, glyceryl monostearate, hydrogenated vegetable oil, and titanium dioxide. The imprints are made with edible black ink. The 1 mg capsules also contain red iron oxide.

[0335] Amrubicin will be administered at 40 mg / m 2 according to the approved regimen and will be administered by intravenous (IV) infusion on Days 1 to 3 of each 21-day cycle. Amrubicin is provided as a yellowish-red powder or mass, packaged in vials containing 20 mg or 50 mg of amrubicin hydrochloride. The excipients include lactose hydrate, L-cysteine hydrochloride, monohydrate, hydrochloric acid, and sodium hydroxide.

[0336] Drugs before and after talazoparib infusion

[0337] Dexamethasone 8 mg IV (or an equivalent dose of other corticosteroids) will be administered only within 1 hour before the talazoparib infusion on Day 1 and Day 8 of Cycle 1.

[0338] For prophylaxis, intravenous (IV) rehydration (1 L of normal saline, 2 to 4 hours) will be administered immediately after all talazoparib doses in Cycle 1 only.

[0339] Example 4

[0340] This example describes a Phase 1b clinical study to evaluate the safety, tolerability, pharmacokinetics, and efficacy of talazoparib in subjects with newly diagnosed or treatment-emergent neuroendocrine prostate cancer (NEPC).

[0341] Prostate cancer is the most commonly diagnosed non - cutaneous cancer in men, with an estimated 191,930 new cases and 33,330 deaths in the United States (US) in 2020 (American Cancer Society, 2020). In the European Union, an estimated 365,000 new cases of prostate cancer were diagnosed in 2015, and an estimated 72,000 and 77,000 deaths in 2012 and 2015, respectively (10% of total cancer deaths) (Crocetti E., Epidemiology of prostate cancer in Europe; publications.jrc.ec.europa.eu / repository / handle / JRC101382 (2015)). NEPC represents an aggressive variant of prostate cancer. Although new cases appear to be rare, less than 2% of patients at initial diagnosis (Parimi et al., Am J Clin Exp Urol., 2:273 - 285 (2014)), NEPC that develops during treatment, characterized by a histological transformation from adenocarcinoma to high - grade neuroendocrine tumor, is increasingly recognized and may develop in 15% to 20% of patients treated with standard therapies for prostate adenocarcinoma, including novel hormonal therapies (Aggarwal et al., J Clin Oncol., 36:2492 - 2503 (2018)). The exact mechanism of this histological transformation is unclear; however, loss of androgen signaling dependence and acquisition of alternative lineage programs are associated with the development of NEPC. Overall, the prognosis of NEPC is poor (Wang et al., J Clin Oncol., 32:3383 - 3390 (2014)), and it is typically treated with platinum - based chemotherapy regimens (Aparicio et al., Clin Cancer Res., 19:3621 - 3630 (2013)). There is currently no standard treatment for NEPC.

[0342] Study design

[0343] An open - label phase 1b study was conducted to evaluate talazoparib monotherapy for NEPC. Talazoparib was administered as a short - term intravenous (IV) infusion every 2 weeks (Q2W) in a 28 - day cycle with escalating doses in subjects with newly diagnosed or treatment - emergent NEPC.

[0344] Due to its known mechanism of action, the risk of first-dose effects (such as cytokine release syndrome (CRS)) in subjects increases after the initial infusion of talazoparib. To reduce these risks, a stepped dosing method was implemented. The study included single-step dose exploration, including a lead-in dose of 1 mg talazoparib on Day 1, followed by a single-step to the target dose of 100 mg talazoparib on Day 15, and then Q2W. In a Phase 1 trial of talazoparib in SCLC subjects, the 100 mg dose of talazoparib was considered the highest dose that was safe and tolerable. Approximately 40 subjects were enrolled in the study.

[0345] Summary of Subject Eligibility Criteria

[0346] Adult subjects (≥18 years old) with metastatic newly diagnosed or treatment-emergent NEPC defined as one or more of the following are eligible for enrollment: Histological diagnosis of small cell NEPC, prostate cancer with neuroendocrine differentiation, as defined by: Positive immunohistochemical staining for chromogranin and / or synaptophysin in the majority of tumor samples or ≥2 alterations in Tp53, RB1, and PTEN (by immunohistochemistry (IHC), or genomic analysis of baseline tumor tissue or circulating tumor DNA (ctDNA)). Subjects are required to have progressed on at least 1 line of prior therapy, including a platinum-containing regimen for newly diagnosed NEPC (if they had not previously been diagnosed or treated for prostate cancer at the time of NEPC diagnosis) or, if treatment-emergent (had been diagnosed with prostate cancer prior to NEPC diagnosis), then including androgen signaling inhibitors (such as abiraterone, enzalutamide, and / or apalutamide). According to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria and Prostate Cancer Working Group 3 (PCWG3) guidelines, subjects must have measurable disease, an Eastern Cooperative Oncology Group (ECOG) performance status ≤2, and adequate organ function.

[0347] Treatment and Procedures

[0348] On Days 1 and 15 of a 28-day cycle (Cycle 2 and later), talazoparib is administered as a short intravenous infusion (approximately 60 minutes, then flushed). To reduce the risk of cytokine release syndrome (CRS), dexamethasone 8 mg IV (or other corticosteroid of equivalent dose) is administered intravenously within 1 hour before all Cycle 1 doses for premedication. Prophylaxis with 1 L of normal saline intravenously (IV) over 4 to 5 hours is required immediately after all doses in Cycle 1.

[0349] Tocilizumab or siltuximab (if tocilizumab is not available) is required at the institutional site for potential treatment of CRS. Talazoparib is administered until disease progression (treatment beyond disease progression is permitted according to PCWG3 guidelines).

[0350] Each subject followed the same treatment protocol and procedural requirements. After obtaining written informed consent, all screening tests and procedures were conducted within 28 days after talquetamab administration (Day 1), unless otherwise specified. A series of clinical safety and research evaluations were performed, including physical examinations, vital signs, clinical laboratory tests, radiological evaluations, tumor biopsies, pharmacokinetic (PK) and biomarker sample collections.

[0351] During all Cycle 1 doses, all subjects were hospitalized for intensive monitoring for 48 hours after talquetamab infusion. Hospitalization was not required for Cycle 2, unless the subject experienced a grade 2 or higher CRS or neurological event during Cycle 1 (if a grade 2 or higher CRS or neurological event was identified during Cycle 1, at least 24 hours of hospitalization was required after talquetamab infusion on Day 1 of Cycle 2). If there were no signs and symptoms of CRS or other acute toxicities, the subject could be discharged after this period. If the subject was not hospitalized during Cycle 2, the subject was observed for 8 hours after talquetamab infusion.

[0352] Conventional radiological imaging (computed tomography [CT] / magnetic resonance imaging [MRI]) and tumor burden assessments were performed. Disease response assessment was determined based on RECIST 1.1 according to the PCWG3 guidelines. To further evaluate the risk of delayed adverse events, subjects returned for a safety follow-up (SFU) visit approximately 42 (±5) days after the last dose of talquetamab.

[0353] For all subjects who did not withdraw consent via an outpatient visit, telephone, or chart review, long-term follow-up was conducted every 3 months for up to 3 years starting from the first dose of talquetamab to evaluate survival and / or the initiation of subsequent cancer treatment.

[0354] Statistical Considerations

[0355] Unless otherwise specified, all subjects who were enrolled and received at least 1 dose of talquetamab were included in the analysis.

[0356] The primary analysis was conducted when the target enrollment was completed and each subject had completed at least 6 months of the study or withdrawn from the study. The final analysis was conducted after the last subject had the opportunity to complete the corresponding end-of-treatment visit / procedure.

[0357] Descriptive statistics of selected demographic, safety, pharmacokinetic, and efficacy data are provided, stratified by dose, dosing regimen, and time (as applicable). Descriptive statistics for continuous data include mean, median, standard deviation, and range, while categorical data are summarized using frequency counts and percentages. The number and percentage of subjects reporting any treatment-emergent adverse events are tabulated. Clinical laboratory test, physical examination findings, and vital sign data are listed. Summaries of laboratory, examination, and vital sign data over time and / or relative to baseline over time are provided.

[0358] Confidence intervals (CIs) for proportions were estimated using the exact method proposed by Clopper-Pearson (Clopper and Pearson, Biometricka, 26:404-413 (1934)). The Kaplan-Meier method was used to estimate the median and percentiles of the time to event endpoint, and the CIs were calculated using the method of Brookmeyer and Crowley (Brookmeyer and Crowley, Biometrics, 38:29-41 (1982)). The Kaplan-Meier method was used to estimate the milestones of the time to event endpoint, and the Greenwood formula (Kalbfleisch and Prentice, The Statistical Analysis of Failure Time Data, John Wiley & Sons, Inc., New York, xi + 321 pp (1980)) was used to estimate the standard error used in the CI calculation.

[0359] Biomarker assessment during the study

[0360] Blood samples were collected for the assessment of circulating biomarkers, including but not limited to PSA and cytokines. Blood samples were also collected for the enumeration and phenotypic analysis of circulating tumor cells (CTCs). These samples were used to help further understand the subjects' disease and their response to treatment.

[0361] Whole blood samples were collected for immunophenotypic flow cytometry to identify changes in T cell subsets and activation status at specific time points. Plasma and / or serum or tissue may also be used for DNA, RNA, and protein expression analysis, including somatic mutations, in order to correlate expression levels with response.

[0362] Baseline DLL3 expression and other exploratory biomarkers that may predict response were evaluated by IHC in formalin-fixed paraffin-embedded (FFPE) archived tumor tissue.

[0363] To meet eligibility requirements, subjects must provide fresh tumor tissue for biomarker discovery and future research, unless archival tissue was collected after the last cancer treatment. For archival tumor samples within the first week after enrollment, archival FFPE tumor tissue collected prior to the study was evaluated.

[0364] Tumor biopsies were collected and pharmacodynamic (PD) changes were analyzed to determine the effect of the study product on one or more targets in the tumor and potentially to analyze molecular mechanisms associated with acquired drug resistance.

[0365] Results

[0366] A total of 40 patients received talazoparib, and 38 patients were included in the efficacy analysis according to RECIST 1.1 criteria. Talazoparib was safe and tolerable in NEPC subjects with adverse event profiles similar to those of AE in SCLC subjects. The complete response rate (CR) was 0 / 38 (0%), the confirmed partial response rate (PR) was 4 / 38 (10.5%), and the disease control rate (DCR) was 12 / 38 (31.6%).

[0367] Thirty-six patient samples were available for DLL3 expression analysis. Immunohistochemical analysis showed that DLL3 expression was detected in 56% (18 / 32 - 4 samples were not evaluable) of treated patient samples. In DLL3+ patient samples, the overall response rate (ORR) was 22.2% (4 / 18), and the DCR was 55.6% (10 / 18).

[0368] All references cited herein (including publications, patent applications, and patents) are hereby incorporated by reference to the extent as if each reference was individually and expressly indicated to be incorporated by reference and set forth in its entirety herein.

[0369] Unless otherwise indicated herein or clearly contradicted by context, in the context of describing the present invention (especially in the context of the appended claims), the use of the terms "a", "an", "the", "at least one", and similar referents shall be construed to cover both the singular and the plural. The use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") shall be construed to mean either one of the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise stated herein or clearly contradicted by context. Unless otherwise described, the terms "comprising", "having", "including", and "containing" shall be regarded as open-ended terms (i.e., meaning "including, but not limited to"). Unless otherwise indicated herein, statements herein regarding ranges of values are merely intended to be a shorthand method of individually referring to each separate value within the range, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise indicated herein or the context otherwise clearly contradicts, all methods described herein may be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to better describe the present invention and does not impose a limitation on the scope of the present invention, unless otherwise required. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0370] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for practicing the present invention. After reading the above description, variations of those preferred embodiments may become apparent to those of ordinary skill in the art. The inventors expect skilled artisans to appropriately employ such variations, and the inventors intend to practice the present invention in a manner different from that specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. In addition, unless otherwise indicated herein or the context otherwise clearly contradicts, the present invention covers any combination of the above elements in all possible variations thereof.

[0371] Sequence

[0372]

[0373]

[0374]

[0375]

[0376]

Claims

1. A method of treating small cell lung cancer (SCLC) in a subject, the method comprising administering to the subject a bispecific antigen-binding molecule that comprises at least a first binding domain that binds to human delta-like ligand 3 (DLL3), wherein at least 25% of SCLC cells express DLL3.

2. The method of claim 1, wherein at least 50% of SCLC cells express DLL3.

3. The method of claim 1, wherein at least 75% of SCLC cells express DLL3.

4. The method of any one of claims 1-3, wherein an immunohistochemistry (IHC) assay is used to determine DLL3 expression.

5. The method of claim 4, wherein a formalin-fixed paraffin-embedded (FFPE) tissue specimen from the subject is used to determine DLL3 expression.

6. The method of any one of claims 1-5, wherein the SCLC has progressed or recurred in the subject after platinum-based therapy.

7. The method of any one of claims 1-5, wherein the SCLC has progressed or recurred in the subject after platinum-based therapy in combination with etoposide and optionally a PD-L1 inhibitor.

8. The method of any one of claims 1-5, wherein the subject (i) has completed up to two cycles of platinum-based therapy in combination with etoposide and optionally a PD-L1 inhibitor, or (ii) has completed four to six cycles of platinum-based therapy in combination with etoposide and optionally a PD-L1 inhibitor and has not experienced disease progression.

9. The method of any one of claims 1-5, the method further comprising administering to the subject a PD-L1 inhibitor and optionally a chemotherapeutic agent.

10. The method of any one of claims 1-9, wherein the bispecific antigen-binding molecule comprises a second binding domain that binds to human CD3.

11. The method of any one of claims 1-10, wherein the bispecific antigen-binding molecule is a protein.

12. The method according to any one of claims 1-11, wherein the bispecific antigen-binding molecule comprises an antibody, a single-chain variable fragment (scFv), a tandem single-chain variable fragment (scFv)2, a bispecific T cell conjugate molecule or a heteromultimer.

13. The method of claim 12, wherein the bispecific antigen-binding molecule comprises the amino acid sequences of SEQ ID NO:14 and SEQ ID NO:

15.

14. The method of claim 13, wherein the bispecific antigen-binding molecule is talquetamab.

15. The method of claim 12, wherein the bispecific antigen-binding molecule comprises a first heterodimer that binds to human DLL3 and a second heterodimer that binds to human CD3, wherein (a) the first heterodimer comprises a heavy chain (HC) having the amino acid sequence of SEQ ID NO:17 and a light chain (LC) having the amino acid sequence of SEQ ID NO:18; and (b) the second heterodimer comprises a heavy chain having the amino acid sequence of SEQ ID NO:19 and a light chain having the amino acid sequence of SEQ ID NO:

20.

16. The method according to any one of claims 1-15, which increases the progression-free survival (PFS) of the subject as compared to a second SCLC subject having less than 50% of SCLC cells expressing DLL3 and treated with the same antigen-binding molecule.

17. The method according to claim 16, which increases one or more of overall survival (OS), objective response rate (ORR), and / or disease control rate (DCR) and duration of response (DOR) as compared to the second subject.

18. The method according to any one of claims 1-17, which results in an objective response rate (ORR) of greater than about 35% in the subject.

19. A method of treating SCLC in a subject, the method comprising administering to the subject a bispecific antigen-binding molecule comprising at least a first binding domain that binds to human delta-like ligand 3 (DLL3), wherein the SCLC has a DLL3 expression level, and wherein at least 25% of SCLC cells express DLL3 at an intensity equal to or greater than 2+, as determined by an IHC assay.

20. The method according to claim 19, wherein the SCLC has a DLL3 expression level, and wherein at least 50% or at least 75% of SCLC cells express DLL3 at an intensity equal to or greater than 2+, as determined by an IHC assay.

21. The method according to claim 19 or claim 20, wherein at least 25% of SCLC cells express DLL3 at an intensity of 2+ or 3+, as determined by an IHC assay.

22. The method according to any one of claims 19-21, wherein a formalin-fixed paraffin-embedded (FFPE) tissue specimen from the subject is used to determine DLL3 expression.

23. The method according to any one of claims 19-22, wherein the SCLC has progressed or recurred in the subject after platinum-based therapy.

24. The method according to any one of claims 19-22, wherein the SCLC has progressed or recurred in the subject after platinum-based therapy in combination with etoposide and optionally a PD-L1 inhibitor.

25. The method according to any one of claims 19-22, wherein the subject (i) has completed at most two cycles of platinum-based therapy in combination with etoposide and optionally a PD-L1 inhibitor, or (ii) has completed four to six cycles of platinum-based therapy in combination with etoposide and optionally a PD-L1 inhibitor and has not experienced disease progression.

26. The method according to any one of claims 19-22, the method further comprising administering to the subject a PD-L1 inhibitor and optionally a chemotherapeutic agent.

27. The method according to any one of claims 19-26, wherein the bispecific antigen-binding molecule comprises a second binding domain that binds to human CD3.

28. The method according to any one of claims 19-27, wherein the bispecific antigen-binding molecule is a protein.

29. The method according to any one of claims 19-28, wherein the bispecific antigen-binding molecule comprises an antibody, a single-chain variable fragment (scFv), a tandem single-chain variable fragment (scFv)2, a bispecific T cell conjugate molecule or a heteromultimer.

30. The method according to claim 29, wherein the bispecific antigen-binding molecule comprises SEQ ID NO:14 and SEQ ID NO:

15.

31. The method according to claim 30, wherein the bispecific antigen-binding molecule is talquetamab.

32. The method according to claim 29, wherein the bispecific antigen-binding molecule comprises a first heterodimer that binds to human DLL3 and a second heterodimer that binds to human CD3, wherein (a) the first heterodimer comprises a heavy chain (HC) having the amino acid sequence of SEQ ID NO:17 and a light chain (LC) having the amino acid sequence of SEQ ID NO:18; and (b) the second heterodimer comprises a heavy chain having the amino acid sequence of SEQ ID NO:19 and a light chain having the amino acid sequence of SEQ ID NO:

20.

33. The method according to any one of claims 19-32, which increases the progression-free survival (PFS) of the subject compared to a second SCLC subject having a lower DLL3 expression level and treated with the same antigen-binding molecule.

34. The method according to claim 33, which increases one or more of OS, ORR, and / or DCR and DOR compared to the second subject.

35. The method according to any one of claims 19-34, which results in an objective response rate (ORR) of the subject greater than about 35%.

36. The method according to any one of claims 19-35, wherein the cancer cells are live cancer cells.

37. The method according to any one of claims 1-14, 16-31, or 33-36, wherein the bispecific antigen-binding molecule is talquetamab, and wherein talquetamab is administered once every two weeks at a dose of 10 mg to 100 mg.

38. The method according to claim 37, wherein talquetamab is administered once every two weeks at a dose of 10 mg.

39. The method according to claim 37, wherein talquetamab is administered once every two weeks at a dose of 100 mg.

40. The method according to any one of claims 37-39, which comprises administering talquetamab once a week at a dose of 10 mg to 100 mg in weeks 1, 2, and 3, and then administering talquetamab once every two weeks.

41. The method according to any one of claims 1-14, 16-31, or 33-36, wherein the bispecific antigen-binding molecule is talquetamab, and wherein talquetamab is administered twice every three weeks at a dose of 10 mg to 100 mg.

42. The method according to claim 41, wherein talquetamab is administered twice every three weeks at a dose of 10 mg, 30 mg, or 100 mg.

43. The method according to claim 41 or 42, wherein talquetamab is administered on day 1 and day 8 of a 21-day cycle.

44. The method according to any one of claims 1-14, 16-31 or 33-36, wherein the bispecific antigen-binding molecule is talazoparib, and wherein talazoparib is administered once every three weeks at a dose of 20 mg to 200 mg.

45. The method according to claim 44, wherein talazoparib is administered once every three weeks at a dose of 20 mg to 100 mg.

46. The method according to claim 44, wherein talazoparib is administered once every three weeks at a dose of 100 mg to 200 mg.

47. The method according to any one of claims 44-46, wherein talazoparib is administered at a dose of 20 mg, 60 mg, 100 mg or 200 mg.

48. The method according to any one of claims 44-47, wherein talazoparib is administered on the first day of a 21-day cycle.

49. The method according to any one of claims 44-47, the method comprising administering talazoparib once a week at a dose of 10 mg to 100 mg in weeks 1 and 2, and then once every three weeks.

50. The method according to claim 5 or claim 22, wherein the FFPE tissue specimens are prepared from core needle biopsy tissue samples.

51. The method according to any one of claims 1-50, wherein the subject is a human.

52. The method according to any one of claims 1-51, wherein the subject has received at least one prior treatment for the cancer and has relapsed.

53. The method according to any one of claims 1-51, wherein the subject has not received prior systemic treatment for the cancer.

54. A method of treating neuroendocrine prostate cancer (NEPC) in a subject, the method comprising administering to the subject a bispecific antigen-binding molecule comprising a first binding domain that binds to human delta-like ligand 3 (DLL3) and a second binding domain that binds to human CD3, wherein the NEPC cells express DLL3 as determined by an immunohistochemistry (IHC) assay.

55. The method according to claim 54, wherein the IHC assay is the Ventana SP347 IHC assay.

56. The method according to claim 54 or claim 55, wherein the bispecific antigen-binding molecule is a protein comprising the amino acid sequences of SEQ ID NO: 14 and SEQ ID NO: 15.

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