WO2025035356A1

WO2025035356A1 - Antibodies against dll3 and uses thereof

Info

Publication number: WO2025035356A1
Application number: PCT/CN2023/112911
Authority: WO
Inventors: Zuoxiang XIAO; Dongwen ZHOU; Wei Zhou
Original assignee: Zhejiang Shimai Pharmaceutical Co Ltd
Current assignee: Zhejiang Shimai Pharmaceutical Co Ltd
Priority date: 2023-08-14
Filing date: 2023-08-14
Publication date: 2025-02-20
Anticipated expiration: 2026-02-14

Abstract

Disclosed herein are antibodies against DLL3 and uses thereof, specifically monoclonal antibodies against DLL3, bispecific antibodies against DLL3 and CD3, nucleic acids comprising nucleotide sequences encoding the antibodies, vectors comprising the nucleic acids, and host cells comprising the nucleic acids or the vectors. Also disclosed are pharmaceutical compositions and conjugates comprising the antibodies, and therapeutic methods for using the antibodies.

Description

ANTIBODIES AGAINST DLL3 AND USES THEREOF

FIELD OF THE INVENTION

The present invention is directed to antibodies against DLL3, and uses of such antibodies, in particular their use in the treatment of cancers.

BACKGROUND OF THE INVENTION

Delta-Like Ligand 3 (DLL3) , a single transmembrane protein that attaches to the cell surface, is a member of the Notch ligand family. The human DLL3 gene is localized on chromosome 19q13 and has an open reading frame length of approximately 1800 bp. The human DLL3 protein consists of 619 amino acids and is characterized by a Delta/Serrate/LAG-2 (DSL) domain, six epidermal growth factor (EGF) -like repeats, and a transmembrane domain. The DSL gene sequence at the extracellular N-terminal domain is highly conserved in the ligand family, which is an essential structure for binding to the Notch receptor. Unlike other Notch ligands, the current researches suggest that DLL3 is an inhibitory Notch ligand. The ligand DLL3 binds to Notch receptors and has an inhibitory effect on the Notch signaling pathway. In addition to the Notch signaling pathway, DLL3 also functions in other signaling pathways. For example, DLL3 activates phosphoinositol-3-kinase/serine-threonine protein kinase B (PI3K/Akt) by inhibiting the Notch signaling pathway. As DLL3 expression was upregulated, the expression levels of Wnt-1 and Wnt-4, as well as Wnt-targeted genes (Axin-2 and Lef-1) were upregulated, suggesting that DLL3 is involved in the activation of the Wnt signaling pathway. Moreover, it has also been demonstrated that DLL3 regulates the Notch/Wnt signaling pathway by regulating the cyclic expression of Nrarp. DLL3 is a highly tumor-selective cell surface target, mainly expressed in neurological or neuroendocrine tumors, especially small cell lung cancer (SCLC) , more than 80%of SCLC have positive expression of DLL3.

The high expression of DLL3 in neuroendocrine tumors shows its potential capability for tumor therapy.

SUMMARY OF THE INVENTION

The present disclosure provides novel antibodies targeting DLL3 or antigen binding fragments thereof, which can be in a form of a monoclonal antibody or bispecific antibody, such as a bispecific T-cell engager (BiTE) . T-cell-dependent efficacy in vitro as well as anti-tumor activity in vivo of the antibodies have been evaluated. Results of these functional assays demonstrate the potent anti-tumor effect of the engineered antibodies, particularly the antibodies in the form of BiTE.

In an aspect, the present disclosure provides an antibody that specifically binds to DLL3, or an antigen binding fragment thereof, comprising a light chain variable region (VL) and a heavy chain variable region (VH) , wherein the VL comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 1-3 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 6-8 respectively.

In some embodiments of the antibody or the antigen binding fragment thereof disclosed herein, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 4, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 9. In other embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 12. In other embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 13.

In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 4 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 13.

In some embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD. In some embodiments, the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab’, F (ab') ₂, Fv, scFv, and ds-scFv.

In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 5 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 10.

In other embodiments, the antibody is a bispecific or a multi-specific antibody. In some embodiments, the antibody is a bispecific antibody which further comprises a second antigen binding region that binds to a second antigen. In some embodiments, the second antigen is a tumor associated antigen or an immune cell antigen. In some embodiments, the second antigen is a T-cell antigen. In some embodiments, the T-cell antigen is selected from the group consisting of T cell receptor (TCR) , CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137) , and NKG2D.

In some embodiments, the second antigen is CD3, and the second antigen binding region comprises a VL and a VH, wherein the VL comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 14-16 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 18-20 respectively.

In some embodiments, the second antigen binding region comprises a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 17 and a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21. In some embodiments, the second antigen binding region comprises a VL comprising an amino acid sequence as set forth in SEQ ID NO: 17 and a VH comprising an amino acid sequence as set forth in SEQ ID NO: 21.

In some embodiments, the antibody comprises a scFv comprising the VL and the VH of the antibody that specifically binds to DLL3, and the scFv is linked to the N terminal of the VL or the VH of the second antigen binding region, optionally via a linker. In some embodiments, the antibody comprises: a first polypeptide chain comprising from the N terminal to C terminal: the scFv, an optional linker, the VL of the second antigen binding region, a light chain constant region (CL) , a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) ; and a second polypeptide chain comprising from the N terminal to C terminal: the VH of the second antigen binding region, a heavy chain constant region 1 (CH1) , a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) .

In some embodiments, the linker comprises an amino acid sequence selected from (G4S) n and GS (G4S) n, wherein n is an integer selected from 1-5, preferably the linker comprises an amino acid sequence as shown in SEQ ID NO: 25 or 26.

In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 22, and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 24. In other embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 23, and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 24.

In some embodiments, the bispecific antibody is a bispecific T-cell engager (BiTE) .

In another aspect, the present disclosure provides a bispecific antibody or an antigen binding fragment thereof, comprising a first antigen binding region that binds to DLL3 comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1) and a second antigen binding region that binds to CD3 comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2) , wherein the VL1 comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 1-3 respectively, and the VH1 comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 6-8 respectively; and the VL2 comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 14-16 respectively, and the VH2 comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 18-20 respectively.

In some embodiments of the bispecific antibody or the antigen binding fragment thereof disclosed herein, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 4 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 9; and the VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 17 and the VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21. In other embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 12; and the VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 17 and the VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21. In other embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 13; and the VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 17 and the VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21.

In some embodiments, the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 4 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 9; and the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 17 and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 21. In some embodiments, the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 12; and the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 17 and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 21. In some embodiments, the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 13; and the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 17 and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 21.

In some embodiments, the first antigen binding region comprises a scFv comprising the VL1 and the VH1, and the scFv is linked to the N terminal of the VL2 or the VH2, optionally via a linker. In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising from the N terminal to C terminal: the scFv, an optional linker, the VL2, a light chain constant region (CL) , a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) ; and a second polypeptide chain comprising from the N terminal to C terminal: the VH2, a heavy chain constant region 1 (CH1) , a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) .

In still another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the antibody or the antigen binding fragment thereof disclosed herein or the bispecific antibody or the antigen binding fragment thereof disclosed herein.

In yet another aspect, the present disclosure provides a vector comprising the nucleic acid disclosed herein.

In another aspect, the present disclosure provides a host cell comprising the nucleic acid disclosed herein or the vector disclosed herein.

In still another aspect, the present disclosure provides a pharmaceutical composition comprising (i) the antibody or the antigen binding fragment thereof disclosed herein, or the bispecific antibody or the antigen binding fragment thereof disclosed herein, and (ii) a pharmaceutically acceptable carrier or excipient.

In some embodiments of the pharmaceutical composition disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from a cytokine, an antibody, a chemotherapeutic agent and a small molecule drug. In some embodiments, the second therapeutic agent is selected from an interleukin (such as IL-2) , a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.

In yet another aspect, the present disclosure provides a conjugate comprising the antibody or the antigen binding fragment thereof disclosed herein or the bispecific antibody or the antigen binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.

In some embodiments of the conjugate disclosed herein, the chemical moiety can be selected from the group consisting of a therapeutic agent, a detectable moiety, and an immune stimulatory molecule.

In another aspect, the present disclosure provides a method of treating a cancer in a subject comprising administering to the subject an effective amount of the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.

In some embodiments of the method disclosed herein, the cancer is a DLL3 positive cancer. In some embodiments, the cancer is a neuroendocrine neoplasm, such as lung cancer, preferably small cell lung cancer.

In some embodiments, the method further comprises administering to the subject a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from a cytokine, an antibody, a chemotherapeutic agent and a small molecule drug. In some embodiments, the second therapeutic agent is selected from an interleukin (such as IL-2) , a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD- 1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.

In another aspect, the present disclosure provides use of the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein in the manufacture of a medicament for treating a cancer in a subject.

In still another aspect, the present disclosure provides the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein for use in treating a cancer in a subject.

In some embodiments of the use disclosed herein, the cancer is a DLL3 positive cancer. In some embodiments, the cancer is a neuroendocrine neoplasm, such as lung cancer, preferably small cell lung cancer. In some embodiments, the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from a cytokine, an antibody, a chemotherapeutic agent and a small molecule drug. In some embodiments, the second therapeutic agent is selected from an interleukin (such as IL-2) , a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

Figure 1 shows binding of anti-DLL3 mAb against recombinant human DLL3 as measured by ELISA.

Figure 2 shows schematic representation of one example of DLL3×CD3 BiTEs of the present invention.

Figure 3 shows binding of DLL3×CD3 BiTEs against recombinant human CD3 as measured by ELISA.

Figure 4 shows binding of DLL3×CD3 BiTEs against recombinant human DLL3 as measured by ELISA.

Figure 5 shows co-binding of DLL3×CD3 BiTEs against recombinant human DLL3 and CD3 as measured by ELISA.

Figure 6A shows binding of DLL3×CD3 BiTEs against DLL3 negative cell line HT55 as measured by flow cytometry.

Figure 6B shows binding of DLL3×CD3 BiTEs against DLL3 positive cell line LS174T-DLL3 as measured by flow cytometry.

Figure 6C shows binding of DLL3×CD3 BiTEs against DLL3 positive cell line H82 as measured by flow cytometry.

Figure 7A shows DLL3×CD3 BiTEs induced T cell activation in the presence of DLL3 negative cell line LS174T.

Figure 7B shows DLL3×CD3 BiTEs induced T cell activation in the presence of DLL3 positive cell line H82.

Figure 7C shows DLL3×CD3 BiTEs induced T cell activation in the presence of DLL3 positive cell line LS174T-DLL3.

Figure 8A shows killing of DLL3×CD3 BiTEs against DLL3 negative LS174T cells in the presence of human T cells.

Figure 8B shows killing of DLL3×CD3 BiTEs against DLL3 positive LS174T-DLL3 cells in the presence of human T cells.

Figure 9A shows tumor volume in the B-NDG mice xenografted with LS174T-DLL3 cells prophylactically treated with 100 or 500 μg/kg DLL3×CD3 BiTEs. The mice treated with physiological saline are used as negative control. Data represent mean tumor volumes ± SEM.

Figure 9B shows body weight of the B-NDG mice xenografted with LS174T-DLL3 cells prophylactically treated with 100 or 500 μg/kg DLL3×CD3 BiTEs. The mice treated with physiological saline are used as negative control. Data represent mean body weight ± SEM.

DETAILED DESCRIPTION OF THE INVENTION

The aforementioned features and advantages of the invention as well as additional features and advantages thereof will be more clearly understood hereafter as a result of a detailed description of the following embodiments when taken in conjunction with the drawings.

The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present invention. The embodiments shall not be construed to limit the scope of the present invention. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions.

Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds., "A multilingual glossary of biotechnological terms: (IUPAC Recommendations) " , Helvetica Chimica Acta (1995) , CH-4010 Basel, Switzerland; Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd Ed. ) , Vols. 1-3, Cold Spring Harbor Laboratory Press (1989) ; F. Ausubel et al, eds., "Current protocols in molecular biology" , Green Publishing and Wiley InterScience, New York (1987) ; Roitt et al., "Immunology (6th Ed. ) , Mosby/Elsevier, Edinburgh (2001) ; and Janeway et al., "Immunobiology" (6th Ed. ) , Garland Science Publishing/Churchill Livingstone, New York (2005) , as well as the general background art cited above.

Definitions

As used herein, singular forms “a” , “and, ” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an antibody” includes a plurality of antibodies and reference to “an antibody” in some embodiments includes multiple antibodies, and so forth.

Unless indicated or defined otherwise, the term "comprise" , and variations such as "comprises" and "comprising" , should be understood to imply the inclusion of a stated elements or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

As used herein, the term “antibody” refers to an immunoglobulin molecule which has ability to specifically bind to a specific antigen. Such molecule often comprises two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains of antibodies contain a binding domain that interacts with an antigen. The constant regions of antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation.

The heavy chain of immunoglobulins can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (fragment crystallizable) . The Fd region comprises the VH and CH1 domains and, in combination with the light chain, forms Fab (antigen-binding fragment) . The Fc fragment is responsible for the immunoglobulin effector functions, which includes, for example, complement fixation and binding to cognate Fc receptors of effector cells. The hinge region, found in IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer that allows the Fab portion to move freely in space relative to the Fc region. The hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses.

According to crystallographic studies, the immunoglobulin hinge region can be further subdivided structurally and functionally into three regions: the upper hinge, the core hinge, and the lower hinge. The upper hinge includes amino acids from the carboxyl end of CH1 to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges. The lower hinge region joins the amino terminal end of, and includes residues in the CH2 domain. Conformational changes permitted by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence may affect the effector functions of the Fc portion of the antibody.

A “light chain variable region” (VL) or “heavy chain variable region” (VH) consists of a “framework” region interrupted by three “complementarity determining regions” or “CDRs” . The framework regions serve to align the CDRs for specific binding to an epitope of an antigen. The CDRs include the amino acid residues of an antibody that are primarily responsible for antigen binding. From amino-terminus to carboxyl-terminus, both VL and VH domains comprise the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of a VL domain are also referred to herein, respectively, as LCDR1, LCDR2, and LCDR3; CDRs 1, 2, and 3 of a VH domain are also referred to herein, respectively, as HCDR1, HCDR2, and HCDR3.

The assignment of amino acids to each VL and VH domain is in accordance with any conventional definition of CDRs. Conventional definitions include, the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991) , the Chothia definition (Chothia &Lesk, J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342: 878-883, 1989) ; a composite of Chothia Kabat CDR in which CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular’s antibody modelling software; and the CONTACT definition of Martin et al. (world wide web bioinfo. org. uk/abs) . Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. The present disclosure can use CDRs defined according to any of these numbering systems, although preferred embodiments use Kabat defined CDRs.

Based on the amino acid sequence of heavy chain constant regions of the antibody, an immunoglobulin molecule can be divided into five classes (isotypes) : IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. The light chain of the antibody can be classified as a lambda (λ) chain or a kappa (κ) chain, based on the amino acid sequence of the light chain.

The term "antibody" as used herein should be understood in its broadest meaning, and includes monoclonal antibodies (including full-length monoclonal antibodies) , polyclonal antibodies, antibody fragments, and multi-specific antibodies containing at least two different antigen binding regions (e.g., bispecific antibodies) . The antibody may contain additional modifications, such as non-naturally occurring amino acids, mutations in Fc regions, and mutations in glycosylation sites. Antibodies also include post-translation modified antibodies, fusion proteins containing the antigenic determinants of the antibody, and immunoglobulin molecules containing any other modifications to antigen recognition sites, as long as these antibodies exhibit desired biological activity.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. That is, each antibodies constituting the population are the same, except for possible naturally occurring mutations in small amount. Monoclonal antibodies are highly specific and are directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced by hybridoma technology, and should not be interpreted as requiring production of antibodies by any specific method.

The term “bispecific antibody” in the context of the present invention is to be understood as an antibody having two different antigen-binding regions defined by different antibody sequences. This can be understood as different target binding but includes as well binding to different epitopes in one target. The term "bispecific antibody" as used herein should be understood in its broadest meaning, and includes full-length bispecific antibodies and antigen binding fragments thereof. The bispecific antibody may contain additional modifications, such as non-naturally occurring amino acids, mutations in Fc regions, and mutations in glycosylation sites. Bispecific antibodies also include post-translation modified antibodies, fusion proteins containing the antigenic determinants of the antibody, and immunoglobulin molecules containing any other modifications to antigen recognition sites, as long as these antibodies exhibit desired biological activity.

As used herein, the term “antigen binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

Examples of antigen binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially an Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vii) a dAb fragment, which consists of a VH domain; (viii) an isolated complementarity determining region (CDR) ; and (ix) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) ) . Such single chain antibodies are also intended to be encompassed within the term "antigen binding fragment" of an antibody. Furthermore, the term also includes a "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) , which forms an antigen binding region together with a complementary light chain polypeptide, and a modified version of any of the foregoing fragments, which retains antigen binding activity.

These antigen binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

As used herein, the term "binding" or "specifically binding" refers to a non-random binding reaction between two molecules, such as between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antibody (KD) , is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antibody: the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antibody. Alternatively, the affinity can also be expressed as the affinity constant (KA) , which is 1/KD.

Avidity is the measure of the strength of binding between an antibody and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antibody and the number of pertinent binding sites present on the antibody. Typically, an antibody will bind to an antigen with a dissociation constant (KD) of 10^-5 to 10 ^-12 M or less, and preferably 10^-7 to 10 ^-12 M or less and more preferably 10 ^-8 to 10 ^-12 M, and/or with a binding affinity of at least 10⁷ M ^-1, preferably at least 10⁸ M ^-1, more preferably at least 10⁹ M ^-1, such as at least 10¹² M ^-1. Any K_D value greater than 10 ^-4 M is generally considered to indicate non-specific binding. Specifically binding of an antibody to an antigen or antigenic determinant can be determined in any suitable manner known, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA) , enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art.

The term “epitope” refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. The epitope defines the smallest binding site of an antibody and therefore is the specific target of the antibody or antigen binding fragment thereof.

As used herein, the term “sequence identity” refers to the extent to which two sequences (amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X%identical to SEQ ID NO: Y” refers to X%identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X%of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs are employed for such calculations. Exemplary programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, 1988) , FASTA (Pearson and Lipman, 1988; Pearson, 1990) and gapped BLAST (Altschul et al., 1997) , BLASTP, BLASTN, or GCG (Devereux et al., 1984) .

Also, in determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

As used herein, the term "tumor associated antigen" refers to an antigen that is differentially expressed in cancer cells compared to normal cells, and therefore can be used to target cancer cells.

As used herein, the term “CD3” refers to the human CD3 protein complex, which has five peptide chains, γ chain, δ chain, ε chain, ζ chain and η chain, and is associated with the T cell receptor α and β chains to form a TCR-CD3 complex. The term includes any CD3 variants, isoforms and species homologs which are naturally expressed by cells, including T cells, or are expressed on cells transfected with genes or cDNA encoding the aforementioned chains.

As used herein, the term “bispecific T-cell engager” or “BiTE” refers to a polypeptide chain molecule having two antigen-binding domains, one of which binds to a T cell antigen and the second of which binds to an antigen present on the surface of target cells (See, PCT Publication WO 05/061547; Baeuerle et al., 2008, Drugs of the Future 33: 137-147; Bargou, et al., 2008, Science 321: 974-977, which are incorporated herein by reference in their entireties) . Thus, the BiTE of the disclosure has an antigen binding region that binds to DLL3 and a second antigen binding region that is directed towards a T cell antigen.

As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

The term “pharmaceutically acceptable” means that the carrier or excipient is compatible with the other ingredients of the composition and not substantially deleterious to the recipient thereof and/or that such carrier or excipient is approved or approvable for inclusion in a pharmaceutical composition for parenteral administration to humans.

As used herein, the terms "treatment" , "treating" , “treat” , and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptom of the disease. "Treatment" , as used herein, may include treatment of a disease or disorder (e.g. cancer) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease) ; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Treating may refer to any indicia of success in the treatment or amelioration or prevention of a cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the antibodies or compositions or conjugates disclosed herein to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with diseases (e.g. cancers) . The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

The term "effective amount" as used herein means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

The term “subject” , as used herein, refers to any mammalian subject for whom diagnosis, treatment or therapy is desired. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc.

The term “neuroendocrine neoplasm (NEN) ” as used herein refers to a neoplasm that originates in neuroendocrine cells. Neuroendocrine cells are a large class of cells that have neuroendocrine phenotypes and can produce a variety of hormones in the body, and are distributed throughout the body. Therefore, the neuroendocrine neoplasm can occur anywhere in the body, but the most common are neuroendocrine neoplasms of the digestive system such as the stomach, intestines, and pancreas, accounting for about 2/3 of all neuroendocrine neoplasms. According to the latest nomenclature by WHO in 2010, “neuroendocrine neoplasm (NEN) ” generally refers to all neoplasms derived from neuroendocrine cells, and the well-differentiated neuroendocrine neoplasm is named neuroendocrine tumor (NET) and the poorly differentiated neuroendocrine tumor is named neuroendocrine carcinoma (NEC) .

Anti-DLL3 antibodies

The present disclosure provides an antibody that specifically binds to DLL3, or an antigen binding fragment thereof, comprising a light chain variable region (VL) and a heavy chain variable region (VH) , wherein the VL comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 1-3 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 6-8 respectively.

In some embodiments, CDR sequences are defined according to Kabat numbering system.

When CDR sequences are defined according to Kabat numbering system, the VL of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 1 (RSSQSIVHSNGDTYLE) , SEQ ID NO: 2 (KVSNRFS) and SEQ ID NO: 3 (FQGSHVPWT) respectively, and the VH of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth in SEQ ID NO: 6 (SYWMN) , SEQ ID NO: 7 (MIHPSDSETRLNQKFKD) and SEQ ID NO: 8 (WDYYDYAWFAY) respectively.

In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 4, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 9. In other embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 12. In other embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 13.

In some embodiments, the VL comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 4 or 11 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to DLL3. In some embodiments, the VH comprises a functional variant of the amino acid sequence as set forth in any one of SEQ ID NOs: 9, 12 and 13 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to DLL3.

The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%sequence identity to the amino acid sequence of the parent polypeptide.

In the context of the functional variant, the number of the inserted, deleted and/or substituted amino acid is preferably no more than 40%of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1-33%, and more preferably 5-30%, more preferably 10-25%, more preferably 15-20%. For example, the number of the inserted, deleted and/or substituted amino acid can be 1-20, preferably 1-10, more preferably 1-7, still more preferably 1-5, and most preferably 1-2. In a preferred embodiment, the number of the inserted, deleted and/or substituted amino acid is 1, 2, 3, 4, 5, 6, or 7.

In some embodiments, the insertion, deletion and/or substitution can be performed at framework (FR) regions, e.g., at FR1, FR2, FR3, and/or FR4.

In some embodiments, the substitution of one or more amino acid (s) can be conservative substitution of one or more amino acid (s) . Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

In a preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 4 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 9. In another preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 12. In another preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 13.

In some embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

Based on the amino acid sequence of heavy chain constant regions of the antibody, a immunoglobulin molecule can be divided into five classes (isotypes) : IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. The light chain of the antibody can be classified as a lambda (λ) chain or a kappa (κ) chain, based on the amino acid sequence of the light chain. The antibodies disclosed herein can be of any classes or subtypes above.

In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD. In some embodiments, the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In a preferred embodiment, the antibody is an IgG1 antibody.

The antibody disclosed herein can be an intact antibody or the antigen binding fragment thereof. The antigen binding fragment can be any fragments of the antibody that retain the ability to specifically bind to DLL3. Examples of antigen binding fragments include but are not limited to a Fab fragment; a F (ab') 2 fragment; a Fab' fragment; a Fd fragment; a Fd' fragment; a Fv fragment; a scFv fragment; a dAb fragment; an isolated complementarity determining region (CDR) ; a nanobody; a linear antibody comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) , and a modified version of any of the foregoing fragments, which retains antigen binding activity.

In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab’, F (ab') ₂, Fv, scFv, and ds-scFv. In a preferred embodiment, the antigen binding fragment is Fab. In another preferred embodiment, the antigen binding fragment is Fv. In another preferred embodiment, the antigen binding fragment is scFv.

In some embodiments, the light chain comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 5 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to DLL3. In some embodiments, the heavy chain comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 10 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to DLL3.

In some embodiments, the number of the inserted, deleted and/or substituted amino acid is preferably no more than 40%of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1-33%, and more preferably 5-30%, more preferably 10-25%, more preferably 15-20%. For example, the number of the inserted, deleted and/or substituted amino acid can be 1-50, preferably 1-20, more preferably 1-10, still more preferably 1-5. In a preferred embodiment, the number of the inserted, deleted and/or substituted amino acid is 1, 2, 3, 4, 5, 6, or 7.

In some embodiments, the insertion, deletion and/or substitution can be performed at framework (FR) regions, e.g., at FR1, FR2, FR3 and/or FR4; and/or constant regions, e.g., CL, CH1, CH2 and/or CH3.

In some embodiments, the substitution of one or more amino acid (s) can be conservative substitution of one or more amino acid (s) . Examples of conservative substitutions are as described above.

In a preferred embodiment, the antibody comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 5 and a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 10.

In other embodiments, the antibody is a bispecific or a multi-specific antibody. In some embodiments, the antibody is a bispecific antibody which further comprises a second antigen binding region that binds to a second antigen. In some embodiments, the second antigen is a tumor associated antigen or an immune cell antigen.

Many tumor associated antigens associated with specific cancers have been identified in the art. In some embodiments, tumor-associated antigens are antigens that can potentially stimulate an obvious tumor-specific immune response. Some of these antigens are encoded by normal cells, but not necessarily expressed by normal cells. These antigens can be characterized as those that are usually silent (i.e., not expressed) in normal cells, those that are expressed only during certain stages of differentiation, and those that are expressed over time, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cell genes such as oncogenes (e.g. activated ras oncogene) , suppressor genes (e.g. mutant p53) , and fusion proteins produced by internal deletions or chromosomal translocations. Other cancer antigens can be encoded by viral genes, such as those carried on RNA and DNA tumor viruses. Many other tumor associated antigens and antibodies against them are known and/or commercially available, and can also be produced by those skilled in the art.

Examples of tumor associated antigens include but are not limited to 5T4, alphafetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, AXL, EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, melanoma-associated antigen, MUC-1, mutated p53, mutated ras, ROR1, GPC3, VEGFR2, and combinations thereof.

In some embodiments, the second antigen is a T-cell antigen. In some embodiments, the T-cell antigen is selected from the group consisting of T cell receptor (TCR) , CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137) , and NKG2D or any combination thereof. In some embodiments, the T-cell antigen is CD3, and the second antigen binding region binds to any of γ chain, δ chain, ε chain, ζ chain and η chain of CD3.

In some embodiments, CDR sequences are defined according to Kabat numbering system. When using Kabat defined CDR sequences, the VL of the second antigen binding region disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as shown in SEQ ID NO: 14 (RSSTGAVTTSNYAN) , SEQ ID NO: 15 (GANKRAP) and SEQ ID NO: 16 (ALWYSNLWV) respectively, and the VH of the second antigen binding region disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as shown in SEQ ID NO: 18 (TYAMN) , SEQ ID NO: 19 (RIRSKYNNYATYYADSVKG) and SEQ ID NO: 20 (HGNFGSSYVSYFAY) respectively.

In some embodiments, the second antigen binding region comprises a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 17 and a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21.

In some embodiments, the VL comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 17 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to CD3. In some embodiments, the VH comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 21 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to CD3.

In some embodiments, the number of the inserted, deleted and/or substituted amino acid is preferably no more than 40%of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1-33%, and more preferably 5-30%, more preferably 10-25%, more preferably 15-20%. For example, the number of the inserted, deleted and/or substituted amino acid can be 1-20, preferably 1-10, more preferably 1-7, still more preferably 1-5, and most preferably 1-2. In a preferred embodiment, the number of the inserted, deleted and/or substituted amino acid is 1, 2, 3, 4, 5, 6, or 7.

In a preferred embodiment, the second antigen binding region comprises a VL comprising an amino acid sequence as set forth in SEQ ID NO: 17 and a VH comprising an amino acid sequence as set forth in SEQ ID NO: 21.

In a preferred embodiment, the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 22 and the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 24. In other preferred embodiment, the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 23 and the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 24.

In some embodiments, the bispecific antibody is a bispecific T-cell engager (BiTE) . In some embodiments, the bispecific antibody is in form of an HBiTE as described in PCT application No. PCT/US2018/016524 (which is incorporated herein by reference in its entirety) . In the HBiTE, the light chain, from N-terminus to C-terminus, comprises an anti-target VL domain, an anti-CD3 VL-CL and a monomeric human IgG1 Fc (e.g., mFc7.2) ; and the heavy chain, from N-terminus to C-terminus, comprises an anti-target VH domain, an anti-CD3 VH-CH1 and a monomeric human IgG1 Fc (e.g., mFc7.2) . Monomeric Fc7.2 contains two amino acid mutations (T366L and Y407H) capable of inhibiting Fc homodimerization.

Bispeicific antibodies

The present disclosure provides a bispecific antibody or an antigen binding fragment thereof, comprising a first antigen binding region that binds to DLL3 comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1) and a second antigen binding region that binds to CD3 comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2) , wherein the VL1 comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 1-3 respectively, and the VH1 comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 6-8 respectively; and the VL2 comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 14-16 respectively, and the VH2 comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 18-20 respectively.

When CDR sequences are defined according to Kabat numbering system, the VL1 of the bispecific antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 1 (RSSQSIVHSNGDTYLE) , SEQ ID NO: 2 (KVSNRFS) and SEQ ID NO: 3 (FQGSHVPWT) respectively, the VH1 of the bispecific antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth in SEQ ID NO: 6 (SYWMN) , SEQ ID NO: 7 (MIHPSDSETRLNQKFKD) and SEQ ID NO: 8 (WDYYDYAWFAY) respectively, the VL2 of the bispecific antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 14 (RSSTGAVTTSNYAN) , SEQ ID NO: 15 (GANKRAP) and SEQ ID NO: 16 (ALWYSNLWV) respectively, and the VH2 of the bispecific antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth in SEQ ID NO: 18 (TYAMN) , SEQ ID NO: 19 (RIRSKYNNYATYYADSVKG) and SEQ ID NO: 20 (HGNFGSSYVSYFAY) respectively.

In some embodiments, the VL1 comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 4 or 11 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to DLL3. In some embodiments, the VH1 comprises a functional variant of the amino acid sequence as set forth in any one of SEQ ID NOs: 9, 12 and 13 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to DLL3. In some embodiments, the VL2 comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 17 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to CD3. In some embodiments, the VH2 comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 21 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to CD3.

In a preferred embodiment, the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 4 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 9; and the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 17 and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 21. In another preferred embodiment, the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 12; and the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 17 and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 21. In another preferred embodiment, the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 13; and the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 17 and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 21.

In some embodiments, the first antigen binding region comprises a scFv comprising the VL1 and the VH1, and the scFv is linked to the N terminal of the VL2 or the VH2, optionally via a linker. In some embodiments, the scFv is linked to the N terminal of the VL2 optionally via a linker. In some embodiments, the scFv is linked to the N terminal of the VH2 optionally via a linker. In some embodiments, the scFv is formed by linking the VL1 and the VH1 via a linker.

In some embodiments, the linker may be any flexible linker. In some embodiments, the linker comprises an amino acid sequence of (G4S) n, wherein n is an integer selected from 1-5. In some embodiments, the linker may comprise an amino acid sequence of GGGGS (SEQ ID NO: 27) . In some embodiments, the linker may comprise an amino acid sequence of GGGGSGGGGS (SEQ ID NO: 28) . In some embodiments, the linker may comprise an amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 25) . In some embodiments, the linker may comprise an amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 29) . In some embodiments, the linker may comprise an amino acid sequence of GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 30) . In a preferred embodiment, the linker comprises an amino acid sequence as shown in SEQ ID NO: 25.

In other embodiments, the linker comprises an amino acid sequence of GS (G4S) n, wherein n is an integer selected from 1-5. In some embodiments, the linker may comprise an amino acid sequence of GSGGGGS (SEQ ID NO: 31) . In some embodiments, the linker may comprise an amino acid sequence of GSGGGGSGGGGS (SEQ ID NO: 26) . In some embodiments, the linker may comprise an amino acid sequence of GSGGGGSGGGGSGGGGS (SEQ ID NO: 32) . In some embodiments, the linker may comprise an amino acid sequence of GSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 33) . In some embodiments, the linker may comprise an amino acid sequence of GSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 34) . In a preferred embodiment, the linker comprises an amino acid sequence as shown in SEQ ID NO: 26.

The bispecific antibody disclosed herein may comprise an Fc region comprising CH2 and CH3 of an antibody.

The Fc region may be of any isotype, including, but not limited to, IgG1, IgG2, IgG3 and IgG4, and may comprise one or more mutations or modifications. In one embodiment, the Fc region is of IgG1 isotype or derived therefrom, optionally with one or more mutations or modifications. In another embodiment, the Fc region is of IgG4 isotype or derived therefrom, optionally with one or more mutations or modifications. In one embodiment, the Fc region is human IgG1 Fc.

In one embodiment, the Fc region has a decreased effector function, e.g., decreased ADCC, ADCP, CDC, and/or C1q, FcγRI, FcγRII, or FcγRIIIA binding. For example, the Fc region may be of an IgG1 isotype, or a non-IgG1 type, e.g. IgG2, IgG3 or IgG4, which has been mutated such that the ability to mediate effector function has been reduced or even eliminated. Such mutations have e.g. been described in Dall'Acqua WF et al., J Immunol. 177 (2) : 1129-1138 (2006) and Hezareh M, J Virol.; 75 (24) : 12161-12168 (2001) . For example, the Fc region may comprise the amino acid sequence having one or more of the following amino acid substitutions: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G, as compared with the wild type sequence.

In one embodiment, the Fc region comprises a mutation removing the acceptor site for Asn-linked glycosylation or is otherwise manipulated to change the glycosylation properties. For example, in an IgG1 Fc region, an N297Q mutation can be used to remove an Asn-linked glycosylation site. Accordingly, in a specific embodiment, Fc region comprises an IgG1 sequence with an N297Q mutation.

In a further embodiment, the Fc region is glyco-engineered to reduce fucose and thus enhance ADCC, e.g. by addition of compounds to the culture media during antibody production as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105: 350 or by using FUT8 knockout cells, e.g. as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87: 614. ADCC may alternatively be optimized using the method described by et al. (1999) Nature Biotech 17: 176. In a further embodiment, the Fc region has been engineered to enhance complement activation, e.g. as described in Natsume et al. (2009) Cancer Sci. 100: 2411.

In some embodiments, the Fc region comprises modifications or mutations that can inhibit Fc homodimerization. In some embodiments, the Fc region comprises a variant of a human IgG1 Fc wildtype sequence. The variant can comprise amino acid substitutions at positions T366 and Y407 of human IgG1 (Kabat numbering) . Preferably, T366 is substituted with L (Leucine) . Preferably, Y407 is substituted with I (Isoleucine) , F (Phenylalanine) , L (Leucine) , M (Methionine) , H (Histidine) , K (Lysine) , S (Serine) , Q (Glutamine) , T (Threonine) , W (Tryptophan) , A (Alanine) , G (Glycine) or N (Asparagine) . More preferably, Y407 is substituted with H. In one embodiment, T366 is substituted with L, and Y407 is substituted with H.

In some embodiments, the Fc region can be a monomeric human IgG1 Fc (e.g., mFc7.2) as described in PCT application No. PCT/US2018/016524, which is incorporated herein by reference in its entirety.

In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising from the N terminal to C terminal: the scFv, an optional linker, the VL2, a light chain constant region (CL) , a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) ; and a second polypeptide chain comprising from the N terminal to C terminal: the VH2, a heavy chain constant region 1 (CH1) , a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) .

The bispecific antibody disclosed herein may further comprise a hinge region of an antibody.

The hinge region of an IgG class antibody refers to a short amino acid sequence region between the CH1 and CH2 portions of the heavy chain that is relatively flexible in the antibody native state. The hinge region may comprise part or all of a wild type hinge sequence or a variant thereof having one or more substitutions.

In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising from the N terminal to C terminal: the scFv, an optional linker, the VL2, a light chain constant region (CL) , a hinge region, a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) ; and a second polypeptide chain comprising from the N terminal to C terminal: the VH2, a heavy chain constant region 1 (CH1) , a hinge region, a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) .

In some embodiments, each of the CH1, CH2, CH3 and hinge region is independently derived from immunoglobulin isotype IgG (e.g. human IgG) , preferably derived from IgG subtype selected from the group consisting of IgG1, IgG2 and IgG4 (e.g. human IgG1, IgG2 and IgG4) . In some embodiments, the CL is derived from λ light chain or κ light chain.

In some embodiments, one or both of the CH2 comprise at least one amino acid mutation that is capable of decreasing the effector function of the bispecific antibody. For example, the CH2 may comprise at least one amino acid substitution selected from E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G, or any combination thereof. In some embodiments, the at least one mutation is selected from L234A, L235A, G237A, P329G or any combination thereof. In a preferred embodiment, the at least one mutation is selected from L234A, L235A, G237A, and P329G. In some embodiments, the at least one mutation is selected from L234F, L235E, P329G or combination thereof. In a preferred embodiment, the at least one mutation is selected from L234F, L235E, and P329G.

In some embodiments, one or both of the CH3 comprise at least one amino acid mutation that is capable of decreasing homodimerization of the first and second polypeptide chains. In preferred embodiments, amino acid T366 of the one or both CH3 is substituted with L (Leucine) , and amino acid Y407 of the one or both CH3 is substituted with I (Isoleucine) , F (Phenylalanine) , L (Leucine) , M (Methionine) , H (Histidine) , K (Lysine) , S (Serine) , Q (Glutamine) , T (Threonine) , W (Tryptophan) , A (Alanine) , G (Glycine) or N (Asparagine) . In one embodiment, amino acid T366 of the one or both CH3 is substituted with L, and amino acid Y407 of the one or both CH3 is substituted with H. In a preferred embodiment, in the CH3 of both the first and second polypeptide chains, T366 is substituted with L and Y407 is substituted with H.

The linker can be those as describe above. For example, the linker may be any flexible linker. In some embodiments, the linker comprises an amino acid sequence selected from (G4S) n and GS (G4S) n, wherein n is an integer selected from 1-5, preferably the linker comprises an amino acid sequence as shown in SEQ ID NO: 25 or 26.

In some embodiments, the first polypeptide chain comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 22 or 23 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to DLL3 and CD3. In some embodiments, the second polypeptide chain comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 24 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to CD3.

In a preferred embodiment, the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 22; and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 24. In other preferred embodiment, the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 23; and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 24.

Nucleic acids

The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the antibody or the antigen binding fragment thereof disclosed herein or the bispecific antibody or the antigen binding fragment thereof disclosed herein.

The term "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. The nucleic acid can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2', 3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

For example, the invention provides nucleic acid molecules encoding any one of the heavy chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%or at least 99%identical to nucleic acids encoding any one of the heavy chain variable region sequences disclosed herein.

For example, the invention provides nucleic acid molecules encoding any one of the light chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%or at least 99%identical to nucleic acids encoding any one of the light chain variable region sequences disclosed herein.

For example, the invention provides nucleic acid molecules encoding a heavy chain variable region sequence that comprises the CDR sequences of any one of the heavy chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that encode a heavy chain variable region sequence that comprises CDR sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR sequences of any one of the heavy chain variable region sequences disclosed herein.

For example, the invention provides nucleic acid molecules encoding a light chain variable region sequence that comprises the CDR sequences of any one of the light chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that encode a light chain variable region sequence that comprises CDR sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR sequences of any one of the light chain variable region sequences disclosed herein.

In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) . In some embodiments, the invention provides a ribonucleic acid (RNA) comprising a nucleotide sequence encoding the antibody disclosed herein. In some embodiments, the invention provides a deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding the antibody disclosed herein.

In some embodiments, the deoxyribonucleic acid (DNA) may be introduced into the cells of a human body in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the invention is comprised in a vector or a delivering agent. In some embodiments, the deoxyribonucleic acid (DNA) of the invention is integrated into the genome of a cell.

In some embodiments, the ribonucleic acid (RNA) may be introduced into the cells of a human body in vivo. In some embodiments, the ribonucleic acid (RNA) of the invention is comprised in a vector or a delivering agent.

Vectors

The present disclosure provides a vector comprising the nucleic acid disclosed herein.

In some embodiments, the vector is an expression vector capable of expressing a polypeptide comprising a heavy or light chain variable region of the antibody. For example, the invention provides expression vectors comprising any of the nucleic acid molecules mentioned above.

Any vector may be suitable for the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, a RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector (AAV) , a lentiviral vector, or any combination thereof. Suitable exemplary vectors include e.g., pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO. 1 GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid) , pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES Luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

An expression vector may be any suitable recombinant expression vector. Suitable vectors comprise those designed for propagation and expansion or for expression or both, such as plasmids and viruses. For example, a vector may be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md. ) , the pBluescript series (Stratagene, LaJolla, Calif. ) , the pET series (Novagen, Madison, Wis. ) , the pGEX series (Pharmacia Biotech, Uppsala, Sweden) , and the pEX series (Clontech, Palo Alto, Calif. ) . Bacteriophage vectors, such as λGT10, λGT11, λZapII (Stratagene) , λEMBL4, and λNM1149, also may be used. Examples of plant expression vectors useful in the context of the disclosure comprise pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech) . Examples of animal expression vectors useful in the context of the disclosure comprise pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech) .

Recombinant expression vectors may be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N. Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley &Sons, NY, 1994. Constructs of expression vectors, which are circular or linear, may be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems may be derived, e.g., from ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, and the like.

For example, the vector may be an adenoviral vector comprising a nucleotide sequence encoding the antibody disclosed herein. The vector may be administered into the body of a subject, and then enter into a cell of the subject in vivo, thereby the nucleotide sequence encoding the antibody disclosed herein is integrated into the genome of the cell, and subsequently the cell expresses the antibody disclosed herein.

Host Cells

The present disclosure provides a host cell comprising the nucleic acid disclosed herein or the vector disclosed herein.

Any cell may be used as a host cell for the nucleic acids or the vectors of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cells such as a mammalian cell. Suitable prokaryotic cells include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae such as Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., Serratia marcescans, and Shigella; Bacilli such as B. subtilis and B. licheniformis; Pseudomonas such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T and HEK293FS.

The host cell of the invention is prepared by introducing the vector disclosed herein or the nucleic acid disclosed herein in vitro or ex vivo. The host cell of the invention may be administered into the body of a subject, and the host cell expresses the antibody disclosed herein in vivo.

The invention provides host cells into which any of the vectors mentioned above have been introduced. The invention further provides a method of preparing the antibody of the invention, wherein the method comprises a) culturing the host cell of the fourth aspect of the invention under a condition suitable for the production of the antibody; and b) obtaining the antibody from the culture.

Pharmaceutical compositions

The present disclosure provides a pharmaceutical composition comprising the antibody or the antigen binding fragment thereof disclosed herein, or the bispecific antibody or the antigen binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.

The antibody or antigen binding fragment thereof or agents of the invention (also referred to herein as “active compounds” ) , and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody or antigen binding fragment thereof or agent and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5%human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from a cytokine, an antibody, a chemotherapeutic agent and a small molecule drug. In some embodiments, the second therapeutic agent is selected from an interleukin (such as IL-2) , a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.

In some embodiments, the second therapeutic agent is a cytokine. The examples of cytokine include, but are not limited to, interleukin (such as IL-2, IL-7, IL-10, IL-12, IL-15) and interferon (such as IFNα, IFNγ) . In some embodiments, the second therapeutic agent is an interleukin. In a preferred embodiment, the second therapeutic agent is IL-2.

In some embodiments, the second therapeutic agent is a chemotherapeutic agent. The chemotherapeutic agents can include, for example, cytotoxic agents, anti-metabolite agents (e.g., folate antagonists, purine analogs, pyrimidine analogs, etc. ) , topoisomerase inhibitors (e.g., camptothecin derivatives, anthracenedione, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc. ) , anti-microtubule agents (e.g., taxanes, vinca alkaloids) , protein synthesis inhibitors (e.g., cephalotaxine, camptothecin derivatives, quinoline alkaloids) , alkylating agents (e.g., alkyl sulfonates, ethylenimines, nitrogen mustards, nitrosoureas, platinum derivatives, triazenes, etc. ) , alkaloids, terpenoids, and kinase inhibitors.

The pharmaceutical composition of the invention can be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation) , transdermal (i.e., topical) , transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA) ; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N. J. ) or phosphate buffered saline (PBS) . In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The active compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

The invention provides therapeutic compositions comprising the antibody or antigen binding fragment thereof of the present invention. Therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM) , DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights) , semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

Conjugates

The present disclosure provides a conjugate comprising the antibody or the antigen binding fragment thereof disclosed herein or the bispecific antibody or the antigen binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.

In the context of the present disclosure, a "conjugate" is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety. The chemical moiety can be, for example, a drug, toxin, therapeutic agent, detectable label, protein, nucleic acid, lipid, nanoparticle, carbohydrate or recombinant virus. An antibody conjugate is often referred to as an "immunoconjugate" . When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent) , the conjugate is often referred to as an "antibody-drug conjugate" or "ADC. "

The term "conjugated" or "linked" may refer to making two polypeptides into one contiguous polypeptide molecule. In one embodiment, an antibody is joined to a chemical moiety. In another embodiment, an antibody joined to a chemical moiety is further joined to a lipid or other molecule to a protein or peptide to increase its half-life in the body. The linkage can be either by chemical or recombinant means. In one embodiment, the linkage is chemical, wherein a reaction between the antibody moiety and the chemical moiety has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the chemical moiety.

A chemical moiety can be linked to the antibody of the invention using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching a chemical moiety to the antibody varies according to the chemical structure of the chemical moiety. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH) , free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the chemical moiety. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of known linker molecules. The linker can be any molecule used to join the antibody to the chemical moiety. The linker is capable of forming covalent bonds to both the antibody and to the chemical moiety. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the chemical moiety are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the chemical moiety from the antibody when the immunoconjugate has reached its target site. Therefore, in these circumstances, immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site.

Cleavage of the linker to release the chemical moiety from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.

In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules) , drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein) . In general, the antibodies or portion thereof is derivatized such that the binding to the target antigen is not affected adversely by the derivatization or labeling. For example, the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bi-specific antibody or a diabody) , a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a strep tavidin core region or a polyhistidine tag) .

One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types) . Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m- maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate) . Such linkers are commercially available.

In some embodiments of the conjugate disclosed herein, the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immune stimulatory molecule.

In some embodiments, the therapeutic agent includes but is not limited to immunomodulators, radioactive compounds, enzymes (for example perforin) , chemotherapeutic agents (for example cis-platin) , or a toxin. In some embodiments, the therapeutic agent can be such as maytansine, geldanamycin, tubulin inhibitors such as tubulin binding agents (e.g., auristatins) , or minor groove binding agents such as calicheamicin.

Other suitable therapeutic agents include such as, small molecule cytotoxic agents, i.e. compounds with the ability to kill mammalian cells having a molecular weight of less than 700 Daltons. Such compounds could also contain toxic metals capable of having a cytotoxic effect. Furthermore, it is to be understood that these small molecule cytotoxic agents also include pro-drugs, i.e. compounds that decay or are converted under physiological conditions to release cytotoxic agents. Examples of such agents include cis-platin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E vincristine and doxorubicin; peptide cytotoxins, i.e. proteins or fragments thereof with the ability to kill mammalian cells, for example, ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNase and RNase; radio-nuclides, i.e. unstable isotopes of elements which decay with the concurrent emission of one or more of a or β particles, or γ rays, for example, iodine-131 , rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225 and astatine-213; chelating agents may be used to facilitate the association of these radionuclides to the molecules, or multimers thereof.

In some embodiments, the detectable moiety can be selected from the group consisting of biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. A detectable moiety for diagnostic purposes includes for instance, fluorescent labels, radiolabels, enzymes, nucleic acid probes and contrast reagents.

The antibody can be conjugated with a detectable marker; for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as computed tomography (CT) , computed axial tomography (CAT) scans, magnetic resonance imaging (MRI) , nuclear magnetic resonance imaging NMRI) , magnetic resonance tomography (MTR) , ultrasound, fiberoptic examination, and laparoscopic examination) . Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI) . For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) .

The antibody or antigen binding fragment can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When a antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. The antibody or antigen binding fragment may also be conjugated with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be conjugated with an enzyme or a fluorescent label.

The antibody may be fused to a self-labelling protein tag (e.g. HaloTag) . For example, the protein tag could be cloned at the end of a constant region. HaloTag is a self-labelling protein tag derived from a bacterial enzyme (ahaloalkane dehalogenase) , designed to covalently bind to a synthetic ligand. In some instances, the synthetic ligand comprises a chloroalkane linker attached to a fluorophore, such as a near-infrared fluorophore (Los et al. (2008) ACS Chem Biol. 3 (6) : 373-82) .

The antibody may be labeled with a magnetic agent, such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) , and manganese.

Paramagnetic particles such as superparamagnetic iron oxide are also of use as labels. The antibody may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) . In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

The antibody can also be labeled with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect expression of a target antigen by x-ray, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I.

In some embodiments, the immune stimulatory molecule is an immune effector molecule which stimulates immune response. For example, the immune stimulatory molecule can be cytokines such as IL-2 and IFN-γ, chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, complement activators; viral/bacterial protein domains, or viral/bacterial peptides.

Therapeutic methods

The present disclosure provides a method of treating a cancer in a subject, comprising administering to the subject an effective amount of the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.

In some embodiments of the method disclosed herein, the cancer is a DLL3 positive cancer. In some embodiments, the cancer is a neuroendocrine neoplasm. The neuroendocrine neoplasm includes neuroendocrine tumor and neuroendocrine carcinoma. Examples of the neuroendocrine neoplasm include, but are not limited to, neuroendocrine tumors of pancreatic origin, neuroendocrine tumors of gastrointestinal origin, pulmonary neuroendocrine tumors, pituitary neuroendocrine tumors, head and neck neuroendocrine tumors, breast neuroendocrine tumors, neuroendocrine tumors of the genitourinary system, adrenal neuroendocrine tumors, and cutaneous neuroendocrine tumors. In a preferred embodiment, the cancer is lung cancer, preferably small cell lung cancer.

In some embodiments, dosage administered to a subject may vary with the embodiment, the medicament employed, the method of administration, and the site and subject being treated. However, a dose should be sufficient to provide a therapeutic response. A clinician may determine the effective amount to be administered to a human or other subject in order to treat a medical condition. The precise amount required to be therapeutically effective may depend upon numerous factors, e.g., such as the activity of the antibody, and the route of administration.

A dose of the antibodies, compositions or conjugates described herein may be administered to a mammal at one time or in a series of sub-doses administered over a suitable period of time, e.g., on a daily, semi-weekly, weekly, bi-weekly, semi-monthly, bi-monthly, semi-annual, or annual basis, as needed. A dosage unit comprising an effective amount of antibodies, compositions or conjugates may be administered in a single daily dose, or the total daily dosage may be administered in two, three, four, or more divided doses administered daily, as needed.

A suitable means of administration may be selected by a medical practitioner. Route of administration may be parenteral, for example, administration by injection, transnasal administration, transpulmonary administration, or transcutaneous administration. Administration may be systemic or local by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibodies, compositions or conjugates are selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, such as orally. Dose and method of administration may vary depending on the weight, age, condition, and the like of the subject, and may be suitably selected.

In some embodiments, the method further comprises administering to the subject a second therapeutic agent. In certain embodiments, the antibody, composition or conjugate disclosed herein is administered prior to, substantially simultaneously with, or after the administration of the second therapeutic agent.

In some embodiments, the second therapeutic agent is selected from a cytokine, an antibody, a chemotherapeutic agent and a small molecule drug. In some embodiments, the second therapeutic agent is selected from an interleukin (such as IL-2) , a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.

Medical uses

The present disclosure provides use of the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein in the manufacture of a medicament for treating a cancer in a subject.

The present disclosure also provides the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein for use in treating a cancer in a subject.

In some embodiments of the use disclosed herein, the cancer is a DLL3 positive cancer. In some embodiments, the cancer is a neuroendocrine neoplasm, such as lung cancer, preferably small cell lung cancer.

In some embodiments, the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from a cytokine, an antibody, a chemotherapeutic agent and a small molecule drug. In some embodiments, the second therapeutic agent is selected from an interleukin (such as IL-2) , a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid. In some embodiments, the second therapeutic agent is a cytokine. The examples of cytokine include, but are not limited to, interleukin (such as IL-2, IL-7, IL-10, IL-12, IL-15) and interferon (such as IFNα, IFNγ) . In some embodiments, the second therapeutic agent is an interleukin. In a preferred embodiment, the second therapeutic agent is IL-2.

Methods for diagnosis and detection

The present disclosure provides a method for detecting DLL3 protein in vitro or in vivo. In some cases, DLL3 expression is detected in a biological sample. The sample can be any sample, including, but not limited to, blood samples, tissue from biopsies, autopsies and pathology specimens. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine. A biological sample is typically obtained from a mammal, such as a human or non-human primate.

The present disclosure also provides a method of determining if a subject has a DLL3 positive cancer by contacting a sample from the subject with anti-DLL3 antibodies disclosed herein; and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample identifies the subject as having the cancer.

In another embodiment, the present disclosure provides a method of diagnosis of a DLL3 positive cancer in a subject by contacting a sample from a subject with anti-DLL3 antibodies disclosed herein; and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample confirms the diagnosis of the cancer in the subject.

In some embodiments, the control sample is a sample from a subject without cancer. In a particular embodiment, the sample is a blood or tissue sample.

In some embodiments of the methods of diagnosis and detection, the anti-DLL3 antibody is directly labeled with a detectable label. In another embodiment, the anti-DLL3 antibody (the first antibody) is unlabeled and a second antibody or other molecule that can bind the first is labeled. As is well known to one of skill in the art, a secondary antibody is chosen that is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody may be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.

Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In an alternative embodiment, DLL3 can be assayed in a biological sample by a competition immunoassay utilizing DLL3 protein standards labeled with a detectable substance and an unlabeled anti-DLL3 antibody. In this assay, the biological sample, the labeled DLL3 protein standards and the anti-DLL3 antibody are combined and the amount of labeled DLL3 protein standard bound to the unlabeled antibody is determined. The amount of DLL3 in the biological sample is inversely proportional to the amount of labeled DLL3 protein standard bound to the anti-DLL3 antibody.

The immunoassays and methods disclosed herein can be used for a number of purposes. In one embodiment, the anti-DLL3 antibody may be used to detect the production of DLL3 in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of DLL3 in a biological sample, such as a tissue sample, or a blood or serum sample. In some examples, the DLL3 is cell-surface DLL3.

Kits

The present disclosure provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as the antibodies or the antigen binding fragment disclosed herein.

In a specific embodiment, the kit comprises a first container containing the antibodies disclosed herein. In a specific embodiment, the kit comprises a first container that is a vial containing the antibodies as a lyophilized sterile powder under vacuum, and the kit further comprises a second container comprising a pharmaceutically acceptable fluid.

In a specific embodiment, provided herein is an injection device containing the antibodies. In a specific embodiment, the injection device comprises the antibody in sterile solution. In a specific embodiment, the injection device is a syringe.

In one embodiment, the kit is provided for detecting DLL3 in a biological sample, such as a blood sample or tissue sample. For example, to confirm a cancer diagnosis in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. Kits for detecting a polypeptide will typically comprise an anti-DLL3 antibody, such as any of the monoclonal antibodies disclosed herein. In a further embodiment, the antibody is labeled (for example, with a fluorescent, radioactive, or an enzymatic label) .

In one embodiment, a kit includes instructional materials disclosing means of use of an anti-DLL3 antibody. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files) . The kits may also include additional components to facilitate the application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like) . The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.

In one embodiment, the diagnostic kit comprises an immunoassay. The method of detecting DLL3 in a biological sample generally includes the steps of contacting the biological sample with an anti-DLL3 antibody. The antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

EXAMPLES

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

293 free style (293FS) cells, CHO-Scells and protein A agarose were purchased from ThermoFisher Scientific. DLL3-positive cell line NCI-H82 (human small cell lung cancer cell) and DLL3-negative cell line LS174T were purchased from National Collection of Authenticated Cell Cultures. DLL3-negative cell line HT55 was purchased from NanJing Cobioer biosciences Co., Ltd.

Human DLL3 protein (His Tag) , rhesus DLL3 protein and human CD3 protein were purchased from ACRO. Mouse DLL3 protein was purchased from Kaika Biology. Anti-human IgG (γ-chain specific) -R-phycoerythrin antibody produced in goat and anti-human IgG (Fc-specific) -peroxidase antibody produced in goat were purchased from Sigma. Mouse monoclonal anti-His tag antibody (HRP) was purchased from Sino Biological.

A stable cell line LS174T-DLL3 was constructed to facilitate in vitro and in vivo efficacy study. Briefly, the commercial DLL3 recombinant plasmid pCMV-DLL3 (Sino Biological) was transiently transfected into LS174T cells with the agent Hieff Trans Liposomal Transfection Reagent (YEASEN) and transfection-specific media Opti-MEMTM I (Gibco) . Cell culture was supplemented with hygromycin B afterwards to select positive clones. 2-3 weeks later, single positive clones were gradually separated and verified with flow-cytometry. A DLL3 positive stable cell line LS174T-DLL3 was obtained.

Example 1. Immunizing and screening of anti-DLL3 antibodies

Antibodies against DLL3 were obtained by immunizing Balb/c mice (6-8 weeks old) using human DLL3 protein as the immunizing conjugates. One week after the fourth immunization, titer of anti-serum collected from the caudal vein of each mouse were determined by ELISA. The mouse with the highest titer of the anti-serum was sacrificed and the spleen removed to fuse with myeloma cells SP2/0 in a ratio of 8: 1.

After culturing for 10 days, hybridoma cells could be obviously observed under the microscope. The binding activity of hybridoma cell supernatant to human DLL3 protein was detected by ELISA. ELISA was performed by using standard protocols. Briefly, human DLL3 protein was coated on Corning EIA/RIA high-binding 96-well plates (Corning Inc. ) at 1000 ng per well overnight at 4℃ and blocked with 3%nonfat milk in PBS (pH7.4) . Thirty microliters hybridoma cell supernatants were added and then incubated at 37℃ for 1 h. The wells were washed with PBS containing 0.05%Tween 20. Bound antibodies were detected by goat anti-mouse IgG-Fc fragment cross-adsorbed antibody HRP conjugated (Bethyl) . The assay was developed at room temperature with TMB substrate (Solarbio) and measured at 450 nm with a microplate reader. By using a similar protocol, the binding activity of hybridoma cell supernatant to rhesus DLL3 protein and mouse DLL3 protein were detected, respectively. The hybridoma cell supernatant that bound to human and rhesus DLL3 protein but not to mouse DLL3 protein was selected for the next assay.

To measure the binding ability of hybridoma cell supernatant to cell surface DLL3, flow cytometry was performed using the DLL3 positive tumor cell line NCI-H82. About 2.5×10⁵ cells were incubated with hybridoma cell supernatant on ice for 1 h. Cells were washed once with PBS containing 0.5%bovine serum albumin (PBSA) and resuspended in 100 μl PBSA. Then 1 μl of goat anti-mouse IgG (H+L) highly gross-adsorbed secondary antibody, Alexa Fluor 633 (Thermo Fisher) was added and incubated for 30 min. Cells were washed once with PBSA and used for flow cytometry analysis. Finally, a specific anti-DLL3 clone 1G2 was identified for the construction of monoclonal and bispecific antibodies.

Example 2. Construction and characterization of anti-DLL3 monoclonal antibody

Anti-DLL3 clone 1G2 was used to construct an intact form of monoclonal antibody IgG1 against human DLL3 (termed as DLL3-1G2 mAb) . The Fab fragment of 1G2 clone was fused to the N-terminus of human IgG1 Fc fragment. The light chain and heavy chain were constructed into the vector pcDNA3.4, respectively. Construction and initial characterization of the DLL3-1G2 mAb were performed as follows.

Cloning of anti-DLL3 monoclonal antibody

To generate the construct of anti-DLL3 monoclonal antibody, the following primers were used:

1G2Mo-mAB-LC-F:

5’ CCTCCTGACTGGGGTGAGGGCCGATGTTTTGATGACCCAAACTCC 3’ (sense) (SEQ ID NO: 35) ;

1G2Mo-mAB-LC-R:

5’ CAGATGGTGCAGCCACCGTACGTTTGATTTCCAGCTTGGTGCCTC 3’ (antisense) (SEQ ID NO: 36) ;

1G2Mo-mAB-HC-F:

5’ TCCTGACTGGGGTGAGGGCCCAGGTCCAACTGCAGCAGCCTGGGG 3’ (sense) (SEQ ID NO: 37) ;

1G2Mo-mAB-HC-R:

5’ GATGGGCCCTTGGTGCTAGCTGCAGAGACAGTGACCAGAGTCCCTT 3’ (antisense) (SEQ ID NO: 38) ;

pBY-vectormAB-LC-FP:

5’ CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCG 3’ (sense) (SEQ ID NO: 39) ;

pBY-vectormAB-HC-FP:

5’ GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGC 3’ (sense) (SEQ ID NO: 40) ;

pBY-SP-RP:

5’ GGCCCTCACCCCAGTCAGGAGGACCAGGCAACAG 3’ (antisense) (SEQ ID NO: 41) .

The gene fragment of VL domain of anti-DLL3 antibody was amplified from anti-DLL3 clone 1G2 with primer pairs 1G2Mo-mAB-LC-F/1G2Mo-mAB-LC-R. The gene fragment of VH domain of anti-DLL3 antibody was amplified from anti-DLL3 clone 1G2 with primer pairs 1G2Mo-mAB-HC-F/1G2Mo-mAB-HC-R. The fragments of light and heavy chain vectors were cloned with primer pairs pBY-vectormAB-LC-FP/pBY-SP-RP and pBY-vectormAB-HC-FP/pBY-SP-RP. The gene fragments of VH and VL domains were then cloned into two light and heavy chain vectors via the Gibson assembly.

Protein expression, purification and initial characterization

DLL3-1G2 mAb was expressed in CHO-Scells. The plasmids and transfection agent PEI were mixed at the ratio of 1: 3 and then dropwise added into CHO-Scell culture. The cells were continued to grow for 5-7 days after transfection. The cell culture was harvested by centrifugation at 8000rpm for 20 min. The culture supernatant containing target proteins was loaded onto Protein A Sepharose 4 Fast Flow column (GE Healthcare) , and purified according to the manufacturer’s instructions.

The purified proteins were subjected to SDS-PAGE. On a non-reduced SDS-PAGE, DLL3-1G2 mAb displays an apparent molecular weight (aMW) of approximately 150 kDa. On a reduced SDS-PAGE, the heavy chain and light chain have an apparent molecular weight of approximately 50 kDa and 25kDa, respectively (data not shown) .

The CDR sequences according to the Kabat numbering system, light chain variable region (VL) and heavy chain variable region (VH) sequences, and the whole light chain (LC) and heavy chain (HC) sequences of DLL3-1G2 mAb are as follows.

DLL3-1G2 mAb LCDR1:

DLL3-1G2 mAb LCDR2:

DLL3-1G2 mAb LCDR3:

DLL3-1G2 mAb HCDR1:

DLL3-1G2 mAb HCDR2:

DLL3-1G2 mAb HCDR3:

DLL3-1G2 mAb VL:

DLL3-1G2 mAb VH:

DLL3-1G2 mAb LC:

DLL3-1G2 mAb HC:

Example 3. Binding of the anti-DLL3 monoclonal antibody to DLL3

ELISA was performed according to standard protocols, to determine binding affinity of DLL3-1G2 mAb to human DLL3 protein. Briefly, human DLL3 protein was coated on Corning EIA/RIA high-binding 96-well plates (Corning Inc. ) at 100 ng per well overnight at 4℃ and blocked with 3%nonfat milk in PBS (pH7.4) . Five-fold serially diluted antibodies from 100 μg/mL were added and incubated at room temperature for 2 h. The plates were washed with PBS containing 1%nonfat milk. Bound antibodies were detected by anti-Fc tag antibody (HRP) (Sigma) . The assay was developed at room temperature with TMB substrate (Solarbio) and monitored at 450 nm with a microplate reader. The half-maximal binding (EC₅₀) was calculated by fitting the data to the Langmuir adsorption isotherm. The results are shown in Figure 1.

The results indicate that DLL3-1G2 mAb binds to human DLL3 with EC₅₀ of 29.53 ng/mL, suggesting that DLL3-1G2 mAb has high affinity to human DLL3 and potentially excellent functions.

Example 4. Construction and characterization of anti-DLL3 bispecific antibodies

Bispecific T cell engager (BiTE) is a novel class of bispecific antibodies that can guide cytotoxic T cells to kill cancer cells by simultaneously binding to a tumor antigen and a T cell antigen, such as CD3 molecule on T cell surface. In this example, a specific format of BiTE consisting of a light chain and a heavy chain forming a heterodimer was designed. The light chain, from N-terminus to C-terminus, comprises an anti-target scFv, an anti-CD3 VL-CL and a monomeric human IgG1 Fc (mFc7.2) . The heavy chain, from N-terminus to C-terminus, comprises an anti-CD3 VH-CH1 and mFc7.2 (Figure 2) . mFc7.2 contains two amino acid mutations (T366L and Y407H) capable of inhibiting Fc homodimerization as described in PCT application No. PCT/US2018/016524, which is incorporated herein by reference in its entirety.

To generate DLL3×CD3 BiTEs, the VL and VH domains of the above DLL3-1G2 mAb were humanized to obtain humanized anti-DLL3 VL and VH domains. One humanized anti-DLL3 VL domain (SEQ ID NO: 11) and two humanized anti-DLL3 VH domains (SEQ ID NOs: 12 and 13) were finally obtained. The humanized VL and VH domains were linked via a linker GGGGSGGGGSGGGGS (SEQ ID NO: 25) to form an anti-DLL3-scFv. The scFv was fused to the N-terminus of the VL domain of an anti-CD3 Fab via a linker GSGGGGSGGGGS (SEQ ID NO: 26) . To obtain the full-length light chain, anti-DLL3 scFv fragment was cloned into the pBY plasmid containing an anti-CD3 VL-CL and a complete engineered Fc by in-fusion cloning. The heavy chain was constructed into a single vector pBY for expression in mammalian cells. The obtained DLL3×CD3 BiTEs were termed as DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1, respectively.

The two plasmids containing heavy chain and light chain gene were co-transfected to 293FS or CHO-Scells. The plasmids and transfection agent PEI were mixed at ratio 1: 3 and then added into 293FS or CHO-Scell culture dropwise. The cells were continued to grow for 5-7 days after transfection. The cell culture was harvested by centrifugation at 8000 rpm for 20 min. The culture supernatant containing target proteins were loaded onto Protein A Sepharose 4 Fast Flow column (GE Healthcare) , and purified according to the manufacturer’s instructions.

The purified proteins were subjected to SDS-PAGE. On a non-reduced SDS-PAGE, DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 displays an apparent molecular weight (aMW) of approximately 124 kDa (data not shown) .

The CDR sequences according to the Kabat numbering system, light chain variable region (VL) and heavy chain variable region (VH) sequences, and the whole light chain (LC) and heavy chain (HC) sequences of DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 are as follows.

LCDR1 against DLL3:

LCDR2 against DLL3:

LCDR3 against DLL3:

HCDR1 against DLL3:

HCDR2 against DLL3:

HCDR3 against DLL3:

LCDR1 against CD3:

LCDR2 against CD3:

LCDR3 against CD3:

HCDR1 against CD3:

HCDR2 against CD3:

HCDR3 against CD3:

VL against CD3:

VH against CD3:

VL against DLL3:

VH against DLL3 of DLL3-1G2H2L1-M6MV1-1:

VH against DLL3 of DLL3-1G2H1L1-M6MV1-1:

LC of DLL3-1G2H2L1-M6MV1-1:

LC of DLL3-1G2H1L1-M6MV1-1:

HC:

Example 5. Binding of DLL3×CD3 BiTEs to DLL3 and CD3

To determine binding affinity of the bispecific antibodies DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 to both human DLL3 and human CD3, ELISA experiments were performed as described in Example 3, with the coating proteins of human DLL3 or human CD3.

Additionally, co-binding assay was performed to further determine binding affinity of DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1. Human CD3 protein (Fc tag) was coated on Corning EIA/RIA high-binding 96-well plates (Corning Inc. ) at 100 ng per well overnight at 4℃ and blocked with 3%nonfat milk in PBS (pH7.4) . Then human DLL3 protein at 100 ng per well and 5-fold serially diluted antibodies from 100 μg/mL were added simultaneously and incubated at room temperature for 2 h. The plates were washed with PBS containing 1%nonfat milk. Bound antibodies were detected by Anti-His tag antibody (HRP) (Sino Biological) . The assay was developed at room temperature with TMB substrate (Solarbio) and monitored at 450 nm with a microplate reader. The half-maximal binding (EC₅₀) was calculated by fitting the data to the Langmuir adsorption isotherm. The results are shown in Figures 3-5.

The results indicate that DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 bind to human CD3 with EC₅₀ of 1453 ng/mL and 891.8 ng/mL, respectively (Figure 3) and bind to human DLL3 with EC₅₀ of 377.9 ng/mL and 267.0 ng/mL, respectively (Figure 4) . Moreover, the results of co-binding analysis show that DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 simultaneously bind to DLL3 and CD3 proteins with high affinity (Figure 5) . These results indicate that DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 can bind to both DLL3 and CD3 with high affinity, suggesting potentially potent anti-tumor activity in vitro and in vivo.

Example 6. Binding of DLL3×CD3 BiTEs to cancer cell lines

To measure the binding ability of DLL3×CD3 BiTEs to cell surface-expressed DLL3, flow cytometry was carried out using the DLL3-positive cell lines H82 and LS174T-DLL3 and DLL3-negative cell line HT55. About 5 × 10⁵ cells were incubated with 10 μg/mL DLL3-1G2H1L1-M6MV1-1 or DLL3-1G2H2L1-M6MV1-1 on ice for 60 min. The cells were washed once with PBS containing 0.5%bovine serum albumin (PBSA) and resuspended in 100 μl PBSA. Then 1 μL /test goat F (ab') 2 anti-human IgG - (Fab') 2 (PE) , pre-adsorbed (Abcam) as secondary antibody was added and incubated for 30 min. The negative control group was added with only secondary antibody. The cells were washed once with 0.5%PBSA and then used for flow cytometry analysis. The results are shown in Figures 6A-6C.

The results show that DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 bind well to the DLL3-positive H82 and LS174T-DLL3 cells, and essentially not bind to DLL3 negative HT55 cells.

Example 7. DLL3×CD3 BiTE-mediated T cell activation

The ability and specificity of DLL3×CD3 BiTEs to activate human T cells in the presence of target cells (LS174T, H82 and LS174T-DLL3 cells) were evaluated by using Bio-Glo^TM Luciferase Assay System with TCR/CD3 effector cells (Jurkat-NFAT-CD3) . Of the tested target cells, LS174T cells did not express human DLL3, while H82 and LS174T-DLL3 cells had a high level of DLL3 expression. The TCR/CD3 effector cells (Jurkat-NFAT-CD3) express endogenous TCR and CD3 receptors. When the effector cells (Jurkat-NFAT-CD3) were engaged with an appropriate TCR/CD3 ligand or anti-TCR/CD3 antibody, the TCR transduces intracellular signals, resulting in TCR-mediated T cell activation and producing an enhanced fluorescence signal.

Target cells (LS174T and LS174T-DLL3 cells) were plated on 96-well plates at a density of 1 × 10⁴ cells in 100 μL DMEM complete medium per well for overnight. After removal of 60 μL of the supernatant, 40 μL antibodies (DLL3-1G2H1L1-M6MV1-1 or DLL3-1G2H2L1-M6MV1-1) were added into each well in a 5-fold gradient dilution at the maximum concentration of 50 μg/mL. At the same time, the above test antibodies were added to new 96-well plates at 40 μL/well, and the target cells (H82 cells) were plated on the new 96-well plates at a density of 1 × 10⁵ cells in 40 μL DMEM complete medium. Then effector cells (Jurkat-NFAT-CD3) were added at a density of 2 × 10⁵ cells in 40 μL DMEM complete medium per well. The plates were incubated for 6 h at 37 ℃ in a humidified incubator. Then, Stable-Lite Luciferase Assay System solution (Vazyme) was added to each well at 120 μL/well, and incubated for 10 min at room temperature in the dark. Luminescence was detected usingELISA reader (Molecular Devices) . The results are shown in Figures 7A-7C.

In the presence of DLL3-negative LS174T cells, effector cells (Jurkat-NFAT-CD3) was not effectively activated by DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 with EC₅₀ of approximately 10569 ng/mL and 20093 ng/mL, respectively (Figure 7A) . In the presence of DLL3-positive H82 or LS174T-DLL3 cells, effector cells (Jurkat-NFAT-CD3) are efficiently activated by DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 with EC₅₀ of approximately 4024 ng/mL, 1971 ng/mL, 5468 ng/mL and 2703 ng/mL, respectively (Figures 7B-7C) . These results suggest that DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 can simultaneously bind to CD3 antigen of effector cells and DLL3 antigen of tumor cells, leading to T cell specific activation.

Example 8. DLL3×CD3 BiTE-mediated killing of human cancer cell lines

Bispecific T cell engager can simultaneously bind to a tumor antigen and a T cell antigen (e.g., CD3 molecular on T cell surface) causing aggregation and activation of T cells, eventually leading to the killing of tumor cells. To evaluate killing efficiency of DLL3×CD3 BiTEs, DLL3-negative LS174T cells and DLL3-positive LS174T-DLL3 cells were used as target cells.

Target cells were plated on 96-well plates at a density of 4×10⁴ cells in 100 μL DMEM complete medium per well and incubated overnight. The next day, 50 μL antibodies 5-fold serially diluted from 4 μg/mL were added into each well. Then effector cells human T effector cells were added at a density of 4 × 10⁴ cells in 50 μL DMEM complete medium per well and incubated for 6hrs. After incubation, the medium was removed and 20 μL CCK8 was added and incubated for 30 minutes in CO₂ incubator. Cell killing activity was measured by using microplate reader according to the manufacturer’s instructions. The results were shown in Figures 8A-8B.

The results show that DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 have no potent T cell-dependent cytotoxicity (CTL) against DLL3-negative LS174T cells (Figure 8A) , but have potent T cell-dependent cytotoxicity against DLL3-positive cell line LS174T-DLL3 with CTL EC₅₀ of 304.8 ng/mL and 103.6 ng/mL, respectively (Figure 8B) , suggesting that the CTL killing potency depends on the binding of the bispecific antibody to tumor antigen DLL3 and T cell antigen CD3. In summary, the results demonstrate DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 substantially possess the lethal effect in vitro that deserves further development.

Example 9. DLL3×CD3 BiTE-mediated inhibition of tumor growth in mice

200 μL 3×10⁶ LS174T-DLL3 cells were subcutaneously inoculated into the right-side abdomen of B-NDG mice, and these mice were randomly divided into five groups (3 mice per group) . The mice in negative control group were dosed intraperitoneally with physiological saline. The mice in experiment group were intraperitoneally treated with 100 or 500 μg/kg of DLL3-1G2H1L1-M6MV1-1 or 100 or 500 μg/kg of DLL3-1G2H2L1-M6MV1-1 for three times weekly, with 100 μL 1000 IU human Interleukin-2 (IL-2) for three times weekly and with 300 μL 2×10⁷ human T cells for one time weekly for a total of 16 days of treatments. The mice received a total of six times treatments with antibodies, six times treatments with IL-2 and two times treatments with T cells. At the same time, tumor volume and body weight of mice were measured three times weekly for 16 days. Tumor growth inhibition rates (TGI) were calculated by using the following formula: TGI (%) = (C-T) /C × 100 (T: average tumor volume of the experimental group; C: average tumor volume of the control group) . The results are shown in Figures 9A-9B.

The results indicated that significant anti-tumor effects were observed by treatment with DLL3-1G2H1L1-M6MV1-1 or DLL3-1G2H2L1-M6MV1-1 at two doses of 100 and 500 μg/kg, and the tumor growth inhibition (TGI) reached 79.79% (for 100 μg/kg group) and 100% (for 500 μg/kg group) , respectively, for DLL3-1G2H1L1-M6MV1-1, and 79.69% (for 100 μg/kg group) and 100% (for 500 μg/kg group) , respectively, for DLL3-1G2H2L1-M6MV1-1 after 16 days of treatment (Figure 9A) . The body weight of all mice groups only has minor variation (Figure 9B) , suggesting low toxicity advantage of DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 in vivo.

In summary, DLL3-1G2H1L1-M6MV1-1 and DLL3-1G2H2L1-M6MV1-1 possess excellent pharmacodynamic functions in vitro and in vivo. Therefore, these bispecific antibodies targeting DLL3 and CD3 are expected to conduct clinical research.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

An antibody that specifically binds to DLL3, or an antigen binding fragment thereof, comprising a light chain variable region (VL) and a heavy chain variable region (VH) , wherein the VL comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 1-3 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 6-8 respectively.
The antibody or the antigen binding fragment thereof according to claim 1, wherein

(i) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 4, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 9; or

(ii) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 12; or

(iii) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 13.
The antibody or the antigen binding fragment thereof according to claim 2, wherein

(i) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 4 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 9; or

(ii) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 12; or

(iii) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 13.
The antibody or antigen binding fragment thereof of any one of claims 1-3, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.
The antibody or the antigen binding fragment thereof according to any one of claims 1-4, wherein the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD.
The antibody or the antigen binding fragment thereof according to any one of claims 1-4, wherein the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
The antibody or the antigen binding fragment thereof according to any one of claims 1-6, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab’, F (ab') ₂, Fv, scFv, and ds-scFv.
The antibody or the antigen binding fragment thereof according to any one of claims 1-7, wherein the antibody is a monoclonal antibody.
The antibody or the antigen binding fragment thereof according to claims 8, wherein the antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 5 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 10.
The antibody or the antigen binding fragment thereof according to any one of claims 1-7, wherein the antibody is a bispecific or a multi-specific antibody.
The antibody or the antigen binding fragment thereof according to claim 10, wherein the antibody is a bispecific antibody which further comprises a second antigen binding region that binds to a second antigen.
The antibody or the antigen binding fragment thereof of according to claim 11, wherein the second antigen is a tumor associated antigen or an immune cell antigen.
The antibody or the antigen binding fragment thereof according to claim 12, wherein the second antigen is a T-cell antigen.
The antibody or the antigen binding fragment thereof according to claim 13, wherein the T-cell antigen is selected from the group consisting of T cell receptor (TCR) , CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137) , and NKG2D.
The antibody or the antigen binding fragment thereof according to claim 11, wherein the second antigen is CD3, and the second antigen binding region comprises a VL and a VH, wherein the VL comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 14-16 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 18-20 respectively.
The antibody or the antigen binding fragment thereof according to claim 15, wherein the second antigen binding region comprises a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 17 and a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21.
The antibody or the antigen binding fragment thereof according to claim 16, wherein the second antigen binding region comprises a VL comprising an amino acid sequence as set forth in SEQ ID NO: 17 and a VH comprising an amino acid sequence as set forth in SEQ ID NO: 21.
The antibody or the antigen binding fragment thereof according to any one of claims 15-17, wherein the antibody comprises a scFv comprising the VL and the VH of the antibody that specifically binds to DLL3, and the scFv is linked to the N terminal of the VL or the VH of the second antigen binding region, optionally via a linker.
The antibody or the antigen binding fragment thereof according to claim 18, wherein the antibody comprises:

a first polypeptide chain comprising from the N terminal to C terminal: the scFv, an optional linker, the VL of the second antigen binding region, a light chain constant region (CL) , a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) ; and

a second polypeptide chain comprising from the N terminal to C terminal: the VH of the second antigen binding region, a heavy chain constant region 1 (CH1) , a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) .
The antibody or the antigen binding fragment thereof according to claim 18 or 19, wherein the linker comprises an amino acid sequence selected from (G4S) n and GS (G4S) n, wherein n is an integer selected from 1-5, preferably the linker comprises an amino acid sequence as shown in SEQ ID NO: 25 or 26.
The antibody or the antigen binding fragment thereof according to claim 19, wherein

(i) the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 22, and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 24; or

(ii) the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 23, and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 24.
The antibody or the antigen binding fragment thereof according to any of claim 11-21, wherein the bispecific antibody is a bispecific T-cell engager (BiTE) .
A bispecific antibody or an antigen binding fragment thereof, comprising a first antigen binding region that binds to DLL3 comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1) and a second antigen binding region that binds to CD3 comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2) , wherein

the VL1 comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 1-3 respectively, and the VH1 comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 6-8 respectively; and

the VL2 comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 14-16 respectively, and the VH2 comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 18-20 respectively.
The bispecific antibody or the antigen binding fragment thereof according to claim 23, wherein

(i) the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 4 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 9; or

(ii) the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 12; or

(iii) the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 13; and wherein

the VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 17 and the VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21.
The bispecific antibody or the antigen binding fragment thereof according to claim 24, wherein

(i) the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 4 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 9; or

(ii) the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 12; or

(iii) the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 13; and wherein

the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 17 and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 21.
The bispecific antibody or the antigen binding fragment thereof according to any one of claims 23-25, wherein the first antigen binding region comprises a scFv comprising the VL1 and the VH1, and the scFv is linked to the N terminal of the VL2 or the VH2, optionally via a linker.
The bispecific antibody or the antigen binding fragment thereof according to claim 26, wherein the bispecific antibody comprises:

a first polypeptide chain comprising from the N terminal to C terminal: the scFv, an optional linker, the VL2, a light chain constant region (CL) , a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) ; and

a second polypeptide chain comprising from the N terminal to C terminal: the VH2, a heavy chain constant region 1 (CH1) , a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) .
The bispecific antibody or the antigen binding fragment thereof according to claim 26 or 27, wherein the linker comprises an amino acid sequence selected from (G4S) n and GS (G4S) n, wherein n is an integer selected from 1-5, preferably the linker comprises an amino acid sequence as shown in SEQ ID NO: 25 or 26.
The bispecific antibody or the antigen binding fragment thereof according to claim 27, wherein

(i) the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 22, and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 24; or

(ii) the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 23, and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 24.
The bispecific antibody or the antigen binding fragment thereof according to any of claims 23-29, wherein the bispecific antibody is a bispecific T-cell engager (BiTE) .
A nucleic acid comprising a nucleotide sequence encoding the antibody or the antigen binding fragment thereof according to any one of claims 1-22 or the bispecific antibody or the antigen binding fragment thereof according to any one of claims 23-30.
A vector comprising the nucleic acid according to claim 31.
A host cell comprising the nucleic acid according to claim 31 or the vector according to claim 32.
A pharmaceutical composition comprising (i) the antibody or the antigen binding fragment thereof according to any one of claims 1-22, or the bispecific antibody or the antigen binding fragment thereof according to any one of claims 23-30; and (ii) a pharmaceutically acceptable carrier or excipient.
The pharmaceutical composition according to claim 34, further comprising a second therapeutic agent.
The pharmaceutical composition according to claim 35, wherein the second therapeutic agent is selected from a cytokine, an antibody, a chemotherapeutic agent and a small molecule drug.
The pharmaceutical composition according to claim 35 or 36, wherein the second therapeutic agent is selected from an interleukin (such as IL-2) , a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.
A conjugate, comprising the antibody or the antigen binding fragment thereof according to any one of claims 1-22 or the bispecific antibody or the antigen binding fragment thereof according to any one of claims 23-30, and a chemical moiety conjugated thereto.
The conjugate according to claim 38, wherein the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immune stimulatory molecule.
A method of treating a cancer in a subject, comprising administering to the subject an effective amount of the antibody or the antigen binding fragment thereof according to any one of claims 1-22, the bispecific antibody or the antigen binding fragment thereof according to any one of claims 23-30, the pharmaceutical composition according to any one of claims 34-37, or the conjugate according to claim 38 or 39.
The method according to claim 40, wherein the cancer is a DLL3 positive cancer.
The method according to claim 41, wherein the cancer is a neuroendocrine neoplasm, such as lung cancer, preferably small cell lung cancer.
The method according to any one of claims 40-42, further comprising administering to the subject a second therapeutic agent.
The method according to claim 43, wherein the second therapeutic agent is selected from a cytokine, an antibody, a chemotherapeutic agent and a small molecule drug.
The method according to claim 43 or 44, wherein the second therapeutic agent is selected from an interleukin (such as IL-2) , a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.