Antibodies and chimeric antigen receptors specific for Delta-like ligand 3 (DLL3)

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 63 / 401,559, filed August 26, 2022, entitled "Antibodies and Chimeric Antigen Receptors Specific for Delta-Like Ligand 3 (DLL3)," the contents of which are incorporated by reference in their entirety.

[0002] Incorporation by Reference of Sequence Listing This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042022240SeqList, created on August 25, 2023, and is 184,247 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] In some aspects, the present disclosure relates to delta-like ligand 3 (DLL3) binding molecules, particularly human antibodies specific to DLL3, including antibody fragments. 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 specific to DLL3. The present disclosure also relates to genetically engineered cells containing such DLL3-binding proteins and receptors, as well as related methods and uses in adoptive cell therapy. [Background technology]

[0004] Delta-like ligand 3 (DLL3) is a type 1 transmembrane delta-like protein and an inhibitory Notch ligand that is normally expressed in intracellular membranes but 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 pulmonary neuroendocrine tumors, melanoma, and glioma. In normal adult tissues, DLL3 is generally expressed at low levels. SCLC often arises in the central pulmonary airways, grows aggressively, and is often metastatic. SCLC has a poor prognosis and few treatment options. The embodiments described herein provide DLL3-binding molecules and targeted therapies useful for treating cancers associated with DLL3 expression. Summary of the Invention [Means for solving the problem]

[0005] Provided herein are binding molecules, such as antibodies or antigen-binding fragments thereof, that bind to Delta-like ligand 3 (DLL3). Also included among the binding molecules are single-chain cell surface proteins containing such antibodies, e.g., polypeptides containing such antibodies, including recombinant receptors such as chimeric antigen receptors (CARs), fusion proteins, or conjugates. Recombinant receptors, such as chimeric antigen receptors (CARs), that bind to DLL3 are also provided. Conjugates, such as immunoconjugates, or fusion proteins, such as T cell engagers, that contain any of the antibodies or antigen-binding fragments described herein are also provided. Polynucleotides containing nucleic acid sequences encoding all or a portion of such antibodies, antigen-binding fragments, recombinant receptors, such as CARs, fusion proteins, or conjugates described herein are also provided. Cells containing any of the provided binding molecules, such as antibodies or antigen-binding fragments thereof, CARs, fusion proteins, or conjugates, are also provided. Also provided are related methods and uses, such as compositions comprising any of the binding molecules, such as an antibody or antigen-binding fragment thereof, a CAR, a fusion protein, a conjugate, a polynucleotide, or a cell, as well as therapeutic uses of any of the described binding molecules, such as an antibody or antigen-binding fragment thereof, a CAR, a fusion protein, a conjugate, a polynucleotide, a cell, or a composition.

[0006] Heavy chain variable (V H ) region and the light chain variable (V L Provided herein is an anti-Delta-like ligand 3 (DLL3) antibody or antigen-binding fragment thereof comprising a V H The regions include heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2) and heavy chain complementarity determining region 3 (CDR-H3) contained within SEQ ID NO: 90, and V L The regions include light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2) and light chain complementarity determining region 3 (CDR-L3) contained within SEQ ID NO:91.

[0007] Heavy chain variable (V H ) region and the light chain variable (VL Provided herein are anti-DLL3 antibodies or antigen-binding fragments thereof comprising a V H The regions include a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in SEQ ID NO: 92, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in SEQ ID NO: 93, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in SEQ ID NO: 94, L The regions include light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in SEQ ID NO: 95, light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in SEQ ID NO: 96, and light chain complementarity determining region 3 (CDR-L3) comprising the sequence set forth in SEQ ID NO: 97.

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

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

[0010] In some optional embodiments, V HThe regions include heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2) and heavy chain complementarity determining region 3 (CDR-H3) contained within SEQ ID NO: 90, and V L The regions include light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2) and light chain complementarity determining region 3 (CDR-L3) contained within SEQ ID NO:91.

[0011] In some optional embodiments, V H The regions include a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in SEQ ID NO: 92, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in SEQ ID NO: 93, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in SEQ ID NO: 94, L The regions include light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in SEQ ID NO: 95, light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in SEQ ID NO: 96, and light chain complementarity determining region 3 (CDR-L3) comprising the sequence set forth in SEQ ID NO: 97.

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

[0013] Heavy chain variable (V H ) region and the light chain variable (V L Provided herein are anti-DLL3 antibodies or antigen-binding fragments thereof comprising a V H The region comprises the sequence shown in SEQ ID NO: 90, L The region comprises the sequence shown in SEQ ID NO:91.

[0014] In some of any of the embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof is recombinant. H Area and V L The domains are human or derived from human proteins.

[0015] In some of the optional embodiments, the antibody comprises a heavy chain constant region (C H ) and / or the light chain constant region (C L In some optional embodiments, C H comprises the sequence set forth in SEQ ID NO: 126 or an amino acid sequence having at least 95%, 96, 97%, 98% or 99% or about this percentage sequence identity to SEQ ID NO: 126, and / or C L comprises the sequence set forth in SEQ ID NO: 127, or an amino acid sequence having at least 95%, 96, 97%, 98%, or 99%, or about this percent sequence identity to SEQ ID NO: 127. In some of any of the 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 of any of the 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 of any of the embodiments, the antibody is a full-length antibody.

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

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

[0019] In some optional embodiments, V H The region is V L It is amino-terminal to the region.

[0020] In some optional embodiments, V H The region is V L It is carboxy-terminal to the region.

[0021] In some optional embodiments, V H Area and V L The regions are connected by a flexible linker. In some of any of the embodiments, the flexible linker comprises the sequence set forth in SEQ ID NO:46.

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

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

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

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

[0026] Also provided are single-chain cell surface proteins comprising any of the anti-DLL3 antibodies or antigen-binding fragments 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 any of the anti-DLL3 antibodies or antigen-binding fragments thereof provided herein, an extracellular antigen-binding domain comprising a transmembrane domain and an intracellular signaling region. In some embodiments, the anti-DLL3 CAR also comprises a spacer between the extracellular antigen-binding domain and the transmembrane domain.

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

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

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

[0032] In some of any of the embodiments, the spacer comprises 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: 1. In some of any of the embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 1.

[0033] In some of any of the embodiments, the spacer comprises 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: 3. In some of any of the embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 3.

[0034] In some embodiments, the spacer comprises at least a portion of the hinge region and at least a portion of the CH3 region of an immunoglobulin or variant thereof. In some embodiments, 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 a length between any of the foregoing. In some optional embodiments, the spacer comprises the sequence set forth 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 of any of the embodiments, the spacer comprises at least a portion of the hinge region, at least a portion of the CH2, and at least a portion of the CH3 region of an immunoglobulin or variant thereof. In some of any of the embodiments, the spacer is 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. In some optional embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 42 or 82, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage sequence identity to SEQ ID NO: 42 or 82.

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

[0037] In some of any of the embodiments, the transmembrane domain comprises a transmembrane domain derived from CD28, such as human CD28. In some of any of the 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 of any of the embodiments, the transmembrane domain comprises 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. In some of any of the embodiments, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 10.

[0039] In some of any of the embodiments, the transmembrane domain comprises 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: 12. In some of any of the embodiments, the transmembrane domain comprises the sequence set forth 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 the cytoplasmic signaling domain of the CD3 zeta (CD3ζ) chain, e.g., the human CD3ζ chain. In some embodiments, the intracellular signaling domain comprises the sequence set forth in SEQ ID NO: 19, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or about this percentage sequence identity to SEQ ID NO: 19. In some embodiments, the transmembrane signaling domain comprises the sequence set forth 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 located between the transmembrane domain and the intracellular signaling domain. In some embodiments, the costimulatory domain comprises the intracellular signaling domain of a T cell costimulatory molecule, or a signaling portion thereof. In some embodiments, the costimulatory domain comprises the intracellular signaling domain of CD28, 4-1BB, or ICOS.

[0042] In some of any of the embodiments, the costimulatory domain comprises the intracellular signaling domain of 4-1BB, such as human 4-1BB. In some of any of the embodiments, the costimulatory domain comprises the sequence set forth 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 of any of the embodiments, the costimulatory domain comprises the intracellular signaling domain of CD28, such as human CD28. In some of any of the embodiments, the costimulatory domain comprises the sequence set forth 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 of any of the embodiments, the CAR comprises an extracellular antigen-binding domain, a spacer, a transmembrane domain, and an intracellular signaling region, in that order from N to C terminus.

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

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

[0047] Heavy chain variable (V H ) region and the light chain variable (V L ) region, H The region comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2) and heavy chain complementarity determining region 3 (CDR-H3) contained within SEQ ID NO: 90, L Provided herein is an anti-DLL3 chimeric antigen receptor (CAR) comprising: an extracellular antigen-binding domain, the regions of which comprise light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3) contained within SEQ ID NO: 91; a spacer comprising the sequence set forth in SEQ ID NO: 1; a transmembrane domain comprising the sequence set forth in SEQ ID NO: 10; an intracellular signaling region comprising the cytoplasmic signaling domain of CD3 zeta (CD3ζ) comprising the sequence set forth in SEQ ID NO: 19; and an intracellular signaling domain of a costimulatory molecule comprising the sequence set forth in SEQ ID NO: 17.

[0048] Heavy chain variable (V H ) region and the light chain variable (V L ) region, H The region comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2) and heavy chain complementarity determining region 3 (CDR-H3) contained within SEQ ID NO: 90, L Provided herein is an anti-DLL3 chimeric antigen receptor (CAR) comprising: an extracellular antigen-binding domain, the regions of which comprise light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3) contained within SEQ ID NO: 91; a spacer comprising the sequence set forth in SEQ ID NO: 3; a transmembrane domain comprising the sequence set forth in SEQ ID NO: 12; an intracellular signaling region comprising the cytoplasmic signaling domain of the CD3 zeta (CD3ζ) chain comprising the sequence set forth in SEQ ID NO: 19; and an intracellular signaling domain of a costimulatory molecule comprising the sequence set forth in SEQ ID NO: 17.

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

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

[0051] In some of any of the embodiments, the anti-DLL3 CAR comprises the sequence set forth 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 set forth in SEQ ID NO: 117. In some of any of the embodiments, the anti-DLL3 CAR comprises the sequence set forth in SEQ ID NO: 117.

[0052] In some of any of the embodiments, the anti-DLL3 CAR comprises or consists of the sequence set forth 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 set forth in SEQ ID NO: 119. In some of any of the embodiments, the anti-DLL3 CAR comprises or consists of the sequence set forth in SEQ ID NO:119.

[0053] Also provided are polynucleotides comprising nucleic acids encoding any of the anti-DLL3 antibodies or antigen-binding domains thereof provided herein.

[0054] Also provided are polynucleotides comprising nucleic acids encoding any of the single chain cell surface proteins provided herein.

[0055] Also provided are polynucleotides comprising nucleic acids encoding any of the conjugates provided herein.

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

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

[0058] In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding a spacer comprising the sequence set forth in 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 to SEQ ID NO: 2. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding a spacer comprising the sequence set forth in SEQ ID NO: 2.

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

[0060] In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding a transmembrane domain comprising the sequence set forth in SEQ ID NO: 11, 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: 11. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding a transmembrane domain comprising the sequence set forth in SEQ ID NO: 11.

[0061] In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding a transmembrane domain comprising the sequence set forth in SEQ ID NO: 13, 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: 13. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding a transmembrane domain comprising the sequence set forth in SEQ ID NO: 13.

[0062] In some of any of the embodiments, the nucleic acid encoding the anti-DLL3 chimeric antigen receptor comprises the sequence set forth 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 set forth in SEQ ID NO: 116. In some of any of the embodiments, the nucleic acid encoding the anti-DLL3 chimeric antigen receptor comprises the sequence set forth in SEQ ID NO: 116.

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

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

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

[0066] Also provided herein are cells 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 are cells comprising any of the anti-DLL3 CARs provided herein.

[0068] Also provided herein are cells comprising any of the polynucleotides provided herein, or any of the vectors provided herein.

[0069] In some embodiments, the cells are lymphocytes. In some embodiments, the cells are NK cells or T cells. In some embodiments, the cells are T cells, and the T cells are CD4+ T cells or CD8+ T cells.

[0070] In some of any of the embodiments, the cells are primary cells obtained from a subject.

[0071] In some of any of the embodiments, among the plurality of cells, at or about 10%, 9% or about 9%, 8% or about 8%, 7% or about 7%, 5% or about 5%, 4% or about 4%, 3% or about 3%, 2% or about 2%, or less than 1% or about 1% of 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 are compositions comprising any of the cells provided herein.

[0073] Also provided herein are compositions comprising any of the anti-DLL3 antibodies or antigen-binding fragments thereof provided herein, any of the single-chain cell surface proteins provided herein, any of the conjugates described herein, or any of the anti-DLL3 CARs provided herein.

[0074] In some optional embodiments, the composition also includes a pharmaceutically acceptable excipient.

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

[0076] In some of any of the embodiments, among the plurality of cells in the composition, at or about 10%, 9% or about 9%, 8% or about 8%, 7% or about 7%, 5% or about 5%, 4% or about 4%, 3% or about 3%, 2% or about 2%, or less than 1% or about 1% of 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 are methods of treatment involving administering any of the anti-DLL3 antibodies or antigen-binding fragments thereof provided herein, any of the single chain cell surface proteins provided herein, any of the conjugates provided herein, any of the anti-DLL3 CARs provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the cells provided herein, or any of the compositions provided herein to a subject having a disease or disorder associated with DLL3.

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

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

[0080] Also provided herein is the use of any of the anti-DLL3 antibodies or antigen-binding fragments thereof provided herein, any of the single chain cell surface proteins provided herein, any of the conjugates provided herein, any of the anti-DLL3 CARs provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the cells provided herein, or any of the compositions 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 of the anti-DLL3 antibodies or antigen-binding fragments thereof provided herein, any of the single chain cell surface proteins provided herein, any of the conjugates provided herein, any of the anti-DLL3 CARs provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the cells provided herein, or any of the compositions provided herein, for the treatment of a disease or disorder associated with DLL3.

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

[0083] Also provided herein are kits that include any of the anti-DLL3 antibodies or antigen-binding fragments thereof provided herein, any of the single chain cell surface proteins provided herein, any of the conjugates provided herein, any of the anti-DLL3 CARs provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the cells provided herein, or any of the compositions provided herein and instructions for use.

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

[0085] Provided herein are articles of manufacture comprising any of the anti-DLL3 antibodies or antigen-binding fragments thereof provided herein, any of the single chain cell surface proteins provided herein, any of the conjugates provided herein, any of the anti-DLL3 CARs provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the cells provided herein, any of the compositions provided herein, or any of the kits provided herein. [Brief explanation of the drawings]

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

[0087] [Figure 2] FIG. 2 shows 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 (intermediate DLL3; also referred to as "H82").

[0088] [Figure 3] Figure 3 shows the levels of IL2 production after 24 hours of co-culture of primary T cells from three different donors engineered to express one of 18 anti-DLL3 CAR candidates with DLL3-expressing H69 (low DLL3) target cells at an effector:target (E:T) ratio of 1:4.

[0089] [Figure 4]Figure 4 shows the levels of IL2 production after primary T cells from three different donors engineered to express one of 18 anti-DLL3 CAR candidates were cocultured with DLL3-expressing H82 (medium DLL3) target cells at an effector:target (E:T) ratio of 1:4 for 24 hours.

[0090] [Figure 5] Figure 5 shows the levels of IL2 production after primary T cells from two different donors engineered to express one of five anti-DLL3 CAR candidates were co-cultured with H69 (low DLL3) or H82 (intermediate DLL3) target cells.

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

[0092] [Figure 7] Figure 7 shows the mean tumor volume over time in a tumor xenograft mouse model in which human H82 cells (medium DLL3) were injected subcutaneously and primary T cells engineered to express one of five anti-DLL3 CAs were administered at two dose levels of 1 x 10 cells (low dose) or 4 x 10 cells (high dose).

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

[0094] [Figure 9]Figure 9 shows the mean tumor volume over time in a tumor xenograft mouse model in which human H69 cells (DLL3 low) were injected subcutaneously and primary T cells engineered to express CAR-A or CAR-B were administered at three dose levels: 1 x 10 cells (low), 2.5 x 10 cells (medium), or 5 x 10 cells (high).

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

[0096] [Figure 11] Figure 11 shows the expansion of administered T cells engineered to express CAR, as assessed by CD3+ cell counts per μL of blood 7, 14, 21, and 28 days after administration, in H69 tumor xenografted mice administered three doses of T cells expressing CAR-A or CAR-B. DETAILED DESCRIPTION OF THE INVENTION

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

[0098] In some embodiments, DLL3 is expressed in cells or tissues associated with certain diseases and conditions, such as malignant tumors, for example, small cell lung cancer (SCLC) cells and high-grade neuroendocrine tumors. In some embodiments, DLL3 is minimally or non-expressed in normal adult tissues.

[0099] Among the embodiments provided are approaches useful for treating diseases and conditions and / or for targeting cell types containing nucleic acid molecules encoding DLL3-binding antibodies, fragments, fusion proteins, or receptors, as well as the encoded antibodies or antigen-binding fragments, fusion proteins, and receptors.

[0100] Among the embodiments provided are approaches useful for treating diseases and conditions associated with DLL3 expression, including adoptive cell therapy involving cells expressing chimeric antigen receptors (CARs) that target DLL3, such as CARs that include a binding molecule as an extracellular antigen binding domain.

[0101] Compositions and articles of manufacture comprising one or more of the polypeptides and nucleic acids provided herein are also provided.

[0102] In some aspects, the provided chimeric antigen receptors (CARs) generally comprise an antibody specific for DLL3 (heavy chain variable (V)) as an antigen binding domain, e.g., an extracellular antigen binding domain. H ) region, single-domain antibody fragments, and single-chain fragments including scFvs. Cells, such as engineered or recombinant cells, that express a DLL3-binding receptor, e.g., an anti-DLL3 chimeric antigen receptor (CAR), and / or contain nucleic acid encoding such a receptor, as well as compositions and articles of manufacture and therapeutic doses containing such cells, are also provided. Methods of making and using the antibodies and fragments, as well as cells that express or contain the antibodies and fragments, such as for the production of antibodies or fragments thereof, are also provided. Compositions, including pharmaceutical compositions containing such antibodies, antigen-binding fragments, receptors, or cells, and conjugates comprising such antibodies or fragments are also provided. In some aspects, the provided compositions, antibodies, antigen-binding fragments, receptors, or cells can be used in connection with methods of therapy or treatment.

[0103] In some situations, the characteristics of the specific target antigen that an antibody or recombinant receptor containing an antigen-binding domain specifically binds to, recognizes, or targets may affect the activity of the receptor. In some situations, DLL3 is expressed by certain cancers and is an attractive therapeutic target for cell therapy. Improved strategies are needed to achieve optimal responses to DLL3-targeted therapy.

[0104] In some situations, optimal response to a therapy such as cell therapy may also depend on the ability of an engineered recombinant receptor, such as a CAR, to be consistently and reliably expressed on the surface of a cell and / or to bind to a target antigen. For example, in some cases, heterogeneity of the transcribed RNA from an introduced transgene (e.g., encoding a recombinant receptor) may affect the expression and / or activity of the recombinant receptor when expressed in cells such as human T cells used in cell therapy. In some situations, the length and type of spacer in a recombinant receptor, such as a CAR, may affect the expression, activity, and / or function of the receptor.

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

[0106] All publications, including patent documents, scientific articles, and databases, referred to 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 a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition 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.

[0108] I.DLL3 binding molecule DLL3-binding molecules, such as DLL3-binding polypeptides, are provided in some embodiments. Such binding molecules include antibodies (including antigen-binding fragments) that specifically bind to DLL3 proteins, such as human DLL3. Also among the binding molecules are polypeptides containing such antibodies, including single-chain cell surface proteins containing such antibodies, e.g., recombinant receptors such as chimeric antigen receptors (CARs), fusion proteins, or conjugates. 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 in some embodiments. Polynucleotides containing nucleic acid sequences encoding all or part of such antibodies, antigen-binding fragments, recombinant receptors such as CARs, fusion proteins, or conjugates, such as those described in Sections IA-IE, are also provided. Examples of such polynucleotides include those described in Section IF. In some embodiments, polynucleotides can be introduced into cells to generate engineered cells containing or expressing 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 provided antibody or antigen-binding fragment thereof specifically binds to the delta-like ligand 3 (DLL3) protein. DLL3 is a type I transmembrane delta-like protein that is typically expressed exclusively on intracellular membranes, such as the Golgi apparatus. DLL3 is a member of the delta protein ligand family and functions as a Notch ligand in the Notch signaling pathway. The Notch pathway is a highly conserved cell signaling pathway involved in various aspects of developmental biology, including somite segmentation and neurogenesis during early embryonic development and the development of pulmonary neuroendocrine cells. Typically, extracellular expression of DLL3 is highest in the fetal brain and lowest in normal adult tissues, except for the brain.

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

[0111] Due to the role of DLL3 in various 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, such as antibodies or receptors, DLL3-binding molecules such as cells expressing DLL3-binding molecules, or compositions thereof, are administered to subjects, such as subjects with or at risk of a disease or condition, such as cancer. In some aspects, administration treats, e.g., ameliorates, one or more symptoms of a disease or condition, such as by reducing the tumor burden of DLL3-expressing cancer.

[0112] In some embodiments, DLL3 expression has been observed to be associated with SCLC migration and invasion through a mechanism involving regulation of the epithelial-mesenchymal transition protein Snail. In some embodiments, DLL3 has also been observed to be expressed 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, other tumors or cancers that may be treated with provided embodiments, such as provided DLL3-binding molecules, include neuroendocrine tumors such as anterior pituitary neuroendocrine tumors, neuroendocrine thyroid tumors, parathyroid tumors, pulmonary neuroendocrine tumors (e.g., bronchial tumors, pulmonary carcinoid tumors), pancreaticoenteroganglionic neuroendocrine tumors (e.g., foregut-pancreaticoenteroganglionic tumors, including pancreatic endocrine tumors, midgut-pancreaticoenteroganglionic tumors, hindgut-pancreaticoenteroganglionic neuroendocrine tumors), liver and gallbladder tumors, adrenal gland tumors (e.g., adrenal medullary tumors), peripheral nervous system tumors (e.g., schwannoma, paraganglioma, neuroblastoma), cervical neuroendocrine tumors, prostate tumors, gliomas (e.g., astrocytoma, central neurocytoma, ependymoma, mixed glioma, oligondroglioma, optic nerve glioma, subependymal giant cell astrocytoma, glioblastoma).

[0113] In some embodiments, diseases or disorders associated with DLL3 expression that can be treated by 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, pancreatic cancer, bladder cancer, prostate cancer, urinary tract carcinoid tumor and neuroendocrine carcinoma, ovarian cancer, testicular cancer, and Merkel cell carcinoma. In some embodiments, a disease or disorder associated with DLL3 expression that can be treated by any of the provided embodiments is large cell neuroendocrine carcinoma (LCNEC). In some embodiments, a disease or disorder associated with DLL3 expression that can be treated by any of the provided embodiments is small cell lung cancer (SCLC).

[0114] A. DLL3-Targeting Antibodies and Antigen-Binding Fragments Thereof Anti-DLL3 polynucleotides, including antibodies and functional antigen-binding fragments, are provided. Among the DLL3-binding polypeptides are heavy chain variable (V H ) region and / or light chain variable (V L In some embodiments, the antibody or antigen-binding fragment is a V 1 -like fragment, such as a single chain antibody (e.g., an antigen-binding antibody fragment), including those containing a V 2 -like fragment, such as a single chain Fv fragment (scFv). H and V L Antibodies include antibodies that specifically bind to DLL3, e.g., human DLL3. Among the anti-DLL3 antibodies provided are human antibodies, or antibodies that are modified forms or 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, and recombinant receptors, such as chimeric receptors including T cell engagers (TCEs) and / or antigen receptors. In some embodiments, 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). The DLL3-binding recombinant receptors can contain provided antibodies (e.g., antigen-binding antibody fragments) that specifically bind to DLL3, such as DLL3 proteins, such as human DLL3. In some embodiments, the provided binding molecules bind to the extracellular portion of DLL3. In some examples, the recombinant receptor is a CAR, such as one that contains an anti-DLL3 antibody or its antigen-binding fragment.

[0116] Polynucleotides containing nucleic acid sequences encoding all or part of such antibodies, antigen-binding fragments, and binding molecules are also provided. The provided polynucleotides can be incorporated into constructs, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) constructs, such as those that can be introduced into cells for expression of the encoded DLL3-binding antibodies, antigen-binding fragments, fusion proteins, conjugates, T cell engagers (TCEs), or receptors, e.g., anti-DLL3 CARs. In some embodiments, the encoded antibodies, antigen-binding fragments, fusion proteins, conjugates, and receptors, such as those containing DLL3-binding polypeptides, compositions, and articles of manufacture, and uses thereof, are also provided. The term "antibody" is used herein in the broadest sense and includes intact antibodies, as well as antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (V) fragments capable of specifically binding to an antigen. H(scFv) regions, single-chain antibody fragments including single-chain variable fragments (sdAb, sdFv, nanobody) fragments, and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses 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 di-scFv, tandem tri-scFv, and the like. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments, also referred to herein as "antigen-binding fragments." The term also encompasses intact 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 that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0117] The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known to refer to non-contiguous sequences of amino acids in an antibody variable region, conferring antigen specificity and / or binding affinity. Generally, 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. The terms "framework region" and "FR" are known to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, 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 may be determined by 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 Pluckthun A, J Mol 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).Details on various numbering schemes can also be found, for example, in Jarasch et al., Proteins, 2017; 85(1):65-71, Martin et al., Bioinformatics tools for antibody engineering. In: Dubel, S. (editor) Handbook of Therapeutic Antibodies, Vol. 1. Wiley-VCH, Weinheim, Germany, Martin, ACR (2010). Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Kontermann, R., Dubel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg, and Martin, ACR, Antibody Information: How to identify the CDRs by looking at a sequence [online]<http: / / www.bioinf.org.uk / abs / info.html> Various prediction algorithm tools are available and known for numbering antibody residues and CDRs (e.g., AbYsis, Abnum, AbYmod, AbRSA, IgBLAST, IMGT, or ANARCI).

[0119] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on a structural algorithm, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes base their numbering on the most common antibody region sequence lengths, sometimes with insertions. Insertions in sequences relative to the standard numbering scheme are indicated using an insertion letter code. For example, residues inserted between residues L30 and L31 are indicated as L31A, L31B, etc. Deletions in sequences relative to the standard numbering scheme are accommodated by skipping numbers. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. For example, the Chothia numbering scheme is nearly identical to the Kabat numbering scheme, except that insertions are placed in structural positions and morphologically equivalent residues are assigned the same number. The Contact scheme is based on the analysis of complex crystal structures and is similar in many aspects to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions based on using Oxford Molecular's AbMd antibody modeling software. The IgBLAST scheme is based on matching germline V, D, and J genes and can be determined using the National Center for Biotechnology Information (NCBI)'s IgBLAST tool.

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

[0121] In some cases, the exact boundary locations of a particular CDR according to the Chothia numbering scheme may vary based on different definitions of a CDR (see, e.g., Martin, ACR, Antibody Information: How to identify the CDRs by looking at a sequence [online]).<http: / / www.bioinf.org.uk / abs / info.html> (See, for example, 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 CDR-L1 boundary position may be L25 to L32 according to the Chothia numbering scheme (Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48). In some cases, the CDR-L2 boundary position may be L50 to L52, and the CDR-L3 boundary position may be L91 to 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 CDR-H1 boundary positions may be H26 to H32 according to the Chothia numbering scheme (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 CDR-H2 boundary position may be H53 to 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 to H55 (Tramontano et al., J Mol Biol, 1990, 215(1):175-82). In some cases, the CDR-H2 boundary position may be H52 to H56 (Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48). In some cases, the CDR-H3 boundary position may be H96 to 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 CDR-H3 boundary position may be H92 to H104 (Morea et al., Biophys Chem, 1997; 68(1-3):9-16 and Morea et al., J Mol Biol., 1998; 275(2):269-944).

[0122] Table 1 below illustrates exemplary numbering and lists exemplary boundary positions for CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, and CDR-H3 identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between the CDRs; for example, FR-L1 precedes CDR-L1, FR-L2 is located between CDR-L1 and CDR-L2, FR-L3 is located between CDR-L2 and CDR-L3, etc. Note that the Kabat numbering scheme shown places the insertion at H35A and H35B, so the ends of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention shown, vary from H32 to H34 depending on the length of the loop. [Table 1]

[0123] Thus, unless otherwise specified, the "CDR" or "complementarity determining region" or each particular CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof, such as a variable region, should be understood to encompass one (or a particular) complementarity determining region as defined by any of the above schemes or other known schemes. For example, if a particular CDR (e.g., CDR-H3) is a CDR of a given V H or V L When a reference is made to a variable region amino acid sequence containing the amino acid sequence of a corresponding CDR, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the above schemes or other known schemes. In some embodiments, the provided binding molecules, e.g., anti-DLL3 antibodies or antigen-binding fragments thereof, or recombinant receptors (e.g., CARs) comprising the antibodies or antigen-binding fragments thereof, contain a predetermined V HCDR-H1, CHR-H2 and CDR-H3, as well as a given V region, are contained within the amino acid sequence and defined by any of the aforementioned schemes or other known schemes, such as the Kabat, Chothia, AbM, IgBLAST, IMGT or Contact methods. L The CDRs are contained within the amino acid sequence and include CDR-L1, CDR-L2, and CDR-L3, as defined by any of the aforementioned schemes or other known schemes, such as Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact methods. In some embodiments, specific CDR sequences are identified. Exemplary CDR sequences of the provided antibodies are described using various numbering schemes, such as Kobat, although it is understood that the provided antibodies may contain CDRs described according to any of the other aforementioned numbering schemes or other known numbering schemes.

[0124] Similarly, unless otherwise specified, it should be understood that the FRs or each particular FR (e.g., FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and / or FR-L4) of a given antibody or region thereof, such as a variable region, encompass one (or particular) framework region defined by any known scheme. In some cases, a scheme for identifying a particular CDR, FR, or FRs or CDRs is specified, such as CDRs defined by the Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact method or other known schemes. In some embodiments, the provided binding molecules, e.g., anti-DLL3 antibodies or antigen-binding fragments thereof, or recombinant receptors (e.g., CARs) comprising the antibodies or antigen-binding fragments thereof, comprise a given V. H FR-H1, FR-H2, FR-H3 and FR-H4, as well as a given V, contained within the region amino acid sequence and defined by any of the aforementioned schemes or other known schemes, such as the Kabat, Chothia, AbM, IgBLAST, IMGT or Contact methods. LThe regions contained within the amino acid sequence include FR-L1, FR-L2, FR-L3, and FR-L4, as defined by any of the aforementioned schemes or other known schemes, such as the Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact methods. In other cases, the specific amino acid sequences of the CDRs or FRs are provided.

[0125] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of a native antibody (V H and V L ) generally have similar structures, with each domain containing four conserved framework regions (FR) and three CDRs. See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). A single V H or V L A V domain may be sufficient to confer antigen-binding specificity. Furthermore, an antibody that binds to a particular antigen may have a V domain that is sufficient to confer antigen-binding specificity. H or V L Domains isolated using complementary V L or V H Libraries of domains can be screened individually. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0126] Among the antibodies provided are antibody fragments. An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than a natural antibody that contains a portion of a natural antibody that binds to the antigen to which the natural antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; heavy chain variable (V) antibodies; and the like. H ) regions, single-chain antibody molecules such as scFvs, and V HIn some embodiments, the antibody comprises a variable heavy chain (VH), ... H ) region and the variable light chain (V L In certain embodiments, the antibody is or comprises an antibody fragment comprising a heavy chain variable (V) region, such as an scFv. H ) region and / or light chain variable (V L ) region.

[0127] A single domain antibody (sdAb) is an antibody fragment that contains 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, a single domain antibody is a human single domain antibody.

[0128] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of natural antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment containing a non-naturally occurring configuration, such as two or more antibody regions or chains connected by a synthetic linker, e.g., a peptide linker, and / or a configuration that cannot be produced by enzymatic digestion of a naturally occurring native antibody. In some aspects, the antibody fragment is an scFv.

[0129] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has undergone humanization, typically to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with the corresponding residues of a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to retain or improve the specificity or affinity of the antibody.

[0130] Among the anti-DLL3 antibodies provided are human antibodies. A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell, or by a non-human source that utilizes a human antibody repertoire, including a human antibody library, or other human antibody coding sequence. The term excludes humanized forms of non-human antibodies that contain 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 immunogens to transgenic animals that have been modified to produce natural human antibodies or natural antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci, which replace endogenous immunoglobulin loci or are extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci are generally inactivated. Human antibodies can also be derived from human antibody libraries, including phage display and cell-free libraries, which contain antibody coding sequences derived from the human repertoire.

[0132] Among the antibodies provided are monoclonal antibodies, including monoclonal antibody fragments. As used herein, the term "monoclonal antibody" refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for variants that contain naturally occurring mutations or that may arise during the production of the monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. This term should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma, recombinant DNA methods, phage display, and other antibody display methods.

[0133] The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains, as well as other polypeptides, such as linker and DLL3-binding polypeptides, can contain amino acid residues, including natural and / or unnatural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, a polypeptide can contain modifications relative to its native or natural sequence, so long as the protein maintains the desired activity. These modifications can be deliberate, such as by site-directed mutagenesis, or can be accidental, such as by mutations of the host producing the protein or errors due to PCR amplification.

[0134] In some of the provided anti-DLL3 antibodies or antigen-binding fragments thereof, the heavy and / or light chain variable (V) H or V LIn some embodiments, the antibody, e.g., the anti-DLL3 antibody, e.g., the antigen-binding antibody fragment, contains the heavy chain variable region (V) sequence described above. 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., anti-DLL3 antibody, e.g., antigen-binding antibody fragment, is a single chain fragment, such as a single chain Fv (scFv) fragment. In some cases, the scFv comprises a V H Area and V L In some embodiments, the antibody, e.g., the anti-DLL3 antibody, e.g., the antigen-binding antibody fragment, comprises a V H and single domain antibodies (sdAbs), such as antibodies containing only the domain.

[0135] In some embodiments, an antibody or antigen-binding fragment, such as an extracellular antigen-binding domain, included in any of the provided recombinant receptors, such as chimeric antigen receptors (CARs, e.g., anti-DLL3 CARs), comprises a heavy chain or a sufficient antigen-binding portion thereof. For example, in some embodiments, an antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) area or V H In some embodiments, an antibody or antigen-binding fragment thereof, such as an extracellular antigen-binding domain included in any of the provided recombinant receptors, such as a chimeric antigen receptor (CAR, e.g., an anti-DLL3 CAR), comprises a light chain or a sufficient antigen-binding portion thereof. For example, in some embodiments, an 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, such as the extracellular antigen-binding domain included in any of the provided recombinant receptors, such as chimeric antigen receptors (CARs, e.g., anti-DLL3 CARs), comprises a sufficient antigen-binding portion of the V H Area or V H A sufficient antigen-binding portion of the region, and V L Area or V L In some embodiments, the recombinant receptor, e.g., an antibody or antigen-binding fragment thereof, such as an antigen-binding fragment of a CAR, comprises a sufficient antigen-binding portion of the VH Area and V L Area, or V H Area and V L In any such embodiment, the V H The domain sequences are those described herein. H In any such embodiment, the V L The domain sequences are those described herein. L In any such embodiment, the V H Any of the region arrays and V L Any of the region sequences can be used in combination. In some of any such embodiments, any one or more of the CDR-H1, CDR-H2, and / or CDR-H3 sequences described herein and any one or more of the 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 a Fab or scFv. In some embodiments, the antibody or antigen-binding fragment further comprises at least a portion of an immunoglobulin constant region or a variant thereof, such as an antigen-binding fragment of a recombinant receptor, e.g., a CAR. In some such embodiments, the antibody is a full-length antibody that also comprises a constant region.

[0136] In some embodiments, the recombinant receptors provided herein, e.g., CARs, are V H In some embodiments, the recombinant receptors, e.g., CARs, provided herein, include antibodies, such as anti-DLL3 antibodies or antigen-binding fragments thereof, that comprise either the V region or a sufficient antigen-binding portion thereof. L For example, in some embodiments, the CAR comprises an antibody, such as an anti-DLL3 antibody or antigen-binding fragment thereof, comprising either the V H Area and V L Area or V H Area and V LIn any such embodiment, the V L The domain sequences are those described herein. L In any such embodiment, the V H The domain sequences are those described herein. H In any such embodiment, the V H One of the region arrays and V L Any of the region sequences may be used in combination. In some of these embodiments, any one or more of the CDR-H1, CDR-H2 and / or CDR-H3 sequences described herein and any one or more of the CDR-L1, CDR-L2 and / or CDR-L3 sequences described herein may be used in combination. In some of these embodiments, the antibody contained in the provided recombinant receptor is an antigen-binding fragment such as Fab or scFv. In some of these embodiments, the receptor, for example, CAR, further comprises a portion of an immunoglobulin constant region or a variant thereof, such as a spacer, such as those described in Section IB below.

[0137] In some embodiments, the anti-DLL3 antibody, e.g., antigen-binding antibody fragment, comprises a V DLL3 antibody having a V DLL3 complementarity determining region 1 (CDR-H1), a V DLL3 complementarity determining region 2 (CDR-H2), and / or a V DLL3 complementarity determining region 3 (CDR-H3) as contained and / or described herein. H In some embodiments, the anti-DLL3 antibody, e.g., antigen-binding antibody fragment, comprises a V domain sequence, including CDR-H1, CDR-H2, and CDR-H3, as contained and / or described herein. H In some embodiments, the anti-DLL3 antibody, e.g., antigen-binding antibody fragment, comprises a V region sequence comprising a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and / or a light chain complementarity determining region 3 (CDR-L3) as contained and / or described herein. LIn some embodiments, the anti-DLL3 antibody, e.g., antigen-binding antibody fragment, comprises a V domain sequence or a sufficient antigen-binding portion thereof, including CDR-L1, CDR-L2, and CDR-L3 as contained and / or described herein. L The domain sequence or a sufficient antigen-binding portion thereof.

[0138] In some embodiments, the anti-DLL3 antibody, e.g., antigen-binding antibody fragment, comprises a V domain comprising the described CDR-H1, CDR-H2, and / or CDR-H3. H V comprising the CDR-L1, CDR-L2 and / or CDR-L3 region sequences as described L In some embodiments, the anti-DLL3 antibody, e.g., antigen-binding antibody fragment, comprises a V region sequence comprising the described CDR-H1, CDR-H2, and CDR-H3. H V comprising the CDR-L1, CDR-L2 and CDR-L3 region sequences as described L The provided antibodies and fragments thereof also include such sequences and, for example, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, V H , V L , scFv sequences, or other sequences of the antibodies or fragments thereof described herein, have a sequence that is at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99% identical to any of the sequences. In some embodiments, the provided antibodies or fragments thereof have at least or about 95% sequence identity to any such sequence. In some embodiments, the provided antibodies or fragments thereof have at least or about 96% sequence identity to any such sequence. In some embodiments, the provided antibodies or fragments thereof have at least or about 97% sequence identity to any such sequence. In some embodiments, the provided antibodies or fragments thereof have at least or about 98% sequence identity to any such sequence. In some embodiments, the provided antibodies or fragments thereof have at least or about 99% sequence identity to any such sequence.

[0139] In some embodiments, the antibody is H The V region sequence alone or as described herein H In some embodiments, the antibody or antigen-binding fragment is an sdAb that contains a sufficient antigen-binding portion, such as any of the sequences (e.g., CDR-H1, CDR-H2, CDR-H3 and / or CDR-H4). L V that is not paired with a region and / or does not have any additional antibody domains or binding sites H These include single domain antibodies, which contain a region.

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

[0141] In some of any of the provided embodiments, a binding molecule, such as an antibody or antigen-binding fragment thereof, comprises a heavy chain variable (V) sequence comprising heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), and / or heavy chain complementarity 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. HIn some of any of the provided embodiments, a binding molecule, such as an antibody or antigen-binding fragment thereof, has a V region that includes CDR-H1, CDR-H2, and CDR-H3 present in SEQ ID NO: 90, such as CDR-H1, CDR-H2, and CDR-H3 according to Kabat, Chothia, AbM, or IMGT numbering or other numbering schemes. H It has.

[0142] In some of any of the provided embodiments, the binding molecule, such as an antibody or antigen-binding fragment thereof, has the amino acid sequence set forth in SEQ ID NO: 90, or the V sequence set forth in SEQ ID NO: 90. H and / or have at least 95%, 96%, 97%, 98% or 99% or about this percentage sequence identity with the amino acid sequence of the V region as described above. H the aforementioned V, including those containing CDR-H1, CDR-H2 and CDR-H3 present in any of the sequences H V having an amino acid sequence including CDR-H1, CDR-H2, and CDR-H3 present in any of the sequences H In some of the embodiments provided, V H The region is V shown in SEQ ID NO: 90 H In some of the provided embodiments, the binding molecule, such as an antibody or antigen-binding fragment thereof, comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% or about this percentage sequence identity with the amino acid sequence set forth in SEQ ID NO: 90, or the V region amino acid sequence set forth in SEQ ID NO: 90. H a V region having an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% or about this percentage sequence identity with the amino acid sequence of the V region, or that contains CDR-H1, CDR-H2, and CDR-H3 present in SEQ ID NO: 90, such as one that contains CDR-H1, CDR-H2, and CDR-H3 present in SEQ ID NO: 90; H In some of the embodiments provided, V H The region is V shown in SEQ ID NO: 90 HIn some of the provided embodiments, the V region amino acid sequence includes CDR-H1, CDR-H2, and CDR-H3, each of which includes the amino acid sequence of CDR-H1, CDR-H2, and CDR-H3 contained within the V region amino acid sequence. H The regions include CDR-H1, CDR-H2 and CDR-H3 contained within SEQ ID NO:90.

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

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

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

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

[0147] In some of the embodiments provided, V H The region includes any of the CDR-H1, CDR-H2 and CDR-H3 described, and is represented by SEQ ID NO: 90. H In some of the provided embodiments, the V region comprises framework region 1 (FR1), FR2, FR3, and / or FR4, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or about this percentage of FR1, FR2, FR3, and / or FR4 contained within the region amino acid sequence. H The region is V shown in SEQ ID NO: 90 H The region comprises FR1, FR2, FR3 and / or FR4 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage of FR1, FR2, FR3 and / or FR4, respectively, contained within the amino acid sequence.

[0148] In some of the embodiments provided, V H The region comprises the amino acid sequence set forth 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 the embodiments provided, V H The region comprises SEQ ID NO: 87 or an amino acid sequence encoded by 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 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 thereofL The V region comprises CDR-L1, CDR-L2 and / or CDR-L3 according to Kabat numbering. L The V region comprises CDR-L1, CDR-L2 and / or CDR-L3 according to Chothia numbering. L The V region comprises CDR-L1, CDR-L2 and / or CDR-L3 according to AbM numbering. L The regions include CDR-L1, CDR-L2 and / or CDR-L3 according to Kabat, Chothia, AbM or IMGT numbering or other numbering schemes.

[0151] In some of any of the provided embodiments, a binding molecule, such as an antibody or antigen-binding fragment thereof, comprises a light chain variable (V) domain comprising a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and / or a light chain complementarity 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. L In some of any of the provided embodiments, a binding molecule, such as an antibody or antigen-binding fragment thereof, has a V region that includes 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. L It has.

[0152] In some of any of the provided embodiments, the binding molecule, such as an antibody or antigen-binding fragment thereof, has the amino acid sequence set forth in SEQ ID NO: 91, or the V LV having an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 or 99% sequence identity with the amino acid sequence of the region L Including the area and / or the aforementioned V L the aforementioned V, including those containing CDR-L1, CDR-L2 and CDR-L3 present in any of the sequences L In some of the provided embodiments, the CDR-L1, CDR-L2, and CDR-L3 are present in any of the V L The region is V shown in SEQ ID NO: 91 L In some of the provided embodiments, the binding molecule, such as an antibody or antigen-binding fragment thereof, comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or about this percentage sequence identity with the amino acid sequence set forth in SEQ ID NO: 91, or the V region amino acid sequence set forth in SEQ ID NO: 91. L V having an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of the region L In some of any of the provided embodiments, V comprises a region and / or comprises the CDR-L1, CDR-L2 and CDR-L3 present in SEQ ID NO: 91, such as comprising the CDR-L1, CDR-L2 and CDR-L3 present in SEQ ID NO: 91. L The region is V shown in SEQ ID NO: 91 L In some of the provided embodiments, the V region comprises a CDR-L1, a CDR-L2, and a CDR-L3, each of which comprises an amino acid sequence of CDR-L1, CDR-L2, and CDR-L3 contained within the V region amino acid sequence. L The regions include CDR-L1, CDR-L2 and CDR-L3 contained within SEQ ID NO:91.

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

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

[0155] In some of the embodiments provided, V LThe region comprises a CDR-L1 comprising the sequence set forth 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 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 set forth 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 regions include CDR-L1, CDR-L2 and CDR-L3, which comprise the sequence shown in SEQ ID NO: 107, the sequence GA shown in SEQ ID NO: 108 and the sequence shown in SEQ ID NO: 109, respectively.

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

[0157] In some of the embodiments provided, V LThe region includes any of the CDR-L1, CDR-L2 and CDR-L3 described and is shown in SEQ ID NO: 91. L In some embodiments, the V region comprises framework region 1 (FR1), FR2, FR3, and / or FR4, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or about this percentage of FR1, FR2, FR3, and / or FR4 contained within the region amino acid sequence. L The region is V shown in SEQ ID NO: 91 L The region comprises FR1, FR2, FR3 and / or FR4 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage of the FR1, FR2, FR3 and / or FR4 contained within the amino acid sequence.

[0158] In some of the embodiments provided, V L The region comprises the amino acid sequence set forth 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 the embodiments provided, V L The region comprises an amino acid sequence encoded by SEQ ID NO: 88 or a nucleic acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or about this percentage 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 an antibody or antigen-binding fragment thereof of any of the provided recombinant receptors, e.g., CARs, conjugates, or binding molecules, HThe region comprises the amino acid sequence of SEQ ID NO: 90 and is a V region of an antibody or antigen-binding fragment thereof of any of the provided recombinant receptors, e.g., CARs, conjugates, or binding molecules. L The region comprises the amino acid sequence of SEQ ID NO:91.

[0161] In some embodiments, the V of an antibody or antigen-binding fragment thereof of any of the provided recombinant receptors, e.g., CARs, conjugates, or binding molecules, H The VL and VL regions comprise the amino acid sequences of SEQ ID NOs: 90 and 91, respectively, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or about this percentage of the VL and VL regions. H and V L For example, the V of an antibody or antigen-binding fragment thereof provided herein includes a sequence having at least or about 90% sequence identity with any of the V, ... H The VL and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 90 and 91, respectively.

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

[0163] In some of any of the provided embodiments, a binding molecule, such as an antibody or antigen-binding fragment thereof, comprises a heavy chain variable (V) sequence comprising heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), and / or heavy chain complementarity 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. H) region, and a light chain variable (V) region comprising light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2) and / or light chain complementarity 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. L In some of any of the provided embodiments, a binding molecule, such as an antibody or antigen-binding fragment thereof, has a V region that includes CDR-H1, CDR-H2, and CDR-H3 present in SEQ ID NO: 90, such as CDR-H1, CDR-H2, and CDR-H3 according to Kabat, Chothia, AbM, or IMGT numbering or other numbering schemes. H and V comprising 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. L It has.

[0164] In some of any of the provided embodiments, the binding molecule, such as an antibody or antigen-binding fragment thereof, has the amino acid sequence set forth in SEQ ID NO: 90, or the V sequence set forth in SEQ ID NO: 90. H and / or have at least 95%, 96%, 97%, 98% or 99% or about this percentage sequence identity with the amino acid sequence of the V region as described above. H the aforementioned V, including those containing CDR-H1, CDR-H2 and CDR-H3 present in any of the sequences H V having an amino acid sequence including CDR-H1, CDR-H2, and CDR-H3 present in any of the sequences H region, and the amino acid sequence shown in SEQ ID NO: 91, or the V L and / or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 or 99% or about this percentage sequence identity with the amino acid sequence of the V Lthe aforementioned V, including those containing CDR-L1, CDR-L2 and CDR-L3 present in any of the sequences L V having an amino acid sequence containing CDR-L1, CDR-L2, and CDR-L3 present in any of the sequences L In some of the embodiments provided, V H The region is V shown in SEQ ID NO: 90 H an amino acid sequence having at least 95%, 96, 97%, 98%, or 99% or about this percentage sequence identity with the amino acid sequence of the V L The region is V shown in SEQ ID NO: 91 L In some of the provided embodiments, the binding molecule, such as an antibody or antigen-binding fragment thereof, comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or about this percentage sequence identity with the amino acid sequence set forth in SEQ ID NO: 90, or the V region amino acid sequence set forth in SEQ ID NO: 90. H a V region having an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% or about this percentage sequence identity with the amino acid sequence of the V region, or that contains CDR-H1, CDR-H2, and CDR-H3 present in SEQ ID NO: 90, such as one that contains CDR-H1, CDR-H2, and CDR-H3 present in SEQ ID NO: 90; H region, and the amino acid sequence shown in SEQ ID NO: 91, or the V L a V region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 or 99% or about this percentage sequence identity with the amino acid sequence of the V region, or having an amino acid sequence that includes CDR-L1, CDR-L2 and CDR-L3 present in SEQ ID NO: 91, such as one that includes CDR-L1, CDR-L2 and CDR-L3 present in SEQ ID NO: 91. L In some of the embodiments provided, V H The region is V shown in SEQ ID NO: 90 HIn some of the provided embodiments, the V region amino acid sequence includes CDR-H1, CDR-H2, and CDR-H3, each of which includes the amino acid sequence of CDR-H1, CDR-H2, and CDR-H3 contained within the V region amino acid sequence. H The region includes CDR-H1, CDR-H2 and CDR-H3 contained within SEQ ID NO: 90, L The region is V shown in SEQ ID NO: 91 L In some of the provided embodiments, the V region comprises a CDR-L1, a CDR-L2, and a CDR-L3, each of which comprises an amino acid sequence of CDR-L1, CDR-L2, and CDR-L3 contained within the V region amino acid sequence. L The regions include CDR-L1, CDR-L2 and CDR-L3 contained within SEQ ID NO:91.

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

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

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

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

[0169] In some of the embodiments provided, V H The region includes any of the CDR-H1, CDR-H2 and CDR-H3 described, and is represented by SEQ ID NO: 90. H In some embodiments, the V region comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of framework region 1 (FR1), FR2, FR3, and / or FR4, respectively, contained within the region amino acid sequence. H The region is V shown in SEQ ID NO: 90 H and V comprises FR1, FR2, FR3 and / or FR4 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage of FR1, FR2, FR3 and / or FR4 contained within the region amino acid sequence, respectively; LThe region includes any of the CDR-L1, CDR-L2 and CDR-L3 described and is shown in SEQ ID NO: 91. L In some embodiments, the V region comprises framework region 1 (FR1), FR2, FR3, and / or FR4, each having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or about this percentage of FR1, FR2, FR3, and / or FR4 contained within the region amino acid sequence. L The region is V shown in SEQ ID NO: 91 L The region comprises FR1, FR2, FR3 and / or FR4 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage of the FR1, FR2, FR3 and / or FR4 contained within the amino acid sequence.

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

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

[0172] Also provided are polynucleotides containing, for example, any of the nucleotide sequences described herein that encode all portions of the provided binding molecules.

[0173] In some embodiments, the antibody or antigen-binding fragment in any of the recombinant receptors provided herein, such as a CAR, is a single-chain antibody fragment, such as a single-chain variable fragment (scFv) or a diabody or a single-domain antibody (dsAb). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable (V H ) region and the light chain variable (V L In some embodiments, single-chain antibody fragments (e.g., scFvs) are multi-domain antibodies, such as scFvs, that comprise a heavy chain variable (V) region. H ) region and the light chain variable (V L The linker comprises one or more linkers connecting two antibody domains or regions, such as the α- and β-domains. The linker is typically a peptide linker, e.g., a flexible and / or soluble peptide linker. Some linkers are rich in glycine and serine, and / or in some cases, threonine. In some embodiments, the linker further comprises charged residues, such as lysine and / or glutamic acid, which may improve solubility. In some embodiments, the linker further comprises one or more prolines.

[0174] Thus, the provided CARs can be used in combination with single-chain antibody fragments such as scFvs and diabodies, particularly V H Area and V LThe present invention also includes an anti-DLL3 antibody comprising a human single-chain antibody fragment that typically includes a linker connecting two antibody domains or regions, such as a region. The linker is typically a peptide linker, e.g., a flexible and / or soluble peptide linker, such as one rich in glycine and serine.

[0175] In some aspects, a linker rich in glycine and serine (and / or threonine) comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of such amino acids. In some embodiments, the linker comprises at least 50%, 55%, 60%, 70%, or 75% or approximately 50%, 55%, 60%, 70%, or 75% glycine, serine, and / or threonine. In some embodiments, the linker is substantially entirely composed of glycine, serine, and / or threonine. Linkers are generally 5-50 amino acids in length, typically 10-30, e.g., 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, 10-25 amino acids in length. Exemplary linkers include linkers having various numbers of repeats of the sequence GGGGS (4GS, SEQ ID NO:44) or GGGS (3GS, SEQ ID NO:45), for example, 2, 3, 4, and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of the sequence set forth in SEQ ID NO:46 (GGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO:31 (GSTSGSGKPGSGEGSTKG). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO:68 (SRGGGGSGGGGSGGGGSLEMA). Exemplary linkers include those having or consisting of the sequence set forth in SEQ ID NO:69 (GSRGGGGSGGGGSGGGGSLEMA). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NOs:29 and 30.

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

[0177] In some embodiments, V H The region is V L In some embodiments, the V H The region is V L In certain embodiments, the fragment, e.g., an scFv, may be carboxy-terminal to the V H A region or a portion thereof followed by a linker, followed by V L In other embodiments, the fragment, e.g., an scFv, may be followed by a V L A region or a portion thereof followed by a linker, followed by V H The region or part thereof may follow.

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

[0179] In some of any of the provided embodiments, the scFv comprises SEQ ID NO: 86 or an amino acid sequence encoded by 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: 86. In some of any of the provided embodiments, the scFv comprises an amino acid sequence encoded by SEQ ID NO: 86. In some of any of the provided embodiments, the scFv has an amino acid sequence encoded by SEQ ID NO: 86. In some embodiments, a provided recombinant receptor, such as a CAR, comprises an extracellular antigen-binding domain comprising an scFv, wherein the scFv comprises SEQ ID NO: 86 or an amino acid sequence encoded by 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: 86. In some embodiments, provided recombinant receptors, such as CARs, comprise an extracellular antigen-binding domain comprising an scFv, wherein the scFv comprises the amino acid sequence encoded by SEQ ID NO: 86. In some embodiments, provided recombinant receptors, such as CARs, comprise an extracellular antigen-binding domain comprising an scFv, wherein the scFv has the amino acid sequence encoded by SEQ ID NO: 86.

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

[0181] In some embodiments, the provided antibodies or antigen-binding fragments thereof comprise a V H Area and V L region and the V region of an antibody or antigen-binding fragment thereof H The region can contain any combination of CDR-H1, CDR-H2 and CDR-H3 amino acid sequences shown in Table 2, and can be used in combination with the V region of an antibody or antigen-binding fragment thereof. L The regions can contain any combination of CDR-L1, CDR-L2, and CDR-L3 amino acid sequences shown in Table 2. In some embodiments, the provided antibodies or antigen-binding fragments thereof contain any combination, orientation, or different linkers of the V, VL1, VL2, and VL3 amino acid sequences shown in Table 2. H Area and / or V LIn some embodiments, the antibody or antigen-binding fragment thereof comprises a V region as set forth in Table 2. H In some embodiments, the antibody or antigen-binding fragment thereof comprises a V region as set forth in Table 2. L In some embodiments, the provided antibodies or antigen-binding fragments thereof comprise a V region as shown in Table 2. H Region or V shown in Table 2 L In some embodiments, the provided antibody or antigen-binding fragment thereof is a single domain antibody (sdAv) comprising a V domain containing CDR-H1, CDR-H2, and / or CDR-H3 as shown in Table 2. H V containing the regions, or CDR-L1, CDR-L2 and / or CDR-L3 shown in Table 2 L It is a single domain antibody (sdAv) containing the domain. [Table 2]

[0182] Among the provided antibodies, e.g., antigen-binding fragments, are human antibodies. In some embodiments of the provided human anti-DLL3 antibodies, e.g., antigen-binding fragments, the human antibody comprises a V segment that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain D segment, and / or a portion that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain J segment. Ha V segment that includes a region and / or a portion that has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence encoded by a germline nucleotide human kappa or lambda chain V segment and / or a portion that has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence encoded by a germline nucleotide human kappa or lambda chain J segment L Includes the area.

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

[0184] In some embodiments, a 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 in a sequence encoded by a germline nucleotide human heavy chain V segment. For example, a human antibody, in some embodiments, comprises a CDR-H1 that has a sequence that is 100% identical or has no more than 1, 2, or 3 amino acid differences compared to the corresponding CDR-H1 region in a sequence encoded by a germline nucleotide human heavy chain V segment.

[0185] In some embodiments, a 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 in a sequence encoded by a germline nucleotide human heavy chain V segment. For example, a human antibody, in some embodiments, comprises a CDR-H2 that has a sequence that is 100% identical or has no more than 1, 2, or 3 amino acid differences compared to the corresponding CDR-H2 region in a sequence encoded by a germline nucleotide human heavy chain V segment.

[0186] In some embodiments, a human antibody or antigen-binding fragment thereof comprises a CDR-H3 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H3 region in a sequence encoded by a germline nucleotide human heavy chain V, D, and J segment. For example, a human antibody, in some embodiments, comprises a CDR-H3 having a sequence that is 100% identical or has no more than 1, 2, or 3 amino acid differences compared to the corresponding CDR-H3 region in a sequence encoded by a germline nucleotide human heavy chain V, D, and J segment.

[0187] In some embodiments, a 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 in a sequence encoded by a germline nucleotide human light chain V segment. For example, a human antibody, in some embodiments, comprises a CDR-L1 that has a sequence that is 100% identical or has no more than 1, 2, or 3 amino acid differences compared to the corresponding CDR-L1 region in a sequence encoded by a germline nucleotide human light chain V segment.

[0188] In some embodiments, a 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 in a sequence encoded by a germline nucleotide human light chain V segment. For example, a human antibody, in some embodiments, comprises a CDR-L2 that has a sequence that is 100% identical or has no more than 1, 2, or 3 amino acid differences compared to the corresponding CDR-L2 region in a sequence encoded by a germline nucleotide human light chain V segment.

[0189] In some embodiments, a 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 in a sequence encoded by a germline nucleotide human light chain V and J segment. For example, a human antibody, in some embodiments, comprises a CDR-L3 that has a sequence that is 100% identical or has no more than 1, 2, or 3 amino acid differences compared to the corresponding CDR-L3 region in a sequence encoded by a germline nucleotide human light chain V 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 segment, wherein the framework regions, e.g., FR1, FR2, FR3, and FR4, have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to framework regions encoded by human germline antibody segments, such as V and / or J segments. H In some embodiments, a human antibody comprises a V segment, in which the framework regions, e.g., FR1, FR2, FR3, and FR4, have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to framework regions encoded by human germline antibody segments, such as V and / or J segments. L For example, in some such embodiments, V H Area and / or V L The framework region sequences contained within the region differ by no more than 10 amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids, compared to the framework region sequences encoded by human germline antibody segments.

[0191] An antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof comprises a light chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a lambda (λ) light chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a kappa (κ) light chain constant region. 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 a heavy chain constant region of an isotype class selected from among IgG, IgM, IgD, IgA, and IgE. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgG heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgG1 heavy chain constant region.

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

[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 kappa light chain constant region comprises the sequence set forth in SEQ ID NO: 127.

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

[0195] In some 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 embodiments, the antibody or antigen-binding fragment thereof comprises a V that comprises the sequence set forth in SEQ ID NO:91, or an amino acid sequence having at least 95%, 96, 97%, 98%, or 99%, or about this percentage sequence identity to SEQ ID NO:91. Land C comprising the sequence set forth in SEQ ID NO: 127, or an amino acid sequence having at least 95%, 96, 97%, 98% or 99% or about this percentage sequence identity to SEQ ID NO: 127. L In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the sequence set forth in SEQ ID NO: 129, or an amino acid sequence having at least 95%, 96, 97%, 98%, or 99%, or about this percentage sequence identity to SEQ ID NO: 129.

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

[0197] 1. Mutants In certain embodiments, the antibody contains one or more amino acid variations, e.g., substitutions, deletions, insertions, and / or mutations, compared to the antibody sequences described herein. Exemplary variations 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, deletions, insertions, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0198] In certain embodiments, the antibody contains one or more amino acid substitutions, for example, compared to the antibody sequences described herein and / or compared to sequences in a natural repertoire, e.g., a human repertoire. Substitution mutagenesis sites of interest include the CDRs and FRs. Amino acid substitutions are introduced into the antibody of interest, and the products can be screened for desired activity, e.g., retention / improvement of antigen binding, reduced immunogenicity, improved half-life, and / or improved effector function, such as the ability to improve antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

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

[0200] In some embodiments, the changes are made in "hotspot" residues of the CDRs that are encoded by codons that undergo high frequency of mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in residues that contact the antigen, and the resulting mutant V H or V L The resulting antibodies are tested for binding affinity. Affinity maturation by construction and reselection from a secondary library is described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some affinity maturation embodiments, diversity is introduced into the various genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-specific approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0201] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may, for example, be outside the antigen-contacting residues of the CDRs. The mutant V provided above may be H and V L In certain embodiments of the sequences, each CDR is either unaltered or contains no more than 1, 2 or 3 amino acid substitutions.

[0202] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to the length of polynucleotides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusion of the antibody to the N- or C-terminus of an enzyme or polypeptide, which increases the serum half-life of the antibody.

[0203] 2. Qualification 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, e.g., by altering the amino acid sequence to remove or insert one or more glycosylation sites and / or by modifying the polysaccharides attached to the glycosylation sites, e.g., using certain cell lines.

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

[0205] Exemplary modifications, variants, and cell lines are described, for example, in U.S. Patent Publication Nos. 2003 / 0157108, 2004 / 0093621, 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent Publication No. 2002 / 0164328; U.S. Patent Publication No. 2004 / 0093621; U.S. Patent Publication No. 2004 / 0132140; U.S. Patent Publication No. 2004 / 0110704; U.S. Patent Publication No. 2004 / 0110282; U.S. Patent Publication No. 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); US Patent Application No. 2003 / 0157108A1, Presta, L; International Publication No. 2004 / 056312A1, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); WO 2003 / 085107; WO 2003 / 011878; U.S. Patent No. 6,602,684; U.S. Patent Publication No. 2005 / 0123546; WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0206] Some modified antibodies have one or more amino acid modifications in the Fc region, such as those having a human Fc region sequence or other portion of the constant region (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes 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 receptor, and / or alter effector function.

[0207] Also among the variants are cysteine ​​engineered antibodies, such as "thioMAbs" and other cysteine ​​engineered variants in which one or more residues of an antibody are replaced with cysteine ​​residues, creating reactive thiol groups at available sites that are used, for example, for conjugation of drugs and linker agents to form immunoconjugates. Cysteine ​​engineered antibodies are described, for example, in U.S. Patent Nos. 7,855,275 and 7,521,541.

[0208] In some embodiments, the antibody is modified to contain an additional non-protein moiety, including a water-soluble polymer. Exemplary polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, propropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde is advantageous for manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. Generally, the number and / or type of polymers 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, such as whether the antibody derivative will be used therapeutically under defined conditions.

[0209] 3. Exemplary Features In some embodiments, the provided antibodies have one or more specific functional characteristics, such as binding properties that include binding to a specific epitope or exhibiting low or reduced binding to a related but non-specific antigen. In some embodiments, the provided antibodies can bind to an epitope that is similar to or overlaps with the epitope of another antibody, such as a reference antibody, and / or can exhibit a specific binding affinity. In some embodiments, the provided antibodies can bind to an epitope that is different from the epitope of the other antibody, for example, binding to a conformational epitope.

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

[0211] In some embodiments, an antibody or antigen-binding fragment thereof is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. An antibody has an equilibrium dissociation constant of ≦10 -7 or ≤ 10 -8 If it is M, it 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, rapidly, for a longer period of time, and / or with greater affinity than alternative antigens. An antibody molecule or antigen-binding fragment thereof specifically or preferentially binds to a target if it binds with greater affinity, avidity, ease, and / or duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to DLL3 is an antibody that binds to DLL3 protein with greater affinity, avidity, ease, and / or duration than it binds to other DLL3 or non-DLL3 antigens from another species. It is also understood that specific or preferential binding does not necessarily require (but 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, such as an anti-DLL3 antibody, eg, a human antibody, generally specifically binds to a particular epitope or region of DLL3, such as an extracellular epitope or region.

[0214] DLL3 is a type I transmembrane delta-like protein that is typically expressed exclusively on intracellular membranes such as the Golgi apparatus. Human DLL3 has an extracellular region with various extracellular domains, including an N-terminal Notch ligand domain, a 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; mature polypeptide without 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 587-619 are substituted with an alanine residue relative to the human DLL3 isoform 1 precursor sequence set forth in SEQ ID NO: 120 (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 has low sequence homology to other cell surface Notch ligands in the family, with 35% homology to DLL1 and 38% homology to DLL4.

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

[0218] In some embodiments, the antibody or antigen-binding fragment thereof 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 set forth 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 set forth in SEQ ID NO: 120). In some embodiments, the one or more epitopes comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acid residues within amino acid residues 27-84 corresponding to 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, relative to the human DLL3 precursor sequence set forth in SEQ ID NO: 120.

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

[0220] In some embodiments, the provided antibodies can bind to DLL3, such as human DLL3, with at least a certain affinity, as measured by any of a number of known methods. In some embodiments, affinity can be measured using the equilibrium dissociation constant (K D In some embodiments, affinity is represented by EC 50 It is expressed by:

[0221] A variety of assays are known to assess binding affinity, including the equilibrium dissociation constant (K D), equilibrium association constant (K A ), E.C. 50 , on-rate (association rate constant, k on or k a , unit 1 / Ms or M -1 s -1 ) and the off-rate (off-rate constant, k off or k d , unit 1 / s or s -1 ), and / or determining 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., a DLL3, such as human DLL3 or cynomolgus monkey DLL3 or mouse DLL3, can be determined, such as by using any of a number of well-known binding assays. For example, in some embodiments, a BIAcore® instrument can be used to determine the binding kinetics and binding constants of a complex of two proteins (e.g., an antibody or antigen-binding fragment thereof and an antigen such as a DLL3 protein) using surface plasmon resonance (SPR) analysis (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 molecular concentration on the sensor surface as molecules bind to or dissociate from the surface. The change in SPR signal is directly proportional to the change in mass concentration near the surface, thereby allowing the measurement of the binding kinetics between two molecules. The dissociation rate constant (k off Or k d ), association rate constant (k on Or k a ), and / or the equilibrium dissociation constant (K D ) and / or the equilibrium association constant (K A) can be determined by monitoring the change in refractive index with respect to time as buffer passes over the chip. Other suitable assays for measuring binding of one protein to another include, for example, immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), or determining binding by monitoring changes in the spectroscopic or optical properties of the protein via fluorescence, UV absorbance, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, gene reporter assays, flow cytometry, and other methods for detecting binding of expressed nucleic acids or proteins.

[0223] In some embodiments, the binding molecule, e.g., an antibody or fragment thereof, is 10 5 M -1 or greater affinity or K A (i.e., units 1 / M or M -1 The equilibrium association constant for a specific binding interaction is given by the on-rate [k on or k a ] vs. dissociation rate (off-rate) [k off or k d In some embodiments, the peptide binding molecule binds, e.g., specifically binds, to an antigen, e.g., a DLL3 protein or epitope therein, at a ratio equal to 10 5 M -1 (In this association reaction, the on-rate [k on ] vs. dissociation rate (off-rate) [k off ]) affinity or K A (i.e., units 1 / M or M -1 In some embodiments, the binding molecule, e.g., antibody or antigen-binding fragment thereof, binds, e.g., specifically binds, to an antigen, e.g., an epitope of human DLL3, with an affinity constant greater than 10 (the equilibrium association constant of the specific binding interaction). 6 M -1 or about 106 M -1 ~10 7 M -1 or about 10 7 M -1 Affinity or K in the range A In some embodiments, the binding affinity can be classified as high affinity or low affinity. For example, in some cases, a binding molecule, such as an antibody or an antigen-binding fragment thereof, that exhibits high affinity binding to a particular epitope exhibits binding affinity of 10 7 M -1 or about 10 7 M -1 K A In some cases, binding molecules that exhibit low affinity binding, such as antibodies or antigen-binding fragments thereof, interact with such epitopes at 10 7 M -1 K up to A In certain embodiments, the aforementioned K A Any of the values ​​or ranges may be determined by SPR. A Either the value or range is determined by SPR as described in Example 3.

[0224] Alternatively, affinity may be expressed in units of M (e.g., 10 -5 M~10 -13 Equilibrium dissociation constant (K M ) of a specific binding interaction D In some embodiments, the antibody or antigen-binding fragment thereof may be defined as 10 -5 K below M D (i.e., the equilibrium dissociation constant of a particular binding interaction in units M; assuming a bimolecular interaction, this association reaction has an off-rate [k off or k d ] vs. binding rate (on-rate) [k on or k a The equilibrium dissociation constant K D is 10 -5 M~10 -8 In certain embodiments, the aforementioned KD Any of the values ​​or ranges may be determined by SPR. D Either the value or range is determined by SPR as described in Example 3.

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

[0226] In some embodiments, the equilibrium dissociation constant (K D ) is 50 nM or about 50 nM to 500 nM or about 500 nM, 50 nM or about 50 nM to 100 nM or about 100 nM, or 100 nM or about 100 nM to 500 nM or about 500 nM. In certain embodiments, the equilibrium dissociation constant (K 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. In some embodiments, the equilibrium dissociation constant K of a binding molecule to a DLL3 protein, such as human DLL3, e.g., an anti-DLL3 antibody or fragment thereof, is D is 50 nM to 500 nM, 50 nM to 100 nM, or 100 nM to 500 nM, or approximately in this range. In certain embodiments, the equilibrium dissociation constant (K 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. D Any of the values ​​or ranges may be determined by SPR. D Either the value or range is determined by SPR as described in Example 3.

[0227] In some embodiments, the K D In certain embodiments, the K of a binding molecule to a DLL3 protein, such as human DLL3, e.g., an anti-DLL3 antibody or fragment thereof, is 50 nM or about 50 nM to 60 nM or about 60 nM. D In certain embodiments, the K of a binding molecule to a DLL3 protein, such as a human DLL3, e.g., an anti-DLL3 antibody or fragment thereof, is 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM, or is approximately this value, or a range defined by any of the foregoing. D is at or about 50 nM, 55 nM, 60 nM, or 65 nM, or in a range defined by any of the foregoing. D Any of the values ​​or ranges may be determined by SPR. D Either the value or range is determined by SPR as described in Example 3.

[0228] In some embodiments, the binding affinity (EC 50 ) is 10 nM or about 10 nM to 15 nM, 10 nM to 20 nM, 10 nM to 30 nM, 50 nM to 500 nM or about 500 nM, 50 nM or about 50 nM to 100 nM or about 100 nM, or 100 nM or about 100 nM to 500 nM or about 500 nM. In certain embodiments, the binding affinity (EC 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. In some embodiments, the binding affinity (EC 50 ) is 50 nM to 500 nM, 50 nM to 100 nM, or 100 nM to 500 nM, or approximately in this range. In certain embodiments, the binding affinity (EC 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. 50 The values ​​or ranges are determined by the experimental conditions described in Example 4.

[0229] In some embodiments, the binding affinity (EC 50 ) is 10 nM or about 10 nM to 20 nM or about 20 nM. In certain embodiments, the EC 50 The values ​​or ranges are 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 related Notch ligand protein such as DLL1 or DLL4, e.g., human DLL1 or human DLL4, to a lesser extent, level, or degree or affinity.

[0231] In some embodiments, the degree of binding of an anti-DLL3 antibody to a different non-DLL3 Notch ligand protein, such as a DLL1 or DLL4 protein, e.g., human DLL1 or human DLL4, or another non-DLL3 protein, is measured to be less than 50%, 40%, 30%, 20%, or 10% of the binding of the antibody to human DLL3, or less than about this percentage. 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, does not cross-react, or binds with a lower level, degree, or affinity to DLL1 or DLL4. In some embodiments, the degree, level, degree, or affinity of binding of a provided anti-DLL3 antibody or antigen-binding fragment thereof to DLL1 or DLL4 is at least 75%, 80%, 90%, 95%, or 99% less than the degree, level, degree, or affinity of binding 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, degree, or affinity. In some embodiments, the degree, level, degree, or affinity of binding of the anti-DLL3 antibody or antigen-binding fragment thereof to human DLL1 is at least 75%, 80%, 90%, 95%, or 99% or about this percent less than the degree, level, degree, or affinity of binding to human DLL3. 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, degree, or affinity. In some embodiments, the degree, level, degree, or affinity of binding of the anti-DLL3 antibody or antigen-binding fragment thereof to human DLL4 is at least 75%, 80%, 90%, 95%, or 99% or about this percent less than the degree, level, degree, or affinity of binding to human DLL3.

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

[0233] In some embodiments, the antibody binds to non-human DLL3, such as DLL3 from monkey, rabbit, rat, mouse, or other species. In some embodiments, the antibody or antigen-binding fragment thereof binds to non-human DLL3, such as cynomolgus monkey (Macaca fascicularis) DLL3, e.g., an epitope or region of 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 mouse (Mus musculus) DLL3, e.g., an epitope or region of mouse DLL3, such as the mouse DLL3 set forth in SEQ ID NO: 124 (Genbank Number: NP_031892.2).

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

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

[0236] In some embodiments, the binding affinity of a binding molecule, such as an anti-DLL3 antibody, 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 of different antigens, e.g., DLL3 proteins from different species, such as human, cynomolgus monkey, or mouse, can be determined. D In some embodiments, the species cross-reactivity of an 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 of human DLL3. D and species mutant DLL3 K D is 1 or about 1. In some embodiments, the anti-DLL3 antibody may have low species cross-reactivity, e.g., an anti-DLL3 antibody has high affinity for human DLL3 but low affinity for species variant DLL3, or vice versa. For example, the K D and human DLL3 K D 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, such as a reference antibody.

[0237] In some embodiments, the provided antibodies or antigen-binding fragments thereof bind to human DLL3 and non-human DLL3 to a similar extent. For example, in some embodiments, the provided antibodies or antigen-binding fragments thereof bind to human DLL3 and non-human DLL3 with a specific equilibrium dissociation constant (K D ) that bind to human DLL3 or its allelic variants or splice variants and have a K that is similar, or approximately the same, or less than a 2-fold difference, or less than a 5-fold difference. D and binds to non-human DLL3, such as cynomolgus monkey DLL3.

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

[0239] B. Immunoconjugates 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, such as, but not limited to, a cytotoxic agent or an imaging agent. Cytotoxic agents include radioisotopes (e.g., 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); chemotherapeutic agents; growth inhibitory agents; enzymes and fragments thereof, such as nucleases; antibiotics; and 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 a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope. Among immunoconjugates are antibody-drug conjugates (ADCs) in which an 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, the ADC comprises the antibody or antigen-binding fragment described herein conjugated to a small molecule compound. In certain embodiments, the ADC comprises the antibody or antigen-binding fragment described herein conjugated to a chemotherapeutic agent. Additionally, some immunoconjugates comprise an antibody conjugated to a toxin, such as an enzymatically active toxin or fragment thereof. In certain embodiments, the immunoconjugate comprises an antibody or antigen-binding fragment thereof described herein and a toxin, such as an enzymatically active toxin or fragment thereof. The immunoconjugate may also comprise an antibody or antigen-binding fragment thereof and a peptide or protein. In certain embodiments, the immunoconjugate comprises an antibody or antigen-binding fragment thereof described herein and a peptide or protein.

[0240] Additionally, in some immunoconjugates, antibodies are conjugated to radioactive atoms to form radioconjugates. 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 such as drugs, small molecules, toxins, proteins, or peptides can be made using any of a number 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 facilitates release of the cytotoxic drug into cells, 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).

[0242] CT Cell Engager In some embodiments, the provided DLL3-binding molecules are molecules for T cell binding therapy. In some embodiments, the provided antibodies or antigen-binding fragments thereof are included in the molecules for T cell binding therapy. In some embodiments, the molecules for T cell binding therapy are or include binding molecules capable of binding to surface molecules expressed on T cells. In some embodiments, the surface molecule is an activation component of T cells, 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 T cell binding therapy are or include antibodies or antigen-binding fragments, such as antibodies or antigen-binding fragments that bind to CD3 or CD2, in addition to any of the antibodies or antigen-binding fragments that bind to DLL3 described herein.

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

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

[0245] A number of 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). Among such exemplary bispecific antibody T cell binding molecules, such as bispecific T cell engager (BiTE) molecules, are tandem scFv molecules fused by a flexible linker (see, e.g., Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); tandem scFv molecules further containing an Fc domain, e.g., composed of a first and a second subunit fused to each other by a flexible linker, allowing for stable association (WO2013026837); 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 diabody formats with C-terminal disulfide bridges; or triomabs, which include all-hybrid mouse / rat IgG molecules (Seimetz et al, Cancer Treat Rev 36, 458-467 (2010)).

[0246] In certain embodiments, the bispecific T cell engager is or comprises a polypeptide construct or fusion protein. In certain embodiments, the polypeptide construct contains a first component comprising an antigen-binding domain that binds to an activating 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 a particular cancer, such as DLL3. In some embodiments, the first and second components are coupled by a linker.

[0247] In some embodiments, the antigen-binding domain of the first component of the bispecific T cell engager binds to a receptor on an endogenous immune cell in the tumor periphery. In some embodiments, the endogenous immune cell is a T cell. In some aspects, engagement of the endogenous T cell receptor redirects the endogenous T cell to the tumor. In some aspects, engagement of the endogenous T cell receptor recruits infiltrating lymphocytes (TILs) toward the tumor. In some aspects, engagement of the endogenous T cell receptor activates the endogenous immune repertoire.

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

[0249] In some aspects, the first component of the bispecific T cell engager is or comprises an antigen-binding domain that binds to a T cell activation component. In some embodiments, the T cell activation component 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, binding of the bispecific T cell binding molecule to the T cell antigen stimulates and / or activates the T cell. In some aspects, the T cell activation component 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, an 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 of the BiTE is anti-CD3. In some aspects, the anti-CD3 domain is an scFv. In some embodiments, the anti-CD3 domain of the BiTE binds to a subunit of the CD3 complex on the T cell receptor. In some aspects, the receptor is on endogenous T cells. In some embodiments, the receptor is on engineered immune cells that further express a recombinant receptor. The effects of CD3 binding on T cells are well known in the art and include, but are not limited to, T cell activation and other downstream cell signaling. Any of these BiTEs can be used in the 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 that bind to DLL3 described herein. In certain embodiments, the antigen-binding domain that binds to DLL3 is an anti-DLL3 scFv comprising (i) a VH domain comprising the sequence set forth in SEQ ID NO: 90, and (ii) a VL domain comprising the sequence set forth in SEQ ID NO: 91. In certain embodiments, the antigen-binding domain that binds to DLL3 is an anti-DLL3 scFv comprising the sequence set forth in SEQ ID NO: 89.

[0253] In some embodiments, both 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 T cells, a peptide linker, and a second component comprising an antigen-binding domain that binds to a surface antigen associated with a particular cancer, such as DLL3. In certain embodiments, the first antigen-binding domain of the BiTE is an anti-CD3 scFv. In certain embodiments, the second antigen-binding domain of the 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 contains a linker that connects a first component comprising an antigen-binding domain that binds to a T cell activating moiety to a second component comprising an antigen-binding domain that binds to DLL3 on cancer cells. In some aspects, the linker is a short, medium, or long linker. In some aspects, the linker can be any peptide linker described herein. In some embodiments, the peptide linker is or includes a cleavable peptide linker.

[0256] D. Multispecific antibodies In certain embodiments, DLL3-binding molecules, such as antibodies or antigen-binding fragments thereof, or fusion proteins containing them, such as recombinant receptors, are multispecific. Multispecific binding molecules include, for example, multispecific antibodies, including bispecific antibodies. Multispecific binding partners, such as antibodies, have binding specificities for at least two different sites, which may be 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, bispecific antibodies can bind to two different epitopes on 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, such as diabodies, triabodies, and tetrabodies, as well as tandem di-scFvs and tandem tri-scFvs. Multispecific chimeric receptors, such as multispecific CARs, containing antibodies are also provided. Multispecific cells containing antibodies or antibody-containing polypeptides are also provided, such as cells containing a cell surface protein that comprises an anti-DLL3 antibody and an additional cell surface protein, such as an additional chimeric receptor that binds to a different antigen or different epitope of DLL3.

[0257] E. Recombinant Receptor Among the provided binding molecules, e.g., DLL3-binding molecules, are cell surface proteins such as recombinant receptors, including those containing one of the provided antibodies or antigen-binding fragments. Polynucleotides encoding all or part of such cell surface proteins, e.g., receptors, are also provided. Receptors include antigen receptors and other chimeric receptors that specifically bind to DLL3, such as the provided anti-DLL3 antibodies, e.g., receptors containing antigen-binding fragments. Among antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). DLL3-binding receptors generally contain antibodies (e.g., antigen-binding fragments) and / or other binding polypeptides that specifically bind to DLL3, such as DLL3 proteins, e.g., human DLL3. Also provided are cells expressing the recombinant receptor, compositions containing such cells, and their uses in adoptive cell therapy, such as treating diseases and disorders associated with DLL3 expression, compositions, and articles of manufacture, as well as their uses.

[0258] Provided are chimeric antigen receptors (CARs) specific for DLL3, and polynucleotides containing nucleic acid sequences encoding all or a portion, fragment, domain, or chain of any of the CARs 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 among the CARs described in Tables E1-E4 and E8 and / or in each row of Table 3.

[0259] Some of the provided polynucleotides encode recombinant receptors, such as antigen receptors, that specifically bind to DLL3. In some aspects, the encoded receptors, such as those containing DLL3-binding polypeptides, compositions and articles of manufacture, and uses thereof, are also provided. The provided polynucleotides can be incorporated into constructs, such as deoxyribonucleic acid (DNA) or RNA constructs, that can be introduced into cells for the expression of the encoded recombinant DLL3-binding receptor. Polynucleotides encoding CARs are also provided herein. In some embodiments, the CAR can be encoded by more than one different polynucleotide, such as two or more polynucleotides. In some of any such embodiments, the two or more polynucleotides can each contain a nucleic acid encoding a portion, fragment, domain, or chain of the CAR.

[0260] 1. Chimeric antigen receptor (CAR) 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 generally contain an extracellular antigen-binding domain and an intracellular signaling region. Among the provided receptors are recombinant receptors such as chimeric antigen receptors (CARs) containing the provided antibodies or antigen-binding fragments thereof, such as one or more of the provided anti-DLL34 antibodies or fragments thereof. In some embodiments, the provided cell surface proteins specifically bind to DLL3, such as human DLL3.

[0261] Among antigen receptors are chimeric and / or functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Chimeric receptors, such as CARs, generally comprise, are, or comprise an extracellular antigen-binding domain comprised in an anti-DLL3 antibody, such as an anti-DLL3 antibody or fragment thereof described herein. In some embodiments, the chimeric receptor, e.g., CAR, comprises an intracellular signaling domain. In some embodiments, the chimeric receptor also comprises a spacer and / or a transmembrane domain. In some embodiments, the spacer is positioned between the extracellular antigen-binding domain and the transmembrane domain. In some embodiments, the CAR comprises an extracellular antigen-binding domain, a spacer, a transmembrane domain, and an intracellular signaling region. Exemplary CARs provided herein include those containing an antigen-binding domain comprising an antibody or antigen-binding fragment thereof described herein, e.g., in Section IA and / or Table 2, or those described in Section IE, Table 3, and / or Tables E1-4 and E8 herein. CARs encoded by a polynucleotide described in Section IF, Table 3, and / or Tables E1-4 and E8 herein are also provided.

[0262] In some cases, CARs are referred to as first-, second-, and / or third-generation CARs. In some embodiments, first-generation CARs provide only a CD3 chain-inducing signal upon antigen binding, in some embodiments, second-generation CARs provide such a signal as well as a costimulatory signal, for example, those that include an intracellular signaling domain derived from a costimulatory receptor such as CD28 or CD137, and in some embodiments, third-generation CARs include multiple costimulatory domains from different costimulatory receptors.

[0263] In some embodiments, the CAR comprises an extracellular antigen-binding domain, in some cases comprising an antibody, e.g., 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 CD3-zeta or a functional variant thereof. In some embodiments, the CAR comprises an extracellular antigen-binding domain, in some cases comprising an antibody, e.g., an antibody fragment that binds 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 CD3-zeta or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer, e.g., a hinge-only spacer, containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g., an IgG4 hinge.

[0264] In some embodiments, the chimeric antigen receptor comprises an extracellular portion containing an antibody or fragment thereof described herein. In some aspects, the chimeric antigen receptor comprises an antibody or fragment thereof described herein and an extracellular portion containing 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 zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain connecting the extracellular domain to the intracellular signaling domain.

[0265] Other exemplary antigen receptors, including CARs, and methods of engineering and introducing such receptors into cells are described in, for example, WO 200014257, WO 2013126726, WO 2012 / 129514, WO 2014031687, WO 2013 / 166321, WO 2013 / 071154, WO 2013 / 123061, U.S. Patent Application Publication Nos. 2002131960, 2013287748, and 201301 49337, 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, European Patent Application No. 2537416, and / or Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some embodiments, antigen receptors include CARs described in U.S. Patent No. 7,446,190 and those described in WO2014055668A1. Exemplary CARs include those disclosed in any of the aforementioned publications, e.g., WO 2014031687, U.S. Pat. No. 8,339,645, U.S. Pat. No. 7,446,179, U.S. Pat. App. Pub. No. 2013 / 0149337, U.S. Pat. No. 7,446,190, U.S. Pat. No. 8,389,282, in which the antigen-binding portion, e.g., scFv, is replaced with, e.g., an antibody provided herein.

[0266] Other DLL3-targeting CARs are described, for example, by Hudecek et al., Clin Cancer Res, 19(12), 3153-3164 (2013) and Baskar et al. MAbs. 4(3): 349-361 (2012). Also see International Publication No. 2014031687, U.S. Patent Application No. 2012 / 20058051.

[0267] A. extracellular antigen-binding domain Among chimeric receptors are chimeric antigen receptors (CARs). Chimeric receptors such as CARs generally comprise an extracellular antigen-binding domain that comprises, is, or is comprised in one or more of the provided anti-DLL3 antibodies or antigen-binding fragments. Thus, chimeric antigen receptors, e.g., CARs, typically comprise one or more DLL3-binding molecules, such as one or more antigen-binding fragments, domains, or portions, or one or more antibody variant domains and / or antibody molecules, such as those described herein, in the extracellular portion of the CAR. In some embodiments, a CAR comprises a variable weight (V) domain of an antibody. H ) chain region and / or variable light (V L In some embodiments, the CAR comprises a DLL3-binding portion or portions of an antibody molecule, such as a variable heavy (V) chain region, e.g., an scFv. H ) chain region and variable light (V L ) chain regions, e.g., scFv, of an antibody molecule. In some embodiments, the CAR comprises one or more of any of the 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 for exemplary antigen-binding domains, such as antibodies or antigen-binding fragments, that can be included in the provided DLL3-binding receptors, such as anti-DLL3 chimeric antigen receptors (CARs). In some aspects, the CAR comprises, e.g., an scFv listed in Table 2 as part of the extracellular antigen-binding domain. In some aspects, the CAR comprises, e.g., an scFv listed in Table 2 as part of the extracellular antigen-binding domain.H In some embodiments, the CAR comprises, for example, a V region as set forth in Table 2 as part of the extracellular antigen-binding domain. L In some embodiments, the DLL3-binding receptor comprises a V region comprising the CDR-H1, CDR-H2, and CDR-H3 sequences set forth in the SEQ ID NOs listed in each row of Table 2. H V region, including CDR-L1, CDR-L2, and CDR-L3 sequences L In some embodiments, the DLL3-binding receptor comprises a DLL3-binding antibody or fragment thereof comprising a V region as set forth in a SEQ ID NO: listed in each row of Table 2. H Region array and V L DLL3-binding antibodies or fragments thereof comprising the domain sequences or V shown in the SEQ ID NOs listed in each row of Table 2. H Region array and V L V having at least 95%, 96%, 97%, 98% or 99% sequence identity with the region sequence H Area and V L In some embodiments, the DLL3-binding receptor comprises an antibody comprising a V region amino acid sequence as set forth in a SEQ ID NO: listed in each row of Table 2. H Region array and V L In some embodiments, the DLL3-binding receptor comprises a DLL3-binding antibody or fragment thereof comprising an scFv sequence set forth in a SEQ ID NO: listed in a respective row of Table 2, or an antibody comprising an scFv amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the scFv sequence set forth in a SEQ ID NO: listed in a respective row of Table 2. In some embodiments, the DLL3-binding receptor comprises a DLL3-binding antibody or fragment thereof comprising an scFv sequence set forth in a SEQ ID NO: listed in a respective row of Table 2.

[0269] In some embodiments, the provided CARs comprise whole antibodies, as well as antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (V) fragments capable of specifically 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, including functional (antigen-binding) antibody fragments. In some embodiments, the antibody or fragment thereof contained in the CAR includes 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 di-scFv, tandem tri-scFv, etc.

[0270] b.Spacer In some embodiments, a recombinant receptor such as a CAR, e.g., an antibody portion thereof, further comprises a spacer (sometimes also referred to as a spacer region), which may comprise a hinge region, e.g., an IgG4 hinge region, and / or a C H 1 / C L , C H 2 and / or C H The scFv fragment may be or may comprise at least a portion of an immunoglobulin constant region, or a mutated or modified form thereof, such as an Fc region. In some embodiments, the constant region or portion is that of a human IgG, such as IgG4, IgG2, or IgG1. In some aspects, the portion of the constant region serves as a spacer between the antigen-recognizing component, e.g., an scFv, and the transmembrane domain.

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

[0272] In some embodiments, the spacer can be of a length that provides increased cellular responsiveness after antigen binding compared to the absence of the spacer or compared to an alternative spacer of a different length (e.g., longer). In some examples, the spacer is 12 amino acids or about 12 amino acids in length, or 12 amino acids or less in length. In some examples, the spacer is 15 amino acids or about 15 amino acids in length, or 15 amino acids or less in length.

[0273] Exemplary spacers include those having at least 10 or about 10 to 300 or about 300 amino acids, 10 or about 10 to 229 or about 229 amino acids, 10 or about 10 to 200 or about 200 amino acids, 10 or about 10 to 175 or about 175 amino acids, 10 or about 10 to 150 or about 150 amino acids, 10 or about 10 to 125 or about 125 amino acids, 10 or about 10 to 100 or about 100 amino acids, 10 or about 10 to 75 or about 75 amino acids, 10 or about 10 to 50 or about 50 amino acids, 10 or about 10 to 40 or about 40 amino acids, 10 or about 10 to 30 or about 30 amino acids, 10 or about 10 to 20 or about 20 amino acids, 10 or about 10 to 15 or about 15 amino acids, and those including any integer between the endpoints of either of the recited ranges. Exemplary spacers include those having at least or about 50 to 175 or about 175 amino acids, 50 or about 50 to 150 or about 150 amino acids, 10 or about 10 to 125 or about 125 amino acids, 50 or about 50 to 100 or about 100 amino acids, 100 or about 100 to 300 or about 300 amino acids, 100 or about 100 to 250 or about 250 amino acids, 125 or about 125 to 250 or about 250 amino acids, or 200 or about 200 to 250 or about 250 amino acids, as well as any integer between either endpoint of the recited range. In some embodiments, the spacer is at least or about 12 amino acids, at least 119 or about 119 amino acids, at least 125 or about 125 amino acids, at least 200 or about 200 amino acids, or at least 220 or about 220 amino acids, or at least 225 or about 225 amino acids in length. In some embodiments, the spacer is at least or about 13 amino acids in length, at least or about 120 amino acids in length, at least or about 120 amino acids in length, at least or about 125 amino acids in length, at least or about 200 amino acids in length, or at least or about 220 amino acids in length, or at least or about 229 amino acids in length.In some embodiments, the spacer is 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids or less in length. In some embodiments, the spacer is at least 100 or about 100 amino acids in length, for example, at least 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, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 7 230, 240, or 250 amino acids, 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 spacer comprises at least 125 or about 125 to 300 or about 300 amino acids, 125 or about 125 to 250 or about 250 amino acids, 125 or about 125 to 230 or about 230 amino acids, 125 or about 125 to 200 or about 200 amino acids, 125 or about 125 to 180 or about 180 amino acids, 125 or about 125 to 150 or about 150 amino acids, 150 or about 150 to 300 or about 300 amino acids, 150 or about 150 to 250 or about 250 amino acids, 150 or about 150 to 230 or about 230 amino acids, 150 or about 150 to 200 or about 200 amino acids, 150 or about 1 The length is 50 to 180 or about 180 amino acids, 180 or about 180 to 300 or about 300 amino acids, 180 or about 180 to 250 or about 250 amino acids, 180 or about 180 to 230 or about 230 amino acids, 180 or about 180 to 200 or about 200 amino acids, 200 or about 200 to 300 or about 300 amino acids, 200 or about 200 to 250 or about 250 amino acids, 200 or about 200 to 230 or about 230 amino acids, 230 or about 230 to 300 or about 300 amino acids, 230 or about 230 to 250 or about 250 amino acids, or 250 or about 250 to 300 or about 300 amino acids. 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.

[0275] Exemplary spacers include an IgG hinge alone, C H2 and C H IgG hinge linked to one or more of the three domains, or C H In some embodiments, the spacer comprises an IgG hinge linked to the C3 domain. In some embodiments, the spacer comprises an IgG hinge alone. In some embodiments, the spacer comprises an IgG hinge, C H 2 and / or C H 3 can be derived from all or a portion of IgG4 or IgG2, such as all or a portion of human IgG4 or human IgG2. In some embodiments, the spacer comprises a hinge, C ... H 2 and / or C H The hinge region may be a chimeric polypeptide containing one or more of the three sequences(s). 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, where the IgG4 hinge region is optionally a human IgG4 hinge region, and the IgG2 hinge region is optionally a human IgG2 hinge region; C H 2 regions, IgG4 C H 2 domain and / or IgG2 C H Contains all or part of the 2 domains, and IgG4 C H The two regions are optionally human IgG4 C H 2 domains, IgG2 C H The two regions are suitably human IgG2 C H 2 regions; and / or C H The three regions are IgG4 C H All or part of the three regions, and / or IgG2 C H Contains all or part of the three regions, IgG4 C H The three regions are, where appropriate, human IgG4 C H 3 regions, IgG2 C H The three regions are suitably human IgG2 C H In some embodiments, the hinge, C H 2 and C H 3 is the hinge region derived from IgG4, C H 2 and C HIn some embodiments, the hinge region is chimeric and comprises hinge regions derived from human IgG4 and human IgG2; H The two regions are chimeric and are derived from human IgG4 and human IgG2. H 2 regions; and / or C H The three regions are chimeric and are derived from human IgG4 and human IgG2. H In some embodiments, the spacer comprises three regions: an IgG4 / 2 chimeric hinge or a modified IgG4 hinge that contains at least one amino acid substitution compared to a human IgG4 hinge region; a human IgG2 / 4 chimeric hinge; H 2 domains; and human IgG4 C H Includes three areas.

[0276] In some embodiments, the spacer comprises or consists of all or a portion of an immunoglobulin hinge, or a modified version thereof. In some embodiments, the spacer is 15 or about 15 amino acids or less in length. In some embodiments, the spacer comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof, and / or comprises about 15 or fewer amino acids. In some embodiments, the spacer is 13 or about 13 amino acids in length and / or comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof. In some embodiments, the spacer is 12 or about 12 amino acids in length and / or comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof. In some embodiments, the spacer comprises the formula X1PPX2P (SEQ ID NO: 32), where 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 certain cases, the spacer has a methionine residue at the C-terminus. In some embodiments, the spacer comprises or consists of a sequence set forth in any 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 set forth in any of SEQ ID NOs: 33-39.

[0277] In some embodiments, the spacer comprises or consists of the 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 to SEQ ID NO:3. In some embodiments, the spacer comprises or consists of the sequence set forth in SEQ ID NO:3. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO:3. In some embodiments, the spacer consists of the sequence set forth in SEQ ID NO:3. In some embodiments, the spacer comprises or consists of SEQ ID NO:4, or an amino acid sequence encoded by 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:4. In some embodiments, the spacer comprises the amino acid sequence encoded by SEQ ID NO:4. In some embodiments, the spacer consists of the 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 to SEQ ID NO:1. In some embodiments, the spacer comprises or consists of the sequence set forth in SEQ ID NO:1. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO:1. In some embodiments, the spacer consists of the sequence set forth in SEQ ID NO:1. In some embodiments, the spacer comprises or consists of SEQ ID NO:2, or an amino acid sequence encoded by 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:2. In some embodiments, the spacer comprises the amino acid sequence encoded by SEQ ID NO:2. In some embodiments, the spacer consists of the amino acid sequence encoded by SEQ ID NO:2.

[0280] In some embodiments, the spacer comprises or consists of SEQ ID NO: 80, or an amino acid sequence encoded by 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: 80. In some embodiments, the spacer comprises the amino acid sequence encoded by SEQ ID NO: 80. In some embodiments, the spacer consists of the amino acid sequence encoded by SEQ ID NO: 80.

[0281] In some embodiments, the spacer is, for example, a C of a human immunoglobulin, e.g., IgG4 and / or IgG2. H or about 124, 125, or 125 amino acids in length, or a length between any of the foregoing. In some embodiments, the spacer is 119 or about 119 or 120 or 120 or about 120 amino acids in length.

[0282] In some embodiments, the spacer comprises or consists of the 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 to SEQ ID NO:5. In some embodiments, the spacer comprises or consists of the sequence set forth in SEQ ID NO:5. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO:5. In some embodiments, the spacer consists of the sequence set forth in SEQ ID NO:5. In some embodiments, the spacer comprises or consists of SEQ ID NO:57 or 81, or an amino acid sequence encoded by 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:57 or 81. In some embodiments, the spacer comprises the amino acid sequence encoded by SEQ ID NO:57 or 81. In some embodiments, the spacer consists of the 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 set forth in SEQ ID NO:58. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO:58. In some embodiments, the spacer consists of the sequence set forth in SEQ ID NO:58. In some embodiments, the spacer comprises or consists of SEQ ID NO:59 or 63, or an amino acid sequence encoded by 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 the amino acid sequence encoded by SEQ ID NO:59 or 63. In some embodiments, the spacer consists of the amino acid sequence encoded by SEQ ID NO:59 or 63.

[0285] In some embodiments, the spacer is a C fragment of a human immunoglobulin, such as, for example, IgG4 and / or IgG2. H 2 domain and C H or about 225, 226 or about 226, 227 or about 227, 228 or about 228, 229 or about 229, 230 or about 230, 231 or about 231, 232 or about 232, 233 or about 233, 234 or about 234, or 235 or about 235 amino acids in length, or a length between any of the foregoing. In some embodiments, the spacer is 228 or about 228 or 229 or about 229 amino acids in length.

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

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

[0288] In some embodiments, the spacer comprises or consists of the 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 an Ig hinge or hinge, chimeric C, such as that shown in SEQ ID NO: 41. H 2 area, and C H 3 regions or are encoded in a sequence such as that shown in SEQ ID NO:40.

[0290] In some embodiments, the spacer comprises or consists 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 thereto. In some embodiments, the spacer is encoded by a sequence comprising or consisting 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 thereto.

[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 set forth in SEQ ID NO: 42. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 42. In some embodiments, the spacer consists of the sequence set forth in SEQ ID NO: 42. In some embodiments, the spacer comprises or consists of SEQ ID NO: 43 or 60, or an amino acid sequence encoded by 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 the amino acid sequence encoded by SEQ ID NO: 43 or 60. In some embodiments, the spacer consists of the 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 set forth in SEQ ID NO:7. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO:7. In some embodiments, the spacer consists of the sequence set forth 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 set forth in SEQ ID NO: 82. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 82. In some embodiments, the spacer consists of the sequence set forth in SEQ ID NO: 82. In some embodiments, the spacer comprises or consists of SEQ ID NO: 83 or 84, or an amino acid sequence encoded by 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 the amino acid sequence encoded by SEQ ID NO: 83 or 84. In some embodiments, the spacer consists of the amino acid sequence encoded by SEQ ID NO:83 or 84.

[0294] Additional exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol. Res., 3(2):125-50, or International Publication No. WO2014031687. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce RNA heterogeneity during expression. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce cryptic splice sites or reduce the likelihood of splicing events at splice sites.

[0295] In some embodiments, the spacer is encoded by a polynucleotide that is optimized for codon usage and / or to reduce RNA heterogeneity, for example, by removing cryptic splice sites. In some embodiments, the spacer is encoded by a polynucleotide that is optimized for codon expression and / or to eliminate splice sites, such as cryptic splice sites. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:2. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:4. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:57. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:59. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:60. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:84.

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

[0297] In some embodiments, the spacer has an amino acid sequence that exhibits 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: 1 or 3, and is encoded by a polynucleotide that is, as appropriate, 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 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, as appropriate, encoded by a polynucleotide that is 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 about this percentage sequence identity to SEQ ID NO: 42 or 82, and is encoded by a polynucleotide that is, where appropriate, optimized for codon usage and / or to reduce RNA heterogeneity.

[0298] C transmembrane domain The antigen recognition component is generally linked to one or more intracellular signaling components, such as an antigen receptor complex, such as a TCR complex in the case of a CAR, and / or a signaling component that mimics activation via a signal through another cell surface receptor. Thus, in some embodiments, a DLL3 binding molecule (e.g., an antibody or its antigen-binding fragment) is linked to an intracellular signaling domain that includes one or more transmembrane domains, such as those described herein, and 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 that is naturally associated with one of the domains in a receptor, such as a CAR, is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

[0299] In some embodiments, the transmembrane domain is derived from either a natural or synthetic source. If the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane domains include those derived from (i.e., at least the transmembrane domain(s) thereof) the alpha, beta, or zeta chain of the T cell receptor, CD3 epsilon, 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., a CAR, is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27 amino acid transmembrane domain of human CD28 (Accession Number: P10747.1), or a 28 amino acid sequence, or is a transmembrane domain comprising the sequence of amino acids set forth in SEQ ID NO: 10 or 12, or a sequence of amino acids 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: 10 or 12.

[0300] In some embodiments, the transmembrane domain comprises or consists of the 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 set forth in SEQ ID NO: 10. In some embodiments, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 10. In some embodiments, the transmembrane domain consists of the sequence set forth 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 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 the amino acid sequence encoded by SEQ ID NO: 11. In some embodiments, the transmembrane domain consists of the 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 set forth in SEQ ID NO: 12. In some embodiments, the transmembrane domain consists of the sequence set forth 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 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 the amino acid sequence encoded by SEQ ID NO: 13. In some embodiments, the transmembrane domain consists of the amino acid sequence encoded by SEQ ID NO: 13. In some embodiments, the transmembrane domain comprises or consists of the 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 the 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 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 optimized for codon usage and / or to reduce RNA heterogeneity, for example, by removing cryptic splice sites, as appropriate. In some embodiments, the transmembrane domain has the amino acid sequence set forth in SEQ ID NO: 10 or 12 and is encoded by the polynucleotide sequence set forth in SEQ ID NO: 11 or 13, or a polynucleotide sequence having at least 90% sequence identity thereto. In certain cases, the transmembrane domain has an N-terminal methionine residue. In some embodiments, the transmembrane domain may be a CD28 transmembrane domain comprising the sequence of amino acids set forth in SEQ ID NO: 10 encoded by the nucleic acid sequence set forth in SEQ ID NO: 11. In some embodiments, the transmembrane domain may be a CD28 transmembrane domain comprising the sequence of amino acids set forth in SEQ ID NO: 12 encoded by the nucleic acid sequence set forth in SEQ ID NO: 13.

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

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

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

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

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

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

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

[0312] In some aspects, the transmembrane domain is synthetic in some embodiments. In some aspects, the synthetic transmembrane domain comprises primarily hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain. In some embodiments, the linkage is via a linker, spacer, and / or transmembrane domain(s).

[0313] D. intracellular signaling components Some intracellular signaling domains mimic or approximate signaling through natural antigen receptors, signaling through such receptors in combination with costimulatory receptors, and / or signaling through costimulatory receptors alone. In some embodiments, a short oligopeptide or polypeptide linker, e.g., a linker 2-10 amino acids in length, such as one containing glycine and serine, e.g., a glycine-serine doublet, is present to form the link between the transmembrane domain and the intracellular signaling domain of the CAR.

[0314] Receptors, e.g., CARs, generally comprise an intracellular signaling region comprising at least one intracellular signaling component(s). In some embodiments, receptors comprise an intracellular component or signaling domain of a TCR complex, such as a TCR CD3 chain, e.g., a CD3 zeta (CD3ζ) chain, which mediates T cell activation and cytotoxicity. Thus, in some aspects, a DLL3-binding antibody is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises 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 additional molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR comprises a chimeric molecule between CD3 zeta (CD3ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0315] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling region of the CAR stimulates and / or activates at least one of the normal effector functions or responses of an immune cell, e.g., a T cell engineered to express the CAR. For example, in some situations, the CAR induces a T cell function, e.g., cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of the entire immunostimulatory chain, e.g., if it transduces an effector function signal. In some embodiments, the intracellular signaling domain(s) comprise the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects, also includes that of a co-receptor that, in its natural context, acts in concert with such receptor to initiate signal transduction after antigen receptor binding, and / or any derivative or variant of such molecule, and / or any synthetic sequence having the same functional capability.

[0316] In the context of natural TCR, full activation generally requires not only TCR-mediated signal transduction but also costimulatory signal.Therefore, in some embodiments, CAR also comprises the component for generating secondary or costimulatory signal to promote full activation.In other embodiments, CAR does not comprise the component for generating costimulatory signal.In some aspects, additional CAR is expressed in the same cell and provides the component for generating secondary or costimulatory signal.

[0317] T cell activation is described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some embodiments, a CAR comprises one or both of these classes of cytoplasmic signaling sequences.

[0318] In some embodiments, CAR comprises a primary cytoplasmic signaling sequence that regulates the primary stimulation and / or activation of TCR complex.The primary cytoplasmic signaling sequence that acts in a stimulatory manner can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.Examples of ITAMs that contain a primary cytoplasmic signaling sequence include those derived from TCR or CD3 zeta, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon.In some embodiments, the intracellular signaling region in CAR contains a cytoplasmic signaling domain, a part thereof, or a sequence derived from CD3 zeta.

[0319] In some embodiments, the intracellular signaling domain comprises a human CD3 zeta stimulatory signaling domain or a functional variant thereof, such as the 112AA cytoplasmic domain of isoform 3 of human CD3ζ (accession number: P20963.2), or a CD3 zeta signaling domain 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 CD3 zeta signaling domain set forth 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 CD3 zeta signaling domain set forth in SEQ ID NO: 19. In some embodiments, the intracellular signaling domain comprises or consists of the human CD3 zeta signaling domain set forth in SEQ ID NO: 19. In some embodiments, the intracellular signaling domain comprises the human CD3 zeta signaling domain set forth in SEQ ID NO: 19. In some embodiments, the intracellular signaling domain consists of the human CD3 zeta signaling domain set forth 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 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 the amino acid sequence encoded by SEQ ID NO: 20. In some embodiments, the intracellular signaling domain consists of the 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 set forth in SEQ ID NO: 19. In some embodiments, the intracellular signaling domain comprises the sequence set forth in SEQ ID NO: 19. In some embodiments, the intracellular signaling domain consists of the sequence set forth 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 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 the amino acid sequence encoded by SEQ ID NO: 20. In some embodiments, the intracellular signaling domain consists of the 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 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 the amino acid sequence encoded by SEQ ID NO: 76. In some embodiments, the intracellular signaling domain consists of the 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 set forth in SEQ ID NO: 21. In some embodiments, the intracellular signaling domain comprises the sequence set forth in SEQ ID NO: 21. In some embodiments, the intracellular signaling domain consists of the sequence set forth 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 set forth in SEQ ID NO: 22. In some embodiments, the intracellular signaling domain comprises the sequence set forth in SEQ ID NO: 22. In some embodiments, the intracellular signaling domain consists of the sequence set forth in SEQ ID NO: 22.

[0326] In some embodiments, CD3 zeta comprises the sequence of amino acids set forth in SEQ ID NO: 19, which is encoded by the nucleic acid sequence set forth in SEQ ID NO: 20. In some embodiments, CD3 zeta is encoded by a polynucleotide that has been optimized for codon usage and / or to reduce RNA heterogeneity, as appropriate, e.g., by removing cryptic splice sites. In some embodiments, CD3 zeta has the amino acid sequence set forth in SEQ ID NO: 19 and is encoded by the polynucleotide sequence set forth in SEQ ID NO: 20.

[0327] In some embodiments, the CAR comprises a signaling domain (e.g., an intracellular or cytoplasmic signaling domain) and / or a transmembrane portion of a costimulatory molecule, such as 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 costimulatory signaling domain of 4-1BB or a functional variant or portion thereof, e.g., the 42 amino acid cytoplasmic domain of human 4-1BB (Accession Number: Q07011.1) or a functional variant or portion thereof, e.g., the sequence of amino acids set forth in SEQ ID NO: 17, or a sequence of amino acids exhibiting 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 set forth 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 set forth in SEQ ID NO: 17. In some embodiments, the intracellular signaling domain comprises or consists of the human 4-1BB costimulatory signaling domain set forth in SEQ ID NO: 17. In some embodiments, the intracellular signaling domain consists of the human 4-1BB costimulatory signaling domain set forth 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 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 the amino acid sequence encoded by SEQ ID NO: 18. In some embodiments, the intracellular signaling domain consists of the 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 set forth in SEQ ID NO: 17. In some embodiments, the intracellular signaling domain comprises the sequence set forth in SEQ ID NO: 17. In some embodiments, the intracellular signaling domain comprises or consists of the nucleic acid sequence encoded by SEQ ID NO: 18, 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: 18. In some embodiments, the intracellular signaling domain comprises the amino acid sequence encoded by SEQ ID NO: 18. In some embodiments, the intracellular signaling domain consists of the 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 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 4-1BB-derived costimulatory molecule is encoded by a polynucleotide that has been optimized for codon usage and / or to reduce RNA heterogeneity, e.g., by removing cryptic splice sites, as appropriate. In some embodiments, the 4-1BB-derived costimulatory domain has the amino acid sequence set forth in SEQ ID NO: 17 and is encoded by the polynucleotide sequence set forth 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, e.g., the 41 amino acid domain thereof and / or such a domain having an LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 15 or 16, or a sequence of amino acids 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 embodiments, the same CAR comprises both a stimulatory or activating component (e.g., a cytoplasmic signaling sequence) and a costimulatory component.

[0335] In some embodiments, the CAR comprises one or more, e.g., two or more, costimulatory domains and a stimulatory or activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components of CD3-zeta, CD28, and 4-1BB.

[0336] In some aspects, the transmembrane domain comprises the transmembrane portion of CD28. The extracellular domain and the transmembrane can be directly or indirectly connected. In some embodiments, the extracellular domain and the transmembrane are connected by a spacer, such as any of those described herein. In some embodiments, the chimeric antigen receptor comprises an intracellular domain of a T cell costimulatory molecule, such as between the transmembrane domain and the intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0337] In some embodiments, the stimulatory or activating component is contained within one CAR, while the costimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, the CAR comprises an activating or stimulatory CAR and a costimulatory CAR, both of which are expressed on the same cell (see International Publication No. WO2014 / 055668). In some aspects, the DLL3-targeting CAR is a stimulatory or activating CAR, and in other aspects, a costimulatory CAR. In some embodiments, the cell further comprises an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013)), such as a CAR that recognizes an antigen other than DLL3, whereby the stimulatory or activating signal delivered via the DLL3-targeting CAR is reduced or inhibited by the binding of the inhibitory CAR to its ligand, for example, reducing off-target effects.

[0338] In some embodiments, the two receptors induce activating and inhibitory signals to cells, respectively, so that the ligation of one receptor to the antigen activates the cell or induces a response, while the ligation of the second inhibitory receptor to the antigen induces a signal that suppresses or attenuates the response.An example is the combination of an activating CAR and an inhibitory CAR (iCAR).This strategy can be used to reduce the possibility of off-target effects, for example, in situations where the activating CAR binds to an antigen that is expressed in disease or condition but also expressed on normal cells, and the inhibitory receptor binds to a separate antigen that is not expressed on disease or condition cells but on normal cells.

[0339] In some embodiments, the chimeric receptor is or comprises an inhibitory CAR (e.g., iCAR), which comprises an intracellular component that attenuates or suppresses immune responses, such as ITAM and / or costimulatory promotion responses, within the cell. Examples of such intracellular signaling components are those found on immune checkpoint molecules such as PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, and EP2 / 4 adenosine receptors, including A2AR. In some embodiments, engineered cells comprise an inhibitory CAR that includes or is derived from the signaling domain of such inhibitory molecules, e.g., to serve to attenuate cellular responses induced by activating and / or costimulatory CARs.

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

[0341] e. Exemplary CAR In some embodiments, the provided anti-DLL3 CARs comprise an extracellular antigen-binding domain containing any of the anti-DLL3 antibodies or antigen-binding fragments described herein, such as in Section IA1 and / or Table 2.

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

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

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

[0345] In some embodiments, provided embodiments of anti-DLL3 CARs include an extracellular antigen-binding domain containing any of the anti-DLL3 antibodies or antigen-binding fragments described herein, such as in Section IA1 and / or Table 2; a modified IgG4 hinge C, such as one that is about 119 amino acids in length; H a spacer comprising: a spacer comprising: 3; a spacer comprising: 3; a spacer comprising: 5; such as that encoded by the nucleotide sequence set forth in SEQ ID NO: 6; a transmembrane domain, such as that derived from human CD28; or a transmembrane domain set forth in SEQ ID NO: 12; such as that 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 that derived from human 4-1BB;

[0346] In some embodiments, provided embodiments of anti-DLL3 CARs include an extracellular antigen-binding domain containing any of the anti-DLL3 antibodies or antigen-binding fragments described herein, such as in Section IA1 and / or Table 2; a modified IgG4 hinge C, such as one that is about 120 amino acids in length; H a spacer comprising: a spacer comprising: 3, or a spacer set forth in SEQ ID NO:58, such as that encoded by the nucleotide sequence set forth in SEQ ID NO:59; a transmembrane domain, such as that derived from human CD28, or a transmembrane domain set forth in SEQ ID NO:10, such as that encoded by the nucleotide sequence set forth 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 that derived from human 4-1BB set forth in SEQ ID NO:17, e.g., that encoded by the nucleotide sequence set forth in SEQ ID NO:18.

[0347] In some embodiments, provided embodiments of anti-DLL3 CARs include an extracellular antigen-binding domain containing any of the anti-DLL3 antibodies or antigen-binding fragments described herein, such as in Section IA1 and / or Table 2; an IgG4 / 2 chimeric hinge or modified IgG4 hinge, an IgG2 / 4 chimeric C H 2 domain, and IgG4 C, such as those of approximately 228 amino acids in length H a spacer comprising three regions, or the spacer set set forth in SEQ ID NO: 42, such as that encoded by the nucleotide sequence set forth in SEQ ID NO: 43; a transmembrane domain, such as that derived from human CD28, or the transmembrane domain set forth in SEQ ID NO: 12, such as that 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 that derived from human 4-1BB, set forth in SEQ ID NO: 17, for example, that encoded by the nucleotide sequence set forth in SEQ ID NO: 18.

[0348] In some embodiments, provided embodiments of anti-DLL3 CARs include an extracellular antigen-binding domain containing any of the anti-DLL3 antibodies or antigen-binding fragments described herein, such as in Section IA1 and / or Table 2; an IgG4 / 2 chimeric hinge or modified IgG4 hinge, an IgG4 / 2 chimeric C H 2 domain, and IgG4 C, such as those of approximately 229 amino acids in length Ha spacer comprising three regions, or the spacer set set forth in SEQ ID NO: 82, such as that encoded by the nucleotide sequence set forth in SEQ ID NO: 83; a transmembrane domain, such as that derived from human CD28, or the transmembrane domain set forth in SEQ ID NO: 10, such as that encoded by the nucleotide sequence set forth 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 that derived from human 4-1BB, set forth in SEQ ID NO: 17, for example, that encoded by the nucleotide sequence set forth in SEQ ID NO: 18.

[0349] In some of any such embodiments, the transmembrane domain is or comprises the sequence set forth in SEQ ID NO: 10. In some of any such embodiments, the transmembrane domain is or comprises the sequence set forth in SEQ ID NO: 12. In some of any such 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 certain embodiments, the intracellular signaling domain is the intracellular signaling domain of human 4-1BB. In some of any such embodiments, the intracellular signaling domain is or comprises the sequence set forth in SEQ ID NO: 17. In some of any such embodiments, the cytoplasmic signaling domain is a human CD3 zeta cytoplasmic signaling domain, such as that set forth in SEQ ID NO: 19. In some of any such embodiments, the intracellular signaling region comprises the sequence set forth in SEQ ID NO: 17 and SEQ ID NO: 19. In some of any such embodiments, as described in Section IE, Table 3, and / or Tables E1-4 and E8 herein. Also provided are CARs encoded by polynucleotides described in Section IF, Table 3, and / or Tables E1-4 and E8 herein. For example, polynucleotides containing any of the nucleotide sequences described herein are also provided, which encode all parts of the provided binding molecules.In certain cases, the transmembrane domain has a methionine residue at its N-terminus.In certain cases, the spacer has a methionine residue at its C-terminus.

[0350] In some of any of the provided embodiments, the anti-DLL3 chimeric antigen receptor is or comprises the sequence set forth 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 set forth in SEQ ID NO: 117. In some of any of the provided embodiments, the anti-DLL3 chimeric antigen receptor is or comprises the sequence set forth 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 set forth 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 set forth in SEQ ID NO: 117. In some embodiments, the anti-DLL3 chimeric antigen receptor consists of the sequence set forth in SEQ ID NO: 117. In some embodiments, the anti-DLL3 chimeric antigen receptor comprises or consists of the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence encoded by a nucleic acid sequence having at least or about this percentage sequence identity to SEQ ID NO: 116. In some embodiments, the anti-DLL3 chimeric antigen receptor comprises the amino acid sequence encoded by SEQ ID NO: 116. In some embodiments, the anti-DLL3 chimeric antigen receptor consists of the amino acid sequence encoded by SEQ ID NO: 116.

[0352] 2. Exemplary Features In some of any of the provided embodiments, a recombinant receptor, e.g., a CAR, containing a DLL3-binding antibody or antigen-binding fragment thereof binds, e.g., specifically binds, to DLL3, such as DLL3 on the surface of cancer cells or tumor cells. In any of the embodiments, the antibody or antigen-binding fragment thereof 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 characteristics of antigen recognition, such as interspecies cross-reactivity, as described herein, such as in Section IA2. In some embodiments, a CAR containing an extracellular antigen-binding domain comprising an antibody or fragment thereof exhibits similar or substantially the same binding properties and characteristics as an antibody or fragment thereof. In some aspects, exemplary properties of the antigen-binding portion of a CAR, e.g., comprising either an antibody or antigen-binding fragment thereof, are similar to the affinities described for antibodies or antigen-binding fragments thereof, such as in Section IA3. In some cases, a CAR containing an extracellular antigen-binding domain comprising an antibody or fragment thereof exhibits binding properties and characteristics that differ from those of an antibody or fragment thereof.

[0353] In some embodiments, the provided CAR specifically binds to the delta-like ligand 3 (DLL3) protein. In some of the embodiments herein, DLL3 refers to human DLL3. The observation that an antibody or other binding molecule, e.g., a CAR, binds to or specifically binds to DLL3 does not necessarily mean that it binds to DLL3 from any species. For example, in some embodiments, DLL3 binding characteristics, such as the ability to specifically bind to DLL3 and / or the ability to compete for binding to DLL3 with a reference receptor, e.g., a reference CAR containing a reference antibody, and / or the ability to bind with a particular affinity or compete to a particular extent, refer to the ability with respect to human DLL3, and the antibody may not have this characteristic with respect to DLL3 of another species, such as a mouse. In some embodiments, the CAR binds to human DLL3 and binds to DLL3 of another species, such as a rhesus monkey or a cynomolgus monkey. In some embodiments, the CAR or its antigen-binding fragment binds to human DLL3 and does not bind to DLL3 of another species, such as a mouse. In some embodiments, the 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 human DLL3, such as the mature human DLL3 sequence set forth in SEQ ID NO: 122 or 123, or an allelic variant or splice variant thereof, e.g., to one or more epitopes or regions of human DLL3. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to one or more epitopes within human DLL3. In some embodiments, the human DLL3 comprises the sequence of amino acids set forth in SEQ ID NO: 120. In some embodiments, the human DLL3 comprises the sequence of amino acids set forth in SEQ ID NO: 121. In some embodiments, a human DLL3 isoform (mature, isoform 1) comprises the sequence of amino acids set forth in SEQ ID NO: 122. In some embodiments, a human DLL3 isoform (mature, isoform 2) comprises the sequence of amino acids set forth in SEQ ID NO: 123. In some embodiments, the antibody or antigen-binding fragment thereof binds to the extracellular domain DLL3, e.g., one or more extracellular epitopes present within the extracellular domain 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 linear epitopes 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 that includes 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 set forth 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 set forth in SEQ ID NO: 120). In some embodiments, the one or more epitopes comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acid residues within amino acid residues 27-84 corresponding to 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, relative to the human DLL3 precursor sequence set forth in SEQ ID NO: 120.

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

[0359] In some embodiments, the CAR binds to a non-human DLL3, such as the DLL3 of monkey, rabbit, rat, mouse, or other species. In some embodiments, the CAR binds to a non-human DLL3, such as the cynomolgus monkey (Macaca fascicularis) DLL3, e.g., an epitope or region of 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 CAR binds to a mouse (Mus musculus) DLL3, e.g., an epitope or region of mouse DLL3, such as the mouse DLL3 set forth in SEQ ID NO: 124 (Genbank Number: NP_031892.2).

[0360] In some embodiments, the provided CAR can bind to DLL3, such as human DLL3, with at least a certain affinity, as measured by any of a number of known methods. In some embodiments, affinity can be measured using the equilibrium dissociation constant (K D In some embodiments, affinity is represented by EC 50 In some embodiments, the provided CAR is represented by the K D or EC 50 Shows.

[0361] In some embodiments, the binding affinity of binding molecules such as anti-DLL3 CARs to different antigens (e.g., DLL3 proteins) from different species can be compared to determine interspecies cross-reactivity.For example, interspecies cross-reactivity can be classified as high cross-reactivity or low cross-reactivity.In some embodiments, the equilibrium dissociation constant K of different antigens (e.g., DLL3 proteins) from different species (e.g., humans, cynomolgus monkeys, or mice) can be determined. D In some embodiments, the species cross-reactivity of an 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 of human DLL3. D and species mutant DLL3 K D is 1 or about 1. In some embodiments, the anti-DLL3 CAR may have low species cross-reactivity, e.g., an anti-DLL3 CAR has high affinity for human DLL3 but low affinity for species variant DLL3, or vice versa. For example, the K D and human DLL3 K D 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] Some of the provided CARs exhibit antigen-dependent activity or signaling, i.e., CARs that exhibit measurably absent or background levels of signaling activity in the absence of an antigen, such as DLL3, and / or in the presence of a non-specific antigen. Thus, in some aspects, the provided CARs exhibit no tonic signaling or antigen-independent activity or signaling, or only background, tolerable, or low levels thereof, in the absence of an antigen, such as DLL3. In some embodiments, the provided anti-DLL3 CAR-expressing cells exhibit biological activities or functions, including cytotoxic activity, cytokine production, and the ability to proliferate. In some embodiments, the provided CAR receptors exhibit the same, substantially the same, or higher antigen-specific and / or antigen-dependent activity or signaling compared to a reference DLL3-specific chimeric antigen receptor under the same or substantially the same conditions or assays, as appropriate.

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

[0364] In some embodiments, engineered cells expressing a provided anti-DLL3 CAR, when administered to a subject in adoptive cell therapy, exhibit improved biological or functional activity, such as anti-tumor activity, tumor growth inhibition, tumor volume reduction, persistence, expansion, or prolonged subject survival. In some embodiments, engineered cells expressing a provided CAR exhibit improved biological or functional activity, such as anti-tumor activity, tumor growth inhibition, tumor volume reduction, persistence, expansion, or prolonged subject survival, compared to engineered cells expressing a reference DLL3-specific chimeric antigen receptor under the same or substantially the same conditions or assays, as appropriate. In some embodiments, the biological or functional activity, e.g., cytotoxic activity, of the chimeric receptor can be measured using any of a number of known methods. Activity can be assessed or determined either in vitro or in vivo. In some embodiments, activity can be assessed once the cells are administered to a subject (e.g., a human). Parameters for evaluation include specific binding of engineered or natural T cells or other immune cells to an antigen, e.g., in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of engineered cells to destroy target cells can be measured using any suitable method, such as the cytotoxicity assays described in, e.g., 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 cells can also be measured by assaying the expression and / or secretion of certain cytokines, such as interleukin-2 (IL-2), interferon-gamma (IFNγ), interleukin-4 (IL-4), TNF-alpha (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD107a, and / or TGF-beta (TGFβ). Assays for measuring cytokines are well known and include, but are not limited to, ELISA, intracellular cytokine staining, cytometric bead arrays, RT-PCR, ELISPOT, flow cytometry, and bioassays in which cells responsive to the relevant cytokine are tested for responsiveness (e.g., proliferation) in the presence of a test sample. In some aspects, biological activity can be measured using animal models of a disease or condition, such as tumor xenograft models, to assess tumor burden or burden and / or reduced survival. In some aspects, biological activity is measured by assessing a clinical outcome, such as a reduction in tumor burden or burden.

[0365] In some embodiments, administration of engineered cells expressing a provided anti-DLL3 CAR exhibits substantially improved tumor growth inhibition, in vivo persistence, and / or prolonged survival of tumor-bearing subjects compared to administration of engineered cells expressing a reference CAR. In some aspects, such improvement is 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 increased persistence and expansion compared to engineered cells expressing a reference DLL3-specific chimeric antigen receptor under the same or substantially the same conditions or assays, as appropriate. In some aspects, the proliferation, expansion, and / or persistence of engineered cells expressing any of the provided receptors, e.g., CARs, can be assessed by determining 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 CD3 + cells, CD4 + cells, CD8 + cells, CD3 + CAR + cells, CD4 + CAR + cells or CD8 + CAR + The exposure, number, or levels of a subset thereof, such as cells, can be assessed in a subject, such as a human or animal subject, to which the engineered cells have been administered. In some embodiments, the exposure, number, concentration, persistence, and proliferation are related to pharmacokinetic parameters. In some cases, pharmacokinetics is measured by measuring the maximum (peak) plasma concentration (C) after administration. max ), peak (i.e., maximum plasma concentration (C max ) occurs when T max ), minimum plasma concentration (i.e., the therapeutic agent, e.g., CAR + Minimum plasma concentration between doses of T cells; C min ), elimination half-life (T 1 / 2 ) and area under the curve (i.e., the CAR of the therapeutic agent + The plasma concentration of a particular therapeutic agent, such as CAR T cells, can be assessed by measuring parameters such as the area under the curve (AUC) generated by plotting the plasma concentration of T cells versus time. + The concentration of T cells can be measured by administering a therapeutic agent, e.g., CAR T cells, to a sample of blood.+ Any known method suitable for assessing the concentration of T cells 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, ELISA or chromatography / mass spectrometry-based assays can be used.

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

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

[0369] In some embodiments, the provided anti-DLL3 CARs exhibit improved expression on the surface of cells, for example, compared to alternative CARs having the same amino acid sequence but encoded by non-splice site elimination and / or codon-optimized nucleotide sequences. In some embodiments, the expression of the recombinant receptor on the surface of cells can be assessed. Techniques for determining the expression of the recombinant receptor on the surface of cells can include the use of chimeric antigen receptor (CAR)-specific antibodies (e.g., Brentjens et al., Sci. Transl. Med. 2013 Mar; 5(177): 177ra38), protein L (Zheng et al., J. Transl. Med. 2012 Feb; 10:29), epitope tags, and monoclonal antibodies that specifically bind to CAR polypeptides (see WO2014190273). In some embodiments, the expression of the recombinant receptor on the surface of cells, e.g., primary T cells, can be assessed by flow cytometry, for example, using a binding molecule that can bind to the recombinant receptor or a portion thereof that can be detected. In some embodiments, the binding molecule used to detect expression of the recombinant receptor is an anti-idiotypic antibody, e.g., an anti-idiotypic agonist antibody specific for a binding domain, e.g., an 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.

[0370] F. Polynucleotides Encoding Binding Molecules Polynucleotides encoding binding molecules such as anti-DLL3 antibodies, antigen-binding fragments thereof, recombinant receptors (e.g., chimeric antigen receptors), and / or portions thereof, e.g., strands or fragments, are also provided. Among the provided polynucleotides are those encoding the anti-DLL3 antibodies (e.g., antigen-binding fragments) or chimeric antigen receptors described herein. Polynucleotides can include those containing naturally occurring and / or non-naturally occurring nucleotides and bases, including those with backbone modifications. The terms "nucleic acid molecule," "nucleic acid," "sequence of nucleotides," and "polynucleotide" can be used interchangeably and refer to a polymer of nucleotides. Such a polymer of nucleotides can contain natural and / or non-natural nucleotides, including, but not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides that comprises a nucleic acid molecule or polynucleotide.

[0371] Polynucleotides containing nucleic acids encoding the anti-DLL3 antibodies or antigen-binding domains thereof described herein, or any portion, fragment, chain, or domain thereof, are provided. In some embodiments, the antibodies or antigen-binding fragments thereof contain multiple domains or chains (e.g., heavy and light chains), and all of the antibodies or antigen-binding fragments thereof are encoded by a single polynucleotide. Polynucleotides containing nucleic acids encoding any of the single-chain cell surface proteins described herein are also provided. Polynucleotides containing nucleic acids encoding any of the conjugates described herein are also provided. Polynucleotides containing nucleic acids encoding any of the anti-DLL3 chimeric antigen receptors described herein are also provided. In some embodiments, binding molecules such as antibodies or antigen-binding fragments thereof or recombinant receptors contain multiple domains or chains (e.g., heavy and light chains), and all of the binding molecules are encoded by more than one polynucleotide, such as two or more polynucleotides. In some embodiments, the polynucleotides are contained within a vector.

[0372] In some aspects, polynucleotides are provided that contain nucleic acid sequences that encode 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 that encode a portion, fragment, strand, or domain of any of the binding molecules provided herein, e.g., in Sections IA and IE.

[0373] In some cases, a polynucleotide encoding a DLL3-binding molecule, such as an antibody or antigen-binding fragment thereof, or a recombinant receptor (e.g., a CAR), comprises a signal sequence encoding a signal peptide, in some cases 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., a CAR), or connected to the 5' end of the nucleic acid sequence encoding the antigen-binding domain. In some cases, a polynucleotide containing a nucleic acid sequence encoding a DLL3-binding molecule, such as an antibody or antigen-binding fragment thereof, or a recombinant receptor (e.g., a CAR), comprises a signal sequence encoding a signal peptide. In some embodiments, the signal sequence may encode a signal peptide derived from a native polypeptide. In other embodiments, the signal sequence may encode a heterologous or non-natural signal peptide. In some embodiments, a non-limiting exemplary signal peptide includes the signal peptide of the IgG kappa chain set forth in SEQ ID NO:50 or encoded by the nucleotide sequence set forth in SEQ ID NO:51. In some embodiments, a non-limiting exemplary signal peptide includes the signal peptide of the GMCSFR alpha chain set forth in SEQ ID NO:52 or encoded by the nucleotide sequence set forth in SEQ ID NO:53. In some embodiments, non-limiting exemplary signal peptides include the signal peptide of the CD8 alpha signal peptide set forth in SEQ ID NO: 54. In some embodiments, non-limiting exemplary signal peptides include the signal peptide of the CD33 signal peptide set forth in SEQ ID NO: 48 and encoded by the nucleotide sequence set forth in SEQ ID NO: 49. In some embodiments, a nucleotide sequence encoding the CD33 signal peptide is set forth in SEQ ID NO: 78 or 79. In some cases, a polynucleotide encoding a DLL3-binding molecule, such as an antibody or antigen-binding fragment thereof or a recombinant receptor (e.g., a CAR), can contain nucleic acid sequences encoding additional molecules, such as surrogate or other markers, or can contain additional components, such as promoters, regulatory elements, and / or multicistronic 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., a CAR), can be operably linked to any of the additional components.

[0374] In some embodiments, the variable heavy chain domain (VH) of an antibody or antigen-binding fragment thereof, or a recombinant receptor containing the antibody or antigen-binding fragment thereof, H In some embodiments, a polynucleotide containing a nucleic acid sequence encoding the variable light chain domain (V) of an antibody or antigen-binding fragment thereof, or a recombinant receptor containing the antibody or antigen-binding fragment thereof, is provided. L In some embodiments, a polynucleotide containing a nucleic acid sequence encoding a variable heavy chain domain (V) of an antibody or antigen-binding fragment thereof, or a recombinant receptor containing the antibody or antigen-binding fragment thereof, is provided. H ) and variable light chain domain (V L A polynucleotide containing a nucleic acid sequence encoding the .

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

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

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

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

[0379] In some embodiments, V H The nucleic acid encoding the region is V L In some embodiments, the V H The nucleic acid encoding the region is V L In certain embodiments, the nucleic acid encoding the scFv may be 3' to the nucleic acid encoding the V HThe nucleic acid encoding the region or a portion thereof is followed by a nucleic acid encoding a linker, followed by a V L In certain embodiments, the nucleic acid encoding the scFv can be followed by a nucleic acid encoding a region or portion thereof. L The nucleic acid encoding the region or a portion thereof is followed by a nucleic acid encoding a linker, followed by a V H Nucleic acid encoding the region or a portion thereof can follow.

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

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

[0382] In some embodiments, the polynucleotide comprises a nucleic acid encoding a spacer comprising the sequence set forth in 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 to SEQ ID NO: 2. In some embodiments, the polynucleotide comprises the sequence set forth 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 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 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 set forth in SEQ ID NO: 59, 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. In some embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NO: 59.

[0386] In some embodiments, the polynucleotide comprises a nucleic acid encoding a spacer comprising the sequence set forth in SEQ ID NO: 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: 60. In some embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NO: 60.

[0387] In some embodiments, the polynucleotide comprises a nucleic acid encoding a spacer comprising the sequence set forth in SEQ ID NO: 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: 84. In some embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NO:84.

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

[0389] In mammalian cells, transcribed nucleic acid sequences are generally processed co-transcriptionally or immediately after transcription. The nascent precursor messenger ribonucleic acid (pre-mRNA) transcribed from genomic deoxyribonucleic acid (DNA) sequences is sometimes edited by splicing to remove introns, and then exons are linked in eukaryotic cells. Although consensus sequences of splice sites are known, in some embodiments, the specific nucleotide information defining a splice site may be complex and may not be readily apparent based on available methods. A cryptic splice site is a splice site that is not predicted based on a standard consensus sequence and is variably activated. Therefore, variable splicing of pre-mRNA at a cryptic splice site results in heterogeneity of transcribed mRNA products when expressed in eukaryotic cells.

[0390] The polynucleotides generated for the expression of transgenes are typically constructed from nucleic acid sequences such as complementary DNA (cDNA) or portions thereof that do not contain introns. Therefore, splicing of such sequences is not expected to occur. However, the presence of hidden splice sites within the cDNA sequence may result in unintended or undesired splicing reactions and heterogeneity of the transcribed mRNA. Such heterogeneity may result in the translation of unintended protein products, such as truncated protein products with variable amino acid sequences that exhibit modified expression and / or activity.

[0391] In some embodiments, the elimination of splice sites, such as cryptic splice sites, can improve or optimize the expression of transgene products, such as polypeptides translated from transgenes, such as anti-DLL3 CAR polypeptides. Splicing of an encoded transgene, such as an encoded DLL3 CAR molecule, at cryptic splice sites can result in reduced protein expression, for example, on the cell surface, and / or reduced function, for example, reduced intracellular signaling. Polynucleotides encoding anti-DLL3 CAR proteins that have been optimized to reduce or eliminate cryptic splice sites are provided herein. Also provided herein are polynucleotides encoding anti-DLL3 CAR proteins that are optimized for codon expression, and / or that contain one or more sequences, such as those identified by the methods or observations herein regarding splice sites, and / or that do not contain any identified splice sites, such as any of the splice sites identified herein. Among the provided polynucleotides are those that, when expressed under certain conditions and / or introduced into certain cell types, such as human T cells, such as primary T cells, exhibit RNA heterogeneity or splice forms that are below a certain degree, as well as cells and compositions and articles of manufacture that contain such polypeptides and / or exhibit such properties. In some embodiments, the RNA heterogeneity of the transcribed RNA is reduced by more than 10%, 15%, 20%, 25%, 30%, 40%, 50% or more, or by more than about this percent or more, compared to a polynucleotide that has not been modified to remove cryptic splice sites and / or by codon optimization. In some embodiments, the provided polynucleotides encoding anti-DLL3 CARs exhibit at least 70%, 75%, 80%, 85%, 90%, or 95% or more RNA heterogeneity of the transcribed RNA.

[0392] RNA heterogeneity can be determined by any of the many methods provided or described herein or known. In some embodiments, the RNA heterogeneity of a transcribed nucleic acid is determined by amplifying the transcribed nucleic acid, such as by reverse transcriptase polymerase chain reaction (RT-PCR), and then detecting one or more differences, such as size differences, in one or more amplification products. In some embodiments, RNA heterogeneity is determined based on the number of amplification products of different sizes or the proportion of amplification products of various different sizes. In some embodiments, RNA, such as total RNA or cytoplasmic polyadenylated RNA, is collected from cells expressing an optimized transgene and amplified by reverse transcription polymerase chain reaction (RT-PCR) using a primer specific to the 5' untranslated region (5' UTR) located upstream of the transgene in the transcribed RNA, which in some cases corresponds to a portion of the promoter sequence in the expression vector, and a primer specific to the 3' untranslated region (3' UTR) located downstream of the transgene expressed in the transcribed RNA sequence, or a primer specific to a sequence within the transgene. In certain embodiments, the transgene is amplified using at least one primer complementary to a sequence in the 5' untranslated region (UTR) and at least one primer complementary to a sequence in the 3' untranslated region (UTR). RNA, such as messenger RNA, can be degraded and its heterogeneity analyzed by several methods. Non-limiting exemplary methods include agarose gel electrophoresis, chip-based capillary electrophoresis, analytical centrifugation, field-flow fractionation, and chromatography, such as size-exclusion chromatography or liquid chromatography.

[0393] In some embodiments, the provided polynucleotides encoding the anti-DLL3 CARs provided herein or the constructs provided herein comprise modifications to remove one or more splice donor and / or acceptor sites that may contribute to splicing events and / or reduced expression and / or increased RNA heterogeneity. In some embodiments, the provided polynucleotides are modified in one or more polynucleotides in the spacer region to eliminate or reduce splicing events.

[0394] In some embodiments, the polynucleotide is a modified polynucleotide, including a polynucleotide modified for codon optimization (O) and / or splice site elimination (SSE). Examples of SEQ ID NOs for such pol...

Claims

1. Heavy chain variable (V Ｈ ) region and the light chain variable (V Ｌ 1. An anti-delta-like ligand 3 (DLL3) antibody or antigen-binding fragment thereof comprising a nucleotide sequence comprising: V Ｈ the regions comprising heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2) and heavy chain complementarity determining region 3 (CDR-H3) contained within SEQ ID NO: 90; V Ｌ the regions comprising light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2) and light chain complementarity determining region 3 (CDR-L3) contained within SEQ ID NO: 91; An anti-DLL3 antibody or antigen-binding fragment thereof.

2. Heavy chain variable (V Ｈ ) region and the light chain variable (V Ｌ ) region, V Ｈ the regions comprise a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in SEQ ID NO: 92, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in SEQ ID NO: 93, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in SEQ ID NO: 94; V Ｌ the regions comprising a light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in SEQ ID NO: 95, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in SEQ ID NO: 96, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence set forth in SEQ ID NO: 97; An anti-DLL3 antibody or antigen-binding fragment thereof.

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

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

5. V Ｈ the regions comprising heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2) and heavy chain complementarity determining region 3 (CDR-H3) contained within SEQ ID NO: 90; V Ｌ the regions comprising light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2) and light chain complementarity determining region 3 (CDR-L3) contained within SEQ ID NO: 91; The anti-DLL3 antibody or antigen-binding fragment thereof according to claim 4.

6. V Ｈ the regions comprise a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in SEQ ID NO: 92, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in SEQ ID NO: 93, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in SEQ ID NO: 94; V Ｌ the regions comprising a light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in SEQ ID NO: 95, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in SEQ ID NO: 96, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence set forth in SEQ ID NO: 97; The anti-DLL3 antibody or antigen-binding fragment thereof according to claim 4 or 5.

7. V Ｈ The region comprises the sequence set forth in SEQ ID NO: 90; V Ｌ The region comprises the sequence set forth in SEQ ID NO: 91; The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. Heavy chain variable (V Ｈ ) region and the light chain variable (V Ｌ ) region, V Ｈ The region comprises the sequence set forth in SEQ ID NO: 90; V Ｌ The region comprises the sequence set forth in SEQ ID NO: 91; An anti-DLL3 antibody or antigen-binding fragment thereof.

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

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

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

13. 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 13. The anti-DLL3 antibody or antigen-binding fragment thereof of claim 11 or 12, comprising:

14. 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 The anti-DLL3 antibody or antigen-binding fragment thereof according to claims 1 to 13, comprising:

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

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

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

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

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

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

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

46.

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

89.

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

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

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

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

27. Approximately 1×10 －１１ M ~ approx. 1 x 10 －７ The equilibrium dissociation constant of M (K Ｄ The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 26, which binds to human DLL3 at the nucleotide sequence (SEQ ID NO: 1).

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

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

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

31. 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 the anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 27, an extracellular antigen-binding domain comprising a transmembrane domain and an intracellular signaling region.

33. The anti-DLL3 CAR of claim 32, further comprising a spacer between the extracellular 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. 36. The anti-DLL3 CAR of claim 35, wherein at least a portion of the hinge region comprises all or a portion of an IgG4 hinge region, optionally a human IgG4 hinge region or a variant thereof, optionally the variant IgG4 hinge region comprising a substitution of amino acids CPSC to CPPC compared to a wild-type IgG4 hinge region.

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

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

39. The anti-DLL3 CAR according to any of claims 33 to 38, wherein the spacer comprises an amino acid sequence having at least or approximately 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 according to any of claims 33 to 39, wherein the spacer comprises a sequence shown in SEQ ID NO:

1.

41. The anti-DLL3 CAR according to any of claims 33 to 38, wherein the spacer comprises an amino acid sequence having at least or approximately 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 according to any one of claims 33 to 38 and 41, wherein the spacer comprises a sequence shown in SEQ ID NO:

3.

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

44. the spacer is 111 or about 111, 112 or about 112, 113 or about 113, 114 or about 114, 115 or about 115, 116 or about 116, 117 or about 117, 118 or about 118, 119 or about 119, 120 or about 120, 121 or about 121, 122 or about 122, 123 or about 123, 124 or about 124, or 125 or about 125 amino acids in length; The anti-DLL3 CAR according to any of claims 33 to 36 and 43, wherein the spacer has a length of 1 or 2, or a length between any of the above, and optionally the spacer comprises the sequence set forth in SEQ ID NO: 5 or 58, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or approximately this percentage, sequence identity to SEQ ID NO: 5 or 58.

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

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

47. The anti-DLL3 CAR according to any of claims 32 to 46, wherein the transmembrane domain comprises a transmembrane domain derived from CD4, CD28, or CD8.

48. The anti-DLL3 CAR according to any one of claims 32 to 47, wherein the transmembrane domain comprises a transmembrane domain derived from CD28, suitably human CD28.

49. The anti-DLL3 CAR according to any of claims 32 to 48, wherein the transmembrane domain comprises SEQ ID NO: 10 or 12, or an amino acid sequence having at least or approximately 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 according to any of claims 33 to 49, wherein the transmembrane domain comprises an amino acid sequence having at least or approximately 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 according to any of claims 33 to 50, wherein the transmembrane domain comprises the sequence shown in SEQ ID NO:

10.

52. The anti-DLL3 CAR according to any of claims 33 to 49, wherein the transmembrane domain comprises an amino acid sequence having at least or approximately 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 according to any one of claims 33 to 49 and 52, wherein the transmembrane domain comprises the sequence shown in SEQ ID NO:

12.

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

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

19.

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

19.

58. The anti-DLL3 CAR according to any of claims 32 to 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 according to any of claims 58 to 60, wherein the costimulatory domain comprises an intracellular signaling domain of CD28, 4-1BB, or ICOS.

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

63. The anti-DLL3 CAR according to any of claims 58 to 62, wherein the costimulatory domain comprises a sequence as set forth in SEQ ID NO: 17, or an amino acid sequence having at least or approximately 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 of claims 58 to 61, wherein the costimulatory domain comprises the intracellular signaling domain of CD28, optionally human CD28.

65. The anti-DLL3 CAR according to any of claims 58 to 61 and 64, wherein the costimulatory domain comprises a sequence set forth in SEQ ID NO: 15 or 16, or an amino acid sequence having at least or approximately 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 according to any of claims 32 to 65, comprising an extracellular antigen-binding domain, a spacer, a transmembrane domain, and an intracellular signaling region in this order from the N-terminus to the C-terminus.

67. extracellular antigen-binding domains, including scFvs; Optionally, a spacer comprising a modified IgG4 hinge comprising the sequence shown in SEQ ID NO: 1; Optionally, a transmembrane domain comprising the sequence set forth in SEQ ID NO: 10, optionally a transmembrane domain derived from human CD28; and an intracellular signaling region comprising the cytoplasmic signaling domain of the CD3 zeta (CD3ζ) chain and, optionally, the intracellular signaling domain of a costimulatory molecule, including the intracellular signaling domain of 4-1BB; in this order from the N-terminus to the C-terminus.

68. extracellular antigen-binding domains, including scFvs; Optionally, a spacer comprising a modified IgG4 hinge comprising the sequence shown in SEQ ID NO: 3; Optionally, a transmembrane domain comprising the sequence set forth in SEQ ID NO: 12, optionally a transmembrane domain derived from human CD28; and an intracellular signaling region comprising the cytoplasmic signaling domain of the CD3 zeta (CD3ζ) chain and, optionally, the intracellular signaling domain of a costimulatory molecule, including the intracellular signaling domain of 4-1BB; in this order from the N-terminus to the C-terminus.

69. An extracellular antigen-binding domain comprising an scFv, Heavy chain variable (V Ｈ ) region and the light chain variable (V Ｌ ) region, V Ｈ the regions comprising heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2) and heavy chain complementarity determining region 3 (CDR-H3) contained within SEQ ID NO: 90; V Ｌ the regions comprising light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2) and light chain complementarity determining region 3 (CDR-L3) contained within SEQ ID NO: 91; extracellular antigen-binding domain; a spacer comprising the sequence shown in SEQ ID NO: 1; A transmembrane domain comprising the sequence set forth in SEQ ID NO: 10; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3 zeta (CD3ζ) chain comprising the sequence set forth in SEQ ID NO: 19 and an intracellular signaling domain of a costimulatory molecule comprising the sequence set forth in SEQ ID NO: 17; An anti-DLL3 chimeric antigen receptor (CAR) comprising:

70. An extracellular antigen-binding domain comprising an scFv, Heavy chain variable (V Ｈ ) region and the light chain variable (V Ｌ ) region, V Ｈ the regions comprising heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2) and heavy chain complementarity determining region 3 (CDR-H3) contained within SEQ ID NO: 90; V Ｌ the regions comprising light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2) and light chain complementarity determining region 3 (CDR-L3) contained within SEQ ID NO: 91; extracellular antigen-binding domain; a spacer comprising the sequence shown in SEQ ID NO: 3; A transmembrane domain comprising the sequence set forth in SEQ ID NO: 12; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3 zeta (CD3ζ) chain comprising the sequence set forth in SEQ ID NO: 19 and an intracellular signaling domain of a costimulatory molecule comprising the sequence set forth in SEQ ID NO: 17; An anti-DLL3 chimeric antigen receptor (CAR) comprising:

71. an extracellular antigen-binding domain comprising an scFv comprising the sequence set forth in SEQ ID NO: 89; a spacer comprising the sequence shown in SEQ ID NO: 1; A transmembrane domain comprising the sequence set forth in SEQ ID NO: 10; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3 zeta (CD3ζ) chain comprising the sequence set forth in SEQ ID NO: 19 and an intracellular signaling domain of a costimulatory molecule comprising the sequence set forth in SEQ ID NO: 17; in this order from the N-terminus to the C-terminus.

72. an extracellular antigen-binding domain comprising an scFv comprising the sequence set forth in SEQ ID NO: 89; a spacer comprising the sequence shown in SEQ ID NO: 3; A transmembrane domain comprising the sequence set forth in SEQ ID NO: 12; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3 zeta (CD3ζ) chain comprising the sequence set forth in SEQ ID NO: 19 and an intracellular signaling domain of a costimulatory molecule comprising the sequence set forth in SEQ ID NO: 17; The anti-DLL3 CAR according to any one of claims 33 to 38, 54 to 63, 66, 68 and 70, comprising, in this order from the N-terminus to the C-terminus.

73. The anti-DLL3 CAR according to any of claims 52 to 72, comprising a sequence set forth in SEQ ID NO: 117, or a sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:

117.

74. The anti-DLL3 CAR according to any one of claims 52 to 73, comprising a sequence shown in SEQ ID NO:

117.

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

76. 29. 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 to 31.

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

79. V comprising the sequence set forth in SEQ ID NO:87 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96, 97%, 98% or 99% or about this percentage sequence identity to SEQ ID NO:

87. Ｈ and a nucleic acid encoding V comprising the sequence set forth in SEQ ID NO:88 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96, 97%, 98% or 99% or about this percentage sequence identity to SEQ ID NO:

88. Ｌ Nucleic acid encoding 79. The polynucleotide of any one of claims 75 to 78, comprising:

80. V comprising the sequence shown in SEQ ID NO: 87 Ｈ and a nucleic acid encoding V comprising the sequence shown in SEQ ID NO: 88 Ｌ Nucleic acid encoding 80. The polynucleotide of any one of claims 75 to 79, comprising:

81. 81. The polynucleotide of any of claims 75 to 80, wherein the antigen-binding fragment is a single-chain Fv (scFv), and 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 about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

86.

82. 82. The polynucleotide of any one of claims 75 to 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 set forth in SEQ ID NO:

86.

83. 83. A polynucleotide according to any of claims 78 to 82, comprising a nucleic acid encoding a spacer comprising the sequence set out in 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 to SEQ ID NO:

2.

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

2.

85. 83. A polynucleotide according to any of claims 78 to 82, comprising 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 about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

4.

86. 86. A polynucleotide according to any one of claims 78 to 82 and 85, comprising a nucleic acid encoding a spacer comprising the sequence shown in SEQ ID NO:

4.

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

11.

88. 88. A polynucleotide according to any one of claims 78 to 84 and 87, comprising a nucleic acid encoding a transmembrane domain comprising the sequence set forth in SEQ ID NO:

11.

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

13.

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

13.

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

116.

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

116.

93. 93. The polynucleotide of any one of claims 75 to 92, which is optimized by splice site elimination.

94. 94. The polynucleotide of any one of claims 75 to 93, which is codon-optimized for expression in a human cell.

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

96. 96. The vector of claim 95, which is a viral vector.

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

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

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

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

101. A cell comprising a polynucleotide according to any one of claims 75 to 94 or a vector according to any one of claims 95 to 98.

102. The cell according to any one of claims 99 to 101, which is a lymphocyte.

103. The cell according to any one of claims 99 to 102, which is a NK cell or a T cell.

104. The cell according to any one of claims 99 to 103, which 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 to 104, which is a primary cell obtained from a subject.

106. 106. The cell of any of claims 100-105, wherein, among the plurality of cells, at or about 10%, 9% or about 9%, 8% or about 8%, 7% or about 7%, 5% or about 5%, 4% or about 4%, 3% or about 3%, 2% or about 2%, or less than 1% or about 1% of the 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 to 106.

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

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

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

1.

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

112. 106. 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 of any of claims 1 to 27, a single chain cell surface protein of claim 28, a conjugate of any of claims 29 to 31, an anti-DLL3 chimeric antigen receptor of any of claims 32 to 74, a polynucleotide of any of claims 75 to 94, a vector of any of claims 95 to 98, a cell of any of claims 99 to 106 or a composition of any of claims 107 to 111.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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