Antibodies and chimeric antigen receptors specific for delta-like ligand 3 (dLL3)

Abstract

Delta-like ligand 3 (DLL3) binding molecules, particularly human antibodies, including antibody fragments, specific for DLL3 are provided. Also provided are recombinant receptors, including chimeric antigen receptors (CARs) comprising such antibodies or fragments, and polynucleotides encoding antibodies, antigen binding fragments or receptors specific for DLL3. Also provided are genetically engineered cells containing such DLL3 binding proteins and receptors, as well as related methods and uses thereof in adoptive cell therapy.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 401,559, filed on August 26, 2022, entitled “ANTIBODIES AND CHIMERIC ANTIGEN RECEPTORS SPECIFIC FORDELTA-LIKE LIGAND 3 (DLL3),” the contents of which are incorporated herein by reference in their entirety. Incorporation by Reference into the Sequence Listing

[0002] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file named 735042022240SeqList, created on August 25, 2023, and is 184,247 bytes in size. The information in the electronic sequence listing is incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates in some aspects to delta-like ligand 3 (DLL3) binding molecules, particularly human antibodies, including antibody fragments, that are specific for DLL3. The present disclosure also relates to recombinant receptors, including chimeric antigen receptors (CARs) containing such antibodies or fragments, and polynucleotides encoding antibodies, antigen-binding fragments, or receptors that are specific for DLL3. The present disclosure also relates to genetically engineered cells containing such DLL3 binding proteins and receptors, as well as related methods and uses thereof in adoptive cell therapy. Background Art

[0004] Delta-like ligand 3 (DLL3) is a type 1 transmembrane delta-like protein and an inhibitory Notch ligand that is normally expressed on cell membranes but is highly expressed on the surface of small cell lung cancer (SCLC) cells and other neuroendocrine cancer cells. DLL3 is abnormally expressed on the cell surface of high-grade lung neuroendocrine tumors, melanomas, and gliomas. In normal adult tissues, DLL3 is usually expressed at low levels. SCLC typically arises in the central lung airways, grows aggressively, and often metastasizes. SCLC has a poor prognosis and limited treatment options. The embodiments described herein provide DLL3-binding molecules and targeted therapies that can be used to treat cancers associated with DLL3 expression. Summary of the Invention

[0005] Provided herein are binding molecules that bind to delta-like ligand 3 (DLL3), such as antibodies or antigen-binding fragments thereof. The binding molecules also include polypeptides containing such antibodies, including single-chain cell surface proteins (e.g., recombinant receptors, such as chimeric antigen receptors (CARs)), fusion proteins, or conjugates containing such antibodies. Also provided are recombinant receptors that bind to DLL3, such as chimeric antigen receptors (CARs). Also provided are conjugates (such as immunoconjugates) or fusion proteins (such as T cell engagers) comprising any of the antibodies or antigen-binding fragments thereof described herein. Also provided are polynucleotides containing nucleic acid sequences encoding all or part of such antibodies, antigen-binding fragments, recombinant receptors (such as CARs), fusion proteins, or conjugates described herein. Also provided are cells comprising any provided binding molecules (such as antibodies or antigen-binding fragments thereof), CARs, fusion proteins, or conjugates. Also provided are compositions comprising any binding molecules (such as antibodies or antigen-binding fragments thereof), CARs, fusion proteins, conjugates, polynucleotides, or cells, and related methods and uses (such as in therapeutic applications) of any of the binding molecules (such as antibodies or antigen-binding fragments thereof), CARs, fusion proteins, conjugates, polynucleotides, cells, or compositions.

[0006] The present invention provides an anti-delta-like ligand 3 (DLL3) antibody or an antigen-binding fragment thereof, wherein the anti-delta-like ligand 3 (DLL3) antibody or an antigen-binding fragment thereof comprises a heavy chain variable (V H ) region and light chain variable (V L ) area, wherein: the V H The V region comprises a heavy chain complementary determining region 1 (CDR-H1), a heavy chain complementary determining region 2 (CDR-H2), and a heavy chain complementary determining region 3 (CDR-H3) contained in SEQ ID NO: 90; and the V L The region comprises the light chain complementary determining region 1 (CDR-L1), light chain complementary determining region 2 (CDR-L2), and light chain complementary determining region 3 (CDR-L3) contained in SEQ ID NO:91.

[0007] Provided herein is an anti-DLL3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable (V H ) region and light chain variable (V L ) area, wherein: the V H The V region comprises a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 92, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 93, and a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 94; and the V LThe region includes a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO:95, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO:96, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO:97.

[0008] In some embodiments, the V H The V region comprises an amino acid sequence having at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 90, and the V L The region comprises an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:91.

[0009] Provided herein is an anti-DLL3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable (V H ) region and light chain variable (V L ) area, wherein: the V H The V region comprises an amino acid sequence having at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 90, and the V L The region comprises an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:91.

[0010] In some embodiments, the V H The V region comprises a heavy chain complementary determining region 1 (CDR-H1), a heavy chain complementary determining region 2 (CDR-H2), and a heavy chain complementary determining region 3 (CDR-H3) contained in SEQ ID NO: 90, and the V L The region comprises the light chain complementary determining region 1 (CDR-L1), light chain complementary determining region 2 (CDR-L2), and light chain complementary determining region 3 (CDR-L3) contained in SEQ ID NO:91.

[0011] In some embodiments, the V HThe V region comprises a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 92, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 93, and a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 94, and the V L The region includes a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO:95, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO:96, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO:97.

[0012] In some embodiments, the V H The region comprises the sequence shown in SEQ ID NO: 90, and the V L The region comprises the sequence shown in SEQ ID NO:91.

[0013] Provided herein is an anti-DLL3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable (V H ) region and light chain variable (V L ) area, wherein: the V H The region comprises the sequence shown in SEQ ID NO: 90, and the V L The region comprises the sequence shown in SEQ ID NO:91.

[0014] In some embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof is recombinant. H area and the V L The region is human or derived from a human protein.

[0015] In some embodiments, the antibody further comprises a heavy chain constant region (C H ) and / or light chain constant region (C L ). In some any embodiments, said C H comprising the sequence shown in SEQ ID NO: 126, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 126; and / or said C Lcomprising the sequence set forth in SEQ ID NO: 127, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 127. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the sequence set forth in SEQ ID NO: 128; and / or a light chain comprising the sequence set forth in SEQ ID NO: 129. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the sequence set forth in SEQ ID NO: 128; and a light chain comprising the sequence set forth in SEQ ID NO: 129.

[0016] In some any embodiments, the antibody is a full-length antibody.

[0017] In some any embodiments, the antibody is an antigen-binding fragment.

[0018] In some any embodiments, the antigen-binding fragment thereof comprises a single-chain Fv (scFv).

[0019] In some embodiments, the V H The V L The amino terminus of the region.

[0020] In some embodiments, the V H The V L The carboxyl terminus of the region.

[0021] In some embodiments, the V H area and the V L The regions are connected by a flexible linker. In some embodiments, the flexible linker comprises the sequence shown in SEQ ID NO:46.

[0022] In some any embodiments, the scFv comprises the sequence shown in SEQ ID NO:89.

[0023] In some embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof specifically binds to human DLL3. In some embodiments, the human DLL3 comprises the amino acid sequence shown in SEQ ID NO: 120 or 121.

[0024] In some embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof does not bind to, does not cross-react with, or binds to a DLL1 or DLL4 protein (such as human DLL1 or human DLL4) at a lower level, degree, or affinity. In some embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof binds to human DLL1 or human DLL4 at a level, degree, or affinity that is at least or about 75%, 80%, 90%, 95%, or 99% less than that of human DLL3.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof is present at about 1×10 -7 M is about 1x10 -8 The equilibrium dissociation constant (K D ) binds to human DLL3.

[0026] Also provided is a single-chain cell surface protein comprising any anti-DLL3 antibody or antigen-binding fragment thereof provided herein.

[0027] Also provided herein is a conjugate comprising any of the anti-DLL3 antibodies or antigen-binding fragments thereof provided herein and a heterologous molecule or moiety. In some embodiments, the heterologous molecule or moiety is a therapeutic moiety. In some embodiments, the heterologous molecule or moiety is a small molecule compound, a protein, a peptide, or a toxin.

[0028] Provided herein is an anti-DLL3 chimeric antigen receptor (CAR), comprising an extracellular antigen binding domain comprising any anti-DLL3 antibody or antigen-binding fragment thereof provided herein, a transmembrane domain, and an intracellular signaling region. In some embodiments, the anti-DLL3 CAR further comprises a spacer located between the extracellular antigen binding domain and the transmembrane domain.

[0029] In some any embodiments, the spacer comprises at least a portion of an immunoglobulin or a variant thereof.

[0030] In some embodiments, the spacer comprises at least a portion of a hinge region of an immunoglobulin or a variant thereof. In some embodiments, at least a portion of the hinge region comprises all or a portion of an IgG4 hinge region (such as a human IgG4 hinge region) or a variant thereof. In some embodiments, the variant IgG4 hinge region comprises a substitution of amino acids CPSC to CPPC compared to a wild-type IgG4 hinge region.

[0031] In some any embodiments, the length of the spacer is less than or less than about 15 amino acids. In some any embodiments, the length of the spacer is between 12 and 15 amino acids.

[0032] In some any embodiments, the spacer comprises an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some any embodiments, the spacer comprises the sequence shown in SEQ ID NO: 1.

[0033] In some any embodiments, the spacer comprises an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In some any embodiments, the spacer comprises the sequence shown in SEQ ID NO: 3.

[0034] In some embodiments, the spacer comprises at least a portion of the hinge region of an immunoglobulin and at least a portion of the CH3 region of an immunoglobulin or a variant thereof. In some embodiments, the length of the spacer is or about 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 or 125 amino acids, or a length between any of the aforementioned values. In some embodiments, the spacer comprises the sequence shown in SEQ ID NO: 5 or 58, or an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5 or 58.

[0035] In some embodiments, the spacer comprises at least a portion of a hinge region of an immunoglobulin, at least a portion of a CH2 region of an immunoglobulin, and at least a portion of a CH3 region of an immunoglobulin, or a variant thereof. In some embodiments, the spacer is at or about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, or 230 amino acids in length, or a length between any of the foregoing values. In some any embodiments, the spacer comprises the sequence shown in SEQ ID NO:42 or 82, or an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:42 or 82.

[0036] In some any embodiments, the transmembrane domain comprises the transmembrane domain of CD4, CD28, or CD8.

[0037] In some embodiments, the transmembrane domain comprises a transmembrane domain from CD28 (such as human CD28). In some embodiments, the transmembrane domain comprises SEQ ID NO: 10 or 12, or an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10 or 12.

[0038] In some any embodiments, the transmembrane domain comprises an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10. In some any embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO: 10.

[0039] In some any embodiments, the transmembrane domain comprises an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12. In some any embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO: 12.

[0040] In some embodiments, the intracellular signaling region comprises an intracellular signaling domain capable of inducing a primary activation signal in a T cell, is a T cell receptor (TCR) component and / or comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of a CD3-ζ (CD3ζ) chain (such as a human CD3ζ chain). In some embodiments, the intracellular signaling domain comprises the sequence shown in SEQ ID NO: 19, or an amino acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 19. In some embodiments, the intracellular signaling domain comprises the sequence shown in SEQ ID NO: 19.

[0041] In some embodiments, the intracellular signaling region further comprises a costimulatory domain. In some embodiments, the costimulatory domain is between the transmembrane domain and the intracellular signaling domain. In some embodiments, the costimulatory domain comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments, the costimulatory domain comprises an intracellular signaling domain of CD28, 4-1BB or ICOS.

[0042] In some embodiments, the costimulatory domain comprises the intracellular signaling domain of 4-1BB (such as human 4-1BB). In some embodiments, the costimulatory domain comprises the sequence shown in SEQ ID NO: 17, or an amino acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17.

[0043] In some embodiments, the costimulatory domain comprises the intracellular signaling domain of CD28 (such as human CD28). In some embodiments, the costimulatory domain comprises the sequence shown in SEQ ID NO: 15 or 16, or an amino acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15 or 16.

[0044] In some any embodiments, the CAR comprises, from its N-terminus to its C-terminus, the extracellular antigen binding domain, the spacer, the transmembrane domain and the intracellular signaling region.

[0045] In some any embodiments, the CAR comprises, in sequence from its N-terminus to the C-terminus: an extracellular antigen binding domain comprising the scFv; a spacer comprising a modified IgG4 hinge, e.g., comprising the sequence shown in SEQ ID NO: 1; a transmembrane domain, such as a transmembrane domain from human CD28, e.g., comprising the sequence shown in SEQ ID NO: 10; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-ζ (CD3ζ) chain and an intracellular signaling domain of a costimulatory molecule, e.g., an intracellular signaling domain comprising 4-1BB.

[0046] In some any embodiments, the CAR comprises, from its N-terminus to the C-terminus: an extracellular antigen binding domain comprising the scFv; a spacer comprising a modified IgG4 hinge, e.g., comprising the sequence shown in SEQ ID NO: 3; a transmembrane domain, such as a transmembrane domain from human CD28, e.g., comprising the sequence shown in SEQ ID NO: 12; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-ζ (CD3ζ) chain and an intracellular signaling domain of a costimulatory molecule, e.g., an intracellular signaling domain comprising 4-1BB.

[0047] The present invention provides an anti-DLL3 chimeric antigen receptor (CAR), which comprises an extracellular antigen binding domain containing a scFv, wherein the scFv comprises a heavy chain variable (V H ) region and light chain variable (V L ) area, wherein: the V H The V region comprises a heavy chain complementary determining region 1 (CDR-H1), a heavy chain complementary determining region 2 (CDR-H2), and a heavy chain complementary determining region 3 (CDR-H3) contained in SEQ ID NO: 90, and the V L The region comprises the light chain complementary determining region 1 (CDR-L1), light chain complementary determining region 2 (CDR-L2), and light chain complementary determining region 3 (CDR-L3) contained in SEQ ID NO:91; a spacer comprising the sequence shown in SEQ ID NO:1; a transmembrane domain comprising the sequence shown in SEQ ID NO:10; and an intracellular signaling region comprising the cytoplasmic signaling domain of the CD3-ζ (CD3ζ) chain and the intracellular signaling domain of a costimulatory molecule, the cytoplasmic signaling domain comprising the sequence shown in SEQ ID NO:19, and the intracellular signaling domain comprising the sequence shown in SEQ ID NO:17.

[0048] The present invention provides an anti-DLL3 chimeric antigen receptor (CAR), which comprises an extracellular antigen binding domain containing a scFv, wherein the scFv comprises a heavy chain variable (V H ) region and light chain variable (V L ) area, wherein: the V H The V region comprises a heavy chain complementary determining region 1 (CDR-H1), a heavy chain complementary determining region 2 (CDR-H2), and a heavy chain complementary determining region 3 (CDR-H3) contained in SEQ ID NO: 90, and the V L The region comprises the light chain complementary determining region 1 (CDR-L1), light chain complementary determining region 2 (CDR-L2), and light chain complementary determining region 3 (CDR-L3) contained in SEQ ID NO:91; a spacer comprising the sequence shown in SEQ ID NO:3; a transmembrane domain comprising the sequence shown in SEQ ID NO:12; and an intracellular signaling region comprising the cytoplasmic signaling domain of the CD3-ζ (CD3ζ) chain and the intracellular signaling domain of a costimulatory molecule, the cytoplasmic signaling domain comprising the sequence shown in SEQ ID NO:19, and the intracellular signaling domain comprising the sequence shown in SEQ ID NO:17.

[0049] In some any embodiments, the CAR comprises, from N-terminus to C-terminus: an extracellular antigen binding domain comprising the scFv comprising the sequence shown in SEQ ID NO: 89; a spacer comprising the sequence shown in SEQ ID NO: 1; a transmembrane domain comprising the sequence shown in SEQ ID NO: 10; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-ζ (CD3ζ) chain and an intracellular signaling domain of a costimulatory molecule, the cytoplasmic signaling domain comprising the sequence shown in SEQ ID NO: 19, and the intracellular signaling domain comprising the sequence shown in SEQ ID NO: 17.

[0050] In some any embodiments, the CAR comprises, from N-terminus to C-terminus: an extracellular antigen binding domain comprising the scFv comprising the sequence shown in SEQ ID NO: 89; a spacer comprising the sequence shown in SEQ ID NO: 3; a transmembrane domain comprising the sequence shown in SEQ ID NO: 12; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-ζ (CD3ζ) chain and an intracellular signaling domain of a costimulatory molecule, the cytoplasmic signaling domain comprising the sequence shown in SEQ ID NO: 19, and the intracellular signaling domain comprising the sequence shown in SEQ ID NO: 17.

[0051] In some any embodiments, the anti-DLL3 CAR comprises the sequence shown in SEQ ID NO: 117, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO: 117. In some any embodiments, the anti-DLL3 CAR comprises the sequence shown in SEQ ID NO: 117.

[0052] In some any embodiments, the anti-DLL3 CAR comprises or consists of the sequence shown in SEQ ID NO: 119, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO: 119. In some any embodiments, the anti-DLL3 CAR comprises or consists of the sequence shown in SEQ ID NO: 119.

[0053] Also provided is a polynucleotide comprising a nucleic acid encoding any of the anti-DLL3 antibodies or antigen-binding domains thereof provided herein.

[0054] Also provided is a polynucleotide comprising a nucleic acid encoding any of the single-chain cell surface proteins provided herein.

[0055] Also provided is a polynucleotide comprising a nucleic acid encoding any of the conjugates provided herein.

[0056] Also provided is a polynucleotide comprising a nucleic acid encoding any of the anti-DLL3 CARs provided herein.

[0057] In some embodiments, the polynucleotide comprises: HA nucleic acid comprising the sequence shown in SEQ ID NO: 87, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 87; and encoding the V L A nucleic acid comprising the sequence shown in SEQ ID NO: 88, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 88. In some embodiments, the polynucleotide comprises: encoding the V H A nucleic acid comprising the sequence shown in SEQ ID NO: 87; and encoding the V L In some embodiments, the antigen-binding fragment is a single-chain Fv (scFv), and the polynucleotide comprises a nucleic acid encoding the scFv, the nucleic acid comprising the sequence shown in SEQ ID NO: 86, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 86. In some embodiments, the antigen-binding fragment is a single-chain Fv (scFv), and the polynucleotide comprises a nucleic acid encoding the scFv, the nucleic acid comprising the sequence shown in SEQ ID NO: 86.

[0058] In some any embodiments, the polynucleotide comprises a nucleic acid encoding the spacer, the nucleic acid comprising the sequence shown in SEQ ID NO: 2, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In some any embodiments, the polynucleotide comprises a nucleic acid encoding the spacer, the nucleic acid comprising the sequence shown in SEQ ID NO: 2.

[0059] In some any embodiments, the polynucleotide comprises a nucleic acid encoding the spacer, the nucleic acid comprising the sequence shown in SEQ ID NO: 4, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In some any embodiments, the polynucleotide comprises a nucleic acid encoding the spacer, the nucleic acid comprising the sequence shown in SEQ ID NO: 4.

[0060] In some any embodiments, the polynucleotide comprises a nucleic acid encoding the transmembrane domain, the nucleic acid comprising the sequence shown in SEQ ID NO: 11, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11. In some any embodiments, the polynucleotide comprises a nucleic acid encoding the transmembrane domain, the nucleic acid comprising the sequence shown in SEQ ID NO: 11.

[0061] In some any embodiments, the polynucleotide comprises a nucleic acid encoding the transmembrane domain, the nucleic acid comprising the sequence shown in SEQ ID NO: 13, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. In some any embodiments, the polynucleotide comprises a nucleic acid encoding the transmembrane domain, the nucleic acid comprising the sequence shown in SEQ ID NO: 13.

[0062] In some any embodiments, the nucleic acid encoding the anti-DLL3 chimeric antigen receptor comprises the sequence shown in SEQ ID NO: 116, or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO: 116. In some any embodiments, the nucleic acid encoding the anti-DLL3 chimeric antigen receptor comprises the sequence shown in SEQ ID NO: 116.

[0063] In some any embodiments, the polynucleotide is optimized by splice site elimination.

[0064] In some any embodiments, the polynucleotide is codon-optimized for expression in human cells.

[0065] Also provided herein is a vector comprising any polynucleotide provided herein. In some any embodiments, the vector is a viral vector. In some any embodiments, the viral vector is a retroviral vector or a lentiviral vector. In some any embodiments, the viral vector is an adeno-associated virus (AAV) vector.

[0066] Also provided herein is a cell comprising any of the anti-DLL3 antibodies or antigen-binding fragments thereof provided herein, any of the single-chain cell surface proteins provided herein, or any of the conjugates provided herein.

[0067] Also provided herein is a cell comprising any of the anti-DLL3 CARs provided herein.

[0068] Also provided herein is a cell comprising any of the polynucleotides provided herein or any of the vectors provided herein.

[0069] In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a NK cell or a T cell. In some embodiments, the cell is a T cell, and the T cell is a CD4+ T cell or a CD8+ T cell.

[0070] In some any embodiments, the cell is a primary cell obtained from a subject.

[0071] In some any embodiments, among a plurality of said cells, less than or less than about 10%, less than or less than about 9%, less than or less than about 8%, less than or less than about 7%, less than or less than about 5%, less than or less than about 4%, less than or less than about 3%, less than or less than about 2%, or less than or less than about 1% of said cells in the plurality of cells comprise an anti-DLL3 chimeric antigen receptor that exhibits tonic signaling and / or antigen-independent activity or signaling.

[0072] Also provided herein is a composition comprising any of the cells provided herein.

[0073] Also provided herein is a composition comprising any anti-DLL3 antibody or antigen-binding fragment thereof provided herein, any single-chain cell surface protein provided herein, any conjugate provided herein, or any anti-DLL3 CAR provided herein.

[0074] In some of any embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0075] In some any embodiments, the composition comprises CD4+ and CD8+ T cells, and the ratio of CD4+ to CD8+ T cells is from at or about 1:3 to 3:1, such as at or about 1:2 to 2:1, such as at or about 1:1.

[0076] In some any embodiments, among the plurality of cells in the composition, less than or less than about 10%, less than or less than about 9%, less than or less than about 8%, less than or less than about 7%, less than or less than about 5%, less than or less than about 4%, less than or less than about 3%, less than or less than about 2%, or less than or less than about 1% of the cells in the plurality of cells comprise an anti-DLL3 chimeric antigen receptor that exhibits tonic signaling and / or antigen-independent activity or signaling.

[0077] Also provided herein is a method of treatment involving administering to a subject having a disease or disorder associated with DLL3 any of the anti-DLL3 antibodies or antigen-binding fragments thereof provided herein, any single-chain cell surface proteins provided herein, any conjugates provided herein, any anti-DLL3 CARs provided herein, any polynucleotides provided herein, any vectors provided herein, any cells provided herein, or any compositions provided herein.

[0078] In some embodiments, the disease or disorder associated with DLL3 is a cancer. In some embodiments, the cancer is a DLL3-expressing cancer. In some embodiments, the disease or disorder associated with DLL3 is selected from a neuroendocrine tumor, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), melanoma, glioma, or glioblastoma. In some embodiments, the disease or disorder associated with DLL3 is a neuroendocrine tumor. In some embodiments, the disease or disorder associated with DLL3 is small cell lung cancer (SCLC). In some embodiments, the disease or disorder associated with DLL3 is large cell neuroendocrine carcinoma (LCNEC).

[0079] Also provided herein are any anti-DLL3 antibodies or antigen-binding fragments thereof provided herein, any single-chain cell surface proteins provided herein, any conjugates provided herein, any anti-DLL3 CARs provided herein, any polynucleotides provided herein, any vectors provided herein, any cells provided herein, or any compositions provided herein for use in treating a disease or disorder associated with DLL3.

[0080] Also provided herein is a use of any anti-DLL3 antibody or antigen-binding fragment thereof provided herein, any single-chain cell surface protein provided herein, any conjugate provided herein, any anti-DLL3 CAR provided herein, any polynucleotide provided herein, any vector provided herein, any cell provided herein, or any composition provided herein for the manufacture of a medicament for treating a disease or disorder associated with DLL3.

[0081] Also provided herein is the use of any anti-DLL3 antibody or antigen-binding fragment thereof provided herein, any single-chain cell surface protein provided herein, any conjugate provided herein, any anti-DLL3 CAR provided herein, any polynucleotide provided herein, any vector provided herein, any cell provided herein, or any composition provided herein for treating a disease or disorder associated with DLL3.

[0082] In some embodiments, the disease or disorder associated with DLL3 is a cancer. In some embodiments, the cancer is a DLL3-expressing cancer. In some embodiments, the disease or disorder associated with DLL3 is selected from a neuroendocrine tumor, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), melanoma, glioma, or glioblastoma. In some embodiments, the disease or disorder associated with DLL3 is a neuroendocrine tumor. In some embodiments, the disease or disorder associated with DLL3 is small cell lung cancer (SCLC). In some embodiments, the disease or disorder associated with DLL3 is large cell neuroendocrine carcinoma (LCNEC).

[0083] Also provided herein is a kit comprising any anti-DLL3 antibody or antigen-binding fragment thereof provided herein, any single-chain cell surface protein provided herein, any conjugate provided herein, any anti-DLL3 CAR provided herein, any polynucleotide provided herein, any vector provided herein, any cell provided herein, or any composition provided herein and instructions for use.

[0084] In some any embodiments, the instructions are for administering the anti-DLL3 antibody or antigen-binding fragment thereof, the single-chain cell surface protein, the conjugate, the anti-DLL3 chimeric antigen receptor, the cell, or the composition to a subject having a disease or disorder associated with DLL3.

[0085] Provided herein is an article of manufacture comprising any anti-DLL3 antibody or antigen-binding fragment thereof provided herein, any single-chain cell surface protein provided herein, any conjugate provided herein, any anti-DLL3 CAR provided herein, any polynucleotide provided herein, any vector provided herein, any cell provided herein, any composition provided herein, or any kit provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Shown are exemplary results of antigen-dependent and antigen-independent signaling based on the Jurkat T cell line, which contains the Nur77 tdTomato knock-in reporter gene and expresses one of several anti-DLL3 chimeric antigen receptors (CARs) containing a DLL3-binding single-chain Fv (scFv) obtained from immunized rats, and in the presence of K562 cells expressing human DLL1, DLL3, DLL4, or cynomolgus macaque DLL3, or the DLL3-expressing target cell NCI-H69.

[0087] Figure 2Shown are DLL3 expression levels in the DLL3-expressing small cell lung cancer target cell lines NCI-H69 (low DLL3; also referred to as "H69") or NCI-H82 (medium DLL3; also referred to as "H82").

[0088] Figure 3 Shown are IL-2 production levels after 24 hours of co-culture of primary T cells from three different donors engineered to express one of 18 candidate anti-DLL3 CARs with DLL3-expressing H69 (low DLL3) target cells at an effector:target (E:T) ratio of 1:4.

[0089] Figure 4 Shown are IL-2 production levels after 24 hours of co-culture of primary T cells from 3 different donors engineered to express one of 18 candidate anti-DLL3 CARs with DLL3-expressing H82 (medium DLL3) target cells at an effector:target (E:T) ratio of 1:4.

[0090] Figure 5 Shown are IL-2 production levels by primary T cells from two different donors engineered to express one of five anti-DLL3 CARs following co-culture with H69 (low DLL3) or H82 (medium DLL3) target cells.

[0091] Figure 6 Shown are target cell death in a spheroid killing assay following co-culture of primary T cells from two different donors engineered to express one of five anti-DLL3 CARs with tumor spheroids of H69 (low DLL3) or H82 (medium DLL3) target cells.

[0092] Figure 7 The results show that human H82 cells (DLL3) were injected subcutaneously and treated with two dose levels of 1×10 6 cells (low dose) or 4x10 6 Mean tumor volume over time in a tumor xenograft mouse model in which primary T cells engineered to express one of five anti-DLL3 CARs were administered at 400 cells (high dose).

[0093] Figure 8 Shown is the expansion of T cells engineered to express CARs administered to H82 tumor xenograft mice at two dose levels to T cells expressing one of five anti-DLL3 CARs, as assessed by CD3+ cell counts per 1 μL of blood, on days 7 and 21 after administration.

[0094] Figure 9 The results show that human H69 cells (low DLL3) were injected subcutaneously and treated with three dose levels of 1×106 cells (low), 2.5x10 6 cells (medium) or 5x10 6 Mean tumor volume over time in a tumor xenograft mouse model administered with primary T cells engineered to express CAR-A or CAR-B.

[0095] Figure 10 Shown are Kaplan-Meier survival curves for mice bearing H69 tumor xenografts administered T cells expressing CAR-A or CAR-B at three dose levels.

[0096] Figure 11 Shown is the expansion of T cells engineered to express CARs administered at three dose levels in H69 tumor xenograft mice, as assessed by CD3+ cell counts per 1 μL of blood, at days 7, 14, 21, and 28 after administration. DETAILED DESCRIPTION

[0097] Delta-like ligand 3 (DLL3) binding molecules are provided. In some aspects, the DLL3 binding molecules provided include antibodies (including antigen-binding antibody fragments, such as single-chain fragments, including single-chain Fv fragments (scFv)), recombinant receptors (including chimeric receptors comprising such antibodies or fragments), and fusion proteins and conjugates (such as immunoconjugates). Polynucleotides encoding such antibodies, fragments, or recombinant receptors are also provided. In some aspects, the embodiments provided relate to antibodies (including antigen-binding fragments) that bind to (or target) DLL3, and other binding molecules (such as fusion proteins, conjugates, T cell engagers (TCEs), and chimeric antigen receptors (CARs)) that incorporate the antibodies (including antigen-binding fragments). In particular, the embodiments provided include CARs that bind to or target DLL3 and engineered T cells expressing the CARs. In some embodiments, the targeted DLL3 is expressed by cells expressing DLL3 (such as those associated with a disease or condition).

[0098] In some aspects, DLL3 is expressed in cells or tissues associated with certain diseases and conditions, such as malignancies, e.g., on small cell lung cancer (SCLC) cells and high-grade neuroendocrine tumors. In some aspects, DLL3 exhibits minimal or no expression in normal adult tissues.

[0099] Provided embodiments include methods useful for treating diseases and disorders and / or targeting such cell types comprising nucleic acid molecules encoding DLL3 binding antibodies, fragments, fusion proteins, or receptors, and the encoded antibodies or antigen-binding fragments, fusion proteins, and receptors.

[0100] Provided embodiments include methods useful for treating diseases and disorders associated with DLL3 expression, including adoptive cell therapy involving cells expressing a chimeric antigen receptor (CAR) targeting DLL3, such as a CAR comprising a binding molecule, such as an extracellular antigen binding domain.

[0101] Also provided are compositions and articles of manufacture comprising any one or more of the polypeptides and nucleic acids provided herein.

[0102] In some aspects, a chimeric antigen receptor (CAR) is provided that generally comprises an antibody (including antigen-binding antibody fragments, such as a heavy chain variable (V H ) domains, single domain antibody fragments and single chain fragments, including scFv), for example as antigen binding domains (such as extracellular antigen binding domains). Also provided are cells, such as engineered or recombinant cells expressing such DLL3 binding receptors (e.g., anti-DLL3 chimeric antigen receptors (CARs)) and / or containing nucleic acids encoding such receptors; and compositions and products and therapeutic doses containing such cells. Also provided are methods for preparing and using antibodies and fragments and cells expressing or containing antibodies and fragments (such as cells for producing antibodies or fragments thereof). Also provided are compositions containing such antibodies, antigen binding fragments, receptors or cells, including pharmaceutical compositions, and conjugates comprising such antibodies or fragments. In some aspects, the provided compositions, antibodies, antigen binding fragments, receptors or cells can be used in combination with a therapy or treatment method.

[0103] In some cases, the properties of a particular target antigen that an antibody or recombinant receptor containing an antigen-binding domain specifically binds to, recognizes, or targets can influence the activity of the receptor. In some cases, DLL3 is expressed by certain cancers and is an attractive therapeutic target for cell-based therapies. Improved strategies are needed to achieve optimal responses to DLL3-targeted therapies.

[0104] In some situations, the best response to therapy (such as cell therapy) can also depend on the ability that the engineered recombinant receptor (such as CAR) is consistently and reliably expressed and / or combined with the target antigen on the cell surface.For example, in some cases, the heterogeneity of the RNA transcribed from the introduced transgenic (for example, encoding recombinant receptor) may affect the expression and / or activity of recombinant receptors in some cases of expression in the cell (such as human T cells) used in cell therapy.In some situations, the length and type of the spacer in recombinant receptor (such as CAR) may affect the expression, activity and / or function of receptor.

[0105] Embodiments are provided that meet such needs. The provided embodiments, such as those incorporating the provided anti-DLL3 antibodies, can be used as effective therapies targeting DLL3, such as in the treatment of cancer and other diseases and disorders, such as those associated with the expression or overexpression of DLL3. In some embodiments, the provided embodiments include cells (e.g., T cells) engineered with CARs that incorporate an anti-DLL3 antibody or antigen-binding fragment into the extracellular antigen-binding domain, which cells (e.g., T cells) can be used to target and kill cells associated with a disease or disorder (e.g., a malignancy, tumor, or cancer) that expresses DLL3 to treat the disease or disorder. In some aspects, the results herein demonstrate that exemplary CAR-engineered T cells exhibit effective antigen-dependent killing of tumor cells expressing DLL3. In some aspects, the provided embodiments are based on the observation that administration of engineered cells expressing the provided DLL3 binding molecules, such as chimeric antigen receptors (CARs), exhibits antigen-specific activity, signaling and function, high anti-tumor activity, antigen-dependent activity or signaling, and / or prolonged in vivo expansion upon administration. Such antibodies, as well as recombinant receptors (such as CARs) incorporating such antibodies, can be used to promote safe and effective treatment of specific diseases and disorders, such as diseases associated with expression of DLL3, such as small cell lung cancer (SCLC).

[0106] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that definitions set forth herein are contrary to or otherwise inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over those incorporated herein by reference.

[0107] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. DLL3 Binding Molecules

[0108] In some aspects, DLL3 binding molecules are provided, such as DLL3 binding polypeptides. Such binding molecules include antibodies (including antigen binding fragments) that specifically bind to DLL3 proteins (such as human DLL3). Binding molecules also include polypeptides containing such antibodies, including single-chain cell surface proteins (e.g., recombinant receptors, such as chimeric antigen receptors (CARs)), fusion proteins, or conjugates containing such antibodies. In some aspects, DLL3 binding recombinant receptors (such as chimeric antigen receptors (CARs) that bind to DLL3 molecules) and polynucleotides encoding DLL3 binding recombinant receptors (e.g., CARs), as well as cells expressing such receptors are provided. Also provided are polynucleotides containing all or part of the nucleic acid sequences encoding such antibodies, antigen binding fragments, recombinant receptors (such as CARs), fusion proteins, or conjugates (such as those described in Sections IA-IE) described herein. Examples of such polynucleotides include those described in Section IF. In some aspects, polynucleotides can be introduced into cells to generate engineered cells that contain or express the provided binding molecules, e.g., DLL3 binding antibodies, fragments, fusion proteins, conjugates, T cell engagers (TCEs), and recombinant receptors, such as CARs.

[0109] In some embodiments, the antibodies or antigen-binding fragments thereof provided specifically bind to the delta-like ligand 3 (DLL3) protein. DLL3 is a type I transmembrane delta-like protein that is normally expressed only on intracellular membranes (such as the Golgi apparatus). DLL3 is a member of the delta protein ligand family that functions as a Notch ligand in the Notch signaling pathway. The Notch pathway is a highly conserved cell signaling pathway that is involved in multiple aspects of developmental biology, including somite segmentation and neurogenesis during early embryonic development and the development of neuroendocrine cells in the lung. Typically, extracellular expression of DLL3 is highest in the fetal brain and is minimal in normal adult tissues, with the exception of the brain.

[0110] DLL3 is an inhibitory Notch pathway ligand that normally inhibits Notch pathway activation by binding to Notch and DLL1 and retaining them in the cytoplasm. Notch signaling is downregulated during neuroendocrine tumor growth and is inhibited by DLL3 expression. DLL3 is one of the direct downstream targets of Achaete-Scute family BHLH transcription factor 1 (ASCL1), a key transcription factor associated with small cell lung cancer (SCLC) carcinogenesis. DLL3 is highly upregulated and aberrantly expressed on the cell surface in SCLC and other high-grade neuroendocrine tumors including large cell neuroendocrine carcinoma (LCNEC), melanoma, glioma, and glioblastoma. In some aspects, DLL3 has been observed to be expressed on the surface of SCLC tumor cells and is associated with tumor and carcinogenesis, e.g., in SCLC or LCNEC. In some embodiments, the disease or disorder associated with DLL3 is cancer. In some embodiments, the cancer is a DLL3-expressing cancer. In some embodiments, the cancer is associated with a solid tumor that expresses DLL3. In some embodiments, the solid tumor expressing DLL3 is a neuroendocrine tumor.

[0111] Due to its role in a variety of diseases and conditions, including various types of cancer, DLL3 is a therapeutic target. Provided embodiments include binding molecules that bind to or target DLL3. In some embodiments, provided embodiments (e.g., DLL3 binding molecules, such as antibodies or receptors, cells expressing DLL3 binding molecules, or compositions thereof) are administered to a subject (e.g., a subject having or at risk for a disease or condition, such as cancer). In some aspects, administration results in the treatment of the disease or condition (e.g., ameliorating one or more symptoms of the disease or condition), such as by reducing tumor burden in a cancer that expresses DLL3.

[0112] In some cases, DLL3 expression has been observed to be associated with SCLC migration and invasion through mechanisms involving control of the epithelial-mesenchymal transition protein Snail. In some aspects, DLL3 expression has also been observed in other tumor types of neuroendocrine origin, including melanoma, glioblastoma multiforme, small cell bladder cancer, metastatic castration-resistant prostate cancer, castration-resistant neuroendocrine prostate cancer, and neuroendocrine lung tumors. In some embodiments, additional tumors or cancers that can be treated using provided embodiments (e.g., provided DLL3-binding molecules) include neuroendocrine tumors, such as anterior pituitary neuroendocrine tumors, thyroid neuroendocrine tumors, parathyroid tumors, lung neuroendocrine tumors (e.g., bronchial tumors, lung carcinoid tumors), gastroenteropancreatic neuroendocrine tumors (e.g., foregut gastroenteropancreatic neuroendocrine tumors, including pancreatic endocrine tumors, midgut gastroenteropancreatic neuroendocrine tumors, hindgut gastroenteropancreatic neuroendocrine tumors), liver and gallbladder tumors, adrenal gland tumors (e.g., adrenal medullary tumors), peripheral nervous system tumors (e.g., schwannomas, paragangliomas, neuroblastomas), cervical neuroendocrine tumors, prostate tumors, gliomas (e.g., astrocytomas, central neurocytomas, ependymomas, mixed gliomas, oligodendrogliomas, optic nerve gliomas, subependymal giant cell astrocytomas, glioblastomas).

[0113] In some embodiments, diseases or disorders associated with the expression of DLL3 that can be treated using the embodiments described herein include medullary carcinoma, lung cancer, extrapulmonary small cell carcinoma, goblet cell carcinoma, breast cancer, gastric cancer, glioma, glioblastoma, thyroid cancer, carcinoid tumor, pancreatic cancer, bladder cancer, prostate cancer, urinary tract carcinoid tumor and neuroendocrine cancer, ovarian cancer, testicular cancer, and Merkel cell carcinoma. In some embodiments, a disease or disorder associated with the expression of DLL3 that can be treated using any of the embodiments provided is large cell neuroendocrine carcinoma (LCNEC). In some embodiments, a disease or disorder associated with the expression of DLL3 that can be treated using any of the embodiments provided is small cell lung cancer (SCLC). A. Antibodies and Antigen-binding Fragments Targeting DLL3

[0114] Anti-DLL3 polypeptides are provided, including antibodies and functional antigen-binding fragments. DLL3 binding polypeptides include antibodies, such as single-chain antibodies (e.g., antigen-binding antibody fragments), such as those containing heavy chain variable (V H ) region and / or light chain variable (V L ) region or a portion thereof. In some embodiments, the antibody or antigen-binding fragment comprises V H and V L, such as single-chain Fv fragments (scFv). Antibodies include antibodies that specifically bind to DLL3 (e.g., human DLL3). Anti-DLL3 antibodies provided include human antibodies, antibodies modified from human antibodies, or antibodies that are variants of human antibodies. Antibodies include isolated antibodies. DLL3-binding molecules containing such antibodies are also provided, such as single-chain proteins, fusion proteins, conjugates, T cell engagers (TCEs), and / or recombinant receptors (e.g., chimeric receptors, including antigen receptors). In some aspects, DLL3-binding molecules include isolated molecules.

[0115] Also provided are DLL3-binding cell surface proteins, such as DLL3-binding recombinant receptors, such as chimeric antigen receptors (CARs). DLL3-binding recombinant receptors can contain provided antibodies (e.g., antigen-binding antibody fragments) that specifically bind to DLL3 (e.g., DLL3 proteins, such as human DLL3). In some aspects, provided binding molecules bind to the extracellular portion of DLL3. In some embodiments, the recombinant receptor is a CAR, such as those containing an anti-DLL3 antibody or an antigen-binding fragment thereof.

[0116] Also provided are polynucleotides containing nucleic acid sequences encoding all or a portion of such antibodies, antigen-binding fragments, and binding molecules. The provided polynucleotides can be incorporated into constructs, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) constructs, such as constructs that can be introduced into cells to express the encoded DLL3 binding antibodies, antigen-binding fragments, fusion proteins, conjugates, T cell engagers (TCEs), or receptors (e.g., anti-DLL3 CARs). In some aspects, also provided are encoded antibodies, antigen-binding fragments, fusion proteins, conjugates, and receptors (e.g., those containing DLL3 binding polypeptides), as well as compositions and articles and uses thereof. The term "antibody" as used herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including whole antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (V) fragments capable of specific binding to an antigen. H) region, single-chain antibody fragments (including single-chain variable fragments (scFv)) and single domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term covers genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific (e.g., bispecific or trispecific) antibodies, diabodies, triabodies and tetrabodies, tandem double scFv, tandem three scFv. Unless otherwise indicated, the term "antibody" should be understood to cover its functional antibody fragments, also referred to herein as "antigen binding fragments". The term also covers complete or full-length antibodies, including antibodies of any class or subclass (including IgG and its subclasses, IgM, IgE, IgA and IgD). Typically, naturally occurring immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain classes, or isotypes: IgM, IgD, IgG, IgA, and IgE, which determine the functional activity of the antibody molecule.

[0117] The terms "complementarity determining region" and "CDR" (synonymous with "hypervariable region" or "HVR") are known to refer to non-contiguous sequences of amino acids within the variable region of an antibody that confer antigen specificity and / or binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" and "FR" are known to refer to the non-CDR portions of the variable region of the heavy and light chains. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.

[0118] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol, 1996; 262:732-745. ("Contact" numbering scheme); Lefranc MP et al., Dev Comp Immunol, 2003; 27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plückthun A, J Mol Biol, 2003; 27(2):56-57 ("IMGT" numbering scheme); Biol, 2001; 309(3): 657-70 ("Aho" numbering scheme); Martin et al., PNAS, 1989; 86(23): 9268-9272 ("AbM" numbering scheme); and Ye et al., Nucleic Acids Res. 2013; 41 (Web Server issue): W34-40 ("IgBLAST" numbering scheme).Detailed information on the various numbering schemes is also described in the following references: for example, Jarasch et al., Proteins, 2017; 85(1): 65-71; Martin et al., Bioinformatics tools for antibody engineering.: Dübel, S. (ed.) Handbook of Therapeutic Antibodies, Volume 1 Wiley-VCH, Weinheim, Germany; Martin, ACR (2010). Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Kontermann, R., Dübel, S. (ed.) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg and Martin, ACR, Antibody Information: How to Identify CDRs by Inspecting Sequences [Online]<http: / / www.bioinf.org.uk / abs / info.html> , all of which are incorporated by reference in their entirety. A variety of prediction algorithm tools (e.g., AbYsis, Abnum, AbYmod, AbRSA, IgBLAST, IMGT, or ANARCI) are available and known for numbering antibody residues and CDRs.

[0119] The boundary of a given CDR or FR may vary according to the scheme used to identify it. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody region sequence length, with insertion in some cases. Insertion in the sequence is indicated using an insertion letter code relative to the standard numbering scheme. For example, the residue inserted between residue L30 and L31 is indicated as L31A, L31B, etc. The disappearance in the sequence relative to the standard scheme is adapted by jumping numbers. These two schemes place certain insertions and deletions ("indels") in different positions, thereby producing different numberings. For example, the Chothia numbering scheme is almost identical to the Kabat numbering scheme, except that insertions are placed in structural positions, and topologically equivalent residues are indeed assigned identical numbers. The Contact scheme is based on the analysis of complex crystal structures, and is similar to the Chothia numbering scheme in many respects. The AbM scheme is a compromise between the Kabat and Chothia definitions, and is the scheme used based on Oxford Molecular's AbM antibody modeling software. The IgBLAST protocol is based on matches to germline V, D, and J genes and can be determined using the IgBLAST tool at the National Center for Biotechnology Information (NCBI).

[0120] In some embodiments, Kabat numbering can be determined by known sequence rules as described, for example, in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Svice, National Institutes of Health, Bethesda, MD. In some embodiments, the Kabat numbering scheme can include, in some aspects, any of the following rules to designate CDRs: CDR-L1 starts at approximately residue 24 of the light chain, always with a C residue preceding, and always with a W residue following; the end of CDR-L1 is defined by a 3-residue stretch, in which the W residue can be followed by a Y, L, or F, followed by a Q or L; the length of CDR-1 is 10 to 17 residues; CDR-L2 always starts 16 residues after the end of CDR-L1; at the end of CDR-L2 The first two residues are I and Y, but can also be V and Y, I and K, or I and F; CDR-L2 is always 7 residues long; CDR-L3 always starts 33 residues after the end of CDR-L2, always has a preceding C residue, and is strictly followed by a FGXG sequence motif, where X is any amino acid; CDR-L3 is 7 to 11 residues long; CDR-H1 starts at approximately position 26 of the heavy chain; the first amino acid in CDR-H1 is always 9 residues after the conserved C residue; CDR-H1 The CDR-H1 is followed by an invariant W residue, followed by V, I, or A. The length of CDR-H1 is 5 to 7 residues. CDR-H2 always begins 15 residues after the end of CDR-H1. The first residue in CDR-H2 is usually preceded by the sequence motif LEWIG, but many variations exist. The end of CDR-H2 is defined by a 3-residue motif—the first residue of the 3-residue motif can be K or R, the second residue of the 3-residue motif can be L, I, V, F, T, or A, and the third residue of the 3-residue motif can be L, I, V, F, T, or A. The base can be T, S, I, or A; CDR-H2 is 16 to 19 residues in length; CDR-H3 always begins 33 residues after the end of CDR-H2 and is always 3 residues after the C residue—the first residue of CDR-H3 is preceded by a conserved C residue and followed by two residues, usually AR; the residue after CDR-H3 is strictly followed by a WGXG sequence motif, where X is any amino acid; CDR-H3 is typically 3 to 25 residues in length; CDR-H3 can be much longer than 25 residues.

[0121] In some cases, the exact boundary positions of certain CDRs may differ due to different definitions of CDRs according to the Chothia numbering scheme (see, e.g., Martin, ACR, Antibody Information: How to Identify CDRs by Viewing Sequences [online]).<http: / / www.bioinf.org.uk / abs / info.html> For example, in some cases, according to Chothia numbering, the boundary positions of CDR-L1 can be L26--L32 (Chothia et al., Science, 1986; 233(4765):755-8 and Chothia C. and Lesk AM J Mol Biol, 1987; 196(4):901-17). In some cases, the boundary positions of CDR-L1 can be L25--L32 (Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48). In some cases, the boundary positions of CDR-L2 can be L50--L52, and the boundary positions of CDR-L3 can be L91--L96 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk AM J Mol Biol, 1987; 196(4):901-17; Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48). In some cases, the boundary positions of CDR-H1 according to Chothia numbering can be H26-H32 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk AM J Mol Biol, 1987; 196(4):901-17; Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48). In some cases, the boundary positions of CDR-H2 can be H53-H55 (Chothia et al., Science, 1986; 233(4765):755-8 and Chothia C. and Lesk AM J Mol Biol, 1987, 196(4):901-17); H52a-H55 (Tramontano et al., J Mol Biol, 1990, 215(1):175-82). In some cases, the boundary positions of CDR-H2 can be H52-H56 (Al-Lazikani et al., J Mol Biol., 1997; 273(4):927-48).In some cases, the boundary positions of CDR-H3 can be H96-H101 (Chothia et al., Science, 1986; 233(4765):755-8 and Chothia C. and Lesk AM J Mol Biol., 1987; 196(4):901-17). In some cases, the boundary positions of CDR-H3 can be H92-H104 (Morea et al., Biophys Chem, 1997; 68(1-3):9-16 and Morea et al., J Mol Biol., 1998; 275(2):269-94).

[0122] Table 1 below illustrates exemplary numbering and lists exemplary position boundaries for CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbers are listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, e.g., FR-L1 is located before CDR-L1, FR-L2 is located between CDR-L1 and CDR-L2, FR-L3 is located between CDR-L2 and CDR-L3, and so on. It should be noted that because the Kabat numbering scheme shown places insertions at H35A and H35B, the ends of the Chothia CDR-H1 loop vary between H32 and H34 depending on the length of the loop when numbering using the Kabat numbering convention shown. 1-Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD 2-Al-Lazikani et al., (1997) JMB 273, 927-948

[0123] Thus, unless otherwise specified, a "CDR" or "complementarity determining region" of a given antibody or region thereof (such as its variable region) or individually designated CDR (e.g., CDR-H1, CDR-H2, CDR-H3) is understood to encompass one (or specific) complementarity determining region as defined by any of the aforementioned schemes or other known schemes. For example, in stating that a particular CDR (e.g., CDR-H3) comprises a given V H or VL In some embodiments, provided binding molecules (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof, or recombinant receptors (e.g., CARs) comprising a given V H CDR-H1, CDR-H2 and CDR-H3 contained in the amino acid sequence of the region and defined by any of the above schemes (such as Kabat, Chothia, AbM, IgBLAST, IMGT or Contact method or other known schemes), and a given V L The CDRs of the present invention are those defined by any of the above schemes (e.g., Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact methods, or other known schemes) as contained within the amino acid sequence of the antibody region. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of the provided antibodies are described using various numbering schemes (e.g., Kabat), but it is understood that the provided antibodies may comprise CDRs described according to any of the above numbering schemes or other known numbering schemes.

[0124] Likewise, unless otherwise specified, a given antibody or region thereof, such as a FR or individually designated one or more FRs (e.g., FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and / or FR-L4) of a variable region thereof, should be understood to encompass one (or specific) framework region as defined by any known scheme. In some cases, an identification scheme is specified for identifying a specific CDR, FR, or multiple specific FRs or CDRs, such as CDRs defined by Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact methods, or other known schemes. In some embodiments, a provided binding molecule (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof, or a recombinant receptor comprising an antibody or antigen-binding fragment thereof (e.g., CAR)) comprises a given V H FR-H1, FR-H2, FR-H3 and FR-H4 contained in the amino acid sequence of the region and defined by any of the above schemes (such as Kabat, Chothia, AbM, IgBLAST, IMGT or Contact method or other known schemes), and a given V LIn some embodiments, the amino acid sequences of CDRs or FRs are defined as FR-L1, FR-L2, FR-L3, and FR-L4 as defined by any of the above schemes (e.g., Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact methods, or other known schemes). In other cases, specific amino acid sequences of CDRs or FRs are given.

[0125] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to the antigen. H and V L ) generally have a similar structure, wherein each domain comprises four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). Single V H or V L The V domain may be sufficient to confer antigen binding specificity. In addition, V domains from antibodies that bind to the antigen may be used. H or V L Domain separation of antibodies that bind to specific antigens to screen for complementary V L or V H Libraries of domains. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0126] The antibodies provided include antibody fragments. An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; heavy chain variable (V H ) region, single-chain antibody molecules (such as scFv) and those containing only V H In some embodiments, the antibody is or comprises a single domain antibody comprising a variable heavy chain (V H ) region and variable light chain (V L In certain embodiments, the antibody is an antibody fragment comprising a heavy chain variable (V H ) region and / or light chain variable (V L ) region of a single-chain antibody fragment (e.g., scFv).

[0127] Single domain antibodies (sdAbs) are antibody fragments comprising all or part of the heavy chain variable region or all or part of the light chain variable region of an antibody.In certain embodiments, the single domain antibody is a human single domain antibody.

[0128] Antibody fragments can be prepared by various techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, antibodies are fragments produced by recombinant production, such as fragments comprising naturally occurring non-existent arrangements (such as those having two or more antibody regions or chains connected by synthetic linkers (e.g., peptide linkers), and / or fragments that can not be produced by enzymatic digestion of naturally occurring intact antibodies. In some aspects, the antibody fragment is scFv.

[0129] "Humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDR and all or substantially all FR amino acid residues are derived from people FR. Humanized antibodies optionally may include at least a portion of an antibody constant region derived from a human antibody. "Humanized form" of a non-human antibody refers to a variant of a non-human antibody that has undergone humanization to generally reduce immunogenicity to people while retaining the specificity and avidity of the parent non-human antibody. In some embodiments, some FR residues in the humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or avidity.

[0130] The anti-DLL3 antibodies provided include human antibodies. A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or a non-human source utilizing a human antibody library or other human antibody encoding sequences, including human antibody libraries. The term does not include humanized forms of non-human antibodies that comprise non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human. The term includes antigen-binding fragments of human antibodies.

[0131] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to an antigen. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus or which is present in an extrachromosomal or randomly integrated chromosome of the animal. In such transgenic animals, the endogenous immunoglobulin locus is typically inactivated. Human antibodies can also be derived from a human antibody library containing antibody coding sequences derived from a human reservoir, including phage display and cell-free libraries.

[0132] The antibody provided includes monoclonal antibodies, including monoclonal antibody fragments. As used herein, the term "monoclonal antibody" refers to a colony of substantially homogeneous antibodies (that is, the independent antibody constituting the colony is identical, except for the possible variants produced during the generation of the monoclonal antibody preparation containing naturally occurring mutations, such variants are usually present in a small amount) obtained or in the colony. Contrast with the polyclonal antibody preparations that generally include the different antibodies for different epitopes, the every kind of monoclonal antibody of the monoclonal antibody preparation is directed to a single epitope on the antigen. The term should not be construed as needing to produce antibody by any ad hoc method. Monoclonal antibodies can be prepared by various techniques, including but not limited to generating from hybridomas, recombinant DNA methods, phage display and other antibody display methods.

[0133] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides (including the provided antibodies and antibody chains and other polypeptides (e.g., linkers and DLL3-binding polypeptides)) can include amino acid residues containing natural and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some aspects, a polypeptide can contain modifications relative to the native or natural sequence, so long as the protein maintains the desired activity. These modifications may be intentional (e.g., through site-directed mutagenesis) or may be accidental (e.g., through mutations in the host producing the protein or errors due to PCR amplification).

[0134] In some of any of the provided anti-DLL3 antibodies or antigen-binding fragments thereof, the heavy and / or light chain variable (V H or V L ) region sequence, or a sufficient antigen-binding portion thereof. In some embodiments, the antibody (e.g., an anti-DLL3 antibody, e.g., an antigen-binding antibody fragment) comprises a heavy chain variable region (V H ) sequence and / or light chain variable region (V L ) sequence, or a sufficient antigen-binding portion thereof. In some embodiments, the antibody (e.g., an anti-DLL3 antibody, e.g., an antigen-binding antibody fragment) is a single-chain fragment, such as a single-chain Fv (scFv) fragment. In some aspects, the scFv comprises a V H Area and V L In some embodiments, the antibody (e.g., an anti-DLL3 antibody, e.g., an antigen-binding antibody fragment) is a single domain antibody (sdAb), e.g., comprising only V H Antibodies to the region.

[0135] In some embodiments, the antibody or antigen binding fragment (e.g., extracellular antigen binding domain) contained in any provided recombinant receptor (e.g., chimeric antigen receptor (CAR; e.g., anti-DLL3 CAR)) comprises a heavy chain or a sufficient antigen binding portion thereof. For example, in some embodiments, the antibody or antigen binding fragment thereof comprises a variable heavy chain (V H ) area or V H In some embodiments, the antibody or antigen binding fragment (e.g., extracellular antigen binding domain) contained in any provided recombinant receptor (e.g., chimeric antigen receptor (CAR; e.g., anti-DLL3 CAR)) comprises a light chain or a sufficient antigen binding portion thereof. For example, in some embodiments, the antibody or antigen binding fragment thereof comprises a variable light chain (V L ) area or V L In some embodiments, the antibody or antigen-binding fragment (e.g., extracellular antigen-binding domain) contained in any provided recombinant receptor (e.g., chimeric antigen receptor (CAR; e.g., anti-DLL3 CAR)) comprises a V H Area or V H The sufficient antigen binding portion of the V L Area or V L In some embodiments, an antibody or antigen-binding fragment thereof (such as an antigen-binding fragment of a recombinant receptor (e.g., CAR)) comprises a V H Area and V L Area or V H Area and V L In any such embodiment, V H The region sequence can be any V region described herein. H In any such embodiment, V L The region sequence can be any V region described herein. L In any such embodiment, any V region sequence described herein H Region sequence and any V L Region sequence can be used in combination.In some any such embodiments, any one or more of CDR-H1, CDR-H2 and / or CDR-H3 sequences described herein and any one or more of CDR-L1, CDR-L2 and / or CDR-L3 sequences described herein can be used in combination.In some such embodiments, the antibody is an antigen binding fragment, such as Fab or scFv.In some embodiments, the antibody or antigen binding fragment also includes at least a portion of an immunoglobulin constant region or a variant thereof, such as an antigen binding fragment of a recombinant receptor (eg, CAR).In some such embodiments, the antibody is a full-length antibody also including a constant region.

[0136] In some embodiments, the recombinant receptor (e.g., a CAR provided herein) comprises an antibody (e.g., an anti-DLL3 antibody) or an antigen-binding fragment thereof comprising any V H In some embodiments, the recombinant receptor (e.g., a CAR as provided herein) comprises an antibody (e.g., an anti-DLL3 antibody) or an antigen-binding fragment thereof comprising any V L For example, in some embodiments, the CAR comprises an antibody or antigen-binding fragment thereof comprising a V region or a sufficient antigen-binding portion thereof. H Area and V L Area, or V H and V L In any such embodiment, V L The region sequence can be any V region described herein. L In any such embodiment, V H The region sequence can be any V region described herein. H In any such embodiment, any V region sequence described herein H Region sequence and any V L Region sequence can be used in combination.In some any such embodiments, any one or more of CDR-H1, CDR-H2 and / or CDR-H3 sequences described herein and any one or more of CDR-L1, CDR-L2 and / or CDR-L3 sequences described herein can be used in combination.In some such embodiments, the antibody contained in the recombinant receptor provided is an antigen binding fragment, such as Fab or scFv.In some such embodiments, receptor (for example, CAR) also includes a spacer, such as a part of an immunoglobulin constant region or a variant thereof, for example, as described in Section 1B below.

[0137] In some embodiments, an anti-DLL3 antibody (eg, antigen-binding antibody fragment) comprises V H region sequence or a sufficient antigen binding portion thereof, said V H The V region sequence or a sufficient antigen-binding portion thereof comprises a heavy chain complementary determining region 1 (CDR-H1), a heavy chain complementary determining region 2 (CDR-H2) and / or a heavy chain complementary determining region 3 (CDR-H3) contained therein and / or as described. In some embodiments, an anti-DLL3 antibody (e.g., an antigen-binding antibody fragment) comprises V H region sequence or a sufficient antigen binding portion thereof, said V HThe V region sequence or a sufficient antigen binding portion thereof comprises CDR-H1, CDR-H2 and CDR-H3 contained therein and / or as described. In some embodiments, the anti-DLL3 antibody (e.g., antigen-binding antibody fragment) comprises V L region sequence or sufficient antigen binding portion, the V L Region sequence or sufficient antigen binding portion thereof comprises a light chain complementary determining region 1 (CDR-L1), a light chain complementary determining region 2 (CDR-L2) and / or a light chain complementary determining region 3 (CDR-L3) contained therein and / or as described. In some embodiments, an anti-DLL3 antibody (e.g., an antigen-binding antibody fragment) comprises V L region sequence or sufficient antigen binding portion, the V L The region sequence or sufficient antigen binding portion comprises CDR-L1, CDR-L2 and CDR-L3 contained therein and / or as described.

[0138] In some embodiments, an anti-DLL3 antibody (eg, antigen-binding antibody fragment) comprises V H Region sequence, the V H The region sequence contains CDR-H1, CDR-H2 and / or CDR-H3 as described; and comprises V L Region sequence, the V L The region sequence contains CDR-L1, CDR-L2 and / or CDR-L3 as described. In some embodiments, the anti-DLL3 antibody (e.g., antigen-binding antibody fragment) comprises V H Region sequence, the V H The region sequence contains CDR-H1, CDR-H2 and CDR-H3 as described; and comprises V L Region sequence, the V L The region sequence contains CDR-L1, CDR-L2 and CDR-L3 as described. The antibodies and fragments thereof provided also include antibodies and fragments thereof having the following sequences, which are similar to such sequences, for example, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, V H 、V L, scFv sequence or other antibodies described herein or the sequence of its fragment is at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98% or at least or at least about 99% identical. In some aspects, the antibody or its fragment provided has at least or at least about 95% sequence identity with any such sequence. In some aspects, the antibody or its fragment provided has at least or at least about 96% sequence identity with any such sequence. In some aspects, the antibody or its fragment provided has at least or at least about 97% sequence identity with any such sequence. In some aspects, the antibody or its fragment provided has at least or at least about 98% sequence identity with any such sequence. In some aspects, the antibody or its fragment provided has at least or at least about 99% sequence identity with any such sequence.

[0139] In some embodiments, the antibody is an sdAb comprising only V H A region sequence or a sufficient antigen-binding portion thereof, such as any V H Sequences (e.g., CDR-H1, CDR-H2, CDR-H3, and / or CDR-H4). In some embodiments, the antibody or antigen-binding fragment comprises a V-containing H Those of single domain antibodies that are not bound to V L The invention can specifically bind to DLL3 in the absence of pairing with the antibody domains and / or in the absence of any additional antibody domains or binding sites.

[0140] In some embodiments, the V of the antibody or antigen-binding fragment thereof is H The V region comprises CDR-H1, CDR-H2 and / or CDR-H3 according to Kabat numbering. In some embodiments, the V region of the antibody or antigen-binding fragment thereof is H The V region comprises CDR-H1, CDR-H2 and / or CDR-H3 according to Chothia numbering. In some embodiments, the V region of the antibody or antigen-binding fragment thereof is H The V region comprises CDR-H1, CDR-H2 and / or CDR-H3 according to AbM numbering. In some embodiments, the V region of an antibody or antigen-binding fragment thereof is H The region comprises CDR-H1, CDR-H2 and / or CDR-H3 according to Kabat, Chothia, AbM or IMGT numbering or other numbering schemes.

[0141] In some embodiments of any of the provided embodiments, the binding molecules (e.g., antibodies or antigen-binding fragments thereof) have a heavy chain variable (V H ) region, the heavy chain variable (V H) region comprises a heavy chain complementary determining region 1 (CDR-H1), a heavy chain complementary determining region 2 (CDR-H2), and / or a heavy chain complementary determining region 3 (CDR-H3) present in SEQ ID NO: 90, such as CDR-H1, CDR-H2, and / or CDR-H3 according to the Kabat, Chothia, AbM, or IMGT numbering schemes, or other numbering schemes. In some of any of the provided embodiments, the binding molecule (e.g., antibody or antigen-binding fragment thereof) has a V H , the V H The CDR-H1, CDR-H2 and CDR-H3 present in SEQ ID NO: 90 are included, such as CDR-H1, CDR-H2 and CDR-H3 according to Kabat, Chothia, AbM or IMGT numbering or other numbering schemes.

[0142] In some of any of the provided embodiments, the binding molecule (e.g., antibody or antigen-binding fragment thereof) comprises V H Area, the V H The region has the amino acid sequence shown in SEQ ID NO: 90, or the V region shown in SEQ ID NO: 90. H The amino acid sequence of the V region has at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity, and / or comprises an amino acid sequence present in any of the aforementioned V H CDR-H1, CDR-H2 and CDR-H3 in the sequence, such as a CDR-H1, CDR-H2 and CDR-H3 present in any of the aforementioned V H In some embodiments of any of the provided embodiments, V H The region comprises the V region shown in SEQ ID NO: 90 H In some embodiments, the binding molecule (e.g., antibody or antigen-binding fragment thereof) comprises a V region amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity. H Area, the V H The region has the amino acid sequence shown in SEQ ID NO: 90, or the V region shown in SEQ ID NO: 90. H an amino acid sequence having at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of the V region; or H A region comprising CDR-H1, CDR-H2 and CDR-H3 present in SEQ ID NO: 90, such as a V region comprising CDR-H1, CDR-H2 and CDR-H3 present in SEQ ID NO: 90 H In some of any of the provided embodiments, VH The region comprises CDR-H1, CDR-H2 and CDR-H3, wherein the CDR-H1, CDR-H2 and CDR-H3 respectively comprise the V sequences shown in SEQ ID NO: 90. H In some embodiments, V H The region comprises CDR-H1, CDR-H2 and CDR-H3 contained within SEQ ID NO:90.

[0143] In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence shown in SEQ ID NO: 92. In some of any of the provided embodiments, V H The region comprises a CDR-H2 comprising the sequence shown in SEQ ID NO: 93. In some of any of the provided embodiments, V H The region comprises a CDR-H3 comprising the sequence shown in SEQ ID NO: 94. In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 92, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 93, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 94. In some of any of the provided embodiments, V H The region comprises CDR-H1, CDR-H2, and CDR-H3 comprising the sequences shown in SEQ ID NOs: 92, 93, and 94, respectively.

[0144] In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence shown in SEQ ID NO: 98. In some of any of the provided embodiments, V H The region comprises a CDR-H2 comprising the sequence shown in SEQ ID NO: 99. In some of any of the provided embodiments, V H The region comprises a CDR-H3 comprising the sequence shown in SEQ ID NO: 100. In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 98, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 99, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 100. In some of any of the provided embodiments, V HThe region comprises CDR-H1, CDR-H2, and CDR-H3 comprising the sequences shown in SEQ ID NOs: 98, 99, and 100, respectively.

[0145] In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence shown in SEQ ID NO: 104. In some of any of the provided embodiments, V H The region comprises a CDR-H2 comprising the sequence shown in SEQ ID NO: 105. In some of any of the provided embodiments, V H The region comprises a CDR-H3 comprising the sequence shown in SEQ ID NO: 106. In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 104, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 105, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 106. In some of any of the provided embodiments, V H The region comprises CDR-H1, CDR-H2, and CDR-H3 comprising the sequences shown in SEQ ID NOs: 104, 105, and 106, respectively.

[0146] In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence shown in SEQ ID NO: 110. In some of any of the provided embodiments, V H The region comprises a CDR-H2 comprising the sequence shown in SEQ ID NO: 111. In some of any of the provided embodiments, V H The region comprises a CDR-H3 comprising the sequence shown in SEQ ID NO: 112. In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 110, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 111, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 112. In some of any of the provided embodiments, V H The region comprises CDR-H1, CDR-H2, and CDR-H3 comprising the sequences shown in SEQ ID NOs: 110, 111, and 112, respectively.

[0147] In some of any of the provided embodiments, V HThe region comprises any one of CDR-H1, CDR-H2 and CDR-H3 as described, and comprises framework region 1 (FR1), FR2, FR3 and / or FR4, wherein the framework region 1 (FR1), FR2, FR3 and / or FR4 are respectively aligned with the V region shown in SEQ ID NO: 90. H The amino acid sequences of the V region of the present invention may have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the FR1, FR2, FR3 and / or FR4 contained therein. H The region comprises FR1, FR2, FR3 and / or FR4, and the FR1, FR2, FR3 and / or FR4 are respectively aligned with the V region shown in SEQ ID NO: 90. H In some embodiments, the amino acid sequences of the FR1, FR2, FR3 and / or FR4 regions have at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0148] In some of any of the provided embodiments, V H Region comprises the amino acid sequence shown in SEQ ID NO: 90. In some of any of the provided embodiments, V H The region has the amino acid sequence shown in SEQ ID NO:90.

[0149] In some of any of the provided embodiments, V H The region comprises an amino acid sequence encoded by SEQ ID NO: 87 or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 87. In some embodiments of any of the provided embodiments, V H The region comprises the amino acid sequence encoded by SEQ ID NO:87.

[0150] In some embodiments, the V of the antibody or antigen-binding fragment thereof is L The V region comprises CDR-L1, CDR-L2 and / or CDR-L3 according to Kabat numbering. In some embodiments, the V region of the antibody or antigen-binding fragment thereof is L The V region comprises CDR-L1, CDR-L2 and / or CDR-L3 according to Chothia numbering. In some embodiments, the V region of the antibody or antigen-binding fragment thereof is LThe V region comprises CDR-L1, CDR-L2 and / or CDR-L3 according to AbM numbering. In some embodiments, the V region of an antibody or antigen-binding fragment thereof is L The region comprises CDR-L1, CDR-L2 and / or CDR-L3 according to Kabat, Chothia, AbM or IMGT numbering or other numbering schemes.

[0151] In some embodiments of any of the provided embodiments, the binding molecules (e.g., antibodies or antigen-binding fragments thereof) have a light chain variable (V L ) region, the light chain variable (V L ) region comprises a light chain complementary determining region 1 (CDR-L1), a light chain complementary determining region 2 (CDR-L2), and / or a light chain complementary determining region 3 (CDR-L3) present in SEQ ID NO: 91, such as CDR-L1, CDR-L2, and / or CDR-L3 according to the Kabat, Chothia, AbM, or IMGT numbering schemes, or other numbering schemes. In some of any of the provided embodiments, the binding molecule (e.g., antibody or antigen-binding fragment thereof) has a V L , the V L Comprising the CDR-L1, CDR-L2 and CDR-L3 present in SEQ ID NO: 91, such as CDR-L1, CDR-L2 and CDR-L3 according to Kabat, Chothia, AbM or IMGT numbering or other numbering schemes.

[0152] In some of any of the provided embodiments, the binding molecule (e.g., antibody or antigen-binding fragment thereof) comprises V L Area, the V L The region has the amino acid sequence shown in SEQ ID NO: 91, or the V region shown in SEQ ID NO: 91. L V domain amino acid sequences having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and / or comprising the amino acid sequences present in any of the aforementioned V L CDR-L1, CDR-L2 and CDR-L3 in the sequence, such as a CDR-L1, CDR-L2 and CDR-L3 present in any of the aforementioned V L In some embodiments of any of the provided embodiments, V L The region comprises the V region shown in SEQ ID NO: 91 LIn some embodiments, the binding molecule (e.g., antibody or antigen-binding fragment thereof) comprises a V region amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. L Area, the V L The region has the amino acid sequence shown in SEQ ID NO: 91, or the V region shown in SEQ ID NO: 91. L or an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the V region amino acid sequence; or L A region comprising CDR-L1, CDR-L2 and CDR-L3 present in SEQ ID NO: 91, such as a V region comprising CDR-L1, CDR-L2 and CDR-L3 present in SEQ ID NO: 91 L In some of any of the provided embodiments, V L The region comprises CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 respectively comprise the V sequences shown in SEQ ID NO: 91. L In some embodiments, V L The region comprises CDR-L1, CDR-L2 and CDR-L3 contained within SEQ ID NO:91.

[0153] In some of any of the provided embodiments, V L The region comprises a CDR-L1 comprising the sequence shown in SEQ ID NO: 95. In some of any of the provided embodiments, V L The region comprises a CDR-L2 comprising the sequence shown in SEQ ID NO: 96. In some of any of the provided embodiments, V L The region comprises a CDR-L3 comprising the sequence shown in SEQ ID NO: 97. In some of any of the provided embodiments, V L The region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 95; a CDR-L2 comprising the sequence set forth in SEQ ID NO: 96; and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 97. In some of any of the provided embodiments, V LThe region comprises CDR-L1, CDR-L2, and CDR-L3 comprising the sequences shown in SEQ ID NOs: 95, 96, and 97, respectively.

[0154] In some of any of the provided embodiments, V L The region comprises a CDR-L1 comprising the sequence shown in SEQ ID NO: 101. In some of any of the provided embodiments, V L The region comprises a CDR-L2 comprising the sequence shown in SEQ ID NO: 102. In some of any of the provided embodiments, V L The region comprises a CDR-L3 comprising the sequence shown in SEQ ID NO: 103. In some of any of the provided embodiments, V L The region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 101; a CDR-L2 comprising the sequence set forth in SEQ ID NO: 102; and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 103. In some of any of the provided embodiments, V L The region comprises CDR-L1, CDR-L2 and CDR-L3 comprising the sequences shown in SEQ ID NOs: 101, 102 and 103, respectively.

[0155] In some of any of the provided embodiments, V L The region comprises a CDR-L1 comprising the sequence shown in SEQ ID NO: 107. In some of any of the provided embodiments, V L The region comprises a CDR-L2 comprising the amino acid sequence GA as set forth in SEQ ID NO: 108. In some of any of the provided embodiments, V L The region comprises a CDR-L3 comprising the sequence shown in SEQ ID NO: 109. In some of any of the provided embodiments, V L The region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 107; a CDR-L2 comprising the amino acid sequence GA set forth in SEQ ID NO: 108; and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 109. In some of any of the provided embodiments, V L The region comprises CDR-L1, CDR-L2, and CDR-L3, comprising the sequence shown in SEQ ID NO: 107, sequence GA shown in SEQ ID NO: 108, and the sequence shown in SEQ ID NO: 109, respectively.

[0156] In some of any of the provided embodiments, V L The region comprises a CDR-L1 comprising the sequence shown in SEQ ID NO: 113. In some of any of the provided embodiments, V L The region comprises a CDR-L2 comprising the sequence shown in SEQ ID NO: 114. In some of any of the provided embodiments, V L The region comprises a CDR-L3 comprising the sequence shown in SEQ ID NO: 115. In some of any of the provided embodiments, V L The region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 113; a CDR-L2 comprising the sequence set forth in SEQ ID NO: 114; and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 115. In some of any of the provided embodiments, V L The region comprises CDR-L1, CDR-L2, and CDR-L3 comprising the sequences shown in SEQ ID NOs: 113, 114, and 115, respectively.

[0157] In some of any of the provided embodiments, V L The region comprises any one of CDR-L1, CDR-L2 and CDR-L3 as described, and comprises framework region 1 (FR1), FR2, FR3 and / or FR4, wherein the framework region 1 (FR1), FR2, FR3 and / or FR4 are respectively aligned with the V region shown in SEQ ID NO: 91. L The amino acid sequences of the V region of the present invention may have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the FR1, FR2, FR3 and / or FR4 contained therein. L The region comprises FR1, FR2, FR3 and / or FR4, and the FR1, FR2, FR3 and / or FR4 are respectively aligned with the V region shown in SEQ ID NO: 91. L In some embodiments, the amino acid sequences of the FR1, FR2, FR3 and / or FR4 regions have at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0158] In some of any of the provided embodiments, V LRegion comprises the amino acid sequence shown in SEQ ID NO: 91. In some of any of the provided embodiments, V L The region has the amino acid sequence shown in SEQ ID NO:91.

[0159] In some of any of the provided embodiments, V L The region comprises an amino acid sequence encoded by SEQ ID NO: 88 or a nucleic acid sequence having at least or at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 88. In some of any of the provided embodiments, V L The region comprises the amino acid sequence encoded by SEQ ID NO:88.

[0160] In some embodiments, the V of any provided recombinant receptor (e.g., CAR), conjugate, or antibody or antigen-binding fragment thereof is H The V region comprises the amino acid sequence of SEQ ID NO: 90, and the V region of any provided recombinant receptor (e.g., CAR), conjugate, or antibody or antigen-binding fragment thereof is L The region comprises the amino acid sequence of SEQ ID NO:91.

[0161] In some embodiments, the V of any provided recombinant receptor (e.g., CAR), conjugate, or antibody or antigen-binding fragment thereof is H and V L The regions comprise the amino acid sequences of SEQ ID NOs: 90 and 91, respectively, or any of the above V H and V L A sequence having at least or at least about 90% sequence identity (e.g., at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity). For example, wherein the V H and V L The regions comprise the amino acid sequences shown in SEQ ID NOs: 90 and 91, respectively.

[0162] In some of any of the provided embodiments, V H Area and V L The regions are or comprise the sequences shown in SEQ ID NOs: 90 and 91, respectively.

[0163] In some embodiments of any of the provided embodiments, the binding molecules (e.g., antibodies or antigen-binding fragments thereof) have a heavy chain variable (V H ) region, the heavy chain variable (VH ) region comprises a heavy chain complementary determining region 1 (CDR-H1), a heavy chain complementary determining region 2 (CDR-H2), and / or a heavy chain complementary determining region 3 (CDR-H3) present in SEQ ID NO: 90, such as CDR-H1, CDR-H2, and / or CDR-H3 according to Kabat, Chothia, AbM, or IMGT numbering, or other numbering schemes; and a light chain variable (V L ) region, the light chain variable (V L ) region comprises a light chain complementary determining region 1 (CDR-L1), a light chain complementary determining region 2 (CDR-L2), and / or a light chain complementary determining region 3 (CDR-L3) present in SEQ ID NO: 91, such as CDR-L1, CDR-L2, and / or CDR-L3 according to Kabat, Chothia, AbM, or IMGT numbering, or other numbering schemes. In some of any of the provided embodiments, the binding molecule (e.g., antibody or antigen-binding fragment thereof) has a V H , the V H comprising CDR-H1, CDR-H2, and CDR-H3 as present in SEQ ID NO:90, such as CDR-H1, CDR-H2, and CDR-H3 as numbered according to Kabat, Chothia, AbM, or IMGT, or other numbering schemes; and V L , the V L Comprising the CDR-L1, CDR-L2 and CDR-L3 present in SEQ ID NO: 91, such as CDR-L1, CDR-L2 and CDR-L3 according to Kabat, Chothia, AbM or IMGT numbering or other numbering schemes.

[0164] In some of any of the provided embodiments, the binding molecule (e.g., antibody or antigen-binding fragment thereof) comprises V H Area, the V H The region has the amino acid sequence shown in SEQ ID NO: 90, or the V region shown in SEQ ID NO: 90. H The amino acid sequence of the V region has at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity, and / or comprises an amino acid sequence present in any of the aforementioned V H CDR-H1, CDR-H2 and CDR-H3 in the sequence, such as a CDR-H1, CDR-H2 and CDR-H3 present in any of the aforementioned V H The sequences of CDR-H1, CDR-H2 and CDR-H3 in the sequence; and V L Area, the V L The region has the amino acid sequence shown in SEQ ID NO: 91, or the V region shown in SEQ ID NO: 91. LV domain amino acid sequences having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and / or comprising the amino acid sequences present in any of the aforementioned V L CDR-L1, CDR-L2 and CDR-L3 in the sequence, such as a CDR-L1, CDR-L2 and CDR-L3 present in any of the aforementioned V L In some embodiments of any of the provided embodiments, V H The region comprises the V region shown in SEQ ID NO: 90 H The amino acid sequence of the V region has at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity, and the V L The region comprises the V region shown in SEQ ID NO: 91 L In some embodiments, the binding molecule (e.g., antibody or antigen-binding fragment thereof) comprises a V region amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. H Area, the V H The region has the amino acid sequence shown in SEQ ID NO: 90, or the V region shown in SEQ ID NO: 90. H an amino acid sequence having at least or about 95%, 96%, 97%, 98%, or 99% sequence identity to a CDR-H1, CDR-H2, and CDR-H3 region of the SEQ ID NO: 90, or comprising the CDR-H1, CDR-H2, and CDR-H3 region of the SEQ ID NO: 90, such as a sequence comprising the CDR-H1, CDR-H2, and CDR-H3 region of the SEQ ID NO: 90; and V L Area, the V L The region has the amino acid sequence shown in SEQ ID NO: 91, or the V region shown in SEQ ID NO: 91. L The amino acid sequence of the V region has at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or comprises the CDR-L1, CDR-L2, and CDR-L3 present in SEQ ID NO: 91, such as a sequence comprising the CDR-L1, CDR-L2, and CDR-L3 present in SEQ ID NO: 91. In some of any of the provided embodiments, V HThe region comprises CDR-H1, CDR-H2 and CDR-H3, wherein the CDR-H1, CDR-H2 and CDR-H3 respectively comprise the V sequences shown in SEQ ID NO: 90. H In some embodiments, V H The region comprises CDR-H1, CDR-H2 and CDR-H3 contained in SEQ ID NO: 90, and V L The region comprises CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 respectively comprise the V sequences shown in SEQ ID NO: 91. L In some embodiments, V L The region comprises CDR-L1, CDR-L2 and CDR-L3 contained within SEQ ID NO:91.

[0165] In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence shown in SEQ ID NO: 92, and V L The region comprises a CDR-L1 comprising the sequence shown in SEQ ID NO: 95. In some of any of the provided embodiments, V H The region comprises CDR-H2 comprising the sequence shown in SEQ ID NO: 93, and V L The region comprises a CDR-L2 comprising the sequence shown in SEQ ID NO: 96. In some of any of the provided embodiments, V H The region comprises CDR-H3 comprising the sequence shown in SEQ ID NO: 94, and V L The region comprises a CDR-L3 comprising the sequence shown in SEQ ID NO: 97. In some of any of the provided embodiments, V H The region comprises CDR-H1 comprising the sequence shown in SEQ ID NO: 92, CDR-H2 comprising the sequence shown in SEQ ID NO: 93, and CDR-H3 comprising the sequence shown in SEQ ID NO: 94, and V L The region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 95, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 96, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 97. In some of any of the provided embodiments, V HThe V region comprises CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences shown in SEQ ID NOs: 92, 93, and 94, respectively, and V L The region comprises CDR-L1, CDR-L2, and CDR-L3 comprising the sequences shown in SEQ ID NOs: 95, 96, and 97, respectively.

[0166] In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence shown in SEQ ID NO: 98, and V L The region comprises a CDR-L1 comprising the sequence shown in SEQ ID NO: 101. In some of any of the provided embodiments, V H The region comprises a CDR-H2 comprising the sequence shown in SEQ ID NO: 99, and V L The region comprises a CDR-L2 comprising the sequence shown in SEQ ID NO: 102. In some of any of the provided embodiments, V H The region comprises a CDR-H3 comprising the sequence shown in SEQ ID NO: 100, and V L The region comprises a CDR-L3 comprising the sequence shown in SEQ ID NO: 103. In some of any of the provided embodiments, V H The region comprises CDR-H1 comprising the sequence shown in SEQ ID NO: 98, CDR-H2 comprising the sequence shown in SEQ ID NO: 99, and CDR-H3 comprising the sequence shown in SEQ ID NO: 100, and V L The region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 101, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 102, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 103. In some of any of the provided embodiments, V H The V region comprises CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences shown in SEQ ID NOs: 98, 99, and 100, respectively, and V L The region comprises CDR-L1, CDR-L2 and CDR-L3 comprising the sequences shown in SEQ ID NOs: 101, 102 and 103, respectively.

[0167] In some of any of the provided embodiments, V HThe region comprises a CDR-H1 comprising the sequence shown in SEQ ID NO: 104, and V L The region comprises a CDR-L1 comprising the sequence shown in SEQ ID NO: 107. In some of any of the provided embodiments, V H The region comprises CDR-H2 comprising the sequence shown in SEQ ID NO: 105, and V L The region comprises a CDR-L2 comprising the amino acid sequence GA as shown in SEQ ID NO: 108. In some of any of the provided embodiments, V H The region comprises CDR-H3 comprising the sequence shown in SEQ ID NO: 106, and V L The region comprises a CDR-L3 comprising the sequence shown in SEQ ID NO: 109. In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence shown in SEQ ID NO: 104, a CDR-H2 comprising the sequence shown in SEQ ID NO: 105, and a CDR-H3 comprising the sequence shown in SEQ ID NO: 106, and V L The region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 107, a CDR-L2 comprising the amino acid sequence GA set forth in SEQ ID NO: 108, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 109. In some of any of the provided embodiments, V H The V region comprises CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences shown in SEQ ID NOs: 104, 105, and 106, respectively, and V L The region comprises CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1, CDR-L2, and CDR-L3 comprise the sequence shown in SEQ ID NO: 107, sequence GA shown in SEQ ID NO: 108, and sequence Q shown in SEQ ID NO: 109, respectively.

[0168] In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence shown in SEQ ID NO: 110, and V L The region comprises a CDR-L1 comprising the sequence shown in SEQ ID NO: 113. In some of any of the provided embodiments, V H The region comprises a CDR-H2 comprising the sequence shown in SEQ ID NO: 111, and V LThe region comprises a CDR-L2 comprising the sequence shown in SEQ ID NO: 114. In some of any of the provided embodiments, V H The region comprises a CDR-H3 comprising the sequence shown in SEQ ID NO: 112, and V L The region comprises a CDR-L3 comprising the sequence shown in SEQ ID NO: 115. In some of any of the provided embodiments, V H The region comprises a CDR-H1 comprising the sequence shown in SEQ ID NO: 110, a CDR-H2 comprising the sequence shown in SEQ ID NO: 111, and a CDR-H3 comprising the sequence shown in SEQ ID NO: 112, and V L The region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 113, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 114, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 115. In some of any of the provided embodiments, V H The V region comprises CDR-H1, CDR-H2 and CDR-H3, wherein the CDR-H1, CDR-H2 and CDR-H3 comprise the sequences shown in SEQ ID NOs: 110, 111 and 112, respectively, and V L The region comprises CDR-L1, CDR-L2, and CDR-L3 comprising the sequences shown in SEQ ID NOs: 113, 114, and 115, respectively.

[0169] In some of any of the provided embodiments, V H The region comprises any one of CDR-H1, CDR-H2 and CDR-H3 as described, and comprises framework region 1 (FR1), FR2, FR3 and / or FR4, wherein the framework region 1 (FR1), FR2, FR3 and / or FR4 are respectively aligned with the V region shown in SEQ ID NO: 90. H The amino acid sequences of the V region of the present invention may have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the FR1, FR2, FR3 and / or FR4 contained therein. H The region comprises FR1, FR2, FR3 and / or FR4, and the FR1, FR2, FR3 and / or FR4 are aligned with the V region shown in SEQ ID NO: 90. HThe amino acid sequences of the V region of the present invention each contain at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to FR1, FR2, FR3 and / or FR4, respectively, and V L The region comprises any of the CDR-L1, CDR-L2 and CDR-L3 as described, and comprises framework region 1 (FR1), FR2, FR3 and / or FR4, and the framework region 1 (FR1), FR2, FR3 and / or FR4 are aligned with the V region shown in SEQ ID NO: 91. L The amino acid sequences of the V region of the present invention each contain at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to FR1, FR2, FR3 and / or FR4, respectively. L The region comprises FR1, FR2, FR3 and / or FR4, and the FR1, FR2, FR3 and / or FR4 are respectively aligned with the V region shown in SEQ ID NO: 91. L In some embodiments, the amino acid sequences of the FR1, FR2, FR3 and / or FR4 regions have at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0170] In some of any of the provided embodiments, V H The region comprises the amino acid sequence shown in SEQ ID NO: 90, and V L Region comprises the amino acid sequence shown in SEQ ID NO: 91. In some of any of the provided embodiments, V H The region has the amino acid sequence shown in SEQ ID NO: 90, and V L The region has the amino acid sequence shown in SEQ ID NO:91.

[0171] In some of any of the provided embodiments, V H The region comprises an amino acid sequence encoded by SEQ ID NO: 87, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 87, and V LThe region comprises an amino acid sequence encoded by SEQ ID NO: 88 or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 88. In some of any of the provided embodiments, V H The region comprises the amino acid sequence encoded by SEQ ID NO: 87, and V L The region comprises the amino acid sequence encoded by SEQ ID NO:88.

[0172] Also provided are polynucleotides comprising any of the nucleotide sequences described herein, eg, encoding all or a portion of a provided binding molecule.

[0173] In some embodiments, the antibody or antigen-binding fragment thereof (e.g., in any recombinant receptor provided herein (e.g., CAR)) is a single-chain antibody fragment, such as a single-chain variable fragment (scFv) or a diabody or a single domain antibody (sdAb). In some embodiments, the antibody or antigen-binding fragment is a multi-domain antibody, such as one comprising a heavy chain variable (V H ) region and light chain variable (V L In some embodiments, a single-chain antibody fragment (e.g., scFv) comprises a single-chain antibody fragment (e.g., scFv) comprising two antibody domains or regions (e.g., heavy chain variable (V H ) region and light chain variable (V L ) region). The linker is typically a peptide linker, for example, a flexible and / or soluble peptide linker. Linkers include those rich in glycine and serine and / or in some cases rich in threonine. In some embodiments, the linker also includes charged residues such as lysine and / or glutamic acid, which can improve solubility. In some embodiments, the linker also includes one or more proline residues.

[0174] Thus, the CARs provided contain anti-DLL3 antibodies, including single-chain antibody fragments, such as scFv and diabodies, particularly human single-chain antibody fragments, which generally comprise two antibody domains or regions (such as V H and V L The linker is typically a peptide linker, such as a flexible and / or soluble peptide linker, such as a peptide linker rich in glycine and serine.

[0175] In some aspects, the joint that is rich in glycine and serine (and / or threonine) comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% such amino acid.In some embodiments, they include at least or at least about 50%, 55%, 60%, 70% or 75% glycine, serine and / or threonine.In some embodiments, the joint is basically made up of glycine, serine and / or threonine completely.The length of joint is usually between about 5 and about 50 amino acid, usually between 10 and 30, for example, 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30, and in some examples length is between 10 and 25 amino acid. Exemplary linkers include linkers with varying numbers of repeats of the sequence GGGGS (4GS; SEQ ID NO: 44) or GGGS (3GS; SEQ ID NO: 45), such as between 2, 3, 4, and 5 repeats of this sequence. Exemplary linkers include those having the sequence shown in SEQ ID NO: 46 (GGGGSGGGGSGGGGS) or consisting thereof. Exemplary linkers also include those having the sequence shown in SEQ ID NO: 31 (GSTSGSGKPGSGEGSTKG) or consisting thereof. Exemplary linkers also include those having the sequence shown in SEQ ID NO: 68 (SRGGGGSGGGGSGGGGSLEMA) or consisting thereof. Exemplary linkers include those having the sequence shown in SEQ ID NO: 69 (GSRGGGGSGGGGSGGGGSLEMA) or consisting thereof. Exemplary linkers also include those having the sequence shown in SEQ ID NO: 29 and 30 or consisting thereof.

[0176] Thus, in some embodiments, provided embodiments include single-chain antibody fragments (e.g., scFv) comprising one or more of the above-described linkers, such as a glycine / serine-rich linker (including a linker having GGGS (SEQ ID NO: 45) or GGGGS (SEQ ID NO: 44) repeats, such as the linker set forth in any one of SEQ ID NOs: 46, 68, or 69). In some embodiments, the linker can be encoded in DNA and comprises the sequence set forth in SEQ ID NO: 47.

[0177] In some embodiments, V H The area can be located in V L In some embodiments, V H The area can be located in V LIn certain embodiments, a fragment (e.g., scFv) may include a V H region or portion thereof, followed by a linker, followed by V L In other embodiments, the fragment (e.g., scFv) may include a V L region or portion thereof, followed by a linker, followed by V H area or part thereof.

[0178] In some of any of the provided embodiments, the scFv comprises the sequence shown in SEQ ID NO: 89, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 89. In some of any of the provided embodiments, the scFv comprises the sequence shown in SEQ ID NO: 89. In some of any of the provided embodiments, the scFv has the sequence shown in SEQ ID NO: 89. In some embodiments, a provided recombinant receptor (such as a CAR) comprises an extracellular antigen binding domain comprising an scFv, and the scFv comprises the sequence shown in SEQ ID NO: 89, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 89. In some embodiments, a provided recombinant receptor (such as a CAR) comprises an extracellular antigen binding domain comprising an scFv, and the scFv comprises the sequence shown in SEQ ID NO: 89, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 89. In some embodiments, a provided recombinant receptor (such as a CAR) comprises an extracellular antigen binding domain comprising an scFv, and the scFv comprises the sequence shown in SEQ ID NO: 89. In some embodiments, a recombinant receptor (such as a CAR) is provided that comprises an extracellular antigen binding domain comprising an scFv, and the scFv has the sequence shown in SEQ ID NO:89.

[0179] In some any of the provided embodiments, the scFv comprises an amino acid sequence encoded by SEQ ID NO: 86 or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 86. In some any of the provided embodiments, the scFv comprises an amino acid sequence encoded by SEQ ID NO: 86. In some any of the provided embodiments, the scFv has an amino acid sequence encoded by SEQ ID NO: 86. In some embodiments, the recombinant receptor (such as CAR) provided comprises an extracellular antigen binding domain comprising an scFv, and the scFv comprises an amino acid sequence encoded by SEQ ID NO: 86 or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 86. In some embodiments, a recombinant receptor (such as a CAR) is provided that comprises an extracellular antigen binding domain comprising an scFv, and the scFv comprises an amino acid sequence encoded by SEQ ID NO: 86. In some embodiments, a recombinant receptor (such as a CAR) is provided that comprises an extracellular antigen binding domain comprising an scFv, and the scFv has an amino acid sequence encoded by SEQ ID NO: 86.

[0180] Table 2 provides SEQ ID NOs of exemplary provided antibodies or antigen-binding fragments (e.g., scFv). In some aspects, exemplary provided antibody fragments can be included in provided DLL3 binding receptors (e.g., anti-DLL3 chimeric antigen receptors (CARs)). In some embodiments, the DLL3 binding antibody or fragment thereof (e.g., scFv) comprises a V sequence comprising CDR-H1, CDR-H2, and CDR-H3. H region and V region containing CDR-L1, CDR-L2 and CDR-L3 sequences L The DLL3 binding antibody or fragment thereof (such as a scFv) comprises a V region as shown in the SEQ ID NO: listed in each row of Table 2 below (according to Kabat, Chothia, AbM, and IMGT numbering schemes). In some embodiments, the DLL3 binding antibody or fragment thereof (such as a scFv) comprises a V region as shown in the SEQ ID NO: listed in each row of Table 2 below. H Region sequence and V L Region sequence, or comprising the V region sequence shown in SEQ ID NO: listed in each row of Table 2 below H Region sequence and V L The V region sequences have at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity. H Area and V LIn some embodiments, the DLL3 binding antibody or fragment thereof (such as scFv) comprises a V sequence as shown in SEQ ID NO: H Region sequence and V L In some embodiments, the DLL3 binding antibody or fragment thereof comprises a scFv sequence as shown in SEQ ID NO: listed in each row of Table 2 below, or an antibody comprising a scFv amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to a scFv sequence as shown in SEQ ID NO: listed in each row of Table 2 below. In some embodiments, the DLL3 binding antibody or fragment thereof comprises a scFv sequence as shown in SEQ ID NO: listed in each row of Table 2 below. In some embodiments, any of the antibodies or antigen-binding fragments thereof (such as scFv) listed in each row of Table 2 can be included in a receptor (such as a chimeric antigen receptor (CAR)) as an extracellular antigen binding domain.

[0181] In some embodiments, provided antibodies or antigen-binding fragments thereof comprise V H Area and V L region, wherein the V H The region may contain any combination of CDR-H1, CDR-H2 and CDR-H3 amino acid sequences shown in Table 2, and the V L The region may contain any combination of CDR-L1, CDR-L2, and CDR-L3 amino acid sequences shown in Table 2. In some embodiments, provided antibodies or antigen-binding fragments thereof comprise the V sequences shown in Table 2. H Area and / or V L In some aspects, the antibody or its antigen-binding fragment comprises the V region described in Table 2. H In some aspects, the antibody or antigen-binding fragment thereof comprises the V region described in Table 2. L In some embodiments, the antibodies or antigen-binding fragments thereof provided are those comprising the V region shown in Table 2. H area or V shown in Table 2 L In some embodiments, the antibodies or antigen-binding fragments thereof provided are single domain antibodies (sdAbs) comprising a V domain comprising CDR-H1, CDR-H2, and / or CDR-H3 as shown in Table 2. H region, or a V region containing CDR-L1, CDR-L2 and / or CDR-L3 shown in Table 2 L district.

[0182] The antibodies (e.g., antigen-binding fragments) provided include human antibodies. In some embodiments of the human anti-DLL3 antibodies (e.g., antigen-binding fragments) provided, the human antibody comprises a V H Area, the V H The region comprises a portion having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence encoded by the germline nucleotide human heavy chain V segment, a portion having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence encoded by the germline nucleotide human heavy chain D segment, and / or a portion having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence encoded by the germline nucleotide human heavy chain J segment; and / or comprises a V L Area, the V L The region comprises a portion having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence encoded by the germline nucleotide human κ or λ chain V segment, and / or a portion having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence encoded by the germline nucleotide human κ or λ chain J segment.

[0183] The antibodies (e.g., antigen-binding fragments) provided include human antibodies. In some embodiments of the human anti-DLL3 antibodies (e.g., antigen-binding fragments) provided, the human antibody comprises a V H Area, the V H The region comprises a portion having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence encoded by the germline nucleotide human heavy chain V segment, a portion having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence encoded by the germline nucleotide human heavy chain D segment, and / or a portion having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence encoded by the germline nucleotide human heavy chain J segment; and comprises V L Area, the V L The region comprises a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain V segment, and / or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain J segment. In some embodiments, the V H The portion of the region corresponds to CDR-H1, CDR-H2 and / or CDR-H3. In some embodiments, VH The portion of the region corresponds to CDR-H1, CDR-H2, and CDR-H3. H The portion of the region corresponds to framework region 1 (FR1), FR2, FR2 and / or FR4. In some embodiments, V L The portion of the region corresponds to CDR-L1, CDR-L2 and / or CDR-L3. In some embodiments, V L The portion of the region corresponds to CDR-L1, CDR-L2, and CDR-L3. In some embodiments, V L The portion of the region corresponds to FR1, FR2, FR2 and / or FR4.

[0184] In some embodiments, the human antibody or antigen-binding fragment thereof comprises a CDR-H1 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H1 region within the sequence encoded by the germline nucleotide human heavy chain V segment. For example, in some embodiments, the human antibody comprises a CDR-H1 having a sequence that is 100% identical or has no more than one, two, or three amino acid differences compared to the corresponding CDR-H1 region within the sequence encoded by the germline nucleotide human heavy chain V segment.

[0185] In some embodiments, the human antibody or antigen-binding fragment thereof comprises a CDR-H2 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H2 region within the sequence encoded by the germline nucleotide human heavy chain V segment. For example, in some embodiments, the human antibody comprises a CDR-H2 having a sequence that is 100% identical or has no more than one, two, or three amino acid differences compared to the corresponding CDR-H2 region within the sequence encoded by the germline nucleotide human heavy chain V segment.

[0186] In some embodiments, the human antibody or antigen-binding fragment thereof comprises a CDR-H3 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H3 region within the sequence encoded by the germline nucleotide human heavy chain V segment, D segment, and J segment. For example, in some embodiments, the human antibody comprises a CDR-H3 having a sequence that is 100% identical or has no more than one, two, or three amino acid differences compared to the corresponding CDR-H3 region within the sequence encoded by the germline nucleotide human heavy chain V segment, D segment, and J segment.

[0187] In some embodiments, the human antibody or antigen-binding fragment thereof comprises a CDR-L1 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-L1 region within the sequence encoded by the germline nucleotide human light chain V segment. For example, in some embodiments, the human antibody comprises a CDR-L1 having a sequence that is 100% identical or has no more than one, two, or three amino acid differences compared to the corresponding CDR-L1 region within the sequence encoded by the germline nucleotide human light chain V segment.

[0188] In some embodiments, the human antibody or antigen-binding fragment thereof comprises a CDR-L2 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-L2 region within the sequence encoded by the germline nucleotide human light chain V segment. For example, in some embodiments, the human antibody comprises a CDR-L2 having a sequence that is 100% identical or has no more than one, two, or three amino acid differences compared to the corresponding CDR-L2 region within the sequence encoded by the germline nucleotide human light chain V segment.

[0189] In some embodiments, the human antibody or antigen-binding fragment thereof comprises a CDR-L3 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-L3 region within the sequence encoded by the germline nucleotide human light chain V segment and J segment. For example, in some embodiments, the human antibody comprises a CDR-L3 having a sequence that is 100% identical or has no more than one, two, or three amino acid differences compared to the corresponding CDR-L3 region within the sequence encoded by the germline nucleotide human light chain V segment and J segment.

[0190] In some embodiments, a human antibody or antigen-binding fragment thereof comprises a framework region comprising a human germline gene segment sequence. For example, in some embodiments, a human antibody comprises a V H In some embodiments, the human antibody comprises a V segment, wherein the framework regions (e.g., FR1, FR2, FR3, and FR4) have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the framework regions encoded by human germline antibody segments (e.g., V segments and / or J segments). L In some embodiments, the framework regions (e.g., FR1, FR2, FR3, and FR4) are at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the framework regions encoded by human germline antibody segments (e.g., V segments and / or J segments). H Area and / or V LThe framework region sequence contained within the region differs from the framework region sequence encoded by the human germline antibody segment by no more than 10 amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid.

[0191] An antibody or its antigen-binding fragment may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domains. In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region. In some embodiments, the antibody or its antigen-binding fragment comprises a lambda (λ) light chain constant region. In some embodiments, the antibody or its antigen-binding fragment comprises a kappa (κ) light chain constant region. In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region. In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region of an isotype class selected from IgG, IgM, IgD, IgA, and IgE. In some embodiments, the antibody or its antigen-binding fragment comprises an IgG heavy chain constant region. In some embodiments, the antibody or its antigen-binding fragment comprises an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the antibody or its antigen-binding fragment comprises an IgG1 heavy chain constant region.

[0192] In some embodiments, the constant region includes a light chain constant region and / or a heavy chain constant region 1 (C H 1). In some embodiments, the antibody comprises at least a portion of the hinge region or a variant thereof. In some embodiments, the antibody comprises C H 2 and / or C H In some embodiments, the Fc region is a human IgG (e.g., IgG1, IgG2, IgG3, or IgG4) Fc region. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG2 Fc region. In some embodiments, the Fc region is a human IgG3 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region.

[0193] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgG1 heavy chain constant region. In some embodiments, an exemplary human IgG1 heavy chain constant region comprises the sequence set forth in SEQ ID NO: 126. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a kappa light chain constant region. In some embodiments, an exemplary human kappa light chain constant region comprises the sequence set forth in SEQ ID NO: 127.

[0194] In some embodiments of any of the provided embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH ) and heavy chain constant region (C H ). In some aspects, the antibody or antigen-binding fragment thereof comprises V H and C H , the V H comprising the sequence shown in SEQ ID NO:90, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:90; said C H comprising the sequence shown in SEQ ID NO: 126, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 126. In some aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the sequence shown in SEQ ID NO: 128, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 128.

[0195] In some embodiments of any of the provided embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (V L ) and the light chain constant region (C L ). In some aspects, the antibody or antigen-binding fragment thereof comprises V L and C L , the V L comprising the sequence shown in SEQ ID NO:91, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:91; said C L comprising the sequence shown in SEQ ID NO: 127, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 127. In some aspects, the antibody or antigen-binding fragment thereof comprises a light chain comprising the sequence shown in SEQ ID NO: 129, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129.

[0196] In some embodiments of any of the provided embodiments, the antibody or antigen-binding fragment thereof comprises a full-length heavy chain and a full-length light chain, wherein the full-length heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region (C H ), wherein the full-length light chain comprises a light chain variable region (V L ) and the light chain constant region (C L In some embodiments, the antibody or antigen-binding fragment thereof comprises V H and CH , the V H comprising the sequence shown in SEQ ID NO:90, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:90, wherein the C H comprising the sequence shown in SEQ ID NO: 126, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 126; and V L and C L , the V L comprising the sequence shown in SEQ ID NO: 91, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 91, wherein the C L comprising the sequence set forth in SEQ ID NO: 127, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 127. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 128, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 128; and a light chain comprising the sequence set forth in SEQ ID NO: 129, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 128 and a light chain comprising the sequence set forth in SEQ ID NO: 129. 1. Variants

[0197] In certain embodiments, the antibody comprises one or more amino acid variations, such as substitutions, deletions, insertions and / or mutations, compared to the sequence of an antibody as described herein. Exemplary variants include those designed to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, residue deletions and / or insertions and / or substitutions within the antibody amino acid sequence. Any combination of deletions, insertions and substitutions can be performed to obtain the final construct, provided that the final construct has desired characteristics, such as antigen binding.

[0198] In certain embodiments, the antibody comprises one or more amino acid substitutions, for example, compared to an antibody sequence as described herein and / or compared to a sequence in a natural library (e.g., a human library). The target site for substitution mutagenesis comprises CDRs and FRs. Amino acid substitutions can be introduced into the target antibody, and the desired activity of the product can be screened, for example, retained / improved antigen binding, reduced immunogenicity, improved half-life, and / or improved effector functions, such as the ability to promote antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0199] In some embodiments, one or more residues within the CDR of a parent antibody (e.g., a humanized or human antibody) are substituted. In some embodiments, substitutions are made to restore a sequence or position in a sequence to a germline sequence, such as an antibody sequence found in a germline (e.g., a human germline), e.g., to reduce the likelihood of immunogenicity, e.g., after administration to a human subject.

[0200] In some embodiments, changes are made in CDR "hotspots" (i.e., residues encoded by codons that undergo high frequency mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that contact antigen), and the resulting variant V H or V L The test of binding affinity is carried out. Affinity maturation carried out by constructing a secondary library and reselecting from the secondary library has been described in, for example, Hoogenboom et al. Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling or oligonucleotide-directed mutagenesis). The secondary library is then set up. The library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity relates to a CDR-guided method, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. The CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are usually targeted.

[0201] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes that do not significantly reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in the CDRs. For example, such changes may be located outside of the antigen contact residues in the CDRs. In the variant V provided above, H and V L In certain embodiments of the sequences, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0202] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions (ranging in length from one residue to polypeptides containing one hundred or more residues), as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusions of the N-terminus or C-terminus of the antibody with an enzyme or polypeptide that increases the serum half-life of the antibody. 2. Modification

[0203] In certain embodiments, the antibody or antigen-binding fragment thereof is altered to increase or decrease the extent to which the antibody or antigen-binding fragment thereof is glycosylated, for example, by altering the amino acid sequence to remove or insert one or more glycosylation sites and / or by modifying one or more oligosaccharides attached to the glycosylation sites, for example, by using certain cell lines.

[0204] In some embodiments, N-linked glycosylation is removed or inserted, which is a glycosylation site occurring at asparagine in the consensus sequence -Asn-Xaa-Ser / Thr. In some embodiments, one or more amino acids are substituted for another amino acid to remove a glycosylation site.

[0205] Exemplary modifications, variants, and cell lines are described, for example, in Patent Publication Nos. US 2003 / 0157108, US 2004 / 0093621, US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107; WO 2003 / 011878; U.S. Patent No. 6,602,684; and US 2005 / 0123546; WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0206] Modified antibodies include those having one or more amino acid modifications in the Fc region, such as those having a human Fc region sequence or other portion of a constant region (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions. Such modifications can be made, for example, to improve half-life, alter binding to one or more types of Fc receptors, and / or alter effector function.

[0207] Variants also include cysteine engineered antibodies, such as "thioMAbs" and other cysteine engineered variants, in which one or more residues of an antibody are substituted with a cysteine residue to create a reactive thiol group at an accessible site, e.g., for conjugation to an agent and a linker to create an immunoconjugate. Cysteine engineered antibodies are described, e.g., in U.S. Patent Nos. 7,855,275 and 7,521,541.

[0208] In some embodiments, modified antibodies are to contain other non-protein moieties, including water-soluble polymers.Exemplary polymers include but are not limited to polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly-(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (for example, glycerol), polyvinyl alcohol and mixtures thereof.Polyethylene glycol propionaldehyde can have advantages in preparation due to its stability in water.Described polymer can have any molecular weight, and can have side chain or unbranched chain.The number of the polymer attached to antibody can change, and if attachment exceeds a kind of polymer, then they can be identical or different molecules. Generally, the amount and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative is to be used for therapy under a defined condition, and the like. 3. Exemplary Features

[0209] In some aspects, the antibodies provided have one or more specified functional characteristics, such as binding properties, including binding to a specific epitope or exhibiting lower or reduced binding to a related but non-specific antigen. In some aspects, the antibodies provided can bind to an epitope that is similar to or overlaps with the epitope of other antibodies, such as a reference antibody, and / or exhibit a specific binding affinity. In some aspects, the antibodies provided can bind to an epitope that is different from the epitope of other antibodies, for example, binding to a conformational epitope.

[0210] In some embodiments, provided antibodies or antigen-binding fragments thereof specifically bind to a delta-like ligand 3 (DLL3) protein. In some embodiments provided herein, the DLL3 is human DLL3. Observing that an antibody or other binding molecule binds to DLL3 or specifically binds to DLL3 does not necessarily mean that it binds to DLL3 from every species. For example, in some embodiments, a characteristic of binding to DLL3 (e.g., the ability to specifically bind to it and / or compete with a reference antibody for its binding and / or bind with a particular affinity or to some extent) refers to the ability to bind to human DLL3, and the antibody may not have this ability to bind to DLL3 from another species (e.g., mouse, rhesus monkey, or cynomolgus monkey).

[0211] In some embodiments, an antibody or antigen-binding fragment thereof is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. -7 or ≤10 -8 When M, the antibody is said to specifically bind to the antigen.

[0212] An antibody molecule or antigen-binding fragment thereof is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular antigen more frequently, more rapidly, for a longer duration, and / or with greater affinity than it reacts or associates with alternative antigens. An antibody molecule or antigen-binding fragment thereof specifically or preferentially binds to a target if it binds with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to DLL3 is one that binds to the DLL3 protein with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other DLL3s from other species or non-DLL3 antigens. It should also be understood that specific or preferential binding does not necessarily require (although it can include) exclusive binding. Methods for determining such specific or preferential binding are also well known, such as immunoassays.

[0213] In some embodiments, an antibody (eg, an anti-DLL3 antibody, eg, a human antibody) specifically binds to a particular epitope or region (eg, a generally extracellular epitope or region) of DLL3.

[0214] DLL3 is a type I transmembrane delta-like protein that is normally expressed only on intracellular membranes, such as the Golgi apparatus. Human DLL3 has an extracellular region with multiple extracellular domains, including the N-terminus of the Notch ligand domain, the Delta, Serrate, Lag2 (DSL) domain, and six epidermal growth factor (EGF)-like repeats (EGF repeats; EGF1-6 in human DLL3); a transmembrane domain, and a cytoplasmic region. With reference to the human DLL3 isoform 1 precursor sequence set forth in SEQ ID NO: 120 (e.g., UNIPROT Q9NYJ7-1; Genbank NP_058637.1; the mature polypeptide without the signal sequence set forth in SEQ ID NO: 122), amino acid residues 1-26 correspond to the signal peptide, amino acid residues 27-84 correspond to the N-terminus of the Notch ligand domain, amino acid residues 176-215 correspond to the DSL domain, amino acid residues 216-249 correspond to the EGF-like 1 domain, amino acid residues 274-310 correspond to the EGF-like 2 domain, amino acid residues 312-351 correspond to the EGF-like 3 domain, amino acid residues 353-389 correspond to the EGF-like 4 domain, amino acid residues 391-427 correspond to the EGF-like 5 domain, amino acid residues 429-465 correspond to the EGF-like 6 domain, amino acid residues 493-513 correspond to the transmembrane domain, and amino acid residues 514-618 correspond to the cytoplasmic domain. Human DLL3 isoform 2 differs from isoform 1 in that amino acid residues 587-618 of the human DLL3 isoform 1 precursor sequence set forth in SEQ ID NO:120 are replaced with alanine residues (human DLL3 isoform 2 precursor sequence set forth in SEQ ID NO:121; e.g., UNIPROT Q9NYJ7-2; Genbank NP_982353.1; mature polypeptide without signal sequence set forth in SEQ ID NO:123). DLL3 shares low sequence homology with other cell surface Notch ligands in the family: 35% homology with DLL1 and 38% homology with DLL4.

[0215] In some embodiments, a DLL3 binding molecule (e.g., an antibody or antigen-binding fragment thereof) binds (e.g., specifically binds) to one or more epitopes or regions of human DLL3, such as human DLL3 (e.g., the mature human DLL3 sequence set forth in SEQ ID NO: 122 or 123), or an allelic variant or splice variant thereof. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to one or more epitopes within human DLL3. In some embodiments, human DLL3 comprises the amino acid sequence set forth in SEQ ID NO: 120. In some embodiments, human DLL3 comprises the amino acid sequence set forth in SEQ ID NO: 121. In some embodiments, a human DLL3 isoform (mature, isoform 1) comprises the amino acid sequence set forth in SEQ ID NO: 122. In some embodiments, a human DLL3 isoform (mature, isoform 2) comprises the amino acid sequence set forth in SEQ ID NO: 123. In some embodiments, the antibody or antigen-binding fragment thereof binds to an extracellular region of DLL3, such as one or more extracellular epitopes present within the extracellular region of mature human DLL3, e.g., corresponding to residues 27-492 of the human DLL3 precursor sequence shown in SEQ ID NO: 120.

[0216] In some embodiments, the antibody or antigen-binding fragment thereof binds to one or more epitopes of DLL3, such as human DLL3. In some embodiments, the antibody or antigen-binding fragment thereof binds to a linear epitope of DLL3, such as human DLL3. In some embodiments, the one or more epitopes comprise a conformational epitope. In some embodiments, the antibody or antigen-binding fragment thereof binds to one or more conformational epitopes of DLL3 (such as human DLL3). In some embodiments, the antibody or antigen-binding fragment thereof binds to one or more conformational epitopes of DLL3 (such as human DLL3).

[0217] In some embodiments, a DLL3 binding molecule (such as an antibody or antigen-binding fragment thereof) binds to an epitope comprising amino acid residues within the N-terminus of a Notch ligand domain, a DSL domain, an EGF-like 1 domain, an EGF-like 2 domain, an EGF-like 3 domain, an EGF-like 4 domain, an EGF-like 5 domain, or an EGF-like 6 domain.

[0218] In some embodiments, the antibody or antigen-binding fragment thereof binds one or more epitopes of human DLL3, such as one or more epitopes comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acid residues within human DLL3 (e.g., the human DLL3 precursor sequence shown in SEQ ID NO: 120), or two or more epitopes, each comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acid residues within human DLL3 (e.g., the human DLL3 precursor sequence shown in SEQ ID NO: 120). In some aspects, with reference to the human DLL3 precursor sequence shown in SEQ ID NO: 120, the one or more epitopes include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acid residues corresponding to amino acid residues 27-84 of the N-terminus of the Notch ligand domain, amino acid residues 176-215 corresponding to the DSL domain, amino acid residues 216-249 corresponding to the EGF-like 1 domain, amino acid residues 274-310 corresponding to the EGF-like 2 domain, amino acid residues 312-351 corresponding to the EGF-like 3 domain, amino acid residues 353-389 corresponding to the EGF-like 4 domain, amino acid residues 391-427 corresponding to the EGF-like 5 domain, or amino acid residues 429-465 corresponding to the EGF-like 6 domain.

[0219] In some embodiments, the antibody or antigen-binding fragment binds to an epitope within amino acid residues 27-84 of SEQ ID NO: 120, which corresponds to the N-terminus of the Notch ligand domain. In some embodiments, the antibody or antigen-binding fragment binds to an epitope within amino acid residues 176-215 of SEQ ID NO: 120, which corresponds to the DSL domain. In some embodiments, the antibody or antigen-binding fragment binds to an epitope within amino acid residues 216-249 of SEQ ID NO: 120, which corresponds to the EGF-like 1 domain. In some embodiments, the antibody or antigen-binding fragment binds to an epitope within amino acid residues 274-310 of SEQ ID NO: 120, which corresponds to the EGF-like 2 domain. In some embodiments, the antibody or antigen-binding fragment binds to an epitope within amino acid residues 312-351 of SEQ ID NO: 120, which corresponds to the EGF-like 3 domain. In some embodiments, the antibody or antigen-binding fragment binds to an epitope within amino acid residues 353-389 of SEQ ID NO: 120, which corresponds to the EGF-like 4 domain. In some embodiments, the antibody or antigen-binding fragment binds to an epitope within amino acid residues 391-427 of SEQ ID NO: 120, which corresponds to the EGF-like 5 domain. In some embodiments, the antibody or antigen-binding fragment binds to an epitope within amino acid residues 429-465 of SEQ ID NO: 120, which corresponds to the EGF-like 6 domain. In some embodiments, the antibody or antigen-binding fragment binds to an epitope within amino acid residues 493-513 of SEQ ID NO: 120, which corresponds to the transmembrane domain.

[0220] In some embodiments, provided antibodies are capable of binding to DLL3 (such as human DLL3) with at least a certain affinity, as measured by any of a variety of known methods. In some embodiments, affinity is determined by the equilibrium dissociation constant (K D In some embodiments, affinity is represented by EC 50 express.

[0221] A variety of assays are known to assess binding affinity, equilibrium dissociation constant (K D ), equilibrium association constant (K A ), EC 50 , association rate (association rate constant; k on or k a ; The unit is 1 / Ms or M -1 s -1 ) and dissociation rate (dissociation rate constant; k off or k d ; The unit is 1 / s or s -1) and / or determine whether a binding molecule (e.g., an antibody or fragment thereof) specifically binds to a particular ligand (e.g., an antigen, such as a DLL3 protein). The binding affinity of a binding molecule (e.g., an antibody or antigen-binding fragment thereof) to an antigen (e.g., DLL3, such as human DLL3 or cynomolgus monkey DLL3 or mouse DLL3) can be determined, such as by using any of a variety of binding assays well known in the art. For example, in some embodiments, surface plasmon resonance (SPR) analysis can be used to determine whether a binding molecule (e.g., an antibody or fragment thereof) specifically binds to a particular ligand (e.g., an antigen, such as a DLL3 protein). The instrument is used to determine the binding kinetics and constants of a complex between two proteins, e.g., an antibody or fragment thereof and an antigen, such as a DLL3 protein (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0222] SPR measures the change in concentration of a molecule at the sensor surface as it binds to or dissociates from the surface. The change in the SPR signal is proportional to the change in mass concentration close to the surface, allowing the binding kinetics between two molecules to be measured. The dissociation rate constant (k off or k d ), association rate constant (k on or k a ) and / or equilibrium dissociation constant (K D ) and / or equilibrium association constant (K A ) can be determined by monitoring the change in refractive index over time as the buffer flows through the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays (such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA)), or by monitoring changes in the spectral or optical properties of the protein by fluorescence, ultraviolet absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blotting, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, genetic reporter assays, flow cytometry, and other methods for detecting expressed nucleic acids or protein binding.

[0223] In some embodiments, a binding molecule (e.g., an antibody or fragment thereof) binds (e.g., specifically binds) to an antigen (e.g., a DLL3 protein or an epitope therein) with an affinity or K of A (i.e., the equilibrium association constant for a specific binding interaction, expressed in 1 / M or M -1 ; is equal to the association rate for this association reaction [k on or ka ] and the dissociation rate [k off or k d ] ratio, assuming bimolecular interactions) is equal to or greater than 10 5 M -1 In some embodiments, the peptide binding molecule binds (e.g., specifically binds) to an epitope of an antigen (e.g., human DLL3) with an affinity or K A Equal to or greater than 10 5 M -1 (i.e., 1 / M or M -1 The equilibrium association constant of a specific binding interaction (which is equal to the association rate [k on ] and the dissociation rate [k off In some embodiments, a binding molecule (e.g., an antibody or antigen-binding fragment thereof) exhibits binding affinity for a T cell epitope of a target polypeptide, wherein the affinity or K A The range is from or about 10 6 M -1 Up to or about 10 7 M -1 In some embodiments, binding affinity can be classified as high affinity or low affinity. For example, in some cases, a binding molecule (e.g., an antibody or antigen-binding fragment thereof) that exhibits high affinity binding to a particular epitope interacts with such an epitope where K A is for or about 10 7 M -1 In some cases, a binding molecule (e.g., an antibody or antigen-binding fragment thereof) that exhibits low affinity binding exhibits at most 10 7 M -1 K A In certain embodiments, any of the aforementioned K A The value or range is determined by SPR. In certain embodiments, any of the aforementioned K A The values or ranges are determined by SPR as described in Example 3.

[0224] Alternatively, affinity can be defined in units of M (e.g., 10 -5 M to 10 -13 The equilibrium dissociation constant (K) of the specific binding interaction D In some embodiments, the antibody or fragment thereof exhibits a binding affinity for an epitope wherein K D is equal to or less than 10 -5 M (i.e., the equilibrium dissociation constant for a specific binding interaction in units of M; equal to the dissociation rate [k off or k d ] and the association rate [kon or k a ] ratio). For example, the equilibrium dissociation constant K D The range can be from 10 -5 M to 10 -8 M. In certain embodiments, any of the foregoing K D The value or range is determined by SPR. In certain embodiments, any of the aforementioned K D The values or ranges are determined by SPR as described in Example 3.

[0225] Association rate (association rate constant; k on or k a ; The unit is 1 / Ms or M -1 s -1 ) and dissociation rate (dissociation rate constant; k off or k d ; The unit is 1 / s or s -1 ) can be determined using any known assay method, such as surface plasmon resonance (SPR) or other methods described herein for measuring binding of one protein to another protein.

[0226] In some embodiments, the equilibrium dissociation constant (K) of the antibody for DLL3 (eg, human DLL3) is D ) is from at or about 50 nM to at or about 500 nM, from at or about 50 nM to at or about 100 nM, or from at or about 100 nM to at or about 500 nM. In certain embodiments, the equilibrium dissociation constant (K) of the antibody for DLL3 (e.g., human DLL3) is from at or about 50 nM to at or about 500 nM, D ) is at or about 100 nM, 90 nM, 80 nM, 70 nM, 60 nM or 50 nM, or a range defined by any of the foregoing values. In some embodiments, the equilibrium dissociation constant, K, of a binding molecule (e.g., an anti-DLL3 antibody or fragment thereof) for a DLL3 protein (e.g., human DLL3) is D is from or about 50 nM to 500 nM, 50 nM to 100 nM, or 100 nM to 500 nM. In certain embodiments, the equilibrium dissociation constant, K, of a binding molecule (e.g., an anti-DLL3 antibody or fragment thereof) for a DLL3 protein (e.g., human DLL3) is D is at or about 1 μM, 500 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM or 50 nM, or a range defined by any of the foregoing values. In certain embodiments, any of the foregoing K D The value or range is determined by SPR. In certain embodiments, any of the aforementioned K D The values or ranges are determined by SPR as described in Example 3.

[0227] In some embodiments, the binding molecule (e.g., an anti-DLL3 antibody or fragment thereof) is directed against the K of a DLL3 protein. D In some embodiments, the K of a binding molecule (e.g., an anti-DLL3 antibody or fragment thereof) to a DLL3 protein (e.g., human DLL3) is between at or about 50 nM and at or about 60 nM. D is at or about 50 nM, 60 nM, 70 nM, 80 nM, 90 nM or 100 nM, or a range defined by any of the foregoing values. In certain embodiments, the K of a binding molecule (e.g., an anti-DLL3 antibody or fragment thereof) to a DLL3 protein (e.g., a human DLL3 protein) is D is at or about 50 nM, 55 nM, 60 nM or 65 nM, or a range defined by any of the foregoing values. In certain embodiments, any of the foregoing K D The value or range is determined by SPR. In certain embodiments, any of the aforementioned K D The values or ranges are determined by SPR as described in Example 3.

[0228] In some embodiments, the binding affinity (EC 50 ) is from at or about 10 nM to 15 nM, 10 nM to 20 nM, 10 nM to 30 nM, 50 nM to at or about 500 nM, from at or about 50 nM to at or about 100 nM, or from at or about 100 nM to at or about 500 nM. In certain embodiments, the binding affinity (EC) of the antibody to DLL3 (such as human DLL3) is from at or about 10 nM to 15 nM, 10 nM to 20 nM, 10 nM to 30 nM, 50 nM to at or about 500 nM, from at or about 50 nM to at or about 100 nM, or from at or about 100 nM to at or about 500 nM. 50 ) is at or about 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM or 10 nM, or a range defined by any of the foregoing values. In some embodiments, the binding affinity (EC) of a binding molecule (e.g., an anti-DLL3 antibody or fragment thereof) to a DLL3 protein (e.g., human DLL3) is 50 ) is from at or about 50 nM to 500 nM, 50 nM to 100 nM, or 100 nM to 500 nM. In certain embodiments, the binding affinity (EC) of a binding molecule (e.g., an anti-DLL3 antibody or fragment thereof) to a DLL3 protein (e.g., human DLL3) is from at or about 50 nM to 500 nM, 50 nM to 100 nM, or 100 nM to 500 nM. 50 ) is at or about 1 μM, 500 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM or 10 nM, or a range defined by any of the foregoing values. In certain embodiments, EC 50 The values or ranges were determined by the experimental conditions described in Example 4.

[0229] In some embodiments, the binding affinity (EC 50 ) is between at or about 10 nM and at or about 20 nM. In certain embodiments, EC 50 The values or ranges were determined by the experimental conditions described in Example 4.

[0230] In some of any of the provided embodiments, the antibody or antigen-binding fragment thereof does not bind, does not cross-react, or binds to a lower degree, level, extent, or affinity to a related Notch ligand protein (such as DLL1 or DLL4, e.g., human DLL1 or human DLL4).

[0231] In some embodiments, the extent to which an anti-DLL3 antibody binds to a different non-DLL3 Notch ligand protein (e.g., a DLL1 or DLL4 protein, e.g., human DLL1 or human DLL4) or other non-DLL3 protein is less than or about 50%, 40%, 30%, 20%, or 10% of the extent to which the antibody binds to human DLL3. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to DLL1 or DLL4. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to, does not cross-react with, or binds to DLL1 or DLL4 at a lower level, degree, or affinity. In some embodiments, provided anti-DLL3 antibodies or antigen-binding fragments thereof bind to DLL1 or DLL4 at a degree, level, degree, or affinity that is at least or about 75%, 80%, 90%, 95%, or 99% less than the degree, level, degree, or affinity to human DLL3. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to human DLL1. In some embodiments, the antibody or antigen-binding fragment thereof does not cross-react with human DLL1. In some embodiments, the antibody or antigen-binding fragment thereof binds to human DLL1 at a lower level, extent, or affinity. In some embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof binds to human DLL1 at a level, extent, or affinity that is at least or about 75%, 80%, 90%, 95%, or 99% less than the level, level, extent, or affinity of the anti-DLL3 antibody. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to human DLL4. In some embodiments, the antibody or antigen-binding fragment thereof does not cross-react with human DLL4. In some embodiments, the antibody or antigen-binding fragment thereof binds to human DLL4 at a lower level, extent, or affinity. In some embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof binds to human DLL4 at a level, extent, or affinity that is at least or about 75%, 80%, 90%, 95%, or 99% less than the level, level, extent, or affinity of the anti-DLL3 antibody or antigen-binding fragment thereof.

[0232] In some aspects, observing that an antibody or other binding molecule binds to DLL3 or specifically binds to DLL3 does not necessarily mean that it binds to DLL3 from every species. For example, in some embodiments, a characteristic of binding to DLL3 (such as the ability to specifically bind to it and / or compete with a reference antibody for its binding and / or bind with a particular affinity or to some extent) refers to the ability to bind to human DLL3, and the antibody may not have this ability to bind to DLL3 from another species (such as mouse, rhesus monkey, or cynomolgus monkey). In some embodiments, the antibody binds to human DLL3 and binds to DLL3 from another species (such as rhesus monkey or cynomolgus monkey). In some embodiments, the antibody or antigen-binding fragment thereof binds to human DLL3 and does not bind to DLL3 from another species (such as mouse). In some embodiments, the antibody binds to human DLL3 and binds to DLL3 from another species (such as mouse).

[0233] In some embodiments, the antibody binds to a non-human DLL3, such as a DLL3 of a monkey, rabbit, rat, mouse, or other species. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope or region of a non-human DLL3, such as a cynomolgus monkey (Macaca fascicularis) DLL3, such as a cynomolgus monkey DLL3 (such as the cynomolgus monkey DLL3 set forth in SEQ ID NO: 125 (NCBI Reference Sequence: XM_005589196.2). In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope or region of a mouse (Mus musculus) DLL3, such as a mouse DLL3 (such as the mouse DLL3 set forth in SEQ ID NO: 124 (GenBank No. NP_031892.2).

[0234] In some embodiments, the antibodies or antigen-binding fragments thereof bind to human DLL3 and bind to cynomolgus monkey DLL3, such as the cynomolgus monkey DLL3 set forth in SEQ ID NO: 125. In some embodiments, some provided anti-DLL3 antibodies or fragments thereof bind to non-human DLL3 (such as cynomolgus monkey DLL3) to a degree that is at least or at least about 75%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150% or more of the degree of binding of the antibodies or antigen-binding fragments thereof to human DLL3.

[0235] In some of any provided embodiments, the antibody or antigen-binding fragment thereof does not bind, does not cross-react, or binds to a lower degree, level, or degree, or affinity, to a non-human DLL3 (e.g., mouse DLL3, such as that set forth in SEQ ID NO: 124). In some embodiments, the extent of binding of the anti-DLL3 antibody to an unrelated non-DLL3 protein or non-human DLL3 (e.g., mouse DLL3 protein) or other non-DLL3 protein is less than or about 50%, 40%, 30%, 20%, or 10% of the extent of binding of the antibody to human DLL3, as measured. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to mouse DLL3, such as that set forth in SEQ ID NO: 124. In some embodiments, the antibody or antigen-binding fragment thereof does not bind, does not cross-react, or binds to mouse DLL3 at a lower level, level, or affinity. In some embodiments, the anti-DLL3 antibodies or antigen-binding fragments thereof provided bind to mouse DLL3 to an extent, level, degree, or affinity that is at least or about 75%, 80%, 90%, 95%, or 99% less than that to human DLL3.

[0236] In some embodiments, the binding affinities of binding molecules (e.g., anti-DLL3 antibodies) for different antigens (e.g., DLL3 proteins from different species) can be compared to determine species cross-reactivity. For example, species cross-reactivity can be classified as high cross-reactivity or low cross-reactivity. In some embodiments, the equilibrium dissociation constants K for different antigens (e.g., DLL3 proteins from different species (e.g., human, cynomolgus monkey, or mouse)) can be compared. D To determine species cross-reactivity. In some embodiments, the species cross-reactivity of the anti-DLL3 antibody can be high, e.g., the anti-DLL3 antibody binds to human DLL3 and species variant DLL3 to a similar extent, e.g., the K for human DLL3 is D Compared with the K for species variant DLL3 D In some embodiments, the species cross-reactivity of the anti-DLL3 antibody can be low, for example, the anti-DLL3 antibody has a high affinity for human DLL3 but a low affinity for species variant DLL3, or vice versa. For example, the K for species variant DLL3 is D K for human DLL3 D The ratio of the species cross-reactivity to that of the anti-DLL3 antibody is greater than 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or more, and the anti-DLL3 antibody has low species cross-reactivity. The degree of species cross-reactivity can be compared to the species cross-reactivity of a known antibody (e.g., a reference antibody).

[0237] In some embodiments, provided antibodies or antigen-binding fragments thereof bind to human DLL3 and non-human DLL3 to similar extents. For example, in some embodiments, provided antibodies or antigen-binding fragments thereof bind to human DLL3 and non-human DLL3 to a similar extent. D ) binds to human DLL3 or its allelic variant or splice variant and has a similar, or substantially identical, or less than 2-fold, or less than 5-fold different K D Binds non-human DLL3, such as cynomolgus monkey DLL3.

[0238] In some embodiments, the total binding capacity (R) as measured using specific surface plasmon resonance (SPR) conditions is max ) is used to determine the binding ability or capacity of a provided antibody or antigen-binding fragment thereof to an antigen (e.g., a DLL3 protein, such as human DLL3). For SPR analysis, the "ligand" is an immobilized target molecule (e.g., a DLL3 protein) on the sensor surface, and the "analyte" is a test molecule (e.g., an antibody) that is used to bind to the "ligand." For example, the "analyte" can be any provided antibody or antigen-binding fragment thereof that binds to a DLL3 protein. For a specific ligand and analyte pair in SPR, R can be determined for specific conditions assuming a 1:1 binding stoichiometry model. max In some embodiments, the binding capacity (R max ):R max (RU) = (analyte molecular weight) / (ligand molecular weight) x immobilized ligand level (RU). In certain aspects of the SPR conditions, the R of the binding between any provided antibody or antigen-binding fragment thereof and a DLL3 protein (such as human DLL3 or cynomolgus monkey DLL3) is max At least or at least about 50 resonance units (RU), such as about 25 RU, 20 RU, 15 RU, 10 RU, 5 RU, or 1 RU. B. Immunoconjugates

[0239] In some embodiments, the DLL3 binding molecule (e.g., an antibody or antigen-binding fragment thereof) is or is part of an immunoconjugate in which the antibody is conjugated to one or more heterologous molecules (e.g., but not limited to, a cytotoxic agent or an imaging agent). Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 , Pb 212and radioisotopes of Lu); chemotherapeutic agents; growth inhibitors; enzymes and fragments thereof (such as nucleolytic enzymes); antibiotics; toxins (such as small molecule toxins or enzymatically active toxins). In some embodiments, the antibody is conjugated to one or more cytotoxic agents (such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof)) or radioisotopes. Immunoconjugates include antibody-drug conjugates (ADCs) in which the antibody is conjugated to one or more drugs. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein can be used in ADCs. In certain embodiments, ADCs comprise antibodies or antigen-binding fragments thereof described herein conjugated to small molecule compounds. In certain embodiments, ADCs comprise antibodies or antigen-binding fragments thereof described herein conjugated to chemotherapeutic agents. Immunoconjugates also include immunoconjugates in which antibodies are conjugated to toxins (such as enzymatically active toxins or fragments thereof). In certain embodiments, immunoconjugates comprise antibodies or antigen-binding fragments thereof described herein and toxins (such as enzymatically active toxins or fragments thereof). Immunoconjugates can also comprise antibodies or antigen-binding fragments thereof and peptides or proteins. In certain embodiments, an immunoconjugate comprises an antibody or antigen-binding fragment thereof described herein and a peptide or protein.

[0240] Immunoconjugates also include immunoconjugates in which the antibody is conjugated to a radioactive atom to form a radioconjugate. Exemplary radioisotopes include At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 , Pb 212 and radioactive isotopes of Lu.

[0241] Conjugates of antibodies and agents (e.g., drugs, small molecule compounds, toxins, proteins, or peptides) can be prepared using any of a variety of known protein coupling agents (e.g., linkers) (see Vitetta et al., Science 238:1098 (1987), WO 94 / 11026). The linker can be a "cleavable linker" that promotes the release of the cytotoxic drug in the cell, such as an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, and a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020). CT cell engager

[0242] In some embodiments, the DLL3 binding molecules provided are molecules for use in T cell engagement therapy. In some embodiments, the antibodies or antigen-binding fragments thereof provided are contained in molecules for use in T cell engagement therapy. In some embodiments, the molecules for use in T cell engagement therapy are or comprise binding molecules capable of binding to surface molecules expressed on T cells. In some embodiments, the surface molecule is an activating component of a T cell, such as a component of the T cell receptor complex. In some embodiments, the surface molecule is CD3 or CD2. In some embodiments, the molecules for use in T cell engagement therapy are or comprise antibodies or antigen-binding fragments thereof, such as antibodies or antigen-binding fragments thereof that bind to CD3 or CD2, and any of the antibodies or antigen-binding fragments thereof that bind to DLL3 as described herein.

[0243] In some embodiments, the molecule used in T cell engagement therapy is a bispecific antibody containing at least one antigen binding domain that binds to an activation component of a T cell (e.g., a T cell surface molecule, such as CD3 or CD2) and at least one antigen binding domain that binds to a surface antigen on a cancer cell (e.g., DLL3). In some embodiments, the molecule used in T cell engagement therapy is a bispecific T cell engager (BiTE). In certain embodiments, the antigen binding domain that binds to a surface antigen on a cancer cell is or comprises any DLL3 binding molecule described herein. In certain embodiments, the antigen binding domain that binds to a surface antigen on a cancer cell is an anti-DLL3 scFv comprising (i) a VH domain comprising the sequence shown in SEQ ID NO: 90 and (ii) a VL domain comprising the sequence shown in SEQ ID NO: 91. In certain embodiments, the antigen binding domain that binds to a surface antigen on a cancer cell is an anti-DLL3 scFv comprising the sequence shown in SEQ ID NO: 89. In some embodiments, simultaneous or nearly simultaneous binding of such an antibody to its two targets can result in a transient interaction between the target cancer cell and the T cell, leading to activation of the T cell (e.g., cytotoxic activity) and subsequent lysis of the target cancer cell.

[0244] In some embodiments, bispecific T cell engagers (BiTEs) are used in conjunction with the provided methods, uses, and articles of manufacture. In some embodiments, bispecific T cell engagers are specific for two specific antigens (or markers or ligands). In some embodiments, the antigens are expressed on the surface of specific cell types. In certain embodiments, the first antigen is associated with an immune cell or engineered immune cell, and the second antigen is DLL3 expressed on the surface of a cancer cell.

[0245] Various methods are known for producing bispecific T cell engagers, including fusion of two different hybridomas (Milstein and Cuello, Nature 1983; 305: 537-540), and chemical tethering via heterobifunctional cross-linkers (Staerz et al. Nature 1985; 314: 628-631). Exemplary such bispecific antibody T cell engaging molecules (e.g., bispecific T cell engager (BiTE) molecules) are those containing: tandem scFv molecules fused via a flexible linker (see, e.g., Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); tandem scFv molecules fused to each other via, e.g., a flexible linker, and further containing an Fc domain composed of a first and a second subunit capable of stably associating (WO 2013026837); diabodies and derivatives thereof, including tandem diabodies (Holliger et al., Prot Eng 9, 299-305 (1996); Kipriyanov et al., J Mol Biol 293, 41-66 (1999)); dual affinity retargeting (DART) molecules, which may include a diabody format with a C-terminal disulfide bridge; or triomabs comprising an intact hybrid mouse / rat IgG molecule (Seimetz et al., Cancer Treat Rev 36,458-467(2010)).

[0246] In certain embodiments, a bispecific T cell engager is or comprises a polypeptide construct or fusion protein. In certain embodiments, the polypeptide construct comprises a first component comprising an antigen binding domain that binds to an activation component of an immune cell or engineered immune cell, and a second component comprising an antigen binding domain that binds to a surface antigen associated with certain cancers (e.g., DLL3). In some embodiments, the first component and the second component are coupled via a linker.

[0247] In some embodiments, the antigen binding domain of the first component of the bispecific T cell engager engages a receptor on an endogenous immune cell around the tumor. In some embodiments, the endogenous immune cell is a T cell. In some aspects, the engagement of the endogenous T cell receptor redirects the endogenous T cell to the tumor. In some aspects, the engagement of the endogenous T cell receptor recruits tumor infiltrating lymphocytes (TIL) to the tumor. In some aspects, the engagement of the endogenous T cell receptor activates the endogenous immune reservoir.

[0248] In some embodiments, simultaneous or nearly simultaneous binding of a bispecific T cell engager to its two targets (e.g., an immune cell and a DLL3-expressing cancer cell) can result in a transient interaction between the DLL3-expressing cancer cell and the T cell, leading to activation of the T cell (e.g., cytotoxic activity, cytokine release) and subsequent lysis of the cancer cell.

[0249] In some embodiments, the first component of the bispecific T cell engager is or comprises an antigen binding domain that binds to an activation component of a T cell. In some embodiments, the activation component of a T cell is a surface molecule. In some embodiments, the surface molecule is or comprises a T cell antigen. Exemplary T cell antigens include but are not limited to CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD44, CD45, CD69, and CD90. In some aspects, the combination of the bispecific T cell engager and the T cell antigen stimulates and / or activates the T cell. In some aspects, the activation component of a T cell is a T cell surface molecule, such as CD3 or CD2.

[0250] In some embodiments, the anti-T cell binding domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

[0251] In some embodiments, the T cell binding domain on the BiTE is anti-CD3. In some aspects, the anti-CD3 domain is a scFv. In some embodiments, the anti-CD3 domain of the BiTE binds to a subunit of the CD3 complex on the receptor of the T cell. In some aspects, the receptor is located on an endogenous T cell. In some embodiments, the receptor is located on an engineered immune cell that also expresses a recombinant receptor. The effect of CD3 engagement of T cells is well known in the art and includes, but is not limited to, T cell activation and other downstream cell signaling. Any such BiTE can be used in the present disclosure provided herein.

[0252] In some embodiments, the second component of the BiTE comprises an antigen binding domain that binds to DLL3 expressed on the surface of a cancer cell. In some embodiments, the surface antigen of the target cell is DLL3. In some embodiments, the second component of the BiTE comprises any of the antibodies or antigen binding domains described herein that bind to DLL3. In certain embodiments, the antigen binding domain that binds to DLL3 is an anti-DLL3 scFv comprising (i) a VH domain comprising the sequence shown in SEQ ID NO: 90 and (ii) a VL domain comprising the sequence shown in SEQ ID NO: 91. In certain embodiments, the antigen binding domain that binds to DLL3 is an anti-DLL3 scFv comprising the sequence shown in SEQ ID NO: 89.

[0253] In some embodiments, the two antigen binding domains (including the first antigen binding domain and the second antigen binding domain) comprise an antibody or antigen binding fragment.

[0254] In some embodiments, the polypeptide is a construct comprising, from N-terminus to C-terminus, a first component comprising an antigen-binding domain that binds to an activation component of a T cell; a peptide linker; and a second component comprising an antigen-binding domain that binds to a surface antigen associated with certain cancers, such as DLL3. In certain embodiments, the first antigen-binding domain of a BiTE is an anti-CD3 scFv. In certain embodiments, the second antigen-binding domain of a BiTE is an anti-DLL3 scFv. In certain embodiments, the anti-DLL3 scFv comprises the sequence set forth in SEQ ID NO: 89.

[0255] In some aspects, the BiTE polypeptide construct comprises a linker connecting a first component comprising an antigen binding domain that binds to an activating portion of a T cell and a second component comprising an antigen binding domain that binds to DLL3 on cancer cells. In some aspects, the linker is a short linker, a medium linker, or a long linker. In some aspects, the linker can be any peptide linker described herein. In some embodiments, the peptide linker is or comprises a cleavable peptide linker. D. Multispecific Antibodies

[0256] In certain embodiments, a DLL3 binding molecule (e.g., an antibody or antigen-binding fragment thereof, or a fusion protein, such as a recombinant receptor comprising the antibody or antigen-binding fragment thereof) is multispecific. Multispecific binding molecules include multispecific antibodies, including, for example, bispecific antibodies. A multispecific binding partner (e.g., an antibody) has binding specificity for at least two different sites, which can be on the same or different antigens. In certain embodiments, one of the binding specificities is for DLL3 and the other is for another antigen. In certain embodiments, a bispecific antibody can bind to two different epitopes of DLL3. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing DLL3. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Multispecific antibodies include multispecific single-chain antibodies (e.g., diabodies, triabodies, and tetrabodies), tandem bi-scFvs, and tandem tri-scFvs. Multispecific chimeric receptors containing antibodies, such as multispecific CARs, are also provided. Also provided are multispecific cells containing antibodies or polypeptides (including antibodies), for example, cells containing a cell surface protein (including an anti-DLL3 antibody) and an additional cell surface protein (such as an additional chimeric receptor) that binds a different antigen or a different epitope on DLL3. E. Recombinant receptor

[0257] The binding molecules provided (e.g., DLL3 binding molecules) are cell surface proteins, such as recombinant receptors, such as receptors comprising one of the provided antibodies or antigen binding fragments. Also provided are polynucleotides encoding all or a portion of such cell surface proteins (e.g., receptors). Receptors include antigen receptors and other chimeric receptors that specifically bind to DLL3, such as receptors containing the provided anti-DLL3 antibodies (e.g., antigen binding fragments). Antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). DLL3 binding receptors typically contain antibodies (e.g., antigen binding fragments) and / or other binding polypeptides that specifically bind to DLL3 (e.g., DLL3 proteins, such as human DLL3). Also provided are cells expressing recombinant receptors, compositions containing such cells, and their use in adoptive cell therapy (e.g., for the treatment of diseases and disorders associated with DLL3 expression), compositions, products, and uses thereof.

[0258] Chimeric antigen receptors (CARs) specific for DLL3 are provided, as well as polynucleotides containing nucleic acid sequences encoding all or a portion, fragment, domain, or chain of any CAR described herein. In some embodiments, the CAR contains one of the DLL3 binding antibody fragments (such as those described in Section IA and / or Table 2). In some embodiments, the CAR is one of the CARs described in each row of Table E1-Table E4 and Table E8 and / or Table 3.

[0259] The polynucleotides provided include polynucleotides encoding recombinant receptors (such as antigen receptors) that specifically bind to DLL3. In some aspects, encoded receptors (such as those containing DLL3 binding polypeptides) and compositions and products thereof and uses are also provided. The polynucleotides provided can be incorporated into constructs (such as deoxyribonucleic acid (DNA) or RNA constructs, such as those that can be introduced into cells to express encoded recombinant DLL3 binding receptors). Also provided herein are polynucleotides encoding CARs. In some embodiments, CARs can be encoded by more than one different polynucleotides (such as two or more polynucleotides). In some any such embodiments, two or more polynucleotides may each contain a nucleic acid encoding a portion, fragment, domain, or chain of a CAR. 1. Chimeric Antigen Receptor (CAR)

[0260] In some aspects, DLL3-binding cell surface proteins, such as receptors, are provided. In some aspects, the provided DLL3-binding cell surface proteins (such as receptors) typically contain an extracellular antigen binding domain and an intracellular signaling domain. Among the provided receptors, for example, recombinant receptors, such as chimeric antigen receptors (CARs) containing provided antibodies or antigen-binding fragments thereof (such as one or more provided anti-DLL3 antibodies or fragments thereof). In some embodiments, the provided cell surface proteins specifically bind to DLL3, such as human DLL3.

[0261] Antigen receptors include chimeric and / or functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Chimeric receptors (such as CARs) generally include an extracellular antigen binding domain, which includes, is or includes an anti-DLL3 antibody, such as an anti-DLL3 antibody or a fragment thereof as described herein. In some embodiments, a chimeric receptor (such as a CAR) includes an intracellular signaling domain. In some embodiments, the chimeric receptor further includes a spacer and / or a transmembrane domain. In some embodiments, the spacer is located between the extracellular antigen binding domain and the transmembrane domain. In some embodiments, CAR includes an extracellular antigen binding domain, a spacer, a transmembrane domain, and an intracellular signaling region. Exemplary CARs provided herein include CARs containing antigen binding domains, which antigen binding domains include antibodies or antigen binding fragments thereof described herein (e.g., in Section IA and / or Table 2), or antibodies or antigen binding fragments thereof described in Section IE, Table 3, and / or Table E1-Table E4 and Table E8 herein. Also provided are CARs encoded by the polynucleotides described in Section IF, Table 3, and / or Tables E1-E4 and E8 of this document.

[0262] In some cases, CAR is referred to as first generation, second generation and / or third generation CAR.In some aspects, first generation CAR is a CAR that provides only the signal induced by CD3 chain when antigen is bound; In some aspects, second generation CAR is a CAR that provides this signal and costimulatory signal, such as a CAR including an intracellular signaling domain from a costimulatory receptor (such as CD28 or CD137); In some aspects, third generation CAR is a CAR including multiple costimulatory domains of different costimulatory receptors.

[0263] In some embodiments, the CAR comprises an extracellular antigen binding domain (in some cases comprising an antibody, such as an antibody fragment that binds to DLL3), a transmembrane domain that is or comprises a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some embodiments, the CAR comprises an extracellular antigen binding domain (in some cases comprising an antibody, such as an antibody fragment that binds to DLL3), a transmembrane domain that is or comprises a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of 4-1BB or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer containing a portion of an Ig molecule (such as a human Ig molecule) (such as an Ig hinge, such as an IgG4 hinge), such as only a hinge spacer.

[0264] In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment thereof as described herein. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment thereof as described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment thereof comprises an scFv, and the intracellular signaling region comprises an ITAM. In some aspects, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-ζ (CD3ζ) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain connecting the extracellular domain and the intracellular signaling domain.

[0265] Other exemplary antigen receptors (including CARs) and methods of engineering and introducing such antigen receptors into cells include, for example, those described in WO 200014257, WO 2013126726, WO 2012 / 129514, WO 2014031687, WO 2013 / 166321, WO 2013 / 071154, WO 2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP2537416; and / or those described in Sadelain et al., Cancer Discov. 2013 Apr; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 Oct; 24(5): 633-39; Wu et al., Cancer, 2012 Mar 18(2): 160-75. In some aspects, the antigen receptor includes a CAR as described in U.S. Patent No. 7,446,190, and those described in WO / 2014055668A1. Examples of CARs include those disclosed in any of the above-mentioned publications (e.g., WO2014031687, US 8,339,645, US 7,446,179, US2013 / 0149337, U.S. Pat. No. 7,446,190, U.S. Pat. No. 8,389,282), for example, and wherein the antigen binding portion (e.g., scFv) is replaced by an antibody (e.g., as provided herein).

[0266] For example, Hudecek et al., Clin Cancer Res, 19(12), 3153-3164 (2013) and Baskar et al. MAbs. 4(3): 349-361 (2012) describe other CARs targeting DLL3. See also WO 2014031687; US 2012 / 20058051. a. Extracellular antigen-binding domain

[0267] Chimeric receptors include chimeric antigen receptors (CARs). Chimeric receptors (such as CARs) typically comprise an extracellular antigen binding domain that comprises, is, or is contained in one or more provided anti-DLL3 antibodies or antigen binding fragments. Thus, a chimeric receptor (such as a CAR) typically comprises one or more DLL3 binding molecules, such as one or more antigen binding fragments, domains, or portions, or one or more antibody variable domains and / or antibody molecules (such as those described herein) in its extracellular portion. In some embodiments, a CAR comprises one or more DLL3 binding portions of an antibody molecule, such as a variable heavy (V) domain of an antibody. H ) chain region and / or variable light (V L In some embodiments, the CAR comprises one or more DLL3 binding portions of an antibody molecule, such as a variable weight (V) H ) chain region and variable light (V L ) chain region, such as scFv. In some aspects, the CAR comprises one or more of any DLL3 binding antibodies or antigen-binding fragments thereof described herein (e.g., in Section IA).

[0268] In some embodiments, Table 2 provides SEQ ID NOs of exemplary antigen binding domains (e.g., antibodies or antigen binding fragments) that can be included in provided DLL3 binding receptors (e.g., anti-DLL3 chimeric antigen receptors (CARs)). In some aspects, the CAR comprises a scFv as described in Table 2, e.g., as part of the extracellular antigen binding domain. In some aspects, the CAR comprises a V described in Table 2. H In some aspects, the CAR comprises the V domains described in Table 2. L In some embodiments, the DLL3 binding receptor comprises a DLL3 binding antibody or fragment thereof comprising a V region comprising CDR-H1, CDR-H2, and CDR-H3 sequences. H region and a V region containing CDR-L1, CDR-L2 and CDR-L3 sequences L Region, as shown in SEQ ID NO: listed in each row of Table 2. In some embodiments, the DLL3 binding receptor comprises a DLL3 binding antibody or fragment thereof comprising a V region as shown in SEQ ID NO: listed in each row of Table 2. H Region sequence and V L or comprising a V region sequence as shown in the SEQ ID NO: listed in each row of Table 2 H Region sequence and V LThe V region sequences have at least 95%, 96%, 97%, 98% or 99% sequence identity. H and V L In some embodiments, the DLL3 binding receptor comprises a DLL3 binding antibody or fragment thereof comprising a V region as shown in SEQ ID NO: ... H Region sequence and V L In some embodiments, the DLL3 binding receptor comprises a DLL3 binding antibody or fragment thereof comprising a scFv sequence as shown in SEQ ID NO: listed in each row of Table 2; or an antibody comprising a scFv amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to a scFv sequence as shown in SEQ ID NO: listed in each row of Table 2. In some embodiments, the DLL3 binding receptor comprises a DLL3 binding antibody or fragment thereof comprising a scFv sequence as shown in SEQ ID NO: listed in each row of Table 2.

[0269] In some embodiments, the CAR provided may comprise an extracellular antigen binding domain comprising all or a portion of an antibody, including polyclonal and monoclonal antibodies, including complete antibodies and functional (antigen binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (V) fragments capable of specific binding to an antigen, and H ) region, single-chain antibody fragments (including single-chain variable fragments (scFv)) and single-domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. In some aspects, the antibody or its fragment included in CAR includes a genetically engineered and / or otherwise modified form of an immunoglobulin, such as an intracellular antibody, a peptibody, a chimeric antibody, a fully human antibody, a humanized antibody and a heteroconjugate antibody, a multispecific (e.g., bispecific or trispecific) antibody, a double antibody, a three-antibody and a four-antibody, a tandem double scFv, a tandem three scFv. b. Spacer

[0270] In some embodiments, the recombinant receptor (such as CAR, such as the antibody portion thereof) further comprises a spacer (also referred to as a spacer region in some cases), which can be or include at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region (e.g., an IgG4 hinge region) and / or a C H 1 / C L 、C H2 and / or C H 3 and / or Fc region. In some embodiments, the constant region or portion is that of human IgG (such as IgG4, IgG2 or IgG1). In some aspects, the portion of the constant region serves as a spacer between the antigen recognition component (e.g., scFv) and the transmembrane domain.

[0271] In some embodiments, the length of the spacer is adjusted to optimize the biophysical synaptic distance between the cell expressing CAR (such as a cell expressing CAR) and the target of CAR (such as a tumor cell expressing DLL3). In some embodiments, CAR is expressed by T cells, and the length of the spacer is adjusted to a length compatible with T cell activation or adjusted to optimize CAR T cell performance.

[0272] In some embodiments, compared with the situation that there is no spacer or compared with the alternative spacer of different length (for example, length is longer), the spacer can have the length that provides the cell responsiveness of increase after antigen binding.In some examples, the length of the spacer is for or about 12 amino acids or the length is not more than or not more than about 12 amino acids.In some examples, the length of the spacer is for or about 15 amino acids or the length is not more than or not more than about 15 amino acids.

[0273] Exemplary spacers include spacers having a length of at least or at least about 10 to at least or at least about 300 amino acids, at least or at least about 10 to at least or at least about 229 amino acids, at least or at least about 10 to at least or at least about 200 amino acids, at least or at least about 10 to at least or at least about 175 amino acids, at least or at least about 10 to at least or at least about 150 amino acids, at least or at least about 10 to at least or at least about 125 amino acids, at least or at least about 10 to at least or at least about 100 amino acids, at least or at least about 10 to at least or at least about 75 amino acids, at least or at least about 10 to at least or at least about 50 amino acids, at least or at least about 10 to at least or at least about 40 amino acids, at least or at least about 10 to at least or at least about 30 amino acids, at least or at least about 10 to at least or at least about 20 amino acids, or at least or at least about 10 to at least or at least about 15 amino acids, and including any integer between the endpoints of any recited ranges. Exemplary spacers include those having a length of at least or at least about 50 to at least or at least about 175 amino acids, at least or at least about 50 to at least or at least about 150 amino acids, at least or at least about 10 to at least or at least about 125 amino acids, at least or at least about 50 to at least or at least about 100 amino acids, at least or at least about 100 to at least or at least about 300 amino acids, at least or at least about 100 to at least or at least about 250 amino acids, at least or at least about 125 to at least or at least about 250 amino acids, or at least or at least about 200 to at least or at least about 250 amino acids, and including any integer between the endpoints of any recited ranges. In some embodiments, the length of the spacer is at least or at least about 12 amino acids, at least or at least about 119 amino acids, at least or at least about 125 amino acids, at least or at least about 200 amino acids, or at least or at least about 220 amino acids, or at least or at least about 225 amino acids. In some embodiments, the length of the spacer is at least or about 13 amino acids, at least or about 120 amino acids, at least or about 125 amino acids, at least or about 200 amino acids, or at least or about 220 amino acids, or at least or about 229 amino acids. In some embodiments, the length of the spacer is at or about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids or less.In some embodiments, the spacer is at least or about 100 amino acids in length, such as at least or about 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids in length.

[0274] In some embodiments, the length of the spacer is at least or at least about 125 to at least or at least about 300 amino acids, at least or at least about 125 to at least or at least about 250 amino acids, at least or at least about 125 to at least or at least about 230 amino acids, at least or at least about 125 to at least or at least about 200 amino acids, at least or at least about 125 to at least or at least about 180 amino acids, at least or at least about 125 to at least or at least about 150 amino acids, at least or at least about 150 to at least or at least about 300 amino acids, at least or at least about 150 to at least or at least about 250 amino acids, at least or at least about 150 to at least or at least about 230 amino acids, at least or at least about 150 to at least or at least about 200 amino acids, at least or at least about 180 amino acids. At least about 150 to at least or at least about 180 amino acids, at least or at least about 180 to at least or at least about 300 amino acids, at least or at least about 180 to at least or at least about 250 amino acids, at least or at least about 180 to at least or at least about 230 amino acids, at least or at least about 180 to at least or at least about 200 amino acids, at least or at least about 200 to at least or at least about 300 amino acids, at least or at least about 200 to at least or at least about 250 amino acids, at least or at least about 200 to at least or at least about 230 amino acids, at least or at least about 230 to at least or at least about 300 amino acids, at least or at least about 230 to at least or at least about 250 amino acids, or 250 to at least or at least about 300 amino acids in length. In some embodiments, the spacer is at least or about 129, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, or 230 amino acids in length, or a length between any of the foregoing values.

[0275] Exemplary spacers include the IgG hinge alone, H 2 and C H One or more of the IgG hinges connected to the C H In some embodiments, the spacer comprises a separate IgG hinge. In some embodiments, the IgG hinge, CH 2 and / or C H 3 can be derived in whole or in part from IgG4 or IgG2, such as derived in whole or in part from human IgG4 or human IgG2. In some embodiments, the spacer can be a spacer containing a hinge, C 3 derived from IgG4, IgG2 and / or IgG2 and IgG4. H 2 and / or C H In some embodiments, the hinge region comprises all or a portion of an IgG4 hinge region. In some embodiments, the hinge region comprises all or a portion of an IgG4 hinge region and / or an IgG2 hinge region, wherein the IgG4 hinge region is optionally a human IgG4 hinge region, and the IgG2 hinge region is optionally a human IgG2 hinge region; H Region 2 contains IgG4 C H Region 2 and / or IgG2 C H All or part of region 2, in which IgG4 C H Region 2 is optionally human IgG4 C H Region 2, and IgG2 C H Region 2 is optionally human IgG2 C H Zone 2; and / or C H Region 3 contains IgG4 C H Region 3 and / or IgG2C H All or part of region 3, in which IgG4 C H Region 3 is optionally human IgG4 C H Region 3, and IgG2 C H Region 3 is optionally human IgG2 C H Region 3. In some embodiments, hinge C H 2 and C H 3 contains the hinge region C from IgG4 H 2 and C H In some embodiments, the hinge region is chimeric and comprises hinge regions from human IgG4 and human IgG2; H Region 2 is chimeric and contains C from human IgG4 and human IgG2. H Zone 2; and / or said C H Region 3 is chimeric and contains C from human IgG4 and human IgG2. H In some embodiments, the spacer comprises an IgG4 / 2 chimeric hinge or a modified IgG4 hinge containing at least one amino acid substitution compared to the human IgG4 hinge region; a human IgG2 / 4 chimeric C H Region 2; and human IgG4 C H Zone 3.

[0276] In some embodiments, the spacer comprises all or a portion of an immunoglobulin hinge or a modified form thereof or consists of them. In some embodiments, the length of the spacer is or is about 15 amino acids or less. In some embodiments, the spacer comprises all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge or a modified form thereof, or consists of them, and / or comprises about 15 or less amino acids. In some embodiments, the length of the spacer is or is about 13 amino acids, and / or comprises all or a portion of an immunoglobulin hinge, optionally an IgG4 or a modified form thereof, or consists of them. In some embodiments, the length of the spacer is or is about 12 amino acids and / or comprises an immunoglobulin hinge, optionally an IgG4, or a modified form thereof or consists of them. In some embodiments, the spacer comprises the formula X1PPX2P (SEQ ID NO: 32), wherein X1 is glycine, cysteine or arginine, and X2 is cysteine or threonine. In some embodiments, the spacer does not comprise the CD28 extracellular region or the CD8 extracellular region. In some cases, the spacer has a methionine residue at the C-terminus. In some embodiments, the spacer comprises or consists of a sequence of SEQ ID NOs: 33-39, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the spacer comprises the amino acid sequence shown in any one of SEQ ID NOs: 33-39.

[0277] In some embodiments, the spacer comprises or consists of a sequence of SEQ ID NO: 1 or 3, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0278] In some embodiments, the spacer comprises or consists of SEQ ID NO: 3, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the spacer comprises or consists of the sequence shown in SEQ ID NO: 3. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 3. In some embodiments, the spacer consists of the sequence shown in SEQ ID NO: 3. In some embodiments, the spacer comprises or consists of the amino acid sequence encoded by SEQ ID NO: 4, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the spacer comprises an amino acid sequence encoded by SEQ ID NO: 4. In some embodiments, the spacer consists of an amino acid sequence encoded by SEQ ID NO: 4.

[0279] In some embodiments, the spacer comprises or consists of SEQ ID NO: 1, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the spacer comprises or consists of the sequence shown in SEQ ID NO: 1. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 1. In some embodiments, the spacer consists of the sequence shown in SEQ ID NO: 1. In some embodiments, the spacer comprises or consists of the amino acid sequence encoded by SEQ ID NO: 2, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the spacer comprises an amino acid sequence encoded by SEQ ID NO: 2. In some embodiments, the spacer consists of an amino acid sequence encoded by SEQ ID NO: 2.

[0280] In some embodiments, the spacer comprises or consists of an amino acid sequence encoded by SEQ ID NO: 80, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 80. In some embodiments, the spacer comprises an amino acid sequence encoded by SEQ ID NO: 80. In some embodiments, the spacer consists of an amino acid sequence encoded by SEQ ID NO: 80.

[0281] In some embodiments, the spacer is or comprises a C sequence that is identical to, for example, a human immunoglobulin (such as IgG4 and / or IgG2). H In some aspects, the spacer is at or about 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125 amino acids in length, or between any of the foregoing values. In some aspects, the spacer is at or about 119 or 120 amino acids in length.

[0282] In some embodiments, the spacer comprises or consists of a sequence of SEQ ID NO: 5 or 58, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0283] In some embodiments, the spacer comprises or consists of SEQ ID NO: 5, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the spacer comprises or consists of the sequence shown in SEQ ID NO: 5. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 5. In some embodiments, the spacer consists of the sequence shown in SEQ ID NO: 5. In some embodiments, the spacer comprises or consists of the amino acid sequence encoded by SEQ ID NO: 57 or 81, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the spacer comprises an amino acid sequence encoded by SEQ ID NO: 57 or 81. In some embodiments, the spacer consists of an amino acid sequence encoded by SEQ ID NO: 57 or 81.

[0284] In some embodiments, the spacer comprises or consists of SEQ ID NO: 58, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58. In some embodiments, the spacer comprises or consists of the sequence shown in SEQ ID NO: 58. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 58. In some embodiments, the spacer consists of the sequence shown in SEQ ID NO: 58. In some embodiments, the spacer comprises or consists of the amino acid sequence encoded by SEQ ID NO: 59 or 63, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 59 or 63. In some embodiments, the spacer comprises an amino acid sequence encoded by SEQ ID NO: 59 or 63. In some embodiments, the spacer consists of an amino acid sequence encoded by SEQ ID NO: 59 or 63.

[0285] In some embodiments, the spacer is or comprises a C sequence that is identical to, for example, a human immunoglobulin (such as IgG4 and / or IgG2). H 2 domains and C HIn some aspects, the spacer is at or about 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, or 235 amino acids in length, or between any of the foregoing values. In some aspects, the spacer is at or about 228 or 229 amino acids in length.

[0286] In some embodiments, the spacer can be derived in whole or in part from IgG4 and / or IgG2 and can contain mutations, such as one or more single amino acid mutations in one or more domains. In some examples, the amino acid modification is a substitution of serine (S) with proline (P) in the hinge region of IgG4. In some embodiments, the amino acid modification is a substitution of asparagine (N) with glutamine (Q) to reduce glycosylation heterogeneity, such as the C sequence of the full-length IgG4 Fc sequence shown in SEQ ID NO: 56. H N177Q mutation at position 177 in region 2, or C of the full-length IgG2 Fc sequence shown in SEQ ID NO: 55 H N176Q at position 176 in zone 2.

[0287] In some embodiments, the spacer is or comprises an IgG4 / 2 chimeric hinge or a modified IgG4 hinge; an IgG2 / 4 chimeric C H Region 2; and IgG4 C H Region 3. In some embodiments, the spacer is about 228 or 229 amino acids in length, such as the spacer shown in SEQ ID NO: 42 or 82.

[0288] In some embodiments, the spacer comprises or consists of a sequence of SEQ ID NO: 42 or 82, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0289] In some embodiments, the spacer is or comprises an Ig hinge or hinge; a chimeric C H Zone 2; and C H Region 3, as shown in SEQ ID NO:41, or encoded by the sequence shown in SEQ ID NO:40.

[0290] In some embodiments, the spacer comprises or consists of a sequence of SEQ ID NO: 41, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In some embodiments, the spacer is encoded by a sequence comprising or consisting of a sequence of SEQ ID NO: 40, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0291] In some embodiments, the spacer comprises or consists of SEQ ID NO: 42, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 42. In some embodiments, the spacer comprises or consists of the sequence shown in SEQ ID NO: 42. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 42. In some embodiments, the spacer consists of the sequence shown in SEQ ID NO: 42. In some embodiments, the spacer comprises or consists of the amino acid sequence encoded by SEQ ID NO: 43 or 60, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 43 or 60. In some embodiments, the spacer comprises an amino acid sequence encoded by SEQ ID NO: 43 or 60. In some embodiments, the spacer consists of an amino acid sequence encoded by SEQ ID NO: 43 or 60.

[0292] In some embodiments, the spacer comprises or consists of an IgD hinge. In some embodiments, the spacer comprises or consists of SEQ ID NO: 7, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7. In some embodiments, the spacer comprises or consists of the sequence shown in SEQ ID NO: 7. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 7. In some embodiments, the spacer consists of the sequence shown in SEQ ID NO: 7.

[0293] In some embodiments, the spacer comprises or consists of SEQ ID NO: 82, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 82. In some embodiments, the spacer comprises or consists of the sequence shown in SEQ ID NO: 82. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 82. In some embodiments, the spacer consists of the sequence shown in SEQ ID NO: 82. In some embodiments, the spacer comprises or consists of the amino acid sequence encoded by SEQ ID NO: 83 or 84, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 83 or 84. In some embodiments, the spacer comprises an amino acid sequence encoded by SEQ ID NO: 83 or 84. In some embodiments, the spacer consists of an amino acid sequence encoded by SEQ ID NO: 83 or 84.

[0294] Additional exemplary spacers include, but are not limited to, those described in the following literature: Hudecek et al. (2013) Clin. Cancer Res., 19: 3153; Hudecek et al. (2015) Cancer Immunol. Res., 3 (2): 125-50 or WO2014031687. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce RNA heterogeneity after expression. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce hidden splice sites or reduce the possibility of splicing events occurring at the splice sites.

[0295] In some embodiments, the spacer is encoded by a polynucleotide that has been optimized for codon usage and / or reducing RNA heterogeneity (e.g., by removing cryptic splice sites). In some embodiments, the spacer is an amino acid sequence encoded by a polynucleotide that has been optimized for codon expression and / or elimination of splice sites (e.g., cryptic splice sites). In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence shown in SEQ ID NO:2. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence shown in SEQ ID NO:4. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence shown in SEQ ID NO:57. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence shown in SEQ ID NO:59. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence shown in SEQ ID NO:60. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence shown in SEQ ID NO:84.

[0296] In some embodiments, the spacer has the amino acid sequence shown in SEQ ID NO: 1 and is encoded by the polynucleotide sequence shown in SEQ ID NO: 2. In some embodiments, the spacer has the amino acid sequence shown in SEQ ID NO: 3 and is encoded by the polynucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the spacer has the amino acid sequence shown in SEQ ID NO: 5 and is encoded by the polynucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the spacer has the amino acid sequence shown in SEQ ID NO: 58 and is encoded by the polynucleotide sequence shown in SEQ ID NO: 59. In some embodiments, the spacer has the amino acid sequence shown in SEQ ID NO: 42 and is encoded by the polynucleotide sequence shown in SEQ ID NO: 43. In some embodiments, the spacer has the amino acid sequence shown in SEQ ID NO: 82 and is encoded by the polynucleotide sequence shown in SEQ ID NO: 84.

[0297] In some embodiments, the spacer has an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or 3 and is encoded by a polynucleotide that has been optionally optimized for codon usage and / or to reduce RNA heterogeneity. In some embodiments, the spacer has an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5 or 58 and is encoded by a polynucleotide that has been optionally optimized for codon usage and / or to reduce RNA heterogeneity. In some embodiments, the spacer has an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 42 or 82 and is encoded by a polynucleotide that has been optionally optimized for codon usage and / or to reduce RNA heterogeneity. c. Transmembrane domain

[0298] The antigen recognition component is typically linked to one or more intracellular signaling components (e.g., a signaling component that mimics activation by an antigen receptor complex (in the case of a CAR, such as a TCR complex) and / or signals via another cell surface receptor). Thus, in some embodiments, a DLL3 binding molecule (e.g., an antibody or antigen binding fragment thereof) is linked to one or more transmembrane domains (such as those described herein) and an intracellular signaling domain comprising one or more intracellular components (such as those described herein). In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain is used that is naturally associated with one of the domains in a receptor (e.g., a CAR). In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domain to a transmembrane domain of the same or different surface membrane protein to minimize interactions with other members of the receptor complex.

[0299] In some embodiments, the membrane-spanning domain is derived from a natural or synthetic source. When the source is natural, in some aspects, the domain can be derived from any membrane-bound protein or transmembrane protein. The membrane-spanning domain includes those derived from the following (i.e., at least one or more membrane-spanning domains comprising the following): α, β or ζ chain of T cell receptor, CD3ε, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 and / or CD154. In some embodiments, the transmembrane domain of the receptor (e.g., CAR) is the transmembrane domain of human CD28 (e.g., the 27 amino acid transmembrane domain or the 28 amino acid sequence of human CD28 (Accession No.: P10747.1)) or a variant thereof, or a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10 or 12, or an amino acid sequence that exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 12.

[0300] In some embodiments, the transmembrane domain comprises or consists of a sequence of SEQ ID NO: 10 or 12, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0301] In some embodiments, the transmembrane domain comprises or consists of SEQ ID NO: 10, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10. In some embodiments, the transmembrane domain comprises or consists of the sequence shown in SEQ ID NO: 10. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO: 10. In some embodiments, the transmembrane domain consists of the sequence shown in SEQ ID NO: 10. In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence encoded by SEQ ID NO: 11, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 11. In some embodiments, the transmembrane domain comprises an amino acid sequence encoded by SEQ ID NO: 11. In some embodiments, the transmembrane domain consists of an amino acid sequence encoded by SEQ ID NO: 11.

[0302] In some embodiments, the transmembrane domain comprises or consists of SEQ ID NO: 12, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the transmembrane domain comprises or consists of the sequence shown in SEQ ID NO: 12. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO: 12. In some embodiments, the transmembrane domain consists of the sequence shown in SEQ ID NO: 12. In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence encoded by SEQ ID NO: 13, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises an amino acid sequence encoded by SEQ ID NO: 13. In some embodiments, the transmembrane domain consists of an amino acid sequence encoded by SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence encoded by SEQ ID NO: 14, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14. In some embodiments, the transmembrane domain comprises an amino acid sequence encoded by SEQ ID NO: 14. In some embodiments, the transmembrane domain consists of the amino acid sequence encoded by SEQ ID NO:14.

[0303] In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence encoded by a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 62, 77, or 85. In some embodiments, the transmembrane domain comprises an amino acid sequence encoded by a nucleic acid sequence encoded by SEQ ID NO: 62, 77, or 85. In some embodiments, the transmembrane domain consists of an amino acid sequence encoded by a nucleic acid sequence encoded by SEQ ID NO: 62, 77, or 85.

[0304] In some embodiments, the transmembrane domain is encoded by a polynucleotide that has been optionally optimized for codon usage and / or reducing RNA heterogeneity (e.g., by removing cryptic splice sites). In some embodiments, the transmembrane domain has the amino acid sequence shown in SEQ ID NO: 10 or 12 and is encoded by the polynucleotide sequence shown in SEQ ID NO: 11 or 13 or a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 11 or 13. In some cases, the transmembrane domain has a methionine residue at the N-terminus. In some embodiments, the transmembrane domain may be a CD28 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10, which is encoded by the nucleic acid sequence shown in SEQ ID NO: 11. In some embodiments, the transmembrane domain may be a CD28 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 12, which is encoded by the nucleic acid sequence shown in SEQ ID NO: 13.

[0305] In some aspects, specific combinations of spacer and transmembrane domain sequences can be used.

[0306] In some embodiments, the spacer of the recombinant receptor (e.g., CAR) comprises the sequence shown in SEQ ID NO: 1, and the transmembrane domain of the recombinant receptor (e.g., CAR) comprises the sequence shown in SEQ ID NO: 10. In some embodiments, the spacer of the recombinant receptor (e.g., CAR) consists of the sequence shown in SEQ ID NO: 1, and the transmembrane domain of the recombinant receptor (e.g., CAR) consists of the sequence shown in SEQ ID NO: 10.

[0307] In some embodiments, the spacer of the recombinant receptor (e.g., CAR) comprises the sequence shown in SEQ ID NO: 3, and the transmembrane domain of the recombinant receptor (e.g., CAR) comprises the sequence shown in SEQ ID NO: 12. In some embodiments, the spacer of the recombinant receptor (e.g., CAR) consists of the sequence shown in SEQ ID NO: 3, and the transmembrane domain of the recombinant receptor (e.g., CAR) consists of the sequence shown in SEQ ID NO: 12.

[0308] In some embodiments, the spacer of the recombinant receptor (e.g., CAR) comprises the sequence shown in SEQ ID NO: 58, and the transmembrane domain of the recombinant receptor (e.g., CAR) comprises the sequence shown in SEQ ID NO: 10. In some embodiments, the spacer of the recombinant receptor (e.g., CAR) consists of the sequence shown in SEQ ID NO: 58, and the transmembrane domain of the recombinant receptor (e.g., CAR) consists of the sequence shown in SEQ ID NO: 10.

[0309] In some embodiments, the spacer of the recombinant receptor (e.g., CAR) comprises the sequence shown in SEQ ID NO: 5, and the transmembrane domain of the recombinant receptor (e.g., CAR) comprises the sequence shown in SEQ ID NO: 12. In some embodiments, the spacer of the recombinant receptor (e.g., CAR) consists of the sequence shown in SEQ ID NO: 5, and the transmembrane domain of the recombinant receptor (e.g., CAR) consists of the sequence shown in SEQ ID NO: 12.

[0310] In some embodiments, the spacer of the recombinant receptor (e.g., CAR) comprises the sequence shown in SEQ ID NO: 82, and the transmembrane domain of the recombinant receptor (e.g., CAR) comprises the sequence shown in SEQ ID NO: 10. In some embodiments, the spacer of the recombinant receptor (e.g., CAR) consists of the sequence shown in SEQ ID NO: 82, and the transmembrane domain of the recombinant receptor (e.g., CAR) consists of the sequence shown in SEQ ID NO: 10.

[0311] In some embodiments, the spacer of the recombinant receptor (e.g., CAR) comprises the sequence shown in SEQ ID NO: 42, and the transmembrane domain of the recombinant receptor (e.g., CAR) comprises the sequence shown in SEQ ID NO: 12. In some embodiments, the spacer of the recombinant receptor (e.g., CAR) consists of the sequence shown in SEQ ID NO: 42, and the transmembrane domain of the recombinant receptor (e.g., CAR) consists of the sequence shown in SEQ ID NO: 12.

[0312] In some aspects, alternatively, in some embodiments, the membrane spaning domain is synthetic. In some aspects, the synthetic membrane spaning domain mainly comprises hydrophobic residues, such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic membrane spaning domain. In some embodiments, connection is achieved through a linker, a spacer and / or one or more membrane spaning domains. d. Intracellular signaling components

[0313] Intracellular signaling domain includes simulation or imitation of the following ones: the signal by natural antigen receptor, the signal by the combination of this receptor and costimulatory receptor and / or the signal by costimulatory receptor only.In some embodiments, short oligopeptide or polypeptide linker (for example, the linker of amino acid between 2 and 10 in length, such as the linker containing glycine and serine, for example glycine-serine doublet) exists and forms the connection between the transmembrane domain of CAR and the intracellular signaling domain.

[0314] Receptors (e.g., CARs) typically include an intracellular signaling region containing at least one or more intracellular signaling components. In some embodiments, the receptor comprises an intracellular component or signaling domain of a TCR complex, such as a TCR CD3 chain that mediates T cell activation and cytotoxicity, such as a CD3 ζ (CD3-ζ) chain. Therefore, in some aspects, DLL3 binding antibodies are connected to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor (e.g., CAR) further comprises a portion of one or more other molecules (e.g., Fc receptor γ, CD8, CD4, CD25, or CD16). For example, in some aspects, CAR comprises a chimeric molecule between CD3-ζ (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0315] In some embodiments, after connecting CAR, the cytoplasmic domain or intracellular signaling region of CAR stimulates and / or activates normal effector functions or reactions of immune cells (e.g., engineered to express CAR T cells). For example, in some contexts, CAR induces the function of T cells, such as cytolytic activity or T helper cell activity, such as the secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or a costimulatory molecule (e.g., if its transduction effector function signal) is used instead of a complete immunostimulatory chain. In some embodiments, one or more intracellular signaling domains include the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also include co-receptors (which act in parallel with this receptor in a natural context to start signal transduction after antigen receptor engagement) and / or any derivatives or variants of such molecules, and / or any synthetic sequence with the same functional capabilities.

[0316] In the case of natural TCR, full activation generally requires not only signal conduction via TCR, but also costimulatory signals. Therefore, in some embodiments, in order to promote full activation, components for generating secondary or costimulatory signals are also included in CAR. In other embodiments, CAR does not include components for generating costimulatory signals. In some aspects, other CARs are expressed in the same cell and are provided for generating components of secondary or costimulatory signals.

[0317] In some aspects, T cell activation is described as being mediated by two categories of cytoplasmic signaling sequences: those (primary cytoplasmic signaling sequences) that initiate antigen-dependent primary activation by TCR, and those (secondary cytoplasmic signaling sequences) that act in an antigen-independent manner to provide secondary or costimulatory signals. In some aspects, CAR includes one or both of such cytoplasmic signaling sequences.

[0318] In some aspects, the CAR includes a primary stimulation and / or activated primary cytoplasmic signaling sequence for regulating the TCR complex. The primary cytoplasmic signaling sequence that works in a stimulating manner can contain a signaling motif (which is referred to as an immunoreceptor tyrosine activation motif or ITAM). The example of the primary cytoplasmic signaling sequence containing ITAM includes those derived from TCR or CD3ζ, FcRγ, CD3γ, CD3δ and CD3ε. In some embodiments, the intracellular signaling region in CAR contains a cytoplasmic signaling domain, a portion thereof or a sequence derived from CD3ζ.

[0319] In some embodiments, the intracellular signaling domain comprises a human CD3 zeta stimulatory signaling domain or a functional variant thereof, such as the 112 AA cytoplasmic domain of isoform 3 of human CD3 zeta (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993.

[0320] In some embodiments, the intracellular signaling domain comprises a human CD3 zeta signaling domain comprising or consisting of the sequence of SEQ ID NO: 19, 21, or 22, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 19, 21, or 22.

[0321] In some embodiments, the intracellular signaling domain comprises or consists of the human CD3z signaling domain shown in SEQ ID NO: 19, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the human CD3z signaling domain shown in SEQ ID NO: 19. In some embodiments, the intracellular signaling domain comprises or consists of the human CD3z signaling domain shown in SEQ ID NO: 19. In some embodiments, the intracellular signaling domain comprises the human CD3z signaling domain shown in SEQ ID NO: 19. In some embodiments, the intracellular signaling domain consists of the human CD3z signaling domain shown in SEQ ID NO: 19. In some embodiments, the intracellular signaling domain comprises or consists of an amino acid sequence encoded by SEQ ID NO: 20, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20. In some embodiments, the intracellular signaling domain comprises an amino acid sequence encoded by SEQ ID NO: 20. In some embodiments, the intracellular signaling domain consists of an amino acid sequence encoded by SEQ ID NO: 20.

[0322] In some embodiments, the intracellular signaling domain comprises or consists of SEQ ID NO: 19, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 19. In some embodiments, the intracellular signaling domain comprises or consists of the sequence shown in SEQ ID NO: 19. In some embodiments, the intracellular signaling domain comprises the sequence shown in SEQ ID NO: 19. In some embodiments, the intracellular signaling domain consists of the sequence shown in SEQ ID NO: 19. In some embodiments, the intracellular signaling domain comprises or consists of an amino acid sequence encoded by SEQ ID NO: 20, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20. In some embodiments, the intracellular signaling domain comprises an amino acid sequence encoded by SEQ ID NO: 20. In some embodiments, the intracellular signaling domain consists of an amino acid sequence encoded by SEQ ID NO: 20.

[0323] In some embodiments, the intracellular signaling domain comprises or consists of an amino acid sequence encoded by SEQ ID NO: 76, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 76. In some embodiments, the intracellular signaling domain comprises an amino acid sequence encoded by SEQ ID NO: 76. In some embodiments, the intracellular signaling domain consists of an amino acid sequence encoded by SEQ ID NO: 76.

[0324] In some embodiments, the intracellular signaling domain comprises or consists of SEQ ID NO: 21, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 21. In some embodiments, the intracellular signaling domain comprises or consists of the sequence shown in SEQ ID NO: 21. In some embodiments, the intracellular signaling domain comprises the sequence shown in SEQ ID NO: 21. In some embodiments, the intracellular signaling domain consists of the sequence shown in SEQ ID NO: 21.

[0325] In some embodiments, the intracellular signaling domain comprises or consists of SEQ ID NO: 22, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 22. In some embodiments, the intracellular signaling domain comprises or consists of the sequence shown in SEQ ID NO: 22. In some embodiments, the intracellular signaling domain comprises the sequence shown in SEQ ID NO: 22. In some embodiments, the intracellular signaling domain consists of the sequence shown in SEQ ID NO: 22.

[0326] In some embodiments, CD3 zeta comprises the amino acid sequence shown in SEQ ID NO: 19, which is encoded by the nucleic acid sequence shown in SEQ ID NO: 20. In some embodiments, CD3 zeta is encoded by a polynucleotide that has been optionally optimized for codon usage and / or reducing RNA heterogeneity (e.g., by removing cryptic splice sites). In some embodiments, CD3 zeta has the amino acid sequence shown in SEQ ID NO: 19 and is encoded by the polynucleotide sequence shown in SEQ ID NO: 20.

[0327] In some embodiments, CAR comprises a signaling domain (e.g., intracellular or cytoplasmic signaling domain) and / or a transmembrane portion of a costimulatory molecule (e.g., a T cell costimulatory molecule). Exemplary costimulatory molecules include CD28, 4-1BB, OX40, DAP10, and ICOS.

[0328] In some embodiments, the intracellular domain comprises the intracellular co-stimulatory signaling domain of 4-1BB or a functional variant or portion thereof, such as the 42 amino acid cytoplasmic domain of human 4-1BB (Accession No. Q07011.1) or a functional variant or portion thereof, such as the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 17.

[0329] In some embodiments, the intracellular signaling domain comprises or consists of the human 4-1BB costimulatory signaling domain shown in SEQ ID NO: 17, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the human 4-1BB costimulatory signaling domain shown in SEQ ID NO: 17. In some embodiments, the intracellular signaling domain comprises or consists of the human 4-1BB costimulatory signaling domain shown in SEQ ID NO: 17. In some embodiments, the intracellular signaling domain comprises the human 4-1BB costimulatory signaling domain shown in SEQ ID NO: 17. In some embodiments, the intracellular signaling domain consists of the human 4-1BB costimulatory signaling domain shown in SEQ ID NO: 17. In some embodiments, the intracellular signaling domain comprises or consists of an amino acid sequence encoded by SEQ ID NO: 18, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the intracellular signaling domain comprises an amino acid sequence encoded by SEQ ID NO: 18. In some embodiments, the intracellular signaling domain consists of an amino acid sequence encoded by SEQ ID NO: 18.

[0330] In some embodiments, the intracellular signaling domain comprises or consists of SEQ ID NO: 17, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 17. In some embodiments, the intracellular signaling domain comprises or consists of the sequence shown in SEQ ID NO: 17. In some embodiments, the intracellular signaling domain comprises the sequence shown in SEQ ID NO: 17. In some embodiments, the intracellular signaling domain consists of the sequence shown in SEQ ID NO: 17. In some embodiments, the intracellular signaling domain comprises or consists of a nucleic acid sequence encoded by SEQ ID NO: 18, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the intracellular signaling domain comprises an amino acid sequence encoded by SEQ ID NO: 18. In some embodiments, the intracellular signaling domain consists of an amino acid sequence encoded by SEQ ID NO: 18.

[0331] In some embodiments, the intracellular signaling domain comprises or consists of a nucleic acid sequence encoded by SEQ ID NO: 67, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 67. In some embodiments, the intracellular signaling domain comprises an amino acid sequence encoded by SEQ ID NO: 67. In some embodiments, the intracellular signaling domain consists of an amino acid sequence encoded by SEQ ID NO: 67.

[0332] In some embodiments, the costimulatory molecules from 4-1BB are encoded by polynucleotides, which have been optionally optimized for codon usage and / or reduction of RNA heterogeneity (e.g., by removing hidden splice sites). In some embodiments, the costimulatory domain from 4-1BB has the amino acid sequence shown in SEQ ID NO: 17 and is encoded by the polynucleotide sequence shown in SEQ ID NO: 18.

[0333] In some embodiments, the costimulatory domain can be derived from CD28. In some aspects, the costimulatory domain can comprise the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the intracellular signaling domain comprises the intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a 41 amino acid domain thereof, and / or such a domain having a LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain can comprise the amino acid sequence set forth in SEQ ID NO: 15 or 16, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 15 or 16.

[0334] In some aspects, the same CAR comprises both a stimulatory or activation component (e.g., a cytoplasmic signaling sequence) and a co-stimulatory component.

[0335] In some embodiments, CAR encompasses one or more (for example, two or more) costimulatory domains and stimulation or activation domains (such as primary activation domains) in cytoplasmic portions. Exemplary CARs include intracellular components of CD3-ζ, CD28, and 4-1BB.

[0336] In some aspects, the membrane-spanning domain comprises the membrane-spanning portion of CD28. Extracellular domain and membrane-spanning domain can be connected directly or indirectly. In some embodiments, extracellular domain and membrane-spanning domain are connected by a spacer (any spacer as described herein). In some embodiments, chimeric antigen receptor comprises the intracellular domain of T cell costimulatory molecule, such as between membrane-spanning domain and intracellular signal transduction domain. In some aspects, T cell costimulatory molecule is CD28 or 4-1BB.

[0337] In some embodiments, stimulation or activation components are included in a CAR, and costimulatory components are provided by another CAR that recognizes another antigen. In some embodiments, CAR includes activation or stimulation CAR and costimulatory CAR expressed on the same cell (see WO 2014 / 055668). In some aspects, the CAR targeting DLL3 is stimulation or activation CAR;In other aspects, it is costimulatory CAR. In some embodiments, the cell also includes inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5 (215) (December 2013)), such as identifying CAR of antigens other than DLL3, thereby the stimulation or activation signal delivered by the CAR targeting DLL3 is reduced or suppressed by the combination of inhibitory CAR and its ligand, for example, to reduce off-target effects.

[0338] In some embodiments, the two receptors induce activation and inhibition signals to the cell, respectively, so that the connection of one receptor to its antigen activates the cell or induces a response, but the connection of the second inhibitory receptor to its antigen induces a signal that inhibits or weakens the response. An example is a combination of an activation CAR and an inhibitory CAR (iCAR). For example, this strategy can be used to reduce the possibility of off-target effects in the following context, in which the activation CAR binds to an antigen that is expressed in a disease or disorder but also expressed on normal cells, and the inhibitory receptor binds to a separate antigen that is expressed on normal cells but not on cells of the disease or disorder.

[0339] In some aspects, the chimeric receptor is or includes inhibitory CAR (for example, iCAR), and includes weakening or suppressing the intracellular component of immune response (such as the response promoted by ITAM and / or costimulation in cells).The example of such intracellular signal transduction components is those components found on immune checkpoint molecules (including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptors, EP2 / 4 adenosine receptors (including A2AR)).In some aspects, engineered cells include inhibitory CAR, and the inhibitory CAR includes the signal transduction domain of this inhibitory molecule or the signal transduction domain derived from this inhibitory molecule, so that it is used to weaken the cell response induced by, for example, activation and / or costimulation CAR.

[0340] In certain embodiments, the intracellular signaling region comprises a CD28 transmembrane and signaling domain connected to a CD3 (e.g., CD3-ζ) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and 4-1BB (CD137; TNFRSF9) costimulatory domain connected to a CD3 ζ intracellular domain. e. Exemplary CARs

[0341] In some embodiments, an anti-DLL3 CAR is provided that comprises an extracellular antigen binding domain comprising any of the anti-DLL3 antibodies or antigen-binding fragments described herein (e.g., in Section IA1 and / or Table 2).

[0342] In some embodiments, embodiments of the anti-DLL3 CARs provided comprise an extracellular antigen binding domain comprising any of the anti-DLL3 antibodies or antigen-binding fragments described herein (e.g., in Section IA1 and / or Table 2); a spacer as shown in SEQ ID NO: 1; a transmembrane domain, such as the transmembrane domain of human CD28 as shown in SEQ ID NO: 10; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain and a costimulatory signaling domain (e.g., a signaling domain of a costimulatory molecule). In some embodiments, embodiments of the anti-DLL3 CARs provided comprise an extracellular antigen binding domain comprising any of the anti-DLL3 antibodies or antigen-binding fragments described herein (e.g., in Section IA1 and / or Table 2); a spacer as shown in SEQ ID NO: 3; a transmembrane domain, such as the transmembrane domain of human CD28 as shown in SEQ ID NO: 12; and an intracellular signaling region comprising a cytoplasmic signaling domain and a costimulatory signaling domain of the CD3-zeta (CD3ζ) chain.

[0343] In some embodiments, an anti-DLL3 CAR is provided that comprises an extracellular antigen binding domain comprising any of the anti-DLL3 antibodies or antigen-binding fragments described herein (e.g., in Section IA1 and / or Table 2); a spacer comprising an IgG4 / 2 chimeric hinge or a modified IgG4 hinge, such as a spacer of about 12 amino acids in length, or the spacer set forth in SEQ ID NO:3, as encoded by the nucleotide sequence set forth in SEQ ID NO:4; a transmembrane domain, such as the transmembrane domain of human CD28, or the transmembrane domain set forth in SEQ ID NO:12, as encoded by the nucleotide sequence set forth in SEQ ID NO:13; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain and a costimulatory signaling domain, such as the costimulatory signaling domain of human 4-1BB, e.g., as set forth in SEQ ID NO:17, as encoded by the nucleotide sequence set forth in SEQ ID NO:18.

[0344] In some embodiments, embodiments of the anti-DLL3 CARs provided comprise an extracellular antigen binding domain comprising any of the anti-DLL3 antibodies or antigen-binding fragments described herein (e.g., in Section IA1 and / or Table 2); a spacer comprising an IgG4 / 2 chimeric hinge or a modified IgG4 hinge, such as a spacer of about 13 amino acids in length, or the spacer set forth in SEQ ID NO: 1, as encoded by the nucleotide sequence set forth in SEQ ID NO: 2; or a transmembrane domain set forth in SEQ ID NO: 10, as encoded by the nucleotide sequence set forth in SEQ ID NO: 11; a transmembrane domain, such as that of human CD28; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain and a costimulatory signaling domain, such as the costimulatory signaling domain of human 4-1BB, e.g., as set forth in SEQ ID NO: 17, as encoded by the nucleotide sequence set forth in SEQ ID NO: 18.

[0345] In some embodiments, embodiments of the anti-DLL3 CARs provided comprise an extracellular antigen binding domain comprising any of the anti-DLL3 antibodies or antigen-binding fragments described herein (e.g., in Section IA1 and / or Table 2); a modified IgG4 hinge-C H 3, such as a spacer of about 119 amino acids in length, or the spacer shown in SEQ ID NO: 5, as encoded by the nucleotide sequence shown in SEQ ID NO: 6; a transmembrane domain, such as the transmembrane domain of human CD28, or the transmembrane domain shown in SEQ ID NO: 12, as encoded by the nucleotide sequence shown in SEQ ID NO: 13; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-ζ (CD3ζ) chain and a costimulatory signaling domain, such as the costimulatory signaling domain of human 4-1BB, e.g., as shown in SEQ ID NO: 17, as encoded by the nucleotide sequence shown in SEQ ID NO: 18.

[0346] In some embodiments, embodiments of the anti-DLL3 CARs provided comprise an extracellular antigen binding domain comprising any of the anti-DLL3 antibodies or antigen-binding fragments described herein (e.g., in Section IA1 and / or Table 2); a modified IgG4 hinge-C H3, such as a spacer of about 120 amino acids in length, or the spacer shown in SEQ ID NO: 58, as encoded by the nucleotide sequence shown in SEQ ID NO: 59; a transmembrane domain, such as the transmembrane domain of human CD28, or the transmembrane domain shown in SEQ ID NO: 10, as encoded by the nucleotide sequence shown in SEQ ID NO: 11; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain and a costimulatory signaling domain, such as the costimulatory signaling domain of human 4-1BB, e.g., as shown in SEQ ID NO: 17, as encoded by the nucleotide sequence shown in SEQ ID NO: 18.

[0347] In some embodiments, embodiments of the anti-DLL3 CARs provided comprise an extracellular antigen binding domain comprising any of the anti-DLL3 antibodies or antigen-binding fragments described herein (e.g., in Section IA1 and / or Table 2); an IgG4 / 2 chimeric hinge or a modified IgG4 hinge, an IgG2 / 4 chimeric C H Region 2 and IgG4C H The invention also provides a spacer comprising a 3 region, such as a spacer of about 228 amino acids in length, or the spacer shown in SEQ ID NO:42, as encoded by the nucleotide sequence shown in SEQ ID NO:43; a transmembrane domain, such as the transmembrane domain of human CD28, or the transmembrane domain shown in SEQ ID NO:12, as encoded by the nucleotide sequence shown in SEQ ID NO:13; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-ζ (CD3ζ) chain and a costimulatory signaling domain, such as the costimulatory signaling domain of human 4-1BB, e.g., as shown in SEQ ID NO:17, as encoded by the nucleotide sequence shown in SEQ ID NO:18.

[0348] In some embodiments, embodiments of the anti-DLL3 CARs provided comprise an extracellular antigen binding domain comprising any of the anti-DLL3 antibodies or antigen-binding fragments described herein (e.g., in Section IA1 and / or Table 2); an IgG4 / 2 chimeric hinge or a modified IgG4 hinge, an IgG2 / 4 chimeric C H Region 2 and IgG4C HThe invention also provides a spacer comprising a 3 region, such as a spacer of about 229 amino acids in length, or the spacer shown in SEQ ID NO:82, as encoded by the nucleotide sequence shown in SEQ ID NO:83; a transmembrane domain, such as the transmembrane domain of human CD28, or the transmembrane domain shown in SEQ ID NO:10, as encoded by the nucleotide sequence shown in SEQ ID NO:11; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-ζ (CD3ζ) chain and a costimulatory signaling domain, such as the costimulatory signaling domain of human 4-1BB, e.g., as shown in SEQ ID NO:17, as encoded by the nucleotide sequence shown in SEQ ID NO:18.

[0349] In some embodiments, the transmembrane domain is or comprises the sequence shown in SEQ ID NO: 10. In some embodiments, the transmembrane domain is or comprises the sequence shown in SEQ ID NO: 12. In some embodiments, the costimulatory signaling domain is the intracellular signaling domain of human CD28, human 4-1BB or human ICOS, or a signaling portion thereof. In specific embodiments, the intracellular signaling domain is the intracellular signaling domain of human 4-1BB. In some embodiments, the intracellular signaling domain is or comprises the sequence shown in SEQ ID NO: 17. In some embodiments, the cytoplasmic signaling domain is the human CD3-ζ cytoplasmic signaling domain, as shown in SEQ ID NO: 19. In some embodiments, the intracellular signaling region comprises the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 19. In some embodiments, the intracellular signaling region comprises the sequences shown in Section IE, Table 3 and / or Table E1-Table E4 and Table E8 herein. Also provided are CARs encoded by the polynucleotides described in Section IF, Table 3 and / or Table E1-Table E4 and Table E8 herein. Also provided are polynucleotides containing any nucleotide sequence described herein, e.g., encoding all or part of the binding molecules provided. In some cases, the transmembrane domain has a methionine residue at the N-terminus. In some cases, the spacer has a methionine residue at the C-terminus.

[0350] In some any of the provided embodiments, the anti-DLL3 chimeric antigen receptor is or comprises the sequence shown in SEQ ID NO: 117, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown in SEQ ID NO: 117. In some any of the provided embodiments, the anti-DLL3 chimeric antigen receptor is or comprises the sequence shown in SEQ ID NO: 117, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown in SEQ ID NO: 117.

[0351] In some embodiments, the anti-DLL3 chimeric antigen receptor comprises or consists of SEQ ID NO: 117, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 117. In some embodiments, the anti-DLL3 chimeric antigen receptor comprises or consists of the sequence shown in SEQ ID NO: 117. In some embodiments, the anti-DLL3 chimeric antigen receptor comprises the sequence shown in SEQ ID NO: 117. In some embodiments, the anti-DLL3 chimeric antigen receptor consists of the sequence shown in SEQ ID NO: 117. In some embodiments, the anti-DLL3 chimeric antigen receptor comprises or consists of an amino acid sequence encoded by SEQ ID NO: 116, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 116. In some embodiments, the anti-DLL3 chimeric antigen receptor comprises an amino acid sequence encoded by SEQ ID NO: 116. In some embodiments, the anti-DLL3 chimeric antigen receptor consists of an amino acid sequence encoded by SEQ ID NO: 116. 2. Exemplary Features

[0352] In some of any of the provided embodiments, a recombinant receptor (e.g., CAR) containing a DLL3 binding antibody or antigen binding fragment thereof binds (e.g., specifically binds) DLL3, such as DLL3 on the surface of a cancer cell or tumor cell. In any embodiment, the antibody or antigen binding fragment in the provided CAR specifically binds to DLL3, such as human DLL3. Exemplary DLL3 binding recombinant receptors (e.g., CARs) can exhibit any binding affinity, binding specificity, and / or other antigen recognition characteristics, such as species cross-reactivity, as described herein (e.g., in Section IA2). In some embodiments, a CAR comprising an extracellular antigen-binding domain containing an antibody or fragment thereof exhibits binding properties and characteristics similar to or substantially identical to those of an antibody or fragment thereof. In some aspects, the exemplary features of the antigen-binding portion of a CAR (e.g., comprising any antibody or antigen-binding fragment thereof) are similar to the affinity described for an antibody or antigen-binding fragment thereof (e.g., in Section IA3). In some cases, a CAR comprising an extracellular antigen-binding domain containing an antibody or fragment thereof exhibits binding properties and characteristics different from those of an antibody or fragment thereof.

[0353] In some embodiments, the CAR provided specifically binds to a delta-like ligand 3 (DLL3) protein. In some embodiments of any of the present invention, DLL3 refers to human DLL3. The observation that an antibody or other binding molecule (e.g., CAR) specifically binds to or binds to DLL3 does not necessarily mean that it binds to DLL3 from every species. For example, in some embodiments, the characteristic of binding to DLL3 (such as the ability to specifically bind to it and / or compete with a reference receptor containing a reference antibody (e.g., a reference CAR) for its binding and / or bind with a specific affinity or compete to some extent) refers to the ability to bind to human DLL3 in some embodiments, and the antibody may not have this function for DLL3 of another species (such as a mouse). In some embodiments, CAR binds to human DLL3 and binds to DLL3 of another species (such as a rhesus monkey or macaque). In some embodiments, CAR or its antigen-binding fragment binds to human DLL3 and does not bind to DLL3 of other species (such as a mouse). In some embodiments, CAR binds to human DLL3 and binds to DLL3 of another species (such as a mouse).

[0354] In some embodiments, the CAR binds (e.g., specifically binds) to one or more epitopes or regions of human DLL3, such as human DLL3 (e.g., the mature human DLL3 sequence set forth in SEQ ID NO: 122 or 123), or an allelic variant or splice variant thereof. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to one or more epitopes within human DLL3. In some embodiments, human DLL3 comprises the amino acid sequence set forth in SEQ ID NO: 120. In some embodiments, human DLL3 comprises the amino acid sequence set forth in SEQ ID NO: 121. In some embodiments, the human DLL3 isoform (mature, isoform 1) comprises the amino acid sequence set forth in SEQ ID NO: 122. In some embodiments, the human DLL3 isoform (mature, isoform 2) comprises the amino acid sequence set forth in SEQ ID NO: 123. In some embodiments, the antibody or antigen-binding fragment thereof binds to an extracellular region of DLL3, such as one or more extracellular epitopes present within the extracellular region of mature human DLL3, e.g., corresponding to residues 27-492 of the human DLL3 precursor sequence set forth in SEQ ID NO: 120.

[0355] In some embodiments, the CAR binds to one or more epitopes of DLL3, such as a linear epitope of DLL3 (such as human DLL3), or one or more conformational epitopes of DLL3 (such as human DLL3).

[0356] In some embodiments, the CAR binds to an epitope comprising amino acid residues within the N-terminus of the Notch ligand domain, the DSL domain, the EGF-like 1 domain, the EGF-like 2 domain, the EGF-like 3 domain, the EGF-like 4 domain, the EGF-like 5 domain, or the EGF-like 6 domain.

[0357] In some embodiments, the CAR binds to one or more epitopes of human DLL3, such as one or more epitopes comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acid residues within human DLL3 (e.g., the human DLL3 precursor sequence shown in SEQ ID NO: 120), or two or more epitopes, each comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acid residues within human DLL3 (e.g., the human DLL3 precursor sequence shown in SEQ ID NO: 120). In some aspects, with reference to the human DLL3 precursor sequence shown in SEQ ID NO: 120, the one or more epitopes include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acid residues corresponding to amino acid residues 27-84 of the N-terminus of the Notch ligand domain, amino acid residues 176-215 corresponding to the DSL domain, amino acid residues 216-249 corresponding to the EGF-like 1 domain, amino acid residues 274-310 corresponding to the EGF-like 2 domain, amino acid residues 312-351 corresponding to the EGF-like 3 domain, amino acid residues 353-389 corresponding to the EGF-like 4 domain, amino acid residues 391-427 corresponding to the EGF-like 5 domain, or amino acid residues 429-465 corresponding to the EGF-like 6 domain.

[0358] In some embodiments, the CAR binds to an epitope within amino acid residues 27-84 of SEQ ID NO: 120, which corresponds to the N-terminus of the Notch ligand domain. In some embodiments, the CAR binds to an epitope within amino acid residues 176-215 of SEQ ID NO: 120, which corresponds to the DSL domain. In some embodiments, the CAR binds to an epitope within amino acid residues 216-249 of SEQ ID NO: 120, which corresponds to the EGF-like 1 domain. In some embodiments, the CAR binds to an epitope within amino acid residues 274-310 of SEQ ID NO: 120, which corresponds to the EGF-like 2 domain. In some embodiments, the CAR binds to an epitope within amino acid residues 312-351 of SEQ ID NO: 120, which corresponds to the EGF-like 3 domain. In some embodiments, the CAR binds to an epitope within amino acid residues 353-389 of SEQ ID NO: 120, which corresponds to the EGF-like 4 domain. In some embodiments, the CAR binds to an epitope within amino acid residues 391-427 of SEQ ID NO: 120, which corresponds to an EGF-like 5 domain. In some embodiments, the CAR binds to an epitope within amino acid residues 429-465 of SEQ ID NO: 120, which corresponds to an EGF-like 6 domain. In some embodiments, the CAR binds to an epitope within amino acid residues 493-513 of SEQ ID NO: 120, which corresponds to a transmembrane domain.

[0359] In some embodiments, the CAR binds to a non-human DLL3, such as a DLL3 of a monkey, rabbit, rat, mouse, or other species. In some embodiments, the CAR binds to an epitope or region of a non-human DLL3, such as a cynomolgus monkey (Macaca fascicularis) DLL3, such as a cynomolgus monkey DLL3 (such as the cynomolgus DLL3 shown in SEQ ID NO: 125 (NCBI Reference Sequence: XM_005589196.2). In some embodiments, the CAR binds to an epitope or region of a mouse (Mus musculus) DLL3, such as a mouse DLL3 (such as the mouse DLL3 shown in SEQ ID NO: 124 (GenBank No. NP 031892.2).

[0360] In some embodiments, the CAR provided is capable of binding to a DLL3 protein (such as a human DLL3 protein) with at least a certain affinity, as measured by any of a variety of known methods. In some embodiments, affinity is determined by the equilibrium dissociation constant (K D In some embodiments, affinity is represented by EC 50In some aspects, the CARs provided exhibit K as described herein (e.g., in Section IA3). D or EC 50 .

[0361] In some embodiments, the binding affinities of binding molecules (e.g., anti-DLL3 CARs) to different antigens (e.g., DLL3 proteins from different species) can be compared to determine species cross-reactivity. For example, species cross-reactivity can be classified as high cross-reactivity or low cross-reactivity. In some embodiments, the equilibrium dissociation constants K for different antigens (e.g., DLL3 proteins from different species (e.g., human, cynomolgus monkey, or mouse)) can be compared. D To determine species cross-reactivity. In some embodiments, the species cross-reactivity of the anti-DLL3 CAR can be high, e.g., the anti-DLL3 CAR binds to human DLL3 and species variant DLL3 to a similar extent, e.g., the K for human DLL3 is D Compared with the K for species variant DLL3 D The ratio of K to Δ is or is about 1. In some embodiments, the species cross-reactivity of the anti-DLL3 CAR can be low, for example, the anti-DLL3 CAR has high affinity for human DLL3 but low affinity for species variant DLL3, or vice versa. For example, the K for species variant DLL3 is D K for human DLL3 D The ratio of the anti-DLL3 CAR to the anti-DLL3 CAR is greater than 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or more, and the anti-DLL3 CAR has low species cross-reactivity. The degree of species cross-reactivity can be compared to the species cross-reactivity of a known CAR (such as a reference CAR).

[0362] The provided CARs include CARs that exhibit antigen-dependent activity or signaling, i.e., signaling activity is measurably absent or at background levels in the absence of antigen (e.g., DLL3) and / or in the presence of non-specific antigens. Thus, in some aspects, the provided CARs do not exhibit tonic signaling or antigen-independent activity or signaling in the absence of the present antigen (e.g., DLL3) or exhibit basal signaling or antigen-independent activity or signaling that is no more than background or tolerable or low levels. In some embodiments, the provided anti-DLL3 CAR-expressing cells exhibit biological activities or functions, including cytotoxic activity, cytokine production, and proliferation capacity. In some embodiments, the provided CAR receptors exhibit the same, substantially the same, or higher antigen-specific signaling and / or antigen-dependent activity or signaling compared to a reference DLL3-specific chimeric antigen receptor, optionally under the same or substantially the same conditions or assays.

[0363] In some embodiments, the CAR provided exhibits missing, reduced or lower levels of activity or signaling in the absence of an antigen (e.g., DLL3). In some embodiments, the CAR provided exhibits reduced, lower or almost absent or completely absent tonic signaling or antigen-independent signaling or activity, such as signaling or activity in the absence of an antigen, such that the signaling or activity is less than or about 30%, 20% or 10%, such as less than or less than about 10%, of the signaling or activity of the CAR in the presence of human DLL3. In some embodiments, the CAR provided exhibits the same, substantially the same or lower tonic signaling and / or antigen-independent activity or signaling, optionally under the same or substantially the same conditions or assays, compared to a reference DLL3-specific chimeric antigen receptor (CAR). In some aspects, in a plurality of cells or a population of cells engineered to express a CAR, less than or less than about 10%, less than or less than about 9%, less than or less than about 8%, less than or less than about 7%, less than or less than about 5%, less than or less than about 4%, less than or less than about 3%, less than or less than about 2%, or less than or less than about 1% of the cells in the plurality of cells comprise a chimeric antigen receptor that exhibits tonic signaling and / or antigen-independent activity or signaling.

[0364] In some embodiments, when administered to a subject for adoptive cell therapy, the engineered cells expressing the provided anti-DLL3 CAR exhibit improved biological activity or functional activity, such as anti-tumor activity, tumor growth inhibition, tumor volume reduction, persistence, expansion, or prolonged survival of the subject. In some embodiments, the engineered cells expressing the provided CAR exhibit improved biological activity or functional activity, such as anti-tumor activity, tumor growth inhibition, tumor volume reduction, persistence, expansion, or prolonged survival of the subject, compared to engineered cells expressing a reference DLL3-specific chimeric antigen receptor, optionally under the same or substantially the same conditions or assays. In some embodiments, the biological activity or functional activity (such as cytotoxic activity) of the chimeric receptor can be measured using any of a variety of known methods. Activity can be assessed or determined in vitro or in vivo. In some embodiments, activity can be assessed after the cells are administered to a subject (e.g., a human). Parameters to be assessed include specific binding of engineered or natural T cells or other immune cells to the antigen, which can be assessed, for example, in vivo, for example, by imaging, or in vitro, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable known method, such as the cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009), and Herman et al., J. Immunological Methods, 285(1):25-40 (2004). In certain embodiments, the biological activity of the cells can also be measured by measuring the expression and / or secretion of certain cytokines such as interleukin-2 (IL-2), interferon-γ (IFNγ), interleukin-4 (IL-4), TNF-α (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF), CD107a, and / or TGF-β (TGFβ). The mensuration of measuring cytokine is well known, and includes but is not limited to ELISA, intracellular cytokine staining, flow cytometry microbead array, RT-PCR, ELISPOT, flow cytometry and bioassay, in which the reactivity (for example, proliferation) of the cell that the relevant cytokine is reacted to is tested in the presence of a test sample. In some respects, the animal model (such as tumor xenograft model) of disease or illness and the minimizing and / or the survival period of assessment tumor load or burden are measured biological activity. In some respects, biological activity is measured by assessing clinical outcome (such as the minimizing of tumor load or burden).

[0365] In some embodiments, administration of engineered cells expressing provided anti-DLL3 CARs exhibits significantly improved tumor growth inhibition, in vivo persistence, and / or prolonged survival of subjects with tumors compared to administration of engineered cells expressing reference CARs. In some aspects, such improvements are observed in subjects with various types of DLL3-expressing cancers, including but not limited to small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), melanoma, glioma, and glioblastoma.

[0366] In some embodiments, engineered cells expressing a provided anti-DLL3 CAR exhibit improved persistence and expansion when administered to a subject for adoptive cell therapy. In some embodiments, engineered cells expressing a provided CAR exhibit the same, substantially the same, or higher persistence and expansion as engineered cells expressing a reference DLL3-specific chimeric antigen receptor, optionally under the same or substantially the same conditions or assays. In some aspects, the proliferation, expansion, and / or persistence of engineered cells expressing any provided receptor (e.g., CAR) can be assessed by measuring the exposure, number, concentration, persistence, and proliferation of engineered cells (e.g., cells administered for adoptive cell therapy). In some embodiments, engineered T cells (e.g., T cells administered for T cell-based therapy) or a subset thereof (e.g., CD3 CAR) from a subject (e.g., a human subject or an animal subject) to which the engineered cells have been administered can be assessed. + cells, CD4 + cells, CD8 + cells, CD3 + CAR + cells, CD4 + CAR + cells or CD8 + CAR + In some aspects, exposure, amount, concentration, persistence, and proliferation are related to pharmacokinetic parameters. In some cases, pharmacokinetics can be assessed by measuring parameters such as: maximum (peak) plasma concentration (C max ), peak time (i.e., the maximum plasma concentration (C max ) time; T max ), minimum plasma concentration (i.e., therapeutic agent (e.g., CAR + T cells) between doses; C min ), elimination half-life after administration (T 1 / 2 ) and the area under the curve (i.e., by plotting time versus therapeutic agent CAR + The area under the curve (AUC) of the plasma concentration of T cells after administration of a specific therapeutic agent (e.g., CAR T cells) is generated.+ T cells) can be concentrated using any known method suitable for evaluating therapeutic agents (e.g., CAR T cells). + The method of measuring the concentration of T cells in a blood sample or any method described herein can be used to measure. For example, nucleic acid-based methods such as quantitative PCR (qPCR) or flow cytometry-based methods or other assays such as immunoassays, ELISAs, or chromatography / mass spectrometry-based assays can be used.

[0367] In some cases, to assess the specificity, cross-reactivity, and / or antigen dependence of a particular activity, signaling, and / or function of a receptor (e.g., a CAR), any of the assays described for assessing the activity, signaling, or biological function of a receptor can be assessed in the presence and absence of a particular target antigen (e.g., human DLL3), or in the presence of a specific target antigen (e.g., human DLL3) and a different non-specific antigen or non-human DLL3 (e.g., mouse DLL3).

[0368] In some embodiments, provided anti-DLL3 CARs exhibit the same, substantially the same, or lower tonic signaling and / or antigen-independent activity or signaling as compared to a reference DLL3-specific CAR, e.g., under the same or substantially the same conditions or assay. For example, in some embodiments, provided anti-DLL3 CARs exhibit a level or extent of tonic signaling and / or antigen-independent activity or signaling that is at least or about 75%, 80%, 90%, 95%, or 99% lower than the level or extent of tonic signaling and / or antigen-independent activity of a reference DLL3-specific CAR, e.g., under the same or substantially the same conditions or assay.

[0369] In some embodiments, as compared to an alternative CAR with the same amino acid sequence but encoded by a nucleotide sequence that has not undergone splice site elimination and / or codon optimization, the anti-DLL3 CAR provided exhibits improved expression on the cell surface. In some embodiments, the expression of recombinant receptors on the cell surface can be assessed. Methods for determining the expression of recombinant receptors on the cell surface can include the use of chimeric antigen receptor (CAR)-specific antibodies (e.g., Brentjens et al., Sci. Transl. Med. 2013 March; 5(177): 177ra38), protein L (Zheng et al., J. Transl. Med. 2012 February; 10: 29), epitope tags, and monoclonal antibodies specifically binding to CAR polypeptides (see WO 2014190273). In some embodiments, the expression of recombinant receptors on the cell (e.g., primary T cells) surface can be assessed, for example, by flow cytometry using a detectable binding molecule that can be bound to the recombinant receptor or a portion thereof. In some embodiments, the binding molecule used to detect recombinant receptor expression is an anti-idiotypic antibody, e.g., an anti-idiotypic agonist antibody specific for a binding domain (e.g., scFv) or a portion thereof. In some embodiments, the binding molecule is or comprises an isolated or purified antigen, e.g., a recombinantly expressed antigen. F. Polynucleotides encoding binding molecules

[0370] Also provided are polynucleotides encoding binding molecules (e.g., anti-DLL3 antibodies, antigen-binding fragments thereof, recombinant receptors (e.g., chimeric antigen receptors), and / or portions thereof, such as chains or fragments). The polynucleotides provided include those encoding the anti-DLL3 antibodies (e.g., antigen-binding fragments) or chimeric antigen receptors described herein. Polynucleotides can include those comprising naturally and / or non-naturally occurring nucleotides and bases, including, for example, those with backbone modifications. The terms "nucleic acid molecule," "nucleic acid," "nucleotide sequence," and "polynucleotide" are used interchangeably and refer to polymers of nucleotides. Such polymers of nucleotides can contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to a linear sequence of nucleotides that constitutes a nucleic acid molecule or polynucleotide.

[0371] Polynucleotides are provided that contain nucleic acids encoding any of the anti-DLL3 antibodies or antigen-binding domains described herein, or any portion, fragment, chain, or domain of the anti-DLL3 antibodies or antigen-binding domains thereof. In some embodiments, the antibody or antigen-binding fragment thereof contains multiple domains or chains (e.g., heavy and light chains), and the antibody or antigen-binding fragment thereof is entirely encoded by a single polynucleotide. Also provided are polynucleotides that contain nucleic acids encoding any of the single-chain cell surface proteins described herein. Also provided are polynucleotides that contain nucleic acids encoding any of the conjugates described herein. Also provided are polynucleotides that contain nucleic acids encoding any of the anti-DLL3 chimeric antigen receptors described herein. In some embodiments, a binding molecule (e.g., an antibody or antigen-binding fragment thereof, or a recombinant receptor) contains multiple domains or chains (e.g., heavy and light chains), and the binding molecule is entirely encoded by more than one polynucleotide (e.g., two or more polynucleotides). In some embodiments, the polynucleotide is contained in a vector.

[0372] In some aspects, polynucleotides are provided that contain nucleic acid sequences encoding any of the binding molecules provided herein (e.g., in Sections IA and IE). In some embodiments, polynucleotides are provided that contain nucleic acid sequences encoding portions, fragments, chains, or domains of any of the binding molecules provided herein (e.g., in Sections IA and IE).

[0373] In some cases, the polynucleotide encoding a DLL3 binding molecule (such as an antibody or antigen-binding fragment thereof, or a recombinant receptor (e.g., CAR)) comprises a signal sequence encoding a signal peptide, which in some cases is encoded upstream of the nucleic acid sequence encoding the DLL3 binding molecule (such as an antibody or antigen-binding fragment thereof, or a recombinant receptor (e.g., CAR)) or is linked to the 5' end of the nucleic acid sequence encoding the antigen-binding domain. In some cases, the polynucleotide containing the nucleic acid sequence encoding the DLL3 binding molecule (such as an antibody or antigen-binding fragment thereof, or a recombinant receptor (e.g., CAR)) comprises a signal sequence encoding a signal peptide. In some aspects, the signal sequence can encode a signal peptide derived from a natural polypeptide. In other aspects, the signal sequence can encode a heterologous or non-natural signal peptide. In some aspects, non-limiting exemplary signal peptides include the signal peptide of the IgG kappa chain, as shown in SEQ ID NO: 50 or encoded by the nucleotide sequence shown in SEQ ID NO: 51. In some aspects, non-limiting exemplary signal peptides include the signal peptide of the GMCSFR alpha chain, as shown in SEQ ID NO: 52 and encoded by the nucleotide sequence shown in SEQ ID NO: 53. In some aspects, non-limiting exemplary signal peptides include the signal peptide of the CD8α signal peptide shown in SEQ ID NO: 54. In some aspects, non-limiting exemplary signal peptides include the signal peptide of the CD33 signal peptide, as shown in SEQ ID NO: 48 and encoded by the nucleotide sequence shown in SEQ ID NO: 49. In some aspects, the nucleotide sequence encoding the CD33 signal peptide is as shown in SEQ ID NO: 78 or 79. In some cases, the polynucleotide encoding a DLL3 binding molecule (such as an antibody or antigen-binding fragment thereof or a recombinant receptor (e.g., CAR)) may contain nucleic acid sequences encoding additional molecules (such as surrogate markers or other markers), or may contain additional components (such as promoters, regulatory elements, and / or polycistronic elements). In some embodiments, the nucleic acid sequence encoding a DLL3 binding molecule (such as an antibody or antigen-binding fragment thereof or a recombinant receptor (e.g., CAR)) may be operably linked to any additional components.

[0374] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a variable heavy chain domain (V H In some embodiments, a polynucleotide comprising the following nucleic acid sequence is provided, wherein the nucleic acid sequence encodes a variable light chain domain (V LIn some embodiments, a polynucleotide comprising the following nucleic acid sequence is provided, wherein the nucleic acid sequence encodes a variable heavy chain domain (V H ) and the variable light chain domain (V L ).

[0375] In some embodiments, the provided polynucleotide comprises a V H A nucleic acid comprising the sequence set forth in SEQ ID NO: 87, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 87. In some embodiments, the polynucleotides provided comprise a nucleic acid encoding a V H A nucleic acid comprising the sequence shown in SEQ ID NO:87.

[0376] In some embodiments, the provided polynucleotide comprises a V L A nucleic acid comprising the sequence set forth in SEQ ID NO: 88, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 88. In some embodiments, the polynucleotides provided comprise a nucleic acid encoding a V L A nucleic acid comprising the sequence shown in SEQ ID NO:88.

[0377] In some embodiments, the provided polynucleotide comprises: encoding the V H A nucleic acid comprising the sequence shown in SEQ ID NO: 87, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 87; and encoding the V L A nucleic acid comprising the sequence set forth in SEQ ID NO: 88, or a nucleic acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 88. In some embodiments, the polynucleotides provided comprise: encoding V H A nucleic acid comprising the sequence shown in SEQ ID NO: 87; and encoding V L A nucleic acid comprising the sequence shown in SEQ ID NO:88.

[0378] In some embodiments, the polynucleotide comprises a sequence encoding an antibody or antigen-binding fragment comprising a heavy chain variable (V H ) region and light chain variable (V L In some embodiments, the polynucleotide comprises a sequence encoding one or more linkers that connect two antibody domains or regions, such as V H Area and V L Exemplary encoded linkers include peptide linkers that are rich in glycine and serine and / or in some cases rich in threonine.

[0379] In some embodiments, encoding V H The nucleic acid in the region may be in the encoding V L In some embodiments, the nucleic acid encoding V H The nucleic acid in the region may be in the encoding V L In certain embodiments, the nucleic acid encoding the scFv may comprise a nucleic acid encoding a V H The nucleic acid encoding the region or part thereof, followed by the nucleic acid encoding the linker, followed by the nucleic acid encoding the V L In certain embodiments, the nucleic acid encoding the scFv may comprise a nucleic acid encoding a V L The nucleic acid encoding the region or part thereof, followed by the nucleic acid encoding the linker, followed by the nucleic acid encoding the V H region or a portion thereof.

[0380] In some any embodiments, the polynucleotide comprises a nucleic acid encoding an scFv comprising the sequence set forth in SEQ ID NO: 86, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 86. In some any embodiments, the polynucleotide comprises a nucleic acid encoding an scFv comprising the sequence set forth in SEQ ID NO: 86.

[0381] In some aspects, the polynucleotide comprises a nucleic acid encoding one or more components of a recombinant receptor (e.g., CAR).

[0382] In some embodiments, the polynucleotide comprises a nucleic acid encoding a spacer comprising the sequence shown in SEQ ID NO: 2, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 2.

[0383] In some embodiments, the polynucleotide comprises a nucleic acid encoding a spacer comprising the sequence set forth in SEQ ID NO: 4, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In some embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NO: 4.

[0384] In some embodiments, the polynucleotide comprises a nucleic acid encoding a spacer comprising the sequence set forth in SEQ ID NO: 6, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NO: 6.

[0385] In some embodiments, the polynucleotide comprises a nucleic acid encoding a spacer comprising the sequence shown in SEQ ID NO: 59, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 59. In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 59.

[0386] In some embodiments, the polynucleotide comprises a nucleic acid encoding a spacer comprising the sequence shown in SEQ ID NO: 60, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 60. In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 60.

[0387] In some embodiments, the polynucleotide comprises a spacer-encoding nucleic acid comprising the sequence shown in SEQ ID NO: 84, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 84. In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 84.

[0388] Also provided are polynucleotides that have been optimized for codon usage and / or elimination of splice sites (such as hidden splice sites). In some embodiments, polynucleotides are modified to optimize codon usage. In some embodiments, polynucleotides are codon optimized for expression in human cells (such as human T cells such as primary human T cells). In some embodiments, the polynucleotides (such as encoding any antibody provided herein, receptors (such as antigen receptors, such as chimeric antigen receptors) and / or DLL3 specific binding proteins) are modified or have been modified to reduce heterogeneity or contain one or more nucleic acid sequences observed herein (such as by the optimization method), thereby resulting in improved features of the polypeptide (such as CAR) compared to those containing different reference sequences or those not yet optimized. In some embodiments, polynucleotides are optimized by splice site elimination. Such characteristics include improved RNA heterogeneity (e.g., due to the presence of one or more splice sites (e.g., one or more cryptic splice sites)) and / or improved expression and / or surface expression of the encoded protein, such as increased expression levels, uniformity, or consistency among cells or different therapeutic cell compositions engineered to express the polypeptide. In some embodiments, the polynucleotides can be codon-optimized for expression in human cells.

[0389] The nucleic acid sequence of transcription in mammalian cells usually undergoes co-transcription processing or processes immediately after transcription, wherein the nascent precursor messenger ribonucleic acid (pre-mRNA) transcribed from genomic deoxyribonucleic acid (DNA) sequence is edited to remove introns by means of splicing in some cases, and then the exons in eukaryotic cells are connected. The consensus sequence of splice site is known, and in some respects, the specific nucleotide information defining splice site may be complicated, and may not be easy to see based on available methods. Concealed splice site is a splice site that is unpredictable and variably activated based on a standard consensus sequence. Therefore, the variable splicing of pre-mRNA at a concealed splice site causes the heterogeneity of the mRNA product transcribed after expression in eukaryotic cells.

[0390] The polynucleotides produced for expressing transgenics are usually constructed from nucleic acid sequences that do not contain introns, such as complementary DNA (cDNA) or portions thereof. Therefore, it is expected that splicing of such sequences does not occur. However, the presence of hidden splice sites within the cDNA sequence may result in unexpected or undesirable splicing reactions and heterogeneity in the transcribed mRNA. This heterogeneity results in the translation of unexpected protein products, such as protein products with truncated variable amino acid sequences that exhibit modified expression and / or activity.

[0391] In some embodiments, elimination of splice sites (such as cryptic splice sites) can improve or optimize expression of transgenic products (such as polypeptides translated from transgenics, such as anti-DLL3 CAR polypeptides). Splicing at cryptic splice sites of encoded transgenics (such as encoded DLL3 CAR molecules) can result in reduced protein expression (e.g., expression on the cell surface) and / or reduced function (e.g., reduced intracellular signaling). Provided herein are polynucleotides encoding anti-DLL3 CAR proteins that have been optimized to reduce or eliminate cryptic splice sites. Also provided herein are polynucleotides encoding anti-DLL3 CAR proteins that have been optimized for codon expression, and / or in which one or more sequences (such as sequences identified by the methods or observations herein regarding splice sites) are present, and / or in which identified splice sites (such as any identified splice sites herein) are absent. The provided polynucleotides include those that exhibit less than a certain degree of RNA heterogeneity or splice form when expressed under certain conditions and / or introduced into specific cell types (such as human T cells, such as primary human T cells) and cells and compositions and products containing such polypeptides and / or exhibiting such characteristics. In some embodiments, the RNA heterogeneity of the transcribed RNA is reduced by greater than or about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more compared to a polynucleotide that has not been modified to remove cryptic splice sites and / or has not been modified by codon optimization. In some embodiments, the provided polynucleoti...

Claims

1. An anti-delta-like ligand 3 (DLL3) antibody or an antigen-binding fragment thereof, wherein the anti-delta-like ligand 3 (DLL3) antibody or an antigen-binding fragment thereof comprises a heavy chain variable (V H ) region and light chain variable (V L ) district, of which: The V H The region comprises the heavy chain complementary determining region 1 (CDR-H1), the heavy chain complementary determining region 2 (CDR-H2), and the heavy chain complementary determining region 3 (CDR-H3) contained in SEQ ID NO: 90, and The V L The region comprises the light chain complementary determining region 1 (CDR-L1), light chain complementary determining region 2 (CDR-L2), and light chain complementary determining region 3 (CDR-L3) contained in SEQ ID NO:

91.

2. An anti-DLL3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable (V H ) region and light chain variable (V L ) district, of which: The V H The region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 92, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 93, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 94, and The V L The region includes a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO:95, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO:96, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO:

97.

3. The anti-DLL3 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The V H The region comprises an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 90, and The V L The region comprises an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

91.

4. An anti-DLL3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable (V H ) region and light chain variable (V L ) district, of which: The V H The region comprises an amino acid sequence having at least or at least about 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 90, and The V L The region comprises an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

91.

5. The anti-DLL3 antibody or antigen-binding fragment thereof according to claim 4, wherein: The V H The region comprises the heavy chain complementary determining region 1 (CDR-H1), the heavy chain complementary determining region 2 (CDR-H2), and the heavy chain complementary determining region 3 (CDR-H3) contained in SEQ ID NO: 90, and The V L The region comprises the light chain complementary determining region 1 (CDR-L1), light chain complementary determining region 2 (CDR-L2), and light chain complementary determining region 3 (CDR-L3) contained in SEQ ID NO:

91.

6. The anti-DLL3 antibody or antigen-binding fragment thereof according to claim 4 or 5, wherein: The V H The region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 92, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 93, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 94, and The V L The region includes a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO:95, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO:96, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO:

97.

7. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein: The V H The region comprises the sequence shown in SEQ ID NO:90, and The V L The region comprises the sequence shown in SEQ ID NO:

91.

8. An anti-DLL3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable (V H ) region and light chain variable (V L ) district, of which: The V H The region comprises the sequence shown in SEQ ID NO:90, and The V L The region comprises the sequence shown in SEQ ID NO:

91.

9. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the anti-DLL3 antibody or antigen-binding fragment thereof is recombinant.

10. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the V H area and the V L The region is human or derived from a human protein.

11. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody further comprises a heavy chain constant region (C H ) and / or light chain constant region (C L ).

12. The anti-DLL3 antibody or antigen-binding fragment thereof according to claim 11, wherein: The C H comprising the sequence shown in SEQ ID NO: 126, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 126; and / or The C L comprising the sequence shown in SEQ ID NO: 127, or an amino acid sequence having at least or at least about 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

127.

13. The anti-DLL3 antibody or antigen-binding fragment thereof according to claim 11 or 12, wherein the antibody or antigen-binding fragment thereof comprises: A heavy chain comprising the sequence shown in SEQ ID NO: 128; and / or A light chain comprising the sequence shown in SEQ ID NO:

129.

14. The anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the sequence shown in SEQ ID NO: 128; and A light chain comprising the sequence shown in SEQ ID NO:

129.

15. The anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the antibody is a full-length antibody.

16. The anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the antibody is an antigen-binding fragment.

17. The anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-10 and 16, wherein the antigen-binding fragment thereof comprises a single-chain Fv (scFv).

18. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1-10, 16 and 17, wherein the V H Area in the V L The amino terminus of the region.

19. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1-10, 16 and 17, wherein the V H Area in the V L The carboxyl terminus of the region.

20. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1-10 and 17-19, wherein the V H area and the V L The regions are connected by flexible linkers.

21. The anti-DLL3 antibody or antigen-binding fragment thereof of claim 20, wherein the flexible linker comprises the sequence shown in SEQ ID NO:

46.

22. The anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 17-21, wherein the scFv comprises the sequence shown in SEQ ID NO:

89.

23. The anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-22, wherein the anti-DLL3 antibody or antigen-binding fragment thereof specifically binds to human DLL3.

24. The anti-DLL3 antibody or antigen-binding fragment thereof of claim 23, wherein the human DLL3 comprises the amino acid sequence shown in SEQ ID NO: 120 or 121.

25. The anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-24, wherein the anti-DLL3 antibody or antigen-binding fragment thereof does not bind to, does not cross-react with, or binds to a DLL1 or DLL4 protein, optionally human DLL1 or human DLL4, at a lower level, degree, or affinity.

26. The anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-25, wherein the anti-DLL3 antibody or antigen-binding fragment thereof binds to human DLL1 or human DLL4 to a degree, level, extent, or affinity that is at least or about 75%, 80%, 90%, 95%, or 99% less than to human DLL3.

27. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 26, wherein the antibody or antigen-binding fragment thereof is expressed at about 1 x 10 -11 M to about 1x 10 -7 The equilibrium dissociation constant (K D ) binds to human DLL3.

28. A single-chain cell surface protein comprising the anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1-27.

29. A conjugate comprising the anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1-27 and a heterologous molecule or moiety.

30. The conjugate of claim 29, wherein the heterologous molecule or moiety is a therapeutic moiety.

31. The conjugate of claim 29 or 30, wherein the heterologous molecule or moiety is a small molecule compound, a protein, a peptide, or a toxin.

32. An anti-DLL3 chimeric antigen receptor (CAR), comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling region comprising the anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1-27.

33. The anti-DLL3 CAR of claim 32, further comprising a spacer between the extracellular antigen binding domain and the transmembrane domain.

34. The anti-DLL3 CAR of claim 33, wherein the spacer comprises at least a portion of an immunoglobulin or a variant thereof.

35. The anti-DLL3 CAR of claim 33 or 34, wherein the spacer comprises at least a portion of a hinge region of an immunoglobulin or a variant thereof.

36. The anti-DLL3 CAR of claim 35, wherein the at least a portion of the hinge region comprises an IgG4 hinge region, optionally all or a portion of a human IgG4 hinge region, or a variant thereof, optionally wherein the variant IgG4 hinge region comprises a substitution of amino acids CPSC to CPPC compared to a wild-type IgG4 hinge region.

37. The anti-DLL3 CAR of any one of claims 33-36, wherein the spacer is less than or less than about 15 amino acids in length.

38. The anti-DLL3 CAR of any one of claims 33-37, wherein the spacer is between 12 and 15 amino acids in length.

39. The anti-DLL3 CAR of any one of claims 33-38, wherein the spacer comprises an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

1.

40. The anti-DLL3 CAR of any one of claims 33-39, wherein the spacer comprises the sequence shown in SEQ ID NO:

1.

41. The anti-DLL3 CAR of any one of claims 33-38, wherein the spacer comprises an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

3.

42. The anti-DLL3 CAR of any one of claims 33-38 and 41, wherein the spacer comprises the sequence shown in SEQ ID NO:

3.

43. The anti-DLL3 CAR of any one of claims 33-36, wherein the spacer comprises at least a portion of a hinge region of an immunoglobulin and at least a portion of a CH3 region of an immunoglobulin, or a variant thereof.

44. The anti-DLL3 CAR of any one of claims 33-36 and 43, wherein the spacer is at or about 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125 amino acids in length, or between any of the foregoing values in length, optionally wherein the spacer comprises the sequence shown in SEQ ID NO: 5 or 58, or an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5 or 58.

45. The anti-DLL3 CAR of any one of claims 33-36, wherein the spacer comprises at least a portion of a hinge region of an immunoglobulin, at least a portion of a CH2 of an immunoglobulin, and at least a portion of a CH3 region of an immunoglobulin, or a variant thereof.

46. The anti-DLL3 CAR of any one of claims 33-36 and 45, wherein the spacer is at or about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 or 230 amino acids in length, or between any of the foregoing values in length, optionally wherein the spacer comprises the sequence shown in SEQ ID NO: 42 or 82, or an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 42 or 82.

47. The anti-DLL3 CAR of any one of claims 32-46, wherein the transmembrane domain comprises the transmembrane domain of CD4, CD28, or CD8.

48. The anti-DLL3 CAR of any one of claims 32-47, wherein the transmembrane domain comprises the transmembrane domain of CD28, optionally human CD28.

49. The anti-DLL3 CAR of any one of claims 32-48, wherein the transmembrane domain comprises SEQ ID NO: 10 or 12, or an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10 or 12.

50. The anti-DLL3 CAR of any one of claims 33-49, wherein the transmembrane domain comprises an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

10.

51. The anti-DLL3 CAR of any one of claims 33-50, wherein the transmembrane domain comprises the sequence shown in SEQ ID NO:

10.

52. The anti-DLL3 CAR of any one of claims 33-49, wherein the transmembrane domain comprises an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

12.

53. The anti-DLL3 CAR of any one of claims 33-49 and 52, wherein the transmembrane domain comprises the sequence shown in SEQ ID NO:

12.

54. The anti-DLL3 CAR of any one of claims 32-53, wherein the intracellular signaling region comprises an intracellular signaling domain capable of inducing a primary activation signal in a T cell, is a T cell receptor (TCR) component and / or comprises an immunoreceptor tyrosine-based activation motif (ITAM).

55. The anti-DLL3 CAR of any one of claims 32-54, wherein the intracellular signaling domain comprises a cytoplasmic signaling domain of a CD3-zeta (CD3ζ) chain, optionally a human CD3ζ chain.

56. The anti-DLL3 CAR of any one of claims 32-55, wherein the intracellular signaling domain comprises the sequence shown in SEQ ID NO: 19, or an amino acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

19.

57. The anti-DLL3 CAR of any one of claims 32-56, wherein the intracellular signaling domain comprises the sequence shown in SEQ ID NO:

19.

58. The anti-DLL3 CAR of any one of claims 32-57, wherein the intracellular signaling region further comprises a costimulatory domain.

59. The anti-DLL3 CAR of claim 58, wherein the costimulatory domain is between the transmembrane domain and the intracellular signaling domain.

60. The anti-DLL3 CAR of claim 58 or 59, wherein the costimulatory domain comprises an intracellular signaling domain of a T cell costimulatory molecule, or a signaling portion thereof.

61. The anti-DLL3 CAR of any one of claims 58-60, wherein the costimulatory domain comprises an intracellular signaling domain of CD28, 4-1BB, or ICOS.

62. The anti-DLL3 CAR of any one of claims 58-61, wherein the costimulatory domain comprises the intracellular signaling domain of 4-1BB, optionally human 4-1BB.

63. The anti-DLL3 CAR of any one of claims 58-62, wherein the costimulatory domain comprises the sequence shown in SEQ ID NO: 17, or an amino acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

17.

64. The anti-DLL3 CAR of any one of claims 58-61, wherein the costimulatory domain comprises the intracellular signaling domain of CD28, optionally human CD28.

65. The anti-DLL3 CAR of any one of claims 58-61 and 64, wherein the costimulatory domain comprises the sequence shown in SEQ ID NO: 15 or 16, or an amino acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15 or 16.

66. The anti-DLL3 CAR of any one of claims 32-65, wherein the CAR comprises, from its N-terminus to its C-terminus, the extracellular antigen binding domain, the spacer, the transmembrane domain, and the intracellular signaling region.

67. The anti-DLL3 CAR of any one of claims 33-38, 54-63, and 66, wherein the CAR comprises, from its N-terminus to its C-terminus: an extracellular antigen-binding domain comprising the scFv; a modified IgG4 hinge-containing spacer, optionally comprising the sequence shown in SEQ ID NO: 1; a transmembrane domain, optionally the transmembrane domain of human CD28, optionally comprising the sequence shown in SEQ ID NO: 10; and An intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain and an intracellular signaling domain of a costimulatory molecule, optionally comprising the intracellular signaling domain of 4-1BB.

68. The anti-DLL3 CAR of any one of claims 33-38, 54-63, and 66, wherein the CAR comprises, from its N-terminus to its C-terminus: an extracellular antigen-binding domain comprising the scFv; a modified IgG4 hinge-containing spacer, optionally comprising the sequence shown in SEQ ID NO: 3; a transmembrane domain, optionally the transmembrane domain of human CD28, optionally comprising the sequence shown in SEQ ID NO: 12; and An intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain and an intracellular signaling domain of a costimulatory molecule, optionally comprising the intracellular signaling domain of 4-1BB.

69. An anti-DLL3 chimeric antigen receptor (CAR), comprising: Contains an extracellular antigen binding domain of a scFv comprising Heavy chain variable (V H ) region and light chain variable (V L ) district, of which: The V H The region comprises the heavy chain complementary determining region 1 (CDR-H1), the heavy chain complementary determining region 2 (CDR-H2), and the heavy chain complementary determining region 3 (CDR-H3) contained in SEQ ID NO: 90, and The V L The region comprises the light chain complementary determining region 1 (CDR-L1), the light chain complementary determining region 2 (CDR-L2), and the light chain complementary determining region 3 (CDR-L3) contained in SEQ ID NO: 91, a spacer comprising the sequence shown in SEQ ID NO: 1; a transmembrane domain comprising the sequence shown in SEQ ID NO: 10; and An intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain comprising the sequence shown in SEQ ID NO: 19, and an intracellular signaling domain of a costimulatory molecule comprising the sequence shown in SEQ ID NO:

17.

70. An anti-DLL3 chimeric antigen receptor (CAR), comprising: Contains an extracellular antigen binding domain of a scFv comprising Heavy chain variable (V H ) region and light chain variable (V L ) district, of which: The V H The region comprises the heavy chain complementary determining region 1 (CDR-H1), the heavy chain complementary determining region 2 (CDR-H2), and the heavy chain complementary determining region 3 (CDR-H3) contained in SEQ ID NO: 90, and The V L The region comprises the light chain complementary determining region 1 (CDR-L1), the light chain complementary determining region 2 (CDR-L2), and the light chain complementary determining region 3 (CDR-L3) contained in SEQ ID NO: 91, a spacer comprising the sequence shown in SEQ ID NO: 3; a transmembrane domain comprising the sequence shown in SEQ ID NO: 12; and An intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain comprising the sequence shown in SEQ ID NO: 19, and an intracellular signaling domain of a costimulatory molecule comprising the sequence shown in SEQ ID NO:

17.

71. The anti-DLL3 CAR of any one of claims 33-38, 54-63, 66, 67, and 69, wherein the CAR comprises, from its N-terminus to its C-terminus: An extracellular antigen-binding domain comprising the scFv, wherein the scFv comprises the sequence shown in SEQ ID NO: 89; a spacer comprising the sequence shown in SEQ ID NO: 1; a transmembrane domain comprising the sequence shown in SEQ ID NO: 10; and An intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain comprising the sequence shown in SEQ ID NO: 19, and an intracellular signaling domain of a costimulatory molecule comprising the sequence shown in SEQ ID NO:

17.

72. The anti-DLL3 CAR of any one of claims 33-38, 54-63, 66, 68, and 70, wherein the CAR comprises, from its N-terminus to its C-terminus: An extracellular antigen-binding domain comprising the scFv, wherein the scFv comprises the sequence shown in SEQ ID NO: 89; a spacer comprising the sequence shown in SEQ ID NO: 3; a transmembrane domain comprising the sequence shown in SEQ ID NO: 12; and An intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain comprising the sequence shown in SEQ ID NO: 19, and an intracellular signaling domain of a costimulatory molecule comprising the sequence shown in SEQ ID NO:

17.

73. The anti-DLL3 CAR of any one of claims 52-72, wherein the anti-DLL3 CAR comprises the sequence shown in SEQ ID NO: 117, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO:

117.

74. The anti-DLL3 CAR of any one of claims 52-73, wherein the anti-DLL3 CAR comprises the sequence shown in SEQ ID NO:

117.

75. A polynucleotide comprising a nucleic acid encoding the anti-DLL3 antibody, or antigen binding domain thereof, of any one of claims 1-27.

76. A polynucleotide comprising a nucleic acid encoding the single-chain cell surface protein of claim 28.

77. A polynucleotide comprising a nucleic acid encoding the conjugate of any one of claims 29-31.

78. A polynucleotide comprising a nucleic acid encoding the anti-DLL3 chimeric antigen receptor of any one of claims 32-74.

79. The polynucleotide of any one of claims 75-78, wherein the polynucleotide comprises: Encode the V H a nucleic acid comprising the sequence shown in SEQ ID NO:87, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:87, and Encode the V L A nucleic acid comprising the sequence shown in SEQ ID NO:88, or a nucleic acid sequence that has at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

88.

80. The polynucleotide of any one of claims 75-79, wherein the polynucleotide comprises: Encode the V H A nucleic acid comprising the sequence shown in SEQ ID NO: 87, and Encode the V L A nucleic acid comprising the sequence shown in SEQ ID NO:

88.

81. The polynucleotide of any one of claims 75-80, wherein the antigen-binding fragment is a single-chain Fv (scFv) and the polynucleotide comprises a nucleic acid encoding the scFv comprising the sequence shown in SEQ ID NO:86, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

86.

82. The polynucleotide of any one of claims 75-81, wherein the antigen-binding fragment is a single-chain Fv (scFv), and the polynucleotide comprises a nucleic acid encoding the scFv comprising the sequence shown in SEQ ID NO:

86.

83. The polynucleotide of any one of claims 78-82, wherein the polynucleotide comprises a nucleic acid encoding the spacer, the nucleic acid comprising the sequence shown in SEQ ID NO: 2, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

2.

84. The polynucleotide of any one of claims 78-83, wherein the polynucleotide comprises a nucleic acid encoding the spacer, the nucleic acid comprising the sequence shown in SEQ ID NO:

2.

85. The polynucleotide of any one of claims 78-82, wherein the polynucleotide comprises a nucleic acid encoding the spacer, the nucleic acid comprising the sequence shown in SEQ ID NO:4, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

4.

86. The polynucleotide of any one of claims 78-82 and 85, wherein the polynucleotide comprises a nucleic acid encoding the spacer, the nucleic acid comprising the sequence shown in SEQ ID NO:

4.

87. The polynucleotide of any one of claims 78-84, wherein the polynucleotide comprises a nucleic acid encoding the transmembrane domain comprising the sequence shown in SEQ ID NO: 11, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

11.

88. The polynucleotide of any one of claims 78-84 and 87, wherein the polynucleotide comprises a nucleic acid encoding the transmembrane domain comprising the sequence shown in SEQ ID NO:

11.

89. The polynucleotide of any one of claims 78-82, 85, and 86, wherein the polynucleotide comprises a nucleic acid encoding the transmembrane domain comprising the sequence shown in SEQ ID NO: 13, or a nucleic acid sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

13.

90. The polynucleotide of any one of claims 78-82, 85, 86, and 89, wherein the polynucleotide comprises a nucleic acid encoding the transmembrane domain comprising the sequence shown in SEQ ID NO:

13.

91. The polynucleotide of any one of claims 78-90, wherein the nucleic acid encoding the anti-DLL3 chimeric antigen receptor comprises the sequence shown in SEQ ID NO: 116, or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO:

116.

92. The polynucleotide of any one of claims 78-91, wherein the nucleic acid encoding the anti-DLL3 chimeric antigen receptor comprises the sequence shown in SEQ ID NO:

116.

93. The polynucleotide of any one of claims 75-92, wherein the polynucleotide is optimized by splice site elimination.

94. The polynucleotide of any one of claims 75-93, wherein the polynucleotide is codon-optimized for expression in human cells.

95. A vector comprising the polynucleotide of any one of claims 75-94.

96. The vector of claim 95, wherein the vector is a viral vector.

97. The vector of claim 96, wherein the viral vector is a retroviral vector or a lentiviral vector.

98. The vector of claim 96, wherein the viral vector is an adeno-associated virus (AAV) vector.

99. A cell comprising the anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-27, the single-chain cell surface protein of claim 28, or the conjugate of any one of claims 29-31.

100. A cell comprising the anti-DLL3 chimeric antigen receptor of any one of claims 32-74.

101. A cell comprising the polynucleotide of any one of claims 75-94 or the vector of any one of claims 95-98.

102. The cell of any one of claims 99-101, wherein the cell is a lymphocyte.

103. The cell of any one of claims 99-102, wherein the cell is a NK cell or a T cell.

104. The cell of any one of claims 99-103, wherein the cell is a T cell, and the T cell is a CD4+ T cell or a CD8+ T cell.

105. The cell of any one of claims 99-104, wherein the cell is a primary cell obtained from a subject.

106. The cell of any one of claims 100-105, wherein, among a plurality of said cells, less than or about 10%, less than or about 9%, less than or about 8%, less than or about 7%, less than or about 5%, less than or about 4%, less than or about 3%, less than or about 2%, or less than or about 1% of said cells in said plurality of cells comprise an anti-DLL3 chimeric antigen receptor that exhibits tonic signaling and / or antigen-independent activity or signaling.

107. A composition comprising the cells of any one of claims 99-106.

108. A composition comprising the anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1-27, the single-chain cell surface protein according to claim 28, the conjugate according to any one of claims 29-31, or the anti-DLL3 chimeric antigen receptor according to any one of claims 32-74.

109. The composition of claim 107 or 108, further comprising a pharmaceutically acceptable excipient.

110. The composition of any one of claims 107-109, wherein the composition comprises CD4+ and CD8+ T cells, and the ratio of CD4+ to CD8+ T cells is from at or about 1:3 to 3:1, optionally at or about 1:2 to 2:1, optionally at or about 1:

1.

111. The composition of any one of claims 107-110, wherein among a plurality of the cells in the composition, less than or about 10%, less than or about 9%, less than or about 8%, less than or about 7%, less than or about 5%, less than or about 4%, less than or about 3%, less than or about 2%, or less than or about 1% of the cells in the plurality of cells comprise an anti-DLL3 chimeric antigen receptor that exhibits tonic signaling and / or antigen-independent activity or signaling.

112. A method of treatment comprising administering to a subject having a disease or disorder associated with DLL3 an anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1-27, a single-chain cell surface protein according to claim 28, a conjugate according to any one of claims 29-31, an anti-DLL3 chimeric antigen receptor according to any one of claims 32-74, a polynucleotide according to any one of claims 75-94, a vector according to any one of claims 95-98, a cell according to any one of claims 99-106, or a composition according to any one of claims 107-111.

113. The method of claim 112, wherein the disease or disorder associated with DLL3 is cancer.

114. The method of claim 113, wherein the cancer is a DLL3-expressing cancer.

115. The method of any one of claims 112-114, wherein the disease or disorder associated with DLL3 is selected from a neuroendocrine tumor, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), melanoma, glioma, or glioblastoma.

116. The method of any one of claims 112-115, wherein the disease or disorder associated with DLL3 is a neuroendocrine tumor.

117. The method of any one of claims 112-116, wherein the disease or disorder associated with DLL3 is small cell lung cancer (SCLC).

118. The method of any one of claims 112-117, wherein the disease or disorder associated with DLL3 is large cell neuroendocrine carcinoma (LCNEC).

119. The anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-27, the single-chain cell surface protein of claim 28, the conjugate of any one of claims 29-31, the anti-DLL3 chimeric antigen receptor of any one of claims 32-74, the polynucleotide of any one of claims 75-94, the vector of any one of claims 95-98, the cell of any one of claims 99-106, or the composition of any one of claims 107-111 for use in treating a disease or disorder associated with DLL3.

120. Use of the anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1-27, the single-chain cell surface protein according to claim 28, the conjugate according to any one of claims 29-31, the anti-DLL3 chimeric antigen receptor according to any one of claims 32-74, the polynucleotide according to any one of claims 75-94, the vector according to any one of claims 95-98, the cell according to any one of claims 99-106, or the composition according to any one of claims 107-111 for the manufacture of a medicament for treating a disease or disorder associated with DLL3.

121. Use of an anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1-27, the single-chain cell surface protein according to claim 28, the conjugate according to any one of claims 29-31, the anti-DLL3 chimeric antigen receptor according to any one of claims 32-74, the polynucleotide according to any one of claims 75-94, the vector according to any one of claims 95-98, the cell according to any one of claims 99-106, or the composition according to any one of claims 107-111 for treating a disease or disorder associated with DLL3.

122. The antibody or antigen-binding fragment thereof, single-chain cell surface protein, conjugate, chimeric antigen receptor, polynucleotide, vector, cell or composition for use or use according to any one of claims 119 to 121, wherein the disease or disorder associated with DLL3 is cancer.

123. The antibody or antigen-binding fragment thereof, single-chain cell surface protein, conjugate, chimeric antigen receptor, polynucleotide, vector, cell or composition for use or use according to claim 122, wherein the cancer is a DLL3-expressing cancer.

124. The antibody or antigen-binding fragment thereof, single-chain cell surface protein, conjugate, chimeric antigen receptor, polynucleotide, vector, cell or composition for use or use according to any one of claims 119 to 123, wherein the disease or disorder associated with DLL3 is a neuroendocrine tumor, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), melanoma, glioma or glioblastoma.

125. The cell, composition, antibody or antigen-binding fragment thereof, single-chain cell surface protein, conjugate, chimeric antigen receptor, polynucleotide or vector for use or use according to any one of claims 119 to 124, wherein the disease or disorder associated with DLL3 is a neuroendocrine tumor.

126. The cell, composition, antibody or antigen-binding fragment thereof, single-chain cell surface protein, conjugate, chimeric antigen receptor, polynucleotide or vector for use or use according to any one of claims 119 to 124, wherein the disease or disorder associated with DLL3 is small cell lung cancer (SCLC).

127. The cell, composition, antibody or antigen-binding fragment thereof, single-chain cell surface protein, conjugate, chimeric antigen receptor, polynucleotide or vector for use or use according to any one of claims 119 to 124, wherein the disease or disorder associated with DLL3 is large cell neuroendocrine carcinoma (LCNEC).

128. A kit comprising an anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1-27, a single-chain cell surface protein according to claim 28, a conjugate according to any one of claims 29-31, an anti-DLL3 chimeric antigen receptor according to any one of claims 32-74, a polynucleotide according to any one of claims 75-94, a vector according to any one of claims 95-98, a cell according to any one of claims 99-106, or a composition according to any one of claims 107-111, and instructions for use.

129. The kit of claim 128, wherein the instructions are for administering the anti-DLL3 antibody or antigen-binding fragment thereof, the single-chain cell surface protein, the conjugate, the anti-DLL3 chimeric antigen receptor, the cell, or the composition to a subject having a disease or disorder associated with DLL3.

130. An article of manufacture comprising the anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-27, the single-chain cell surface protein of claim 28, the conjugate of any one of claims 29-31, the anti-DLL3 chimeric antigen receptor of any one of claims 32-74, the polynucleotide of any one of claims 75-94, the vector of any one of claims 95-98, the cell of any one of claims 99-106, the composition of any one of claims 107-111, or the kit of claims 128 or 129.

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