Proteins containing a delta-like ligand 3 (DLL3) antigen-binding domain and their use

Abstract

Antibodies and antigen-binding regions that bind to Delta-like protein 3 (DLL3) are described. Multispecific antigen-binding constructs, e.g., bispecific antibodies, that contain antigen-binding regions that bind to DLL3 are also described. The present application also describes methods of treatment or detection using anti-DLL3 antibodies, antigen-binding fragments thereof, or multispecific antigen-binding constructs, as well as related molecules, compositions, and methods.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 63 / 094,933 and U.S. Provisional Application No. 63 / 094,934, filed on 22 October 2020, and the disclosures of each of these applications are incorporated herein by reference in their entirety.

[0002] (Reference to electronically submitted sequence listings) This application includes a sequence listing submitted electronically via EFS-Web as an ASCII-formatted sequence listing, which has the filename "sequence listing JBI6411", a creation date of October 7, 2021, and a size of 275KB. The sequence listing submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety.

[0003] (Field of Invention) This application relates to a protein containing an antigen-binding domain that binds to delta-like canonical Notch ligand 3 (DLL3), as well as related compositions and methods. [Background technology]

[0004] Prostate cancer is the second most common cancer, the sixth leading cause of cancer death in men, and accounts for 14% (903,500) of all new cancer cases and 6% (258,400) of all cancer deaths in men worldwide. Metastatic prostate cancer is the second leading cause of cancer death in men in the United States. The progression of prostate cancer from diagnosis to death is best classified into a series of clinical stages based on the severity of the disease, hormonal status, and the presence or absence of detectable metastasis: localized disease, elevated prostate-specific antigen (PSA) levels without detectable metastasis after radiotherapy or surgery, and clinical metastasis in the uncastrated or castrated stage. Surgery, radiation therapy, or a combination of both can be curative for patients with localized disease, but a significant proportion of these patients have recurrent disease, evidenced by elevated PSA levels, which can also lead to metastasis formation and progression to the terminal stage of the disease, particularly in high-risk groups.

[0005] Androgen depletion therapy (ADT) is the standard treatment, and the generally predictable outcomes are a decrease in PSA, a stable period with no tumor growth, followed by an increase in PSA and regrowth as a castration-resistant disease. For many years, ADT has been the standard treatment for patients with metastatic prostate cancer.

[0006] However, recent clinical data suggest that androgen depletion therapy may lead to the emergence of an androgen-independent tumor phenotype known as neuroendocrine prostate cancer (NEPC) through the process of cellular redifferentiation. Delta-like canonical Notch ligand 3 (DLL3) has been shown to be abundant in NEPC tumors at both the RNA and protein levels. Therefore, strategies designed to target DLL3 may have clinical utility in the NEPC / small cell carcinoma patient population.

[0007] Small cell lung cancer accounts for approximately 20% of all lung cancers. Because lymph node or distant metastasis often occurs by the time of diagnosis, small cell lung cancer progresses rapidly and is difficult to remove surgically. This cancer shows a high early response rate to anticancer drugs. Therefore, chemotherapy is considered the first-line treatment option. However, the cancer quickly becomes resistant to chemotherapy, recurs, and the 3-year survival rate falls below 5%.

[0008] Therefore, new therapies are needed to treat cancers such as NEPC, small cell carcinoma, or small cell lung cancer.

[0009] In normal cells, DLL3 regulates intracellular notch signaling. In cancer cells, DLL3 is expressed extracellularly; for example, in humans, it has eight extracellular domains containing 618 amino acids and six EGF-like repeats. Human DLL3 is highly homologous to that of cynomolgus monkeys and mice / rats, sharing 96% and 83% amino acid sequence identity, respectively, but has less than 40% identity with DLL1 and DLL4. DLL3 has low to undetectable expression in normal tissues, but is highly expressed on the cell surface of neuroendocrine tumors, including small cell lung cancer, prostate cancer, large cell carcinoma, and bladder cancer, and is a target for T cell redirection for the treatment of neuroendocrine cancers. [Overview of the project] [Means for solving the problem]

[0010] In a general embodiment, the present disclosure relates to an isolated protein comprising an antigen-binding region that binds to delta-like protein 3 (DLL3), wherein the antigen-binding region binds to an epitope within residues 429-618 of human DLL3 as described in SEQ ID NO: 263.

[0011] In some embodiments, the isolated protein comprises a) a heavy chain variable region (VH) having heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3 of VH having the amino acid sequence of SEQ ID NO: 1, and a light chain variable region (LCDR) 1, LCDR2, and LCDR3 of VL having the amino acid sequence of SEQ ID NO: 2 region, VL), b) VH having HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence of SEQ ID NO: 3, and VL having LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence of SEQ ID NO: 4, c) VH having HCDR1, HCDR2, and HCDR3 of VH having SEQ ID NO: 5, and VL having LCDR1, LCDR2, and LCDR3 of VL having SEQ ID NO: 6, d) VH having HCDR1, HCDR2, and HCDR3 of VH having SEQ ID NO: 7, and VL having LCDR1, LCDR2, and LC DR3 includes antigen-binding regions that compete for binding to DLL3 with the reference antibody, including e) HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 9, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 10, f) HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 11, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 12, or g) HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 13, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 14. Optionally, the reference antibody includes HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 3, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 4.

[0012] The proteins isolated by random selection were: a) SEQ ID NOs. 15, 16, 17, 33, 34, 35, respectively; b) SEQ ID NOs. 18, 19, 20, 36, 37, 38, respectively; c) SEQ ID NOs. 21, 22, 23, 39, 37, 40, respectively; d) SEQ ID NOs. 24, 25, 26, 41, 42, 43, respectively; e) SEQ ID NOs. 18, 28, 29, 44, 45, 46, respectively; f) SEQ ID NOs. 30, 31, 32, 47, 48, 49, respectively; g) SEQ ID NOs. 50, 51, 17, 33, 34, 3 5, h) containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of 5, 51, 17, 33, 34, 35 respectively, i) containing HCDR1, 54, 20, 36, 37, 38 respectively, j) containing HCDR1, 56, 23, 39, 37, 40 respectively, k) containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of 57, 58, 26, 41, 42, 43 respectively, l) containing HCDR1, 60, 29, 44, 45, 46 respectively, or m) containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of 61, 62, 32, 47, 48, 49 respectively. Optionally, isolated proteins may contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of 15, 16, 17, 33, 34, and 35 respectively. Optionally, the antigen-binding region that binds to DLL3 is scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH. Optionally, the antigen-binding region that binds to DLL3 is Fab. Optionally, the antigen-binding region that binds to DLL3 is scFv. Optionally, scFv contains VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH), from the N-terminus to the C-terminus. Optionally, L1 contains a) approximately 5-50 amino acids, b) approximately 5-40 amino acids, c) approximately 10-30 amino acids, and d) approximately 10-20 amino acids. Optionally, L1 contains the amino acid sequence of SEQ ID NOs. 27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139. Optionally, L1 contains the amino acid sequence of SEQ ID NO. 120.

[0013] The disclosure also provides antigen-binding regions that bind to DLL3, including the VH of SEQ ID NOs: 1, 3, 5, 7, 9, 11, or 13 and the VL of SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14. Optionally, the antigen-binding regions that bind to DLL3 include a) the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2, b) the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 4, c) the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 6, d) the VH of SEQ ID NO: 7 and the VL of SEQ ID NO: 8, e) the VH of SEQ ID NO: 9 and the VL of SEQ ID NO: 10, f) the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12, and / or g) the VH of SEQ ID NO: 13 and the VL of SEQ ID NO: 14. Optionally, the antigen-binding region that binds to DLL3 includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to the VH of SEQ ID NO: 3, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to the VL of SEQ ID NO: 4. Optionally, the antigen-binding region that binds to DLL3 includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to the amino acid sequence of SEQ ID NO: 63 or 64.

[0014] This disclosure provides isolated proteins that are monospecific proteins or multispecific antigen-binding constructs. Optionally, the isolated protein is a multispecific antigen-binding construct. Optionally, the multispecific antigen-binding construct is a bispecific protein. Optionally, the multispecific antigen-binding construct is a tripspecific protein. Optionally, the multispecific antigen-binding construct includes an antigen-binding region that binds to an antigen on a lymphocyte. Optionally, the lymphocyte is a T cell. Optionally, the T cell is CD8 +These are T cells. Optionally, the lymphocytes are natural killer (NK) cells. Optionally, in the multispecific antigen-binding construct, the antigens on the lymphocytes are CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C. Optionally, the antigen on the lymphocytes is CD3ε.

[0015] In some embodiments, the multispecific antigen-binding construct includes an antigen-binding region that binds to CD3ε, a) heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 98, HCDR2 of SEQ ID NO: 99, HCDR3 of SEQ ID NO: 100, light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 106, LCDR2 of SEQ ID NO: 107, and LCDR3 of SEQ ID NO: 108, and b) VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85. In some embodiments, the antigen-binding region that binds to CD3ε in the multispecific antigen-binding construct includes HCDR1 of SEQ ID NO: 98, HCDR2 of SEQ ID NO: 99, HCDR3 of SEQ ID NO: 100, LCDR1 of SEQ ID NO: 106, LCDR2 of SEQ ID NO: 107, and LCDR3 of SEQ ID NO: 108. In some embodiments, the multispecific antigen-binding construct includes an antigen-binding region that binds to CD3ε, which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to the VH of SEQ ID NO: 84, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to the VL of SEQ ID NO: 85.

[0016] In some embodiments, the multispecific antigen-binding construct includes an antigen-binding region that binds to CD3ε, comprising a) heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 96, HCDR3 of SEQ ID NO: 97, light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 101, LCDR2 of SEQ ID NO: 102, and LCDR3 of SEQ ID NO: 104, or b) VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80. In some embodiments, the multispecific antigen-binding construct includes an antigen-binding region that binds to CD3ε, comprising a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to the VH of SEQ ID NO: 77 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to the VL of SEQ ID NO: 80.

[0017] This disclosure also relates to isolated multispecific antigen-binding constructs comprising an antigen-binding region that binds to delta-like protein 3 (DLL3), wherein the antigen-binding region that binds to DLL3 corresponds to a) SEQ ID NOs. 15, 16, 17, 33, 34, 35, respectively; b) SEQ ID NOs. 18, 19, 20, 36, 37, and 38, respectively; c) SEQ ID NOs. 21, 22, 23, 39, 37, and 40, respectively; d) SEQ ID NOs. 24, 25, 26, 41, 42, and 43, respectively; e) SEQ ID NOs. 18, 28, 29, 44, 45, and 46, respectively; f) SEQ ID NOs. 30, 31, 32, 47, 48, and 49, respectively; g) SEQ ID NOs. 50, 51, 17, 33, 34, and 35, respectively; h) SEQ ID NOs. 52, 51, 17, 33, 34, and 35, respectively; i) SEQ ID NOs. 53, 54, 20, 36, respectively. The present invention provides a multispecific antigen-binding construct comprising: 37, 38, j) SEQ ID NOs. 55, 56, 23, 39, 37, and 40, respectively; k) SEQ ID NOs. 57, 58, 26, 41, 42, and 43, respectively; l) SEQ ID NOs. 59, 60, 29, 44, 45, and 46, respectively; m) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NOs. 61, 62, 32, 47, 48, and 49; n) VH of SEQ ID NO. 1 and VL of SEQ ID NO. 2; o) VH of SEQ ID NO. 3 and VL of SEQ ID NO. 4; p) VH of SEQ ID NO. 5 and VL of SEQ ID NO. 6; q) VH of SEQ ID NO. 7 and VL of SEQ ID NO. 8; r) VH of SEQ ID NO. 9 and VL of SEQ ID NO. 10; s) VH of SEQ ID NO. 11 and VL of SEQ ID NO. 12; or t) VH of SEQ ID NO. 13 and VL of SEQ ID NO. 14. Optionally, the multispecific antigen-binding constructs include binding domains that bind to DLL3, containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 15, 16, 17, 33, 34, and 35, respectively.

[0018] In certain embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising an antigen-binding region that binds to delta-like protein 3 (DLL3), wherein the antigen-binding region that binds to DLL3 comprises the heavy chain complementarity-determining regions (HCDRs) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 3, and the light chain complementarity-determining regions (LCDRs) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 4.

[0019] The disclosure also provides an isolated multispecific antigen-binding construct comprising an antigen-binding region that binds to delta-like protein 3 (DLL3), wherein the antigen-binding region that binds to DLL3 comprises the heavy chain variable region (VH) of SEQ ID NO: 3 and the light chain variable region (VL) of SEQ ID NO: 4.

[0020] Optionally, the protein is conjugated to an isolated half-life extension region (moiety). Optionally, the half-life extension region is immunoglobulin (Ig), an Ig fragment, an Ig constant region, an Ig constant region fragment, an Fc region, transferrin, albumin, an albumin-binding domain, or polyethylene glycol. Optionally, an Ig constant region fragment contains an Fc region. Optionally, an antigen-binding region that binds to DLL3 is conjugated to the N-terminus of the Ig constant region or an Ig constant region fragment. Optionally, an antigen-binding region that binds to DLL3 is conjugated to the C-terminus of the Ig constant region or an Ig constant region fragment via a second linker (L2). Optionally, L2 contains the amino acid sequence of SEQ ID NOs. 27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139. Optionally, the Ig constant region or fragment of the Ig constant region is the IgG1, IgG2, IgG3, or IgG4 isotype. Optionally, the Ig constant region or fragment of the Ig constant region is the IgG1 isotype. Optionally, the Ig constant region or fragment of the Ig constant region contains at least one mutation resulting in reduced binding of the protein to the Fcγ receptor (FcγR).Optionally, at least one mutation resulting in reduced binding to FcγR is found in F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331 The group was selected from A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, with residue numbering following the EU index. Optionally, the mutation resulting in reduced protein binding to FcγR is L234A_L235A_D265S.

[0021] This disclosure provides an isolated protein comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region comprises a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively; b) VH of SEQ ID NO. 1 and VL of SEQ ID NO. 2; c) VH of SEQ ID NO. 3 and VL of SEQ ID NO. 4; d) scFv of SEQ ID NO. 63; and / or e) scFv of SEQ ID NO. 64. Optionally, the isolated protein comprises an antigen-binding region that binds to DLL3, wherein the antigen-binding region comprises a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively; and / or b) VH of SEQ ID NO. 1 and VL of SEQ ID NO. 2. Optionally, isolated proteins include antigen-binding regions that bind to DLL3, and these antigen-binding regions include a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 18, 19, 20, 36, 37, and 38, respectively; b) VH of SEQ ID NO. 5 and VL of SEQ ID NO. 6, and / or scFv of SEQ ID NO. 65. Optionally, isolated proteins include antigen-binding regions that bind to DLL3, and these antigen-binding regions include a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 21, 22, 23, 39, 37, and 40, respectively; b) VH of SEQ ID NO. 7 and VL of SEQ ID NO. 8, and / or scFv of SEQ ID NO. 66. Optionally, an isolated protein containing an antigen-binding region that binds to DLL3, wherein the antigen-binding region comprises a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 24, 25, 26, 41, 42, and 43, respectively, and b) VH of SEQ ID NO. 9 and VL of SEQ ID NO. 10, and / or scFv of SEQ ID NO. 68. Optionally, an isolated protein containing an antigen-binding region that binds to DLL3, wherein the antigen-binding region comprises a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 27, 28, 29, 44, 45, and 46, respectively, and b) VH of SEQ ID NO. 11 and VL of SEQ ID NO. 12, and / or scFv of SEQ ID NO. 68.Optionally, isolated proteins include antigen-binding regions that bind to DLL3, the antigen-binding regions being a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 30, 31, 32, 47, 48, and 49, respectively, and b) VH of SEQ ID NO. 13 and VL of SEQ ID NO. 14, and / or scFv of SEQ ID NO. 69.

[0022] Optionally, the isolated protein is a multispecific antigen-binding construct containing an antigen-binding region that binds to CD3ε. Optionally, the multispecific antigen-binding construct contains an antigen-binding region that binds to CD3ε, including the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 98, HCDR2 of SEQ ID NO: 99, HCDR3 of SEQ ID NO: 100, light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 106, LCDR2 of SEQ ID NO: 107, and LCDR3 of SEQ ID NO: 108, and / or b) the VH of SEQ ID NO: 84 and the VL of SEQ ID NO: 85. Optionally, the multispecific antigen-binding construct includes a) an antigen-binding region that binds to CD3ε, comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 96, HCDR3 of SEQ ID NO: 97, light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 101, LCDR2 of SEQ ID NO: 102, and LCDR3 of SEQ ID NO: 104, and / or b) an antigen-binding region that binds to CD3ε, comprising VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80.

[0023] This disclosure provides an isolated anti-DLL3 / anti-CD3 protein comprising a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to a lymphocyte antigen. Optionally, the lymphocyte antigen is a T cell antigen. Optionally, the T cell antigen is CD8 + This is a T cell antigen. Optionally, the lymphocyte antigen is an NK cell antigen. Optionally, the lymphocyte antigens are CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C. Optionally, the lymphocyte antigen is CD3ε.

[0024] Optionally, in isolated anti-DLL3 / anti-CD3 proteins, the first antigen-binding region that binds to DLL3 and / or the second antigen-binding region that binds to lymphocyte antigens includes scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH. Optionally, the first antigen-binding region that binds to DLL3 and / or the second antigen-binding region that binds to lymphocyte antigens includes Fab. Optionally, the first antigen-binding region that binds to DLL3 and / or the second antigen-binding region that binds to lymphocyte antigens includes scFv. Optionally, the first antigen-binding region that binds to DLL3 includes scFv, and the second antigen-binding region that binds to lymphocyte antigens includes Fab. Optionally, the first antigen-binding region that binds to DLL3 includes Fab, and the second antigen-binding region that binds to lymphocyte antigens includes scFv. Optionally, scFv contains VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH), from the N-terminus to the C-terminus. Optionally, L1 contains a) approximately 5-50 amino acids, b) approximately 5-40 amino acids, c) approximately 10-30 amino acids, or d) approximately 10-20 amino acids. Optionally, L1 contains the amino acid sequence of SEQ ID NOs. 27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139. Optionally, L1 contains the amino acid sequence of SEQ ID NO. 120.

[0025] Optionally, in isolated anti-DLL3 / anti-CD3 proteins, the first antigen-binding region that binds to DLL3 includes HCDR1 of SEQ ID NOs. 15, 18, 21, 24, 27, 30, 50, 52, 53, 55, 57, 59, or 61; HCDR2 of SEQ ID NOs. 16, 19, 22, 25, 28, 31, 51, 54, 56, 58, 60, or 62; HCDR3 of SEQ ID NOs. 17, 20, 23, 26, 29, 32, 17, 20, 23, 26, 29, or 32; LCDR1 of SEQ ID NOs. 33, 36, 39, 41, 44, or 47; LCDR2 of SEQ ID NOs. 34, 37, 42, 45, or 48; and LCDR3 of SEQ ID NOs. 35, 38, 40, 43, 46, or 49. Optionally, the first antigen-binding region to bind to DLL3 is: a. SEQ ID NOs. 15, 16, 17, 33, 34, 35, b. SEQ ID NOs. 18, 19, 20, 36, 37, 38, c. SEQ ID NOs. 21, 22, 23, 39, 37, 40, d. SEQ ID NOs. 24, 25, 26, 41, 42, 43, e. SEQ ID NOs. 18, 28, 29, 44, 45, 46, f. SEQ ID NOs. 30, 31, 32, 47, 48, 49, g. SEQ ID NOs. 50, 51, 17, 33, 34, 35, h. contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 52, 51, 17, 33, 34, 35 respectively; i. contain HCDR1, 54, 20, 36, 37, 38 respectively; j. contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 55, 56, 23, 39, 37, 40 respectively; k. contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 57, 58, 26, 41, 42, 43 respectively; l. contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 59, 60, 29, 44, 45, 46 respectively; or m. contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 61, 62, 32, 47, 48, 49 respectively. Optionally, the first antigen-binding region that binds to DLL3 contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 15, 16, 17, 33, 34, 35 respectively.

[0026] In some embodiments, the first antigen-binding region that binds to DLL3 includes a. the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2, b. the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 4, c. the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 6, d. the VH of SEQ ID NO: 7 and the VL of SEQ ID NO: 8, e. the VH of SEQ ID NO: 9 and the VL of SEQ ID NO: 10, f. the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12, or g. the VH of SEQ ID NO: 13 and the VL of SEQ ID NO: 14. Optionally, the first antigen-binding region that binds to DLL3 includes the amino acid sequence of SEQ ID NO: 63 or 64. Optionally, the first antigen-binding region that binds to DLL3 includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 64. Optionally, the first antigen-binding region that binds to DLL3 includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to the VH of SEQ ID NO: 3, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 99%, at least 95%, or at least 100%) identical to the VL of SEQ ID NO: 4. Optionally, the second antigen-binding region that binds to CD3 includes an HCDR1 of SEQ ID NO: 95 or 98, an HCDR2 of SEQ ID NO: 96 or 99, an HCDR3 of SEQ ID NO: 97 or 100, an LCDR1 of SEQ ID NO: 101 or 106, an LCDR2 of SEQ ID NO: 102 or 107, and an LCDR3 of SEQ ID NO: 104 or 108. Optionally, the second antigen-binding region that binds to CD3 includes HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 96, HCDR3 of SEQ ID NO: 97, LCDR1 of SEQ ID NO: 101, LCDR2 of SEQ ID NO: 102, and LCDR3 of SEQ ID NO: 104. Optionally, the second antigen-binding region that specifically binds to CD3 includes HCDR1 of SEQ ID NO: 98, HCDR2 of SEQ ID NO: 99, HCDR3 of SEQ ID NO: 100, LCDR1 of SEQ ID NO: 106, LCDR2 of SEQ ID NO: 107, and LCDR3 of SEQ ID NO: 108.Optionally, the second antigen-binding region that binds to CD3 includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to the VH of SEQ ID NO: 77 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to the VL of SEQ ID NO: 80. Optionally, the second antigen-binding region that specifically binds to CD3 includes a VH of SEQ ID NO: 77 and a VL of SEQ ID NO: 80. Optionally, the second antigen-binding region that binds to CD3 includes VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to VH of SEQ ID NO: 84, and VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to VL of SEQ ID NO: 85. Optionally, the second antigen-binding region that binds to lymphocyte antigens includes VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85.

[0027] In some embodiments, a first antigen-binding region that binds to DLL3 is conjugated to a first immunoglobulin (Ig) constant region or a fragment of the first Ig constant region, and / or a second antigen-binding region that binds to a lymphocyte antigen is conjugated to a second immunoglobulin (Ig) constant region or a fragment of the second Ig constant region. Optionally, the isolated anti-DLL3 / anti-CD3 protein further includes a second linker (L2) between the first antigen-binding region that binds to DLL3 and the first Ig constant region or the first Ig constant region, and between the second antigen-binding region that binds to a lymphocyte antigen and the second Ig constant region or a fragment of the second Ig constant region. Optionally, L2 contains the amino acid sequence of SEQ ID NOs. 27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, or 138. Optionally, the Ig constant region fragment contains the Fc region. Optionally, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region are IgG1, IgG2, IgG3, or IgG4 isotypes. Optionally, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region are IgG1 isotypes. Optionally, the first Ig constant region or fragment of the first Ig constant region and the second Ig constant region or fragment of the second Ig constant region contain at least one mutation resulting in reduced binding of the multispecific antigen-binding construct to FcγR.Optionally, at least one mutation resulting in reduced binding of the multispecific antigen-binding construct to FcγR is present in the following variants: F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A32 The group was selected from the following: 7G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, with residue numbering following the EU index. Optionally, the mutation that results in reduced binding of the multispecific antigen-binding construct to FcγR is L234A_L235A_D265S. Optionally, the protein contains at least one mutation in the CH3 domain of the Ig constant region. Optionally, the at least one mutation in the CH3 domain of the Ig constant region is one of the following: T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y The group is selected from 407V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, and T350V / T366L / K392L / T394W, and the residue numbering follows the EU index.

[0028] In a general embodiment, this application is, (1) A first antigen-binding region that binds to DLL3, wherein the first antigen-binding region includes a first VH having HCDR1, HCDR2, and HCDR3, and a first VL having LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are (a) Sequence numbers 15, 16, 17, 33, 34, 35 respectively (b) Sequence numbers 18, 19, 20, 36, 37, 38 respectively (c) Sequence numbers 21, 22, 23, 39, 37, 40 respectively, (d) Sequence numbers 24, 25, 26, 41, 42, 43 respectively, (e) Sequence numbers 18, 28, 29, 44, 45, 46 respectively, (f) Sequence numbers 30, 31, 32, 47, 48, 49 respectively, (g) Sequence numbers 50, 51, 17, 33, 34, 35 respectively, (h) Sequence numbers 52, 51, 17, 33, 34, 35 respectively, (i) Sequence numbers 53, 54, 20, 36, 37, 38 respectively, (j) Sequence numbers 55, 56, 23, 39, 37, 40 respectively, (k) Sequence numbers 57, 58, 26, 41, 42, 43 respectively, (l) Sequence numbers 59, 60, 29, 44, 45, 46, or (m) A first antigen-binding region that binds to DLL3, containing the amino acid sequences of SEQ ID NOs. 61, 62, 32, 47, 48, and 49, respectively. (2) A second antigen-binding region that binds to CD3ε, (a) A second VH having the HCDR1, HCDR2, and HCDR3 amino acid sequences of SEQ ID NOs. 95, 96, and 97, respectively, and a second VL having the LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NOs. 101, 102, and 104, respectively, or (b) relating to a bispecific antigen-binding construct comprising a second antigen-binding region, which includes a second VH having the HCDR1, HCDR2, and HCDR3 amino acid sequences of SEQ ID NOs. 98, 99, and 100, respectively, and a second VL having the LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NOs. 106, 107, and 108, respectively.

[0029] The bispecific antigen-binding construct is referred to herein as the "anti-DLL3 / anti-CD3 construct" or "anti-DLL3 / anti-CD3."

[0030] In some embodiments, the isolated anti-DLL3 / anti-CD3 protein comprises a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3, a) the first antigen-binding domain that binds to DLL3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, 35, respectively, and the second domain that binds to lymphocyte antigens comprises SEQ ID NOs. 95, 96, 97, 101, 10 2, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of 104, and / or b) a first antigen-binding region that binds to DLL3 comprising Fab comprising VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2, and a second antigen-binding region that binds to CD3 comprising scFv of SEQ ID NO: 105, and / or c) isolated anti-DLL3 / anti-CD3 protein comprising HC1 of SEQ ID NO: 109, LC1 of SEQ ID NO: 110, and HC1 of SEQ ID NO: 112.

[0031] In some embodiments, the isolated anti-DLL3 / anti-CD3 protein comprises a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3, a) the first antigen-binding domain that binds to DLL3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 comprises SEQ ID NOs. 95, 96, 97, 101, and 102, respectively. a) comprising 104 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and / or b) a first antigen-binding region that binds to DLL3 comprising Fab comprising VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2, and a second antigen-binding region that binds to CD3 comprising scFv of SEQ ID NO: 119, and / or c) an isolated anti-DLL3 / anti-CD3 protein comprising HC1 of SEQ ID NO: 109, LC1 of SEQ ID NO: 110, and HC1 of SEQ ID NO: 113.

[0032] In some embodiments, the isolated anti-DLL3 / anti-CD3 protein comprises a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3, a. The first antigen-binding domain that binds to DLL3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 comprises SEQ ID NOs. 98, 99, 100, 106, and 107, respectively. b. The first antigen-binding region that binds to DLL3 comprises 108 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and / or c. The isolated anti-DLL3 / anti-CD3 protein comprises HC1 of SEQ ID NO: 111, HC2 of SEQ ID NO: 116, and LC2 of SEQ ID NO: 117.

[0033] In some embodiments, an isolated anti-DLL3 / anti-CD3 protein comprises a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3, wherein the first antigen-binding domain that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes SEQ ID NOs. 95, 96, 97, 101, and 10 2. The first antigen-binding region that binds to DLL3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and / or b. The first antigen-binding region that binds to DLL3 comprises scFv of SEQ ID NO: 63, and the second antigen-binding region that binds to CD3 comprises Fab, which comprises VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80, and / or c. The isolated anti-DLL3 / anti-CD3 protein comprises HC1 of SEQ ID NO: 111, HC2 of SEQ ID NO: 114, and LC2 of SEQ ID NO: 115.

[0034] In some embodiments, an isolated anti-DLL3 / anti-CD3 protein comprises a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3, wherein the first antigen-binding domain that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes SEQ ID NOs. 98, 99, 100, 106, and 107, respectively. b. The first antigen-binding region that binds to DLL3 comprises 108 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and / or c. The isolated anti-DLL3 / anti-CD3 protein comprises HC1 of SEQ ID NO: 71, HC2 of SEQ ID NO: 118, and LC2 of SEQ ID NO: 117.

[0035] In some embodiments, an isolated anti-DLL3 / anti-CD3 protein comprising a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3, wherein a. The first antigen-binding domain that binds to DLL3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 98, 99, 100, 106, 107, and 108, respectively, and / or b. The first antigen-binding domain that binds to DLL3 comprises an scFv that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the scFv of SEQ ID NO. 64, and the second antigen-binding domain that binds to CD3 is at least identical to the VH of SEQ ID NO. 84. c. The isolated anti-DLL3 / anti-CD3 protein comprises a Fab containing VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to VH of SEQ ID NO: 85 and / or c. the isolated anti-DLL3 / anti-CD3 protein comprises HC1 that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to HC1 of SEQ ID NO: 71, HC2 that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to HC2 of SEQ ID NO: 118 and LC2 that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 117.

[0036] In some embodiments, an isolated anti-DLL3 / anti-CD3 protein comprises a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3, wherein the first antigen-binding domain that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes SEQ ID NOs. 98, 99, 100, 106, and 107, respectively. b. The first antigen-binding region that binds to DLL3 comprises 108 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and / or c. The isolated anti-DLL3 / anti-CD3 protein comprises HC1 of SEQ ID NO: 229, HC2 of SEQ ID NO: 230, and LC2 of SEQ ID NO: 117.

[0037] In some embodiments, an isolated anti-DLL3 / anti-CD3 protein comprising a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3, wherein the first antigen-binding domain that binds to DLL3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 98, 99, 100, 106, 107, and 108, respectively, and / or b. The first antigen-binding domain that binds to DLL3 comprises an scFv that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the scFv of SEQ ID NO. 64, and the second antigen-binding domain that binds to CD3 comprises at least 8% identical to the VH of SEQ ID NO. 84. c. The isolated anti-DLL3 / anti-CD3 protein comprises a Fab containing VH that is 0% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to VH of SEQ ID NO: 85 and / or c. the isolated anti-DLL3 / anti-CD3 protein comprises HC1 that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to HC1 of SEQ ID NO: 229, HC2 that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to HC2 of SEQ ID NO: 230 and LC2 that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 117.

[0038] This disclosure also provides an immunoconjugate comprising an isolated antigen-binding region that binds to DLL3 of this disclosure.

[0039] The disclosure also provides an immunoconjugate comprising an isolated protein containing an antigen-binding region that binds to DLL3 of the disclosure.

[0040] The disclosure also provides an immunoconjugate comprising an isolated, multispecific antigen-binding construct containing an antigen-binding region that binds to DLL3 of the disclosure.

[0041] The Disclosure also provides a pharmaceutical composition comprising an isolated antigen-binding region bound to DLL3 of the Disclosure.

[0042] The disclosure also provides a pharmaceutical composition comprising an isolated protein having an antigen-binding region that binds to DLL3 of the disclosure.

[0043] The Disclosure also provides a pharmaceutical composition comprising an isolated, multispecific antigen-binding construct containing an antigen-binding region that binds to DLL3 of the Disclosure.

[0044] This disclosure also provides isolated polynucleotides encoding isolated antigen-binding regions that bind to DLL3 of this disclosure.

[0045] The Disclosure also provides isolated polynucleotides encoding isolated proteins that include an antigen-binding region that binds to DLL3 of the Disclosure.

[0046] The Disclosure also provides isolated polynucleotides encoding isolated multispecific antigen-binding constructs comprising an antigen-binding region that binds to DLL3 of the Disclosure.

[0047] This disclosure also provides vectors comprising the polynucleotides of this disclosure.

[0048] Furthermore, the present invention provides a host cell comprising the polynucleotide or vector of the present disclosure.

[0049] The Disclosure also provides a method for treating DLL3-expressing cancer in a subject, comprising administering a therapeutically effective amount of a DLL3-binding antigen-binding domain, a protein comprising a DLL3-binding antigen-binding domain, a multispecific antigen-binding construct comprising a DLL3-binding antigen-binding domain, an immunoconjugate of the Disclosure, or a pharmaceutical composition of the Disclosure to a subject in need for a period of time sufficient to treat the DLL3-expressing cancer.

[0050] The Disclosure also provides a method for reducing the amount of DLL3-expressing tumor cells in a subject, comprising administering to the subject for a period of time sufficient to reduce the amount of DLL3-expressing tumor cells the following: an antigen-binding region that binds to DLL3, a protein comprising an antigen-binding region that binds to DLL3, a multispecific antigen-binding construct comprising an antigen-binding region that binds to DLL3, an immunoconjugate of the Disclosure, or a pharmaceutical composition of the Disclosure.

[0051] The Disclosure also provides a method for preventing the establishment of DLL3-expressing cancer in a subject, comprising administering to a subject in need of such a subject an antigen-binding domain that binds to DLL3, a protein comprising an antigen-binding domain that binds to DLL3, a multispecific antigen-binding construct comprising an antigen-binding domain that binds to DLL3, an immune conjugate of the Disclosure, or a pharmaceutical composition of the Disclosure, to prevent the establishment of DLL3-expressing cancer in the subject.

[0052] The Disclosure also provides a method for treating a non-cancerous condition in a subject at risk of developing a DLL3-expressing cancerous condition, comprising administering to a subject in need of an antigen-binding region that binds to DLL3, a protein comprising an antigen-binding region that binds to DLL3, a multispecific antigen-binding construct comprising an antigen-binding region that binds to DLL3, an immunoconjugate of the Disclosure, or a pharmaceutical composition of the Disclosure, the non-cancerous condition.

[0053] The Disclosure also provides a method for treating prostate cancer in a subject, comprising administering a therapeutically effective amount of a DLL3-binding antigen-binding domain, a protein comprising a DLL3-binding antigen-binding domain, a multispecific antigen-binding construct comprising a DLL3-binding antigen-binding domain, an immunoconjugate of the Disclosure, or a pharmaceutical composition of the Disclosure to a subject in need for a period of time sufficient to treat prostate cancer.

[0054] The Disclosure also provides a method for treating small cell lung cancer in a subject, comprising administering a therapeutically effective amount of a DLL3-binding antigen-binding domain, a protein comprising a DLL3-binding antigen-binding domain, a multispecific antigen-binding construct comprising a DLL3-binding antigen-binding domain, an immunoconjugate of the Disclosure, or a pharmaceutical composition of the Disclosure to a subject in need for a period of time sufficient to treat the small cell lung cancer.

[0055] The Disclosure also provides a method for detecting prostate cancer or small cell lung cancer in a subject, comprising administering the immunoconjugate of the Disclosure to the subject and detecting the binding of the immunoconjugate to DLL3, thereby detecting prostate cancer or small cell lung cancer.

[0056] The Disclosure also provides a kit comprising an antigen-binding region that binds to DLL3, a protein comprising an antigen-binding region that binds to DLL3, a multispecific antigen-binding construct comprising an antigen-binding region that binds to DLL3, an immunoconjugate of the Disclosure, or a pharmaceutical composition of the Disclosure.

[0057] This disclosure also provides an anti-idiotype antibody that binds to an antigen-binding region that binds to DLL3 of this disclosure.

[0058] As shown in the examples, the isolated multispecific antigen-binding constructs disclosed herein may be particularly effective in mediating T cell-mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release, and / or exhibiting enhanced antitumor efficacy. [Brief explanation of the drawing]

[0059] The foregoing will become clear from a more specific description of the exemplary embodiments, as shown in the attached drawings. [Figure 1] A schematic diagram of the DLL3 extracellular domain, including the DSL domain and six EGF domains, is shown. The amino acid sequences shown represent residues 176-215 of the DSL domain (SEQ ID NO: 246), residues 216-249 of the EGF-1 domain (SEQ ID NO: 247), residues 274-310 of the EGF-2 domain (SEQ ID NO: 248), residues 312-351 of the EGF-3 domain (SEQ ID NO: 249), residues 353-389 of the EGF-4 domain (SEQ ID NO: 250), residues 391-427 of the EGF-5 domain (SEQ ID NO: 251), residues 429-465 of the EGF-6 domain (SEQ ID NO: 252), and residues 429-618 of the EGF-6 domain + C-terminal domain (SEQ ID NO: 263). [Figure 2A] Figure 2A shows the cell binding of a bispecific anti-DLL3×CD3 antibody to DLL3+ tumor cell lines. Figure 2B shows the cell binding of a bispecific anti-DLL3×CD3 antibody to SHP77 cells, a DLL3+ tumor cell line. Figure 2B shows the cell binding of a bispecific anti-DLL3×CD3 antibody to HCC1833 cells, a DLL3+ tumor cell line. [Figure 2B] Figure 2A shows the cell binding of a bispecific anti-DLL3×CD3 antibody to DLL3+ tumor cell lines. Figure 2B shows the cell binding of a bispecific anti-DLL3×CD3 antibody to SHP77 cells, a DLL3+ tumor cell line. Figure 2B shows the cell binding of a bispecific anti-DLL3×CD3 antibody to HCC1833 cells, a DLL3+ tumor cell line. [Figure 3] This shows the binding of a bispecific anti-DLL3×CD3 antibody to human pan-T cells using FACS. [Figure 4] This shows tumor lysis of anti-DLL3 × CD3 bispecific antibodies with and without optimized anti-DLL3 sequences, as evaluated in an IncuCyte-based cytotoxicity assay. [Figure 5A]Figure 5A shows in vitro target cell injury by a bispecific anti-DLL3×CD3 antibody, measured in real time by the incuCyte imaging system to quantify targeted cell death. Isolated pan-T cells were co-incubated with DLL3+SHP77 cells for 120 hours in the presence of the bispecific anti-DLL3×CD3 antibody. Figure 5B shows in vitro target cell injury by a bispecific anti-DLL3×CD3 antibody, measured in real time by the incuCyte imaging system to quantify targeted cell death. Isolated pan-T cells were co-incubated with DLL3-HEK293 cells for 120 hours in the presence of the bispecific anti-DLL3×CD3 antibody. [Figure 5B] Figure 5A shows in vitro target cell injury by a bispecific anti-DLL3×CD3 antibody, measured in real time by the incuCyte imaging system to quantify targeted cell death. Isolated pan-T cells were co-incubated with DLL3+SHP77 cells for 120 hours in the presence of the bispecific anti-DLL3×CD3 antibody. Figure 5B shows in vitro target cell injury by a bispecific anti-DLL3×CD3 antibody, measured in real time by the incuCyte imaging system to quantify targeted cell death. Isolated pan-T cells were co-incubated with DLL3-HEK293 cells for 120 hours in the presence of the bispecific anti-DLL3×CD3 antibody. [Figure 6] This shows that isolated pan-T cells were co-incubated for 120 hours with DLL3+SHP77 cells in the presence of a bispecific anti-DLL3 / CD3 antibody. [Figure 7] This shows in vitro T-cell IFN-γ release induced by a bispecific anti-DLL3×CD3 antibody. IFN-γ concentration was measured from the supernatant collected at the indicated time points. [Figure 8A] Figure 8A shows cytotoxicity against DLL3+ target cell lines in PBMCs mediated by a bispecific anti-DLL3×CD3 antibody, with an E:T ratio of 10:1. Figure 8B shows cytotoxicity against DLL3+ target cell lines in PBMCs mediated by a bispecific anti-DLL3×CD3 antibody, with an E:T ratio of 5:1. Figure 8C shows cytotoxicity against DLL3+ target cell lines in PBMCs mediated by a bispecific anti-DLL3×CD3 antibody, with an E:T ratio of 1:1. [Figure 8B] Figure 8A shows cytotoxicity against DLL3+ target cell lines in PBMCs mediated by a bispecific anti-DLL3×CD3 antibody, with an E:T ratio of 10:1. Figure 8B shows cytotoxicity against DLL3+ target cell lines in PBMCs mediated by a bispecific anti-DLL3×CD3 antibody, with an E:T ratio of 5:1. Figure 8C shows cytotoxicity against DLL3+ target cell lines in PBMCs mediated by a bispecific anti-DLL3×CD3 antibody, with an E:T ratio of 1:1. [Figure 8C] Figure 8A shows cytotoxicity against DLL3+ target cell lines in PBMCs mediated by a bispecific anti-DLL3×CD3 antibody, with an E:T ratio of 10:1. Figure 8B shows cytotoxicity against DLL3+ target cell lines in PBMCs mediated by a bispecific anti-DLL3×CD3 antibody, with an E:T ratio of 5:1. Figure 8C shows cytotoxicity against DLL3+ target cell lines in PBMCs mediated by a bispecific anti-DLL3×CD3 antibody, with an E:T ratio of 1:1. [Figure 9] This shows the proliferation of CD3+ T cells in response to a bispecific anti-DLL3×CD3 antibody in a whole PBMC cytotoxicity assay. [Figure 10A] Figure 10A shows the activation of T cells in response to the bispecific anti-DLL3×CD3 antibody, with CD25+ cell percentage. Figure 10B shows the activation of T cells in response to the bispecific anti-DLL3×CD3 antibody, with CD69+ cell percentage. Figure 10C shows the activation of T cells in response to the bispecific anti-DLL3×CD3 antibody, with CD71+ cell percentage. [Figure 10B] Figure 10A shows the activation of T cells in response to the bispecific anti-DLL3×CD3 antibody, with CD25+ cell percentage. Figure 10B shows the activation of T cells in response to the bispecific anti-DLL3×CD3 antibody, with CD69+ cell percentage. Figure 10C shows the activation of T cells in response to the bispecific anti-DLL3×CD3 antibody, with CD71+ cell percentage. [Figure 10C] Figure 10A shows the activation of T cells in response to the bispecific anti-DLL3×CD3 antibody, with CD25+ cell percentage. Figure 10B shows the activation of T cells in response to the bispecific anti-DLL3×CD3 antibody, with CD69+ cell percentage. Figure 10C shows the activation of T cells in response to the bispecific anti-DLL3×CD3 antibody, with CD71+ cell percentage. [Figure 11A] The dose-response curve for IFNγ concentration after 48 hours is shown. [Figure 11B] The dose-response curve for IFNγ concentration at 120 hours is shown. [Figure 12A] The dose-response curve for CD8+CD25+ T cells as a percentage of all CD8+ T cells after 48 hours is shown. [Figure 12B] The dose-response curve for CD8+CD25+ T cells as a percentage of all CD8+ T cells at 120 hours is shown. [Figure 13A] The dose-response curve for CD8+ T cell proliferation at 72 hours is shown. [Figure 13B] The dose-response curve for CD8+ T cell proliferation at 120 hours is shown. [Modes for carrying out the invention]

[0060] Various publications, articles, patents, and patent applications are cited or described throughout this specification and in the background art, and each of these references is incorporated herein by reference in its entirety. The considerations of documents, operations, materials, devices, articles, etc., included herein are for the purpose of providing the background to the present invention. Such considerations do not constitute an endorsement that any or all of these things constitute part of the prior art to any invention disclosed or claimed.

[0061] Any methods and materials similar to or equivalent to those described herein may be used to carry out the tests of the present invention, but exemplary materials and methods are described herein.

[0062] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which the present invention pertains. Otherwise, certain terms used herein have the meanings set forth herein. All patents, published patent applications and publications referenced herein are incorporated by reference as if they were included herein in their entirety. When used herein and in the appended claims, it should be noted that the singular forms “a,” “an,” and “the” refer to multiple objects unless otherwise clearly indicated by the context. For example, “a cell” includes a combination of two or more cells, etc.

[0063] Where a list is presented, it should be understood that, unless otherwise specified, each individual element of that list and all combinations of that list constitute a distinct embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0064] Unless otherwise specified, all numerical values, such as concentrations or concentration ranges, described herein should be understood in all cases as being modified by the word “approximately.” Therefore, numerical values ​​typically include ±10% of the stated value. For example, a dosage of 10 mg includes 9 mg to 11 mg. When used herein, the use of numerical ranges explicitly includes all possible subranges, including integers and fractions of values ​​within that range, and all individual numerical values ​​within that range, unless otherwise explicitly specified in the context.

[0065] When used herein, the connecting term "and / or" between multiple enumerated elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is included in the meaning and therefore satisfies the requirements of the term "and / or" when used herein. The simultaneous applicability of two or more of the options is also included in the meaning and therefore satisfies the requirements of the term "and / or."

[0066] The transitional phrases “comprising,” “consisting essentially of,” and “consisting of” are intended to imply the generally accepted meanings in patent terminology, namely, (i) “comprising” is synonymous with “containing,” “containing,” or “characterizing,” and is comprehensive or non-restrictive, not excluding other unlisted elements or process steps; (ii) “consisting of” excludes any elements, processes, or components not specified in the claims; and (iii) “consisting essentially of” limits the claims to specified materials or processes, and those that “do not substantially affect the basic and novel features” of the claimed invention. Embodiments described with the phrase “comprising” (or its equivalent) are also provided as embodiments described independently with “consisting” and “consisting essentially of.”

[0067] "Approximately" means that a particular value is within the acceptable margin of error as determined by those skilled in the art, which in part depends on how that value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular assay, result, or embodiment, unless otherwise expressly stated in the example or elsewhere in the specification, "approximately" means within the range of one standard deviation or up to 5%, whichever is greater, for the practice of the art.

[0068] "Activation," "stimulation," "activated," or "stimulated" refers to the induction of a change in the biological state of a cell that results in the expression of activation markers, cytokine production, or mediation of target cell proliferation or cytotoxicity. Cells can be activated by primary stimulus signals.

[0069] An "alternative scaffold" refers to a single-chain protein framework containing a structured core that associates with a highly conformable variable domain. Because the variable domain allows for polymorphisms to be introduced without compromising the integrity of the scaffold, the variable domain can be genetically engineered and selected to bind to a specific antigen.

[0070] "Antibody-dependent cell-mediated cytotoxicity," or "ADCC," refers to a mechanism that induces cell death in which antibody-coated target cells interact with effector cells with lytic activity, such as natural killer cells (NK), monocytes, macrophages, and neutrophils, via the Fc-gamma receptor (FcγR) expressed on the effector cells.

[0071] "Antibody-dependent phagocytosis" or "ADCP" refers to a mechanism by which antibody-coated target cells are eliminated through uptake by phagocytic cells such as macrophages or dendritic cells.

[0072] An "antigen" refers to any molecule (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, part thereof, or combination thereof) that can mediate an immune response or be bound by an antigen-binding domain or T cell receptor. Examples of immune responses include antibody production and activation of immune cells such as T cells, B cells, or NK cells. Antigens may be expressed by genes from biological samples such as tissue samples, tumor samples, cells, or fluids, organisms, protein / antigen subunits, dead or inactivated whole cells, or lysates, synthesized from such samples, or purified from such samples.

[0073] The terms "antigen-binding region," "antigen-binding fragment," or "antigen-binding domain" each refer to the portion of a full-length antibody that binds to an antigen. The antigen-binding region may be a synthetic polypeptide, an enzymatically obtained polypeptide, or a genetically modified polypeptide. Typically, the antigen-binding region includes at least one or more portions of the VH region. The antigen-binding fragment is a polyvalent molecule containing one, two, three, or more antigen-binding portions of an antibody and a single-chain construct, where the VL and VH regions, or selected portions thereof, are joined by a synthetic linker or by genetic recombination to form a functional antigen-binding molecule. The antigen-binding fragment may also be a single-domain antibody (sdAb), also known as a nanobody, which is an antibody fragment consisting of a single monomeric variable antibody domain (VHH). Examples of antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv, see, e.g., Bird et al., Science 1988;242:423-426 and Huston et al. PNAS 1988;85:5879-5883), dsFv, Fd (typically the VH and CH1 domains), and dAb (typically the VH domain) fragments; VH, VL, VHH, and V-NAR domains; monovalent molecules containing a single VH chain and a single VL chain; minibodies, diabodies, triabodies, tetrabodies, and kappabodies (see, e.g., Ill et al., Protein Eng See 1997;10:949-57); Examples include camel IgG; IgNAR; and one or more isolated CDRs or functional paratopes, the isolated CDRs or antigen-binding residues or polypeptides may be associated or linked together to form a multispecific antigen-binding construct comprising functional antibody fragments, minimal recognition units consisting of amino acid residues mimicking antibody CDRs such as the FR3-CDR3-FR4 moiety, HCDR1, HCDR2 and / or HCDR3 and LCDR1, LCDR2 and / or LCDR3, an alternative scaffold for binding to the antigen, and an antigen-binding region.Various types of antibody fragments are described or reviewed, for example, in Holliger and Hudson, Nat Biotechnol 2005;23:1126-1136, International Publication No. 2005040219, and published U.S. Patent Applications Nos. 20050238646 and 20020161201. Antibody fragments can be obtained using conventional recombinant or protein engineering techniques, and these fragments can be screened for antigen binding or other functions in the same way as intact antibodies. Various methods have been developed for the production of antibody fragments. Conventionally, these fragments are obtained from the proteolytic digestion of full-length antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods, 24:107-117 (1992), and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Alternatively, Fab'-SH fragments can be recovered directly from E. coli and chemically conjugated to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). By another approach, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See International Publication No. 1993 / 16185, U.S. Patent Nos. 5,571,894 and 5,587,458. The antibody fragment may also be a “linear antibody,” as described, for example, in U.S. Patent No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific. Antigen-binding regions (such as VH and VL) can be linked together via a synthetic linker to form various types of single-chain antibody designs, where the VH / VL domains can form intramolecular or intermolecular pairs when the VH and VL domains are expressed as separate single chains, to form monovalent antigen-binding regions such as single-chain Fv(scFv) or diabodies.The antigen-binding region can also be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, which may be monospecific or multispecific, in order to genetically engineer bispecific and multispecific antigen-binding constructs.

[0074] "Antibody" in a broad sense includes polyclonal antibodies, monoclonal antibodies including mouse, human, humanized, and chimeric monoclonal antibodies, multispecific antibodies such as those with antigen-binding domains, bispecificity, tripspecificity, and tetraspecificity, dimers, tetramers, or multimers, single-chain antibodies, domain antibodies, and immunoglobulin molecules including any other modified configurations of immunoglobulin molecules containing antigen-binding sites of the required specificity. "Full-length antibody" consists of two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds, and a multimer thereof (e.g., IgM). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinge, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further classified into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with framework regions (FRs). Each VH and VL consists of three CDR and four FR segments arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five main classes, IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be assigned to one of two distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. The general principles of antibody molecular structure and various techniques for antibody production are provided, for example, in Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).

[0075] The isolated proteins or constructs of this application may also include antibody derivatives. As used herein, the term “antibody derivative” refers to a molecule comprising a full-length antibody or an antigen-binding fragment thereof, wherein one or more amino acids are chemically modified or substituted. Chemical modifications that may be used in antibody derivatives include, for example, alkylation, PEGylation, acylation, esterification, or amide formation for linking the antibody to a second molecule. Exemplary modifications include PEGylation (e.g., cysteine ​​PEGylation), biotinylation, radiolabeling, and conjugation with a second reagent (e.g., a cytotoxic agent).

[0076] The antibodies described herein include “amino acid sequence variants” having modified antigen-binding or biological activity. Examples of such amino acid modifications include antibodies with enhanced affinity for an antibody (e.g., “affinity-matured” antibodies), and antibodies with modified Fc regions (if present), such as antibodies with modified (increased or decreased) antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) (see, for example, International Publication No. 00 / 42072 (Presta, L.) and International Publication No. 99 / 51642 (Iduosogie et al)). And / or antibodies with increased or decreased serum half-life (see, for example, International Publication No. 00 / 42072, Presta, L.).

[0077] A "bispecific antigen-binding construct" or "bispecific construct" refers to a construct that specifically binds to two different antigens or two different epitopes within the same antigen. A bispecific antigen-binding construct may be a protein, a protein complex, or an antibody. A bispecific antibody may cross-react to other related antigens, such as the same antigen (homolog) from other species, such as humans or monkeys, e.g., cynomolgus monkeys (Macaca cynomolgus) (cynomolgus, cyno) or chimpanzees (Pan troglodytes), or it may bind to epitopes shared between two or more different antigens.

[0078] "Bispecific anti-DLL3 / anti-CD3 antibody," "anti-DLL3×CD3," "DLL3 / CD3 antibody," "DLL3×CD3 antibody," and "anti-DLL3 / anti-CD3 protein" refer to constructs or antibodies that include at least one binding domain that binds to DLL3 and CD3 and specifically binds to DLL3, and at least one binding domain that specifically binds to CD3. The domains that specifically bind to DLL3 and CD3 are typically V H / V L They are paired. A bispecific anti-DLL3×CD3 antibody may be monovalent with respect to binding to either DLL3 or CD3.

[0079] As used herein, the term "hypervariable region" refers to the amino acid residue of an antibody responsible for antigen binding. The hypervariable region generally consists of amino acid residues derived from the "complementarity-determining region" or "CDR" (residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242), and / or residues derived from the "hypervariable loop" (residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain); Chothia and This includes Lesk, J. Mol. Biol. 1987; 196: 901-917). Typically, the numbering of amino acid residues in this region is carried out by the method described above in Kabat et al. Expressions such as “Kabat position,” “Kabat variable domain residue numbering,” and “by Kabat” herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to the shortening or insertion into the FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of the CDR H2 (residue 52a by Kabat) and a residue inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c by Kabat). The Kabat numbering of residues can be determined for a given antibody by aligning its sequence with a "standard" Kabat numbered sequence in homologous regions.

[0080] A "reference antibody" and an "antibody that binds to the same epitope" refer to an antibody that blocks 50% or more of the binding of the reference antibody to that antigen in a competitive assay. Conversely, a reference antibody blocks 50% or more of the binding of other antibodies to that antigen in a competitive assay.

[0081] In relation to the methods of the present invention, the term "administration" means a method of preventing, treating, or relieving, therapeutically or preventively, a syndrome, disorder, or disease described herein by using the complex, or its constituents, compositions, or agents of the present invention. Such methods include administering effective amounts of the above-mentioned antibodies, their antigen-binding fragments, or their conjugates, or constituents, compositions, or agents at different points in time during the course of treatment, or simultaneously in a combination. The methods of the present invention are understood to encompass all known therapeutic treatment regimens.

[0082] The ability of a target antibody to "block" the binding of a target molecule to a natural target ligand means that, in assays using soluble or cell surface-bound target and ligand molecules, the antibody detectably reduces the binding of the target molecule to its ligand in a dose-dependent manner, and the target molecule then detectably binds to the ligand in the absence of the antibody.

[0083] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and can metastasize to distal parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors.

[0084] Complement-dependent cell injury (CDC) refers to a mechanism by which the Fc effector domain of a target-bound protein binds to and activates complement component C1q, which in turn activates the complement cascade, leading to the death of the target cell. Complement activation can also cause the deposition of complement components on the target cell surface, which facilitates CDC through the binding of complement receptors (e.g., CR3) to leukocytes.

[0085] The "complementarity-determining region" (CDR) is the region of an antibody that binds to an antigen. There are three CDRs in the VH (Very High) antibody (HCDR1, HCDR2, HCDR3) and three CDRs in the VL (Very Low) antibody (LCDR1, LCDR2, LCDR3). CDR can be defined using various descriptions, such as: Kabat (Wu et al. (1970) J Exp Med 132:211-50, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thornton J Bmol Biol 263:800-15, 1996). The correspondence between various descriptions and variable area numbering is described (see, for example, Lefranc et al. (2003) Dev Comp Immunol 27:55-77, Honegger and Pluckthun (2001), J Mol Biol 309:657-70; see the International ImMunoGeneTics (IMGT) database, web resource, http: / / www_imgt_org). CDRs can be depicted using available programs such as abYsis by UCL Business PLC. As used herein, the terms “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2”, and “LCDR3” include CDRs as defined by any of the Kabat, Chothia, IMGT, or AbM methods described above, unless otherwise expressly stated in the specification.For example, the correspondence between numbering systems, including Kabat numbering and IMGT-specific numbering systems, is well known to those skilled in the art (see, for example, Kabat, Chothia, Martin, and Lefranc mentioned above).

[0086] [Table 1]

[0087] "CD3" refers to an antigen expressed on T cells as part of a multimolecular T cell receptor (TCR) complex, consisting of homodimers or heterodimers formed from the association of two or four receptor chains: CD3 epsilon, CD3 delta, CD3 zeta, and CD3 gamma. Human CD3 epsilon contains the amino acid sequence of SEQ ID NO: 253. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide, or protein fragment unless expressly specified as being from a non-human species. Thus, "CD3" means human CD3 unless specified as being from a non-human species, such as "mouse CD3" or "monkey CD3."

[0088] Throughout this specification, "CD3-specific," "specifically binding to CD3," or "anti-CD3 antibody" refers to an antibody that specifically binds to the CD3-ε polypeptide (SEQ ID NO: 253), including antibodies that specifically bind to the CD3-ε extracellular domain (ECD) (SEQ ID NO: 254). CD3-ε, together with CD3-γ, -δ, and -ζ, and the T cell receptor α / β and γ / δ heterodimers, forms the T cell receptor-CD3 complex. This complex plays a crucial role in antigen binding, recognizing several intracellular signaling pathways. The CD3 complex mediates signal transduction, leading to T cell activation and proliferation. CD3 is essential for the immune response.

[0089] As used herein, “conjugate” refers to a protein covalently bound to one or more heterologous molecules, including but not limited to therapeutic peptides or proteins, antibodies, labels, or neurotoxic agents. When one protein is conjugated to another protein, it is also referred to as the two proteins being fused together. In a non-limiting example, an antibody or antigen-binding fragment of this application may be conjugated to another polypeptide to form a fusion protein. In certain embodiments, an antibody or antigen-binding fragment of this application may be fused or conjugated to another polypeptide via a linker.

[0090] "Decrease," "decrease," "lower," "reduce," or "mitigate" generally refers to the ability of a test molecule to mediate a reduced response (i.e., a downstream effect) compared to a response mediated by a control or vehicle. Exemplary responses include T cell expansion, T cell activation, or T cell-mediated tumor cell death, or the binding of a protein to its antigen or receptor, enhanced binding to Fcγ, or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. A decrease may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or an increase in the measured response, such as an increase of approximately 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 times or more, for example, 500, 600, 700, 800, 900, or 1000 times or more (including all integers greater than 1 and decimals in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0091] "Delta-like protein 3" or "DLL3" refers to a known protein also known as delta-like 3, delta 3, or Drosophila delta homolog 3. Unless otherwise specified, DLL3 as used herein refers to human DLL3. All DLL3 isoforms and variants are encompassed by "DLL3". Amino acid sequences of various isoforms are available from databases such as NCBI accession numbers NP_058637.1 (isoform 1 precursor, 618 amino acids) and NP_982353.1 (isoform 2 precursor, 587 amino acids). The full-length amino acid sequence of human DLL3 is shown in SEQ ID NO: 255. The DLL3 sequence includes the DSL domain (residues 176-215), EGF-1 domain (residues 216-249), EGF-2 domain (residues 274-310), EGF-3 domain (residues 312-351), EGF-4 domain (residues 353-389), EGF-5 domain (residues 391-427), EGF-6 domain (residues 429-465), and the C-terminal domain (residues 466-618) (Figure 1). The amino acid sequence of the DLL3 DSL domain is shown in SEQ ID NO: 246. The amino acid sequence of the DLL3 EGF-1 domain is shown in SEQ ID NO: 247. The amino acid sequence of the DLL3 EGF-2 domain is shown in SEQ ID NO: 248. The amino acid sequence of the DLL3 EGF-3 domain is shown in SEQ ID NO: 249. The amino acid sequence of the DLL3 EGF-4 domain is shown in SEQ ID NO: 250. The amino acid sequence of the DLL3 EGF-5 domain is shown in SEQ ID NO: 251. The amino acid sequence of the DLL3 EGF-6 domain is shown in SEQ ID NO: 252. The amino acid sequence of the DLL3 EGF-6+ C-terminal domain is shown in SEQ ID NO: 263.

[0092] "Differentiation" refers to a method of reducing the capacity or proliferation of cells, or moving them into a more developmentally restricted state.

[0093] "Code" or "coding" refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which has either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties that arise therefrom, serving as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, in a cell or other biological system, if the transcription and translation of mRNA corresponding to a gene produces a protein, then that gene, cDNA, or RNA codes for a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence, and the non-coding strand used as a template for the transcription of a gene or cDNA, may be referred to as coding for a protein or other product of that gene or cDNA.

[0094] The terms “enhance,” “promote,” “increase,” “expand,” or “improve” generally refer to the ability of a test molecule to mediate a greater response (i.e., a downstream effect) compared to a response mediated by a control or vehicle. Exemplary responses include T cell expansion, T cell activation, or T cell-mediated tumor cell death, or the binding of a protein to its antigen or receptor, enhanced binding to Fcγ, or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. Enhancement may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or an increase in the measured response such as an increase of approximately 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 times or more, for example, 500, 600, 700, 800, 900, or 1000 times or more (including all integers greater than 1 and decimals in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0095] An "epitope" refers to a portion of an antibody, or a part of an antigen to which the antibody-binding portion specifically binds. Typically, an epitope consists of a surface population of chemically active (polar, nonpolar, or hydrophobic, etc.) moieties, such as amino acids or polysaccharide side chains, and may possess specific three-dimensional structural and charge properties. Epitopes can consist of continuous and / or discontinuous amino acids forming higher-order structural spatial units. In discontinuous epitopes, amino acids from different parts of the antigen's linear sequence come into very close proximity in three-dimensional space due to protein molecule folding. Antibody "epitopes" differ depending on the methodology used to identify them.

[0096] "Expansion" refers to the result of cell division and cell death.

[0097] "Express" and "expression" refer to the well-known transcription and translation that occur intracellularly or in vitro. Therefore, the expression product, for example, a protein, may be expressed by a cell or in vitro, and may be intracellular, extracellular, or transmembrane protein.

[0098] An "expression vector" refers to a vector that can be used in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0099] "dAb" or "dAb fragment" refers to an antibody fragment composed of a VH domain (Ward et al. (1989), Nature 341:544 546).

[0100] "Fab" or "Fab fragment" refers to an antibody fragment composed of VH, CH1, VL, and CL domains.

[0101] "F(ab')2" or "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide crosslinks within a hinge region.

[0102] "Fd" or "Fd fragment" refers to an antibody fragment composed of VH and CH1 domains.

[0103] "Fv" or "Fv fragment" refers to an antibody fragment consisting of a VH domain and a VL domain derived from a single arm of the antibody. Fv fragments lack the constant region of the Fab(CH1 and CL) domain. The VH and VL in the Fv fragment are held together by non-covalent interactions.

[0104] The "framework region" or "FR" residues are these VH or VL residues other than the CDR as defined herein.

[0105] A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, as well as their polymers (e.g., IgM). Each heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant domain, the heavy chain constant domain consisting of subdomains CH1, hinge, CH2, and CH3. Each light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). The VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with framework regions (FRs). Each VH and VL consists of three CDRs and four FR segments arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0106] "Genetic modification" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence and produces a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may also be called a "cloned" or "foreign" gene or sequence and may include regulatory or control sequences that are manipulably ligated to a polynucleotide encoding a chimeric antigen receptor, such as an initiator, terminater, promoter, signaling, secretion, or other sequence used by the cell's genetic mechanism. The gene or sequence may include non-functional sequences that do not have a known function. A host cell that receives and expresses the introduced DNA or RNA is "genetically engineered." The DNA or RNA introduced into a host cell may originate from any source, including cells of the same genus or species as the host cell, or from a different genus or species.

[0107] "Heterogeneous" refers to two or more polynucleotides or two or more polypeptides that are not found in nature in the same relationship to one another.

[0108] "Heterogeneic polynucleotide" refers to a polynucleotide that does not exist in nature and encodes two or more neoantigens as described herein.

[0109] "Heterogeneous polypeptide" refers to polypeptides that do not exist in nature and include two or more neoantigen polypeptides as described herein.

[0110] A "host cell" refers to any cell containing heterologous nucleic acids. An example of heterologous nucleic acid is a vector (e.g., an expression vector).

[0111] "Human antibody" refers to an antibody optimized to produce a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody contains a constant region or a portion of a constant region, that constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is obtained from a system using human germline immunoglobulin or a rearranged immunoglobulin gene, the human antibody will contain heavy-chain and light-chain variable regions "derived" from a human sequence. Exemplary such systems include human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals possessing human immunoglobulin loci, such as mice or rats. "Human antibody" typically involves amino acid differences when compared to immunoglobulin expressed in humans, due to differences in the system used to obtain the human antibody and the human immunoglobulin locus, intentional introduction of somatic mutations or substitutions into the framework or CDR, or both. Typically, a “human antibody” is at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a “human antibody” may contain a consensus framework sequence derived from human framework sequence analysis, for example, as described in Knappik et al., (2000) J Mol Biol 296:57-86, or synthetic HCDR3 incorporated into a human immunoglobulin gene library presented on phages, for example, as described in Shi et al., (2010) J Mol Biol 397:385-96 and International Publication No. 2009 / 085462. Antibodies in which at least one CDR originates from a non-human species are not included in the definition of "human antibodies."

[0112] A "humanized antibody" means an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Because humanized antibodies can contain substitutions in their framework, the framework may not be an exact copy of the expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0113] "In combination with ~" means administering two or more therapeutic agents to the subject together as a mixture, simultaneously as individual agents, or sequentially as individual agents in any order.

[0114] "Isolated" refers to a homogeneous population of molecules (e.g., synthetic polynucleotides or polypeptides) that have been substantially separated and / or purified from other components of the system in which the molecules are produced, such as in recombinant cells, and proteins that have undergone at least one purification or isolation step. "Isolated" refers to molecules that are substantially free from other cellular material and / or chemicals, and includes molecules isolated to higher purities, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. "Isolated" antibodies are those that have been isolated from components of their natural environment. In some embodiments, antibodies are purified to a purity of over 95% or 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For an overview of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B, 848:79-87 (2007).

[0115] As used herein, “linker” refers to a chemical linker or a single-chain peptide linker that covalently links two different entities. A linker can link any two of the antibodies or their fragments, fusion proteins, and conjugates of the present invention. For example, a linker can link VH and VL in scFv or a monoclonal antibody or its antigen-binding fragment to a therapeutic molecule such as a second antibody. A single-chain peptide linker can be used that consists of 1 to 25 amino acids linked by peptide bonds, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In certain embodiments, the amino acids are selected from 20 natural amino acids. In certain other embodiments, one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Chemical linkers such as hydrocarbon linkers, polyethylene glycol (PEG) linkers, polypropylene glycol (PPG) linkers, polysaccharide linkers, polyester linkers, hybrid linkers consisting of PEG and an embedded heterocycle, and hydrocarbon chains can also be used.

[0116] "Modifying" refers to either an enhanced or reduced ability of a test molecule to mediate a greater or lesser response (i.e., a downstream effect) compared to the response mediated by the control or vehicle.

[0117] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules that is identical except for possible known modifications such as removal of C-terminal lysine from the antibody heavy chain, isomerization or deamidement of amino acids, oxidation of methionine, or deamidementation of asparagine or glutamine; in other words, an individual antibody that constitutes a population. A bispecific monoclonal antibody binds to two different antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, such as bispecific, and may be monovalent, bivalent, or polyvalent.

[0118] A “multispecific antigen-binding construct” or “multispecific molecule” refers to a construct that specifically binds to two or more different antigens or two or more different epitopes within the same antigen. A multispecific antigen-binding construct may be a protein, a protein complex, or an antibody. It includes an antibody or its antigen-binding fragment that associates with or binds to at least one other functional molecule (e.g., another peptide or protein, such as a ligand for another antibody or receptor), thereby forming a molecule that binds to at least two different binding sites or target molecules. A multispecific molecule may also be cross-reactive to other relevant antigens, e.g., the same antigen (homolog) from other species such as humans or monkeys, e.g., cynomolgus monkeys (Macaca fascicularis) (cynomolgus, cyno) or chimpanzees (Pan troglodytes), and may bind to epitopes shared among two or more different antigens. Exemplary multispecific molecules include tripspecific or bispecific antibodies, and antibodies linked to soluble receptor fragments or ligands.

[0119] "Natural killer cells" and "NK cells" are used interchangeably and synonymously in this specification. In this specification, NK cells refer to CD16 + CD56 + and / or CD57 + TCR -NK cells refer to differentiated lymphocytes that exhibit a specific phenotype. NK cells are characterized by their ability to bind to and kill cells that cannot express "self" MHC / HLA antigens through the activation of specific cytolytic enzymes, their ability to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and their ability to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0120] When used in relation to nucleic acids or amino acids, "operably linked" and similar terms refer to the operational linkage of nucleic acid sequences or amino acid sequences, respectively, that are arranged in a functional relationship with one another. For example, operable linked promoter, enhancer element, open reading frame, 5' and 3' UTR, and terminator sequences result in the precise production of nucleic acid molecules (e.g., RNA) and, in some cases, the production of polypeptides (i.e., expression of the open reading frame). An operable linked peptide refers to a peptide in which the functional domains of the peptide are arranged at appropriate distances from each other to confer the intended function of each domain.

[0121] The term "paratope" refers to a region or area of ​​an antibody molecule containing residues that are involved in antigen binding and interact with the antigen. Paratopes can consist of continuous and / or discontinuous amino acids that form higher-order structural spatial units. The paratopes of a given antibody can be defined and characterized in detail at various levels using a variety of experimental and computational methods. Experimental methods include hydrogen / deuterium exchange mass spectrometry (HX-MS). Paratopes are defined differently depending on the mapping method used. Paratopes may include amino acid residues directly involved in epitope binding (some of which are typically located within the CDR) and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked by the specifically bound antigen (i.e., amino acid residues located within the "solvent-excluded surface" and / or footprint" of the specifically bound antigen).

[0122] A "pharmaceutical combination" refers to a combination of two or more active ingredients administered together or separately.

[0123] A "pharmaceutical composition" refers to a composition obtained by combining an active ingredient with a pharmaceutically acceptable carrier.

[0124] A "pharmaceutically acceptable carrier" or "excipient" refers to a component in a pharmaceutical composition other than the active ingredient that is non-toxic to the target. Exemplary pharmaceutically acceptable carriers are buffers, stabilizers, or preservatives.

[0125] A "polynucleotide" or "nucleic acid" refers to a synthetic molecule containing nucleotide chains covalently bonded by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide. Polynucleotides can be DNA or RNA molecules.

[0126] "Preventing," "preventing," "prevention," or "preventive measures" for a disease or disability means preventing the occurrence of the disability in the subject.

[0127] "Proliferation" refers to an increase in cell division, which is either symmetrical or asymmetrical division of cells.

[0128] A "promoter" refers to the minimum sequence required to initiate transcription. Promoters may also contain enhancer or repressor elements that enhance or suppress transcription, respectively.

[0129] In this specification, the terms “protein” and “polypeptide” are used interchangeably and refer to molecules containing one or more polypeptides, each consisting of at least two amino acid residues linked by peptide bonds. Proteins may be monomers or protein complexes of two or more identical or different subunits. Small polypeptide molecules consisting of fewer than 50 amino acids may be referred to as “peptides.” Proteins may be heterofusion proteins, glycoproteins, or proteins modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation, or biotinylation. Proteins may be recombinantly expressed.

[0130] "Recombinant organism" refers to polynucleotides, polypeptides, vectors, viruses, and other macromolecules prepared, expressed, produced, or isolated by recombinant means.

[0131] A "regulatory element" refers to any cis- or trans-acting genetic element that controls a particular aspect of nucleic acid sequence expression.

[0132] "Recurrent" refers to the recurrence of the disease or its signs and symptoms after a period of improvement following prior treatment with medication.

[0133] "Refractory" refers to a disease that does not respond to treatment. A refractory disease may be resistant to treatment before or at the start of treatment, or it may become resistant during treatment.

[0134] When used in relation to amino acid sequences, the terms “sequence identity,” “sequence identity percentage (%),” or “identity %” refer to the number of identical amino acid matches ("hits") in two or more aligned amino acid sequences compared to the number of amino acid residues that make up the entire length of the amino acid sequence. In other terms using alignment for two or more sequences, the percentage of identical amino acid residues (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence) can be determined when the sequences are compared and aligned for maximum identity, as measured using sequence comparison algorithms known in the art, or when manually aligned and visually inspected. Therefore, the sequences compared to determine sequence identity may differ due to amino acid substitutions, additions, or deletions. Suitable programs for aligning protein sequences are known to those skilled in the art. The percentage of sequence identity of protein sequences can be determined using programs such as CLUSTALW, Clustal Omega, FASTA, or BLAST, for example, using the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).

[0135] "Single-chain Fv" or "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region (VL) and at least one antibody fragment containing a heavy chain variable region (VH), wherein the VL and VH are sequentially linked via a polypeptide linker and can be expressed as a single-chain polypeptide. Unless otherwise specified, as used herein, scFv may have the VL and VH variable regions in any order, for example, with respect to the N-terminus and C-terminus of the polypeptide, scFv may contain VL-linker-VH or VH-linker-VL.

[0136] The "(scFv)2", "tandem scFv", or "bis-scFv" fragment refers to a fusion protein containing two light chain variable regions (VL) and two heavy chain variable regions (VH), where the two VLs and the two VHs are continuously linked via a polypeptide linker and can be expressed as a single-chain polypeptide. The two VLs and the two VHs are fused by a peptide linker to form a bivalent molecule VL A -linker-VH A -linker-VL B -linker-VH B to form two binding sites that can simultaneously bind to two different antigens or epitopes.

[0137] "Specifically binds", "specific binding", "specifically bound", or "binds" refers to a proteinaceous molecule binding to an antigen or an epitope within the antigen with a higher affinity than its affinity for other antigens. Typically, the proteinaceous molecule binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (K -7 M or less, such as about 5×10 -8 M or less, about 1×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, or about 1×10 -12 M or less, and typically, K D is at least 100-fold smaller than K D for binding to non-specific antigens (e.g., BSA, casein). D

[0138] "Subject" includes any human or non-human animal. "Non-human animals" include all vertebrates, such as mammals and non-mammals like non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "subject" and "patient" may be used interchangeably herein.

[0139] The terms "T cell" and "T lymphocyte" are interchangeable and are used synonymously herein. T cells include thymocytes, naive T lymphocytes, memory T cells, immature T lymphocytes, mature T lymphocytes, quiescent T lymphocytes, or activated T lymphocytes. T cells may also be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells may also be helper T cells (HTL, CD4) + T cells), CD4 + T cells, cytotoxic T cells (CTL, CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL, CD8 + T cells), CD4 + CD8 + This can be a T cell, or any other subset of T cells. It also includes "NKT cells," which refer to a special population of T cells that not only express the semi-variant αβ T cell receptor but also various molecular markers typically associated with NK cells, such as NK1.1. NKT cells include NK1.1 + and NK1.1 - , and CD4 + CD4 - CD8 + , and CD8 - The cells included are NKT cells, which are unique in that their TCR recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or detrimental effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also included are "gamma delta T cells (γδ T cells)," which refer to a special population of a small subset of T cells that have different TCRs on their surface. Unlike most T cells, whose TCR consists of two glycoprotein chains denoted as α and β-TCR chains, the TCR in γδ T cells consists of a γ chain and a δ chain. γδ T cells can play a role in immune surveillance and immunomodulation, are an important source of IL-17, and have a robust CD8 +It has been found that it induces a cytotoxic T cell response. It also includes "regulatory T cells" or "Tregs," which refer to T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs are typically Foxp3-positive CD4 cells. + T cells and IL-10 producing CD4 + This may also include T cells, specifically Foxp3-negative regulatory T cells.

[0140] In this specification, the terms “therapeutic effective dose” or “effective dose” as used interchangeably refer to the amount effective in obtaining the desired therapeutic outcome in the required dosage and duration. The therapeutic effective dose may vary depending on factors such as the individual’s condition, age, sex, and weight, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improvement in the patient’s health, reduction in tumor burden, cessation or slowing of tumor growth, and / or absence of metastasis of cancer cells to other parts of the body.

[0141] "Transduction" refers to the introduction of foreign nucleic acids into cells using a viral vector.

[0142] "To treat," "to treat," or "treatment" of a disease or disability such as cancer means achieving one or more of the following: reducing the severity and / or duration of the disability; suppressing the worsening of symptoms characteristic of the disability being treated; limiting or preventing the recurrence of the disability in a person who previously had the disability; or limiting or preventing the recurrence of symptoms in a person who previously had the symptomatic disability.

[0143] "Tumor cells" or "cancer cells" refer to cancerous, precancerous, or transformed cells that exhibit spontaneous or induced phenotypic changes in vivo, ex vivo, or tissue culture. These changes do not necessarily involve the uptake of new genetic material. Transformation can be induced by infection with transforming viruses and the incorporation of new genomic nucleic acids, or by the uptake of exogenous nucleic acids, and may occur spontaneously or after exposure to carcinogens, thereby resulting in mutations of endogenous genes. Transformation / cancer is exemplified by morphological changes, cell immortalization, abnormal growth control, lesion formation, proliferation, malignant lesions, regulation of tumor-specific marker levels, invasiveness, and tumor growth in suitable animal hosts such as nude mice, in vitro, in vivo, and ex vivo.

[0144] "Variant," "mutant," or "altered" means a polypeptide or polynucleotide that is different from a reference polypeptide or reference polynucleotide by one or more modifications, such as one or more substitutions, insertions, or deletions.

[0145] Throughout this specification, unless otherwise explicitly stated herein, the numbering of amino acid residues in the constant region of an antibody follows the EU index described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

[0146] "VHH" refers to a single-domain antibody or nanobody composed solely of the antigen-binding region of the heavy chain. VHH single-domain antibodies lack the CH1 domains of the light and heavy chains in the conventional Fab region.

[0147] composition of a substance Antigen-binding region that binds to DLL3 This disclosure provides antigen-binding regions that bind to DLL3, monospecific and multispecific antigen-binding constructs comprising antigen-binding regions that bind to DLL3, encoding polynucleotides, vectors, host cells, and methods for constructing and using them. The antigen-binding regions that bind to DLL3 identified herein have shown improved properties in terms of improved thermal stability. The multispecific antigen-binding constructs disclosed herein may be particularly effective in mediating T cell-mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release, and / or exhibiting enhanced antitumor effects.

[0148] This disclosure provides an isolated protein comprising an antigen-binding region that binds to delta-like protein 3 (DLL3), wherein the antigen-binding region that binds to DLL3 binds to an epitope in the EGF-6+ C-terminal domain of DLL3 (residues 429-618 of DLL3), as shown in SEQ ID NO: 263. As shown in the examples, multispecific antigen-binding constructs targeting epitopes in the EGF-6 domain or closer to the C-terminus of DLL3 achieved potent levels of antitumor cytotoxicity.

[0149] In consideration of this disclosure, any known method in the art can be used to identify the region within DLL3 to which the antibody of this application binds. For example, an ELISA assay can be used to identify the domain within DLL3 to which the antibody binds. Anti-DLL3 antibodies were evaluated for binding to recombinant DLL3 domain antigens across the N-terminal DSL fusion domain (DL3W44, SEQ ID NO: 189), EGF-1+2 fusion (DL3W42, SEQ ID NO: 187), EGF-2 (DL3W41, SEQ ID NO: 186), EGF-3 (DL3W40, SEQ ID NO: 185), EGF-4 (DL3W39, SEQ ID NO: 184), EGF-5 (DL3W38, SEQ ID NO: 183), EGF-6 (DL3W37, SEQ ID NO: 182), and EGF-6+ C-terminal domain fusion (DL3W36, SEQ ID NO: 181) in a domain mapping ELISA assay. MesoScale Discovery high-binding plates were coated overnight at 4°C with 20 nM antigen. The plates were washed with PBS containing 0.1% Tween and then blocked with Starting Block solution for 30 minutes. Antibody was added and incubated at ambient temperature for 60 minutes, after which excess antibody was removed by washing three times with PBS (Gibco, #14190-136). Antigen-binding antibodies were detected using sulfotagged anti-human antibody (Meso Scale Discovery, R32AJ) at ambient temperature for 60 minutes, followed by another PBS wash. Signal acquisition was performed on an MSD Sector 600 imager with appropriate plate settings in the presence of 1× MSD read buffer T (MSD, catalog no. R92TC-1). Data were analyzed for the highest binding signal per domain indicating preferred domain binding.

[0150] The residues in DLL3 to which antibodies bind can be determined using an H / D exchange assay. In the H / D exchange assay, recombinantly expressed soluble DLL3 is incubated in deuterated water for a predetermined time, either in the presence or absence of antibody, resulting in deuterium incorporation with exchangeable hydrogen atoms not protected by the antibody. This is followed by protease digestion of the protein and analysis of the peptide fragment using LC-MS. The H / D exchange assay can be performed using known protocols. In some embodiments, the H / D exchange mixture is quenched by adding a quenching buffer (e.g., 8M urea, 1M TCEP, pH 3.0) and then passed through an equilibrated immobilized pepsin / FPXIII column at room temperature (e.g., 600 μL / min). Next, the digestible fragments are packed into a reverse-phase trap column (e.g., 600 μL / min), desalted (e.g., 600 μL for 1 minute), separated (e.g., on a C18 column), and analyzed by mass spectrometry (e.g., using an LTQ® Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) with a capillary temperature of 275°C, a resolution of 150,000, and a mass range (m / z) of 300–1,800).

[0151] In some embodiments, the application provides isolated proteins, such as antibodies, comprising an antigen-binding region, wherein the antigen-binding region that binds to DLL3 competes with a reference antibody disclosed herein for binding to DLL3. In some embodiments, the reference antibody includes VH having HCDR1, HCDR2, and HCDR3, and VL having LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are a. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 2, b. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 3, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 4, c. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 5, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 6, d. HCDR1, HCDR2, and HCDR3 of VH in sequence number 7, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 8, e. HCDR1, HCDR2, and HCDR3 of VH in sequence number 9, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 10, f. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 11, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 12, or g. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 13, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 14.

[0152] In certain such embodiments, the reference antibody includes HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 3 (VH), and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 4 (VL).

[0153] Competition for binding between the test antibody and the reference antibody of this application to soluble DLL3 (SEQ ID NO: 263) can be assayed in vitro using well-known methods in consideration of this disclosure. For example, the binding of a labeled antibody to DLL3, e.g., the membrane-proximal region of DLL3, in the presence of an unlabeled reference antibody can be evaluated by ELISA. Competition can be demonstrated using Bioacore analysis or flow cytometry. The test antibody competes with the reference antibody for binding to DLL3 when the test antibody inhibits the binding of the reference antibody to soluble DLL3 by 85% or more, e.g., 90% or more, or 95% or more.

[0154] In some embodiments, the present application relates to an isolated protein, such as an antibody, comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region that binds to DLL3 comprises VH having HCDR1, HCDR2, and HCDR3, and VL having LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 1 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 2, or HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 3 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 4, or HCDR1, HCDR2 of VH of SEQ ID NO: 5 The invention provides isolated proteins, including HCDR3, and VL LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 6, or VH LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 7, and VL LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 8, or VH LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 9, and VL LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 10, or VH LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 11, and VL LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 12, or VH LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 13, and VL LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 14. In certain embodiments, the isolated protein includes an antigen-binding region that binds to DLL3, the antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 3, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 4.

[0155] In some embodiments, the present application relates to an isolated protein, such as an antibody, comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region that binds to DLL3 is Sequence numbers 15, 16, 17, 33, 34, 35, Sequence numbers 18, 19, 20, 36, 37, 38, respectively Sequence numbers 21, 22, 23, 39, 37, 40, Sequence numbers 24, 25, 26, 41, 42, 43, respectively Sequence numbers 18, 28, 29, 44, 45, 46, Sequence numbers 30, 31, 32, 47, 48, 49, The sequence numbers are 50, 51, 17, 33, 34, and 35 respectively. The sequence numbers are 52, 51, 17, 33, 34, and 35 respectively. Sequence numbers 53, 54, 20, 36, 37, 38, The sequence numbers are 55, 56, 23, 39, 37, and 40, respectively. Sequence numbers 57, 58, 26, 41, 42, 43, respectively Each of these corresponds to sequence numbers 59, 60, 29, 44, 45, 46, or The present invention provides isolated proteins, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 61, 62, 32, 47, 48, and 49, respectively.

[0156] In another embodiment, the disclosure provides an isolated protein comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region that binds to DLL3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 15, 16, 17, 33, 34, and 35.

[0157] In another embodiment, the disclosure provides an isolated protein comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region that binds to DLL3 comprises a VH having the amino acid sequence of SEQ ID NOs. 1, 3, 5, 7, 9, 11, or 13 and a VL having the amino acid sequence of SEQ ID NOs. 2, 4, 6, 8, 10, 12, or 14.

[0158] This disclosure relates to an isolated protein comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region that binds to DLL3 is The VH of the amino acid sequence of SEQ ID NO: 1 and the VL of the amino acid sequence of SEQ ID NO: 2 (also referred to as VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2), VH of SEQ ID NO: 1 and VL of SEQ ID NO: 4, VH of SEQ ID NO: 1 and VL of SEQ ID NO: 6 VH of SEQ ID NO: 1 and VL of SEQ ID NO: 8, VH of SEQ ID NO: 1 and VL of SEQ ID NO: 10, VH of SEQ ID NO: 1 and VL of SEQ ID NO: 12, VH of SEQ ID NO: 1 and VL of SEQ ID NO: 14, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 2, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 6 VH of SEQ ID NO: 3 and VL of SEQ ID NO: 8, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 10, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 12, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 14, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 2, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 6 VH of SEQ ID NO: 3 and VL of SEQ ID NO: 8, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 10, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 12, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 14, VH of SEQ ID NO: 5 and VL of SEQ ID NO: 2 VH of SEQ ID NO: 5 and VL of SEQ ID NO: 4, VH of SEQ ID NO: 5 and VL of SEQ ID NO: 6, VH of SEQ ID NO: 5 and VL of SEQ ID NO: 8 VH of SEQ ID NO: 5 and VL of SEQ ID NO: 10 VH of SEQ ID NO: 5 and VL of SEQ ID NO: 12 VH of SEQ ID NO: 5 and VL of SEQ ID NO: 14 VH of sequence number 7 and VL of sequence number 2, VH of sequence number 7 and VL of sequence number 4, VH of sequence number 7 and VL of sequence number 6, VH of sequence number 7 and VL of sequence number 8, VH of sequence number 7 and VL of sequence number 10, VH of sequence number 7 and VL of sequence number 12, VH of sequence number 7 and VL of sequence number 14, VH of sequence number 9 and VL of sequence number 2, VH of sequence number 9 and VL of sequence number 4, VH of sequence number 9 and VL of sequence number 6, VH of sequence number 9 and VL of sequence number 8, VH of sequence number 9 and VL of sequence number 10, VH of sequence number 9 and VL of sequence number 12, VH of sequence number 9 and VL of sequence number 14, VH of SEQ ID NO: 11 and VL of SEQ ID NO: 2 VH of sequence number 11 and VL of sequence number 4, VH of sequence number 11 and VL of sequence number 6, VH of SEQ ID NO: 11 and VL of SEQ ID NO: 8, VH of sequence number 11 and VL of sequence number 10, VH of sequence number 11 and VL of sequence number 12, VH of sequence number 11 and VL of sequence number 14, VH of SEQ ID NO: 13 and VL of SEQ ID NO: 2, VH of SEQ ID NO: 13 and VL of SEQ ID NO: 4, VH of SEQ ID NO: 13 and VL of SEQ ID NO: 6, VH of SEQ ID NO: 13 and VL of SEQ ID NO: 8, VH of sequence number 13 and VL of sequence number 10, VH of SEQ ID NO: 13 and VL of SEQ ID NO: 12, or This provides isolated proteins, including VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14.

[0159] In certain embodiments, the disclosure provides an isolated protein comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region that binds to DLL3 comprises VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4.

[0160] The disclosure also provides isolated proteins comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region that binds to DLL3 comprises VH, which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 3, and VL, which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VL of SEQ ID NO: 4.

[0161] In some embodiments, the antigen-binding region that binds to DLL3 includes, for example, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 3.

[0162] In some embodiments, the antigen-binding region that binds to DLL3 includes, for example, the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 4, which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL.

[0163] In some embodiments, the antigen-binding region that binds to DLL3 includes a VH that is at least 95% identical to the VH of SEQ ID NO: 3 and a VL that is at least 95% identical to the VL of SEQ ID NO: 4.

[0164] In some embodiments, the antigen-binding region that binds to DLL3 includes a VH that is at least 95% identical to the VH of SEQ ID NO: 3 and a VL that is at least 99% identical to the VL of SEQ ID NO: 4.

[0165] In some embodiments, the antigen-binding region that binds to DLL3 includes a VH that is at least 99% identical to the VH of SEQ ID NO: 3 and a VL that is at least 99% identical to the VL of SEQ ID NO: 4.

[0166] In some embodiments, the antigen-binding region that binds to DLL3 includes a VH that is at least 99% identical to the VH of SEQ ID NO: 3 and a VL that is at least 95% identical to the VL of SEQ ID NO: 4.

[0167] This disclosure provides an isolated protein comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region that binds to DLL3 comprises the amino acid sequence of SEQ ID NOs. 63, 64, 65, 66, 67, 68, or 69.

[0168] In certain embodiments, the disclosure provides an isolated protein comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region that binds to DLL3 comprises the amino acid sequence of SEQ ID NO: 63.

[0169] In certain embodiments, the disclosure provides an isolated protein comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region that binds to DLL3 comprises the amino acid sequence of SEQ ID NO: 64.

[0170] The disclosure also provides an isolated protein comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region that binds to DLL3 comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 63.

[0171] The disclosure also provides an isolated protein comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region that binds to DLL3 comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 64.

[0172] In some embodiments, the antigen-binding region that binds to DLL3 is scFv.

[0173] In some embodiments, the antigen-binding region that binds to DLL3 is (scFv)2.

[0174] In some embodiments, the antigen-binding region that binds to DLL3 is Fv.

[0175] In some embodiments, the antigen-binding region that binds to DLL3 is Fab.

[0176] In some embodiments, the antigen-binding region that binds to DLL3 is F(ab')2.

[0177] In some embodiments, the antigen-binding region that binds to DLL3 is Fd.

[0178] In some embodiments, the antigen-binding region that binds to DLL3 is dAb.

[0179] In some embodiments, the antigen-binding region that binds to DLL3 is VHH.

[0180] In certain embodiments, the antigen-binding region that binds to DLL3 is scFv.

[0181] DLL3 binding scFv Any of the VH domains and VL domains identified herein that bind to DLL3, or any of their components, can be genetically engineered into either the VH-linker-VL or VL-linker-VH formatted scFv. Any of the VH and VL identified herein can be used to generate sc(Fv)2 structures such as VH-linker-VL-linker-VL-linker-VH, VH-linker-VL-linker-VH-linker-VL, VH-linker-VH-linker-VH-linker-VL, VL-linker-VH-linker-VH-linker-VH, or VL-linker-VL-linker-VH-linker-VH.

[0182] VH and VL or their components identified herein can be incorporated into the scFv format, and the binding and thermal stability of the resulting scFv to DLL3 can be evaluated using methods known in light of this disclosure. Binding can be evaluated using ProteOn XPR36, Biacore3000, or KinExA instruments, ELISA, or competitive binding assays known to those skilled in the art. Binding can be evaluated using purified scFv or E. coli supernatant or lysed cells containing expressed scFv. Measured affinity of test scFv to DLL3 may differ when measured under different conditions (e.g., molar osmotic concentration, pH). Therefore, affinity and other binding parameters (e.g., K) may differ. D , K on , K off The measurement of thermal stability is typically performed using standardized conditions and standardized buffers. Thermal stability can be evaluated by heating the test scFv at a high temperature such as 50°C, 55°C, or 60°C for a period of time such as 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes, and measuring the binding of the test scFv to DLL3. An scFv that retains equivalent binding to DLL3 when compared to an unheated scFv sample is said to be thermally stable.

[0183] In recombinant expression systems, the linker is a peptide linker and may contain any naturally occurring amino acids. Exemplary amino acids that may be included in the linker are Gly, Ser Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker needs to be of an appropriate length to link VH and VL in such a way that they form precise higher-order structures relative to each other, in order to maintain desired activities such as binding to DLL3.

[0184] The linker can be approximately 5 to 50 amino acids long. In some embodiments, the linker is approximately 10 to 40 amino acids long. In some embodiments, the linker is approximately 10 to 35 amino acids long. In some embodiments, the linker is approximately 10 to 30 amino acids long. In some embodiments, the linker is approximately 10 to 25 amino acids long. In some embodiments, the linker is approximately 10 to 20 amino acids long. In some embodiments, the linker is approximately 15 to 20 amino acids long. In some embodiments, the linker is 6 amino acids long. In some embodiments, the linker is 7 amino acids long. In some embodiments, the linker is 8 amino acids long. In some embodiments, the linker is 9 amino acids long. In some embodiments, the linker is 10 amino acids long. In some embodiments, the linker is 11 amino acids long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker is 13 amino acids long. In some embodiments, the linker is 14 amino acids long. In some embodiments, the linker is 15 amino acid lengths. In some embodiments, the linker is 16 amino acid lengths. In some embodiments, the linker is 17 amino acid lengths. In some embodiments, the linker is 18 amino acid lengths. In some embodiments, the linker is 19 amino acid lengths. In some embodiments, the linker is 20 amino acid lengths. In some embodiments, the linker is 21 amino acid lengths. In some embodiments, the linker is 22 amino acid lengths. In some embodiments, the linker is 23 amino acid lengths. In some embodiments, the linker is 24 amino acid lengths. In some embodiments, the linker is 25 amino acid lengths. In some embodiments, the linker is 26 amino acid lengths. In some embodiments, the linker is 27 amino acid lengths. In some embodiments, the linker is 28 amino acid lengths. In some embodiments, the linker is 29 amino acid lengths.In some embodiments, the linker is 30 amino acid lengths. In some embodiments, the linker is 31 amino acid lengths. In some embodiments, the linker is 32 amino acid lengths. In some embodiments, the linker is 33 amino acid lengths. In some embodiments, the linker is 34 amino acid lengths. In some embodiments, the linker is 35 amino acid lengths. In some embodiments, the linker is 36 amino acid lengths. In some embodiments, the linker is 37 amino acid lengths. In some embodiments, the linker is 38 amino acid lengths. In some embodiments, the linker is 39 amino acid lengths. In some embodiments, the linker is 40 amino acid lengths. Exemplary linkers that can be used are glycy-rich linkers, glycy and ser-containing linkers, glycy and ala-containing linkers, ala and ser-containing linkers, and other flexible linkers.

[0185] Other linker sequences may include immunoglobulin hinge regions, CL, or CH1 portions derived from any immunoglobulin heavy or light chain isotype. Alternatively, various non-protein polymers, including polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol, may be found to be used as linkers. Exemplary linkers that may be used are shown in Table 2. Additional linkers are described, for example, in International Publication No. 2019 / 060695.

[0186] In some embodiments, scFv includes VH, a first linker (L1), and VL (VH-L1-VL) from the N-terminus to the C-terminus.

[0187] In some embodiments, the scFv comprises VL, L1, and VH (VL-L1-VH) from the N-terminus towards the C-terminus. In some embodiments, L1 comprises the amino acid sequence of SEQ ID NO: 120, SEQ ID NO: 27, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, or SEQ ID NO: 139.

[0188]

Table 2

[0189] In certain embodiments, L1 comprises or consists of the amino acid sequence of SEQ ID NO: 120.

[0190] In some embodiments, scFv is the heavy chain complementarity determination regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 1, and the light chain complementarity determination regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 2, or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 3, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 4, or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 5, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 6, or SEQ ID NO: 7 VH includes HCDR1, HCDR2, and HCDR3, and VL includes LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 8, or VH includes HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 9, and VL includes LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 10, or VH includes HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 11, and VL includes LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 12, or VH includes HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 13, and VL includes LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 14. In a particular embodiment, scFv includes VH includes HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 3, and VL includes LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 4.

[0191] In some embodiments, scFv includes VH having HCDR1, HCDR2, and HCDR3, and VL having LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are Sequence numbers 15, 16, 17, 33, 34, 35, Sequence numbers 18, 19, 20, 36, 37, 38, respectively Sequence numbers 21, 22, 23, 39, 37, 40, Sequence numbers 24, 25, 26, 41, 42, 43, respectively Sequence numbers 18, 28, 29, 44, 45, 46, Sequence numbers 30, 31, 32, 47, 48, 49, The sequence numbers are 50, 51, 17, 33, 34, and 35 respectively. The sequence numbers are 52, 51, 17, 33, 34, and 35 respectively. Sequence numbers 53, 54, 20, 36, 37, 38, The sequence numbers are 55, 56, 23, 39, 37, and 40, respectively. Sequence numbers 57, 58, 26, 41, 42, 43, respectively Each of these corresponds to sequence numbers 59, 60, 29, 44, 45, 46, or Each contains the amino acid sequences of sequence numbers 61, 62, 32, 47, 48, and 49, respectively.

[0192] In a particular embodiment, scFv includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 15, 16, 17, 33, 34, and 35.

[0193] In some embodiments, scFv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2.

[0194] In some embodiments, scFv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 4.

[0195] In some embodiments, scFv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 6.

[0196] In some embodiments, scFv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 8.

[0197] In some embodiments, scFv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 10.

[0198] In some embodiments, scFv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 12.

[0199] In some embodiments, scFv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 14.

[0200] In some embodiments, the scFv comprises VH of SEQ ID NO: 3 and VL of SEQ ID NO: 2.

[0201] In some embodiments, the scFv comprises VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4.

[0202] In some embodiments, the scFv comprises VH of SEQ ID NO: 3 and VL of SEQ ID NO: 6.

[0203] In some embodiments, the scFv comprises VH of SEQ ID NO: 3 and VL of SEQ ID NO: 8.

[0204] In some embodiments, the scFv comprises VH of SEQ ID NO: 3 and VL of SEQ ID NO: 10.

[0205] In some embodiments, the scFv comprises VH of SEQ ID NO: 3 and VL of SEQ ID NO: 12.

[0206] In some embodiments, the scFv comprises VH of SEQ ID NO: 3 and VL of SEQ ID NO: 14.

[0207] In some embodiments, the scFv comprises VH of SEQ ID NO: 5 and VL of SEQ ID NO: 2.

[0208] In some embodiments, the scFv comprises VH of SEQ ID NO: 5 and VL of SEQ ID NO: 4.

[0209] In some embodiments, the scFv comprises VH of SEQ ID NO: 5 and VL of SEQ ID NO: 6.

[0210] In some embodiments, the scFv comprises VH of SEQ ID NO: 5 and VL of SEQ ID NO: 8.

[0211] In some embodiments, the scFv comprises VH of SEQ ID NO: 5 and VL of SEQ ID NO: 10.

[0212] In some embodiments, the scFv comprises VH of SEQ ID NO: 5 and VL of SEQ ID NO: 12.

[0213] In some embodiments, scFv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 14.

[0214] In some embodiments, scFv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 2.

[0215] In some embodiments, scFv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 4.

[0216] In some embodiments, scFv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 6.

[0217] In some embodiments, scFv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 8.

[0218] In some embodiments, scFv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 10.

[0219] In some embodiments, scFv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 12.

[0220] In some embodiments, scFv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 14.

[0221] In some embodiments, scFv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 2.

[0222] In some embodiments, scFv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 4.

[0223] In some embodiments, scFv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 6.

[0224] In some embodiments, scFv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 8.

[0225] In some embodiments, scFv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 10.

[0226] In some embodiments, scFv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 12.

[0227] In some embodiments, scFv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 14.

[0228] In some embodiments, scFv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 2.

[0229] In some embodiments, scFv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 4.

[0230] In some embodiments, scFv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 6.

[0231] In some embodiments, scFv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 8.

[0232] In some embodiments, scFv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 10.

[0233] In some embodiments, scFv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 12.

[0234] In some embodiments, scFv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 14.

[0235] In some embodiments, scFv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 2.

[0236] In some embodiments, scFv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 4.

[0237] In some embodiments, scFv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 6.

[0238] In some embodiments, scFv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 8.

[0239] In some embodiments, scFv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 10.

[0240] In some embodiments, scFv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 12.

[0241] In some embodiments, scFv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14.

[0242] In some embodiments, scFv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4.

[0243] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 1 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 2.

[0244] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 3, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 4.

[0245] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 5, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 6.

[0246] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 7, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 8.

[0247] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 9, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 10.

[0248] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 11, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 12.

[0249] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 13, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 14.

[0250] In some embodiments, scFv includes VH and VL of SEQ ID NO: 4, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 3.

[0251] In some embodiments, scFv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical.

[0252] In some embodiments, scFv includes a VH that is at least 95% identical to the VH of SEQ ID NO: 3 and a VL that is at least 95% identical to the VL of SEQ ID NO: 4.

[0253] In some embodiments, scFv includes a VH that is at least 99% identical to the VH of SEQ ID NO: 3 and a VL that is at least 95% identical to the VL of SEQ ID NO: 4.

[0254] In some embodiments, scFv includes a VH that is at least 99% identical to the VH of SEQ ID NO: 3 and a VL that is at least 99% identical to the VL of SEQ ID NO: 4.

[0255] In some embodiments, scFv includes a VH that is at least 95% identical to the VH of SEQ ID NO: 3 and a VL that is at least 99% identical to the VL of SEQ ID NO: 4.

[0256] In some embodiments, the linker includes the amino acid sequence of SEQ ID NOs. 63, 64, 65, 66, 67, 68, or 69.

[0257] In some embodiments, scFv includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 63, 64, 65, 66, 67, 68, or 69.

[0258] In some embodiments, scFv includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 63.

[0259] In some embodiments, scFv includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 64.

[0260] In certain embodiments, scFv includes the amino acid sequence of SEQ ID NO: 63.

[0261] In certain embodiments, scFv includes the amino acid sequence of SEQ ID NO: 64.

[0262] Other antigen-binding regions that bind to DLL3 Any of the VH and VL compounds or their components identified herein that bind to DLL3 can be genetically engineered into Fab, F(ab')2, Fd, or Fv formats, and their binding to DLL3 and thermal stability can be evaluated using assays described herein.

[0263] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH having HCDR1, HCDR2, and HCDR3, and VL having LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 2, or HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 3, and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 4, or HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 5, and This includes LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 6, or HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 8, or HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 9, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 10, or HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 11, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 12, or HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 13, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 14.

[0264] In certain embodiments, Fab, F(ab')2, Fd, or Fv includes HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 3, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 4.

[0265] In some embodiments, Fab, F(ab')2, Fd, or Fv are Sequence numbers 15, 16, 17, 33, 34, and 35, respectively Sequence numbers 18, 19, 20, 36, 37, and 38, respectively Sequence numbers 21, 22, 23, 39, 37, and 40, respectively. Sequence numbers 24, 25, 26, 41, 42, and 43, respectively Sequence numbers 18, 28, 29, 44, 45, and 46, respectively. Sequence numbers 30, 31, 32, 47, 48, and 49, respectively Sequence numbers 50, 51, 17, 33, 34, and 35, respectively. Sequence numbers 52, 51, 17, 33, 34, and 35, respectively. Sequence numbers 53, 54, 20, 36, 37, and 38, respectively Sequence numbers 55, 56, 23, 39, 37, and 40, respectively. Sequence numbers 57, 58, 26, 41, 42, and 43, respectively. Sequence IDs 59, 60, 29, 44, 45, and 46, respectively, or This includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 61, 62, 32, 47, 48, and 49, respectively.

[0266] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2.

[0267] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 4.

[0268] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 6.

[0269] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 8.

[0270] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 10.

[0271] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 12.

[0272] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 14.

[0273] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 2.

[0274] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4.

[0275] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 6.

[0276] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 8.

[0277] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 10.

[0278] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 12.

[0279] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 14.

[0280] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 2.

[0281] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 4.

[0282] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 6.

[0283] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 8.

[0284] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 10.

[0285] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 12.

[0286] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 14.

[0287] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 2.

[0288] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 4.

[0289] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 6.

[0290] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 8.

[0291] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 10.

[0292] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 12.

[0293] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 14.

[0294] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 2.

[0295] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 4.

[0296] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 6.

[0297] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 8.

[0298] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 10.

[0299] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 12.

[0300] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 14.

[0301] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 2.

[0302] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 4.

[0303] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 6.

[0304] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 8.

[0305] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 10.

[0306] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 12.

[0307] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 14.

[0308] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 2.

[0309] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 4.

[0310] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 6.

[0311] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 8.

[0312] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 10.

[0313] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 12.

[0314] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14.

[0315] In certain embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2.

[0316] In some embodiments, Fab, F(ab')2, Fd, or Fv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 1, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VL of SEQ ID NO: 2.

[0317] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH and VL of SEQ ID NO: 2, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 1.

[0318] In some embodiments, Fab, F(ab')2, Fd, or Fv includes a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2.

[0319] In some embodiments, Fab, F(ab')2, Fd, or Fv includes a VH that is at least 95% identical to VH of SEQ ID NO: 1 and a VL that is at least 95% identical to VL of SEQ ID NO: 2.

[0320] In some embodiments, Fab, F(ab')2, Fd, or Fv includes a VH that is at least 99% identical to VH of SEQ ID NO: 1 and a VL that is at least 95% identical to VL of SEQ ID NO: 2.

[0321] In some embodiments, Fab, F(ab')2, Fd, or Fv includes a VH that is at least 99% identical to VH of SEQ ID NO: 1 and a VL that is at least 99% identical to VL of SEQ ID NO: 2.

[0322] In some embodiments, Fab, F(ab')2, Fd, or Fv includes a VH that is at least 99% identical to VH of SEQ ID NO: 1 and a VL that is at least 95% identical to VL of SEQ ID NO: 2.

[0323] In certain embodiments, Fab, F(ab')2, Fd, or Fv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4.

[0324] In some embodiments, Fab, F(ab')2, Fd, or Fv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 3, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VL of SEQ ID NO: 4.

[0325] In some embodiments, Fab, F(ab')2, Fd, or Fv includes VH and VL of SEQ ID NO: 4, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 3.

[0326] In some embodiments, Fab, F(ab')2, Fd, or Fv includes a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4.

[0327] In some embodiments, Fab, F(ab')2, Fd, or Fv includes a VH that is at least 95% identical to VH of SEQ ID NO: 3 and a VL that is at least 95% identical to VL of SEQ ID NO: 4.

[0328] In some embodiments, Fab, F(ab')2, Fd, or Fv includes a VH that is at least 99% identical to VH of SEQ ID NO: 3 and a VL that is at least 95% identical to VL of SEQ ID NO: 4.

[0329] In some embodiments, Fab, F(ab')2, Fd, or Fv includes a VH that is at least 99% identical to VH of SEQ ID NO: 3 and a VL that is at least 99% identical to VL of SEQ ID NO: 4.

[0330] In some embodiments, Fab, F(ab')2, Fd, or Fv includes a VH that is at least 99% identical to VH of SEQ ID NO: 3 and a VL that is at least 95% identical to VL of SEQ ID NO: 4.

[0331] The VH and VL of Fab containing an antigen-binding region that binds to DLL3 can be genetically engineered into Fab-Fc HC (VH-CH1-hinge-CH2-CH3) and Fab-Fc LC (VL-CL) forms, respectively. In certain such embodiments, Fab-Fc HC contains an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 109.

[0332] In some embodiments, Fab-Fc LC contains an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 110. In certain embodiments, Fab-Fc LC contains an amino acid sequence that is identical to SEQ ID NO: 110.

[0333] As shown in the examples, particularly suitable antigen-binding regions for binding to DLL3 for incorporation into a multispecific construct include Fab-Fc HC having the amino acid sequence of SEQ ID NO: 109 and Fab-Fc LC having the amino acid sequence of SEQ ID NO: 110.

[0334] In some embodiments, F(ab')2 includes the amino acid sequence of SEQ ID NO: 63.

[0335] In some embodiments, F(ab')2 includes the amino acid sequence of SEQ ID NO: 64.

[0336] In some embodiments, F(ab')2 includes the amino acid sequence of SEQ ID NO: 65.

[0337] In some embodiments, F(ab')2 includes the amino acid sequence of SEQ ID NO: 66.

[0338] In some embodiments, F(ab')2 includes the amino acid sequence of SEQ ID NO: 67.

[0339] In some embodiments, F(ab')2 includes the amino acid sequence of SEQ ID NO: 68.

[0340] In some embodiments, F(ab')2 includes the amino acid sequence of SEQ ID NO: 69.

[0341] In some embodiments, Fv includes the amino acid sequence of SEQ ID NO: 63.

[0342] In some embodiments, Fv includes the amino acid sequence of SEQ ID NO: 64.

[0343] In some embodiments, Fv includes the amino acid sequence of SEQ ID NO: 65.

[0344] In some embodiments, Fv includes the amino acid sequence of SEQ ID NO: 66.

[0345] In some embodiments, Fv includes the amino acid sequence of SEQ ID NO: 67.

[0346] In some embodiments, Fv includes the amino acid sequence of SEQ ID NO: 68.

[0347] In some embodiments, Fv includes the amino acid sequence of SEQ ID NO: 69.

[0348] Antigen-binding regions having homologous antigen-binding regions and conservative substitutions Variants of the antigen-binding region that binds to DLL3 are within the scope of this disclosure. For example, a variant may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acid substitutions in the antigen-binding region that binds to DLL3, insofar as it retains or has improved functional properties compared to the parent antigen-binding region. In some embodiments, sequence identity may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with respect to the antigen-binding region that binds to DLL3 of this disclosure. In some embodiments, the diversity resides in the framework region. In some embodiments, variants are generated by conservative substitution.

[0349] In some embodiments, the isolated protein containing the antigen-binding region that binds to DLL3 includes VH and VL which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH and VL of the antigen-binding region that binds to DLL3 disclosed herein.

[0350] Also, VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2 VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, VH of SEQ ID NO: 5 and VL of SEQ ID NO: 6, VH of sequence number 7 and VL of sequence number 8, VH of sequence number 9 and VL of sequence number 10, VH of SEQ ID NO: 11 and VL of SEQ ID NO: 12, or An antigen-binding region that binds to DLL3 is also provided, which includes VH and VL that are at least 80% identical to VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14.

[0351] In some embodiments, the identity is 85%. In some embodiments, the identity is 90%. In some embodiments, the identity is 91%. In some embodiments, the identity is 91%. In some embodiments, the identity is 92%. In some embodiments, the identity is 93%. In some embodiments, the identity is 94%. In some embodiments, the identity is 94%. In some embodiments, the identity is 95%. In some embodiments, the identity is 96%. In some embodiments, the identity is 97%. In some embodiments, the identity is 98%. In some embodiments, the identity is 99%.

[0352] In some embodiments, the antigen-binding region that binds to DLL3 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 1 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 2.

[0353] In some embodiments, the antigen-binding region that binds to DLL3 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 3 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 4.

[0354] In some embodiments, the antigen-binding region that binds to DLL3 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 5, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 6.

[0355] In some embodiments, the antigen-binding region that binds to DLL3 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 7, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 8.

[0356] In some embodiments, the antigen-binding region that binds to DLL3 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 9, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 10.

[0357] In some embodiments, the antigen-binding region that binds to DLL3 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 11 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 12.

[0358] In some embodiments, the antigen-binding region that binds to DLL3 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 13 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 14.

[0359] In some embodiments, the antigen-binding region that binds to DLL3 includes VH, which is at least 85% identical to VH of SEQ ID NO: 3, and VL of SEQ ID NO: 4.

[0360] In some embodiments, the antigen-binding region that binds to DLL3 includes VH, which is at least 90% identical to VH of SEQ ID NO: 3, and VL of SEQ ID NO: 4.

[0361] In some embodiments, the antigen-binding region that binds to DLL3 includes VH, which is at least 91% identical to VH of SEQ ID NO: 3, and VL of SEQ ID NO: 4.

[0362] In some embodiments, the antigen-binding region that binds to DLL3 includes VH, which is at least 92% identical to VH of SEQ ID NO: 3, and VL of SEQ ID NO: 4.

[0363] In some embodiments, the antigen-binding region that binds to DLL3 includes VH, which is at least 93% identical to VH of SEQ ID NO: 3, and VL of SEQ ID NO: 4.

[0364] In some embodiments, the antigen-binding region that binds to DLL3 includes VH, which is at least 94% identical to VH of SEQ ID NO: 3, and VL of SEQ ID NO: 4.

[0365] In some embodiments, the antigen-binding region that binds to DLL3 includes VH, which is at least 95% identical to VH of SEQ ID NO: 3, and VL of SEQ ID NO: 4.

[0366] In some embodiments, the antigen-binding region that binds to DLL3 includes VH, which is at least 96% identical to VH of SEQ ID NO: 3, and VL of SEQ ID NO: 4.

[0367] In some embodiments, the antigen-binding region that binds to DLL3 includes VH, which is at least 97% identical to VH of SEQ ID NO: 3, and VL of SEQ ID NO: 4.

[0368] In some embodiments, the antigen-binding region that binds to DLL3 includes VH, which is at least 98% identical to VH of SEQ ID NO: 3, and VL of SEQ ID NO: 4.

[0369] In some embodiments, the antigen-binding region that binds to DLL3 includes VH, which is at least 99% identical to VH of SEQ ID NO: 3, and VL of SEQ ID NO: 4.

[0370] In some embodiments, the antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which is at least 85% identical.

[0371] In some embodiments, the antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which is at least 90% identical.

[0372] In some embodiments, the antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which is at least 91% identical.

[0373] In some embodiments, the antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which is at least 92% identical.

[0374] In some embodiments, the antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which is at least 93% identical.

[0375] In some embodiments, the antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which is at least 94% identical.

[0376] In some embodiments, the antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which is at least 95% identical.

[0377] In some embodiments, the antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which is at least 96% identical.

[0378] In some embodiments, the antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which is at least 97% identical.

[0379] In some embodiments, the antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which is at least 98% identical.

[0380] In some embodiments, the antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which is at least 99% identical.

[0381] The identity percentage between two arrays is a function of the number of identical positions shared by the arrays, taking into account the number of gaps that need to be introduced for optimal alignment of the two arrays and the length of each gap (i.e., identity % = number of identical positions / total number of positions × 100).

[0382] The percentage of identity between two amino acid sequences can be determined using the algorithm by E. Meyers and W. Miller (Comput Appl Biosci 4:11-17 (1988)), which is incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4. In addition, the percentage of identity between two amino acid sequences can also be determined using the algorithm by Needleman and Wunsch (J Mol Biol 48:444-453 (1970)), which is incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, gap weightings 16, 14, 12, 10, 8, 6, or 4, and length weightings 1, 2, 3, 4, 5, or 6.

[0383] In some embodiments, variants of the antigen-binding region that bind to DLL3 include one or two conservative substitutions in any of the CDR regions while retaining the desired functional properties of the parent antigen-binding fragment that binds to DLL3.

[0384] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding properties of an antibody, including amino acid modifications. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative amino acid substitution is a substitution in which an amino acid is replaced by an amino acid residue with a similar side chain. The families of amino acid residues with similar side chains are clearly defined and include amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), non-charged side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), β-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Furthermore, any native residue in the polypeptide may also be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67; ​​Sasaki et al., (1988) Adv Biophys 35:1-24). Amino acid substitutions for the antibodies of this application can be carried out by known methods, e.g., PCR mutagenesis (U.S. Patent No. 4,683,195). Alternatively, a library of variants may be generated, for example, using random codons (NNK) or non-random codons (e.g., DVK codons encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp)). The resulting variants can be tested for their characteristics using the assays described herein.

[0385] Method for producing antigen-binding fragments that bind to DLL3 The antigen-binding regions that bind to DLL3 provided in this disclosure can be produced using a variety of techniques. For example, the Kohler and Milstein hybridoma method can be used to identify VH / VL pairs that bind to DLL3. In the hybridoma method, mice or other host animals, such as hamsters, rats, or chickens, are immunized with human and / or cynomolgus monkey DLL3, and then hybridoma cells are formed by fusing spleen cells of animals immunized with myeloma cells using standard methods. Colonies arising from a single immortalized hybridoma cell can be screened for the production of antibodies containing antigen-binding regions that bind to DLL3 with desired properties such as binding specificity, cross-reactivity or lack thereof, affinity for the antigen, and any desired functionality.

[0386] Antigen-binding regions that bind to DLL3 produced by immunizing non-human animals can be humanized. Exemplary humanization techniques, including the selection of a human acceptor framework, include CDR transplantation (U.S. Patent No. 5,225,539), SDR transplantation (U.S. Patent No. 6,818,749), resurfacing (Padlan, (1991) Mol Immunol 28:489-499), specificity-determining residue resurfacing (U.S. Patent Application Publication No. 2010 / 0261620), human framework adaptation (U.S. Patent No. 8,748,356), or hyperhumanization (U.S. Patent No. 7,709,226). These methods involve transplanting a CDR or a subset of CDR residues from a parent antibody into a human framework that can be selected based on overall homology to the parent framework, based on similarity in CDR length, identity of canonical structure, or a combination thereof.

[0387] The humanized antigen-binding region can be further optimized to improve its selectivity or affinity for a desired antigen by incorporating altered framework-supporting residues to maintain binding affinity (reverse mutation) using techniques such as those described in International Publication Nos. 1090 / 007861 and 1992 / 22653, or by introducing diversity into either the CDR to improve the affinity of the antigen-binding region, for example.

[0388] Antigen-binding fragments that bind to DLL3 can be produced using transgenic animals such as mice, rats, or chickens that possess a human immunoglobulin (Ig) locus in their own genome, as described, for example, in U.S. Patent No. 6,150,584, International Publication No. 1999 / 45962, 2002 / 066630, 2002 / 43478, 2002 / 043478, and 1990 / 04036. The endogenous immunoglobulin locus of such animals may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the animal genome using homologous or non-homologous recombination, using a transchromosome, or using a minigene. Companies such as Regeneron (http: / / _www_regeneron_com), Harbour Antibodies (http: / / _www_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http: / / _www_omtinc_net), KyMab (http: / / _www_kymab_com), Trianni (http: / / _www.trianni_com), and Ablexis (http: / / _www_ablexis_com) may be working to provide human antibodies targeting selected antigens using the above technologies. In some embodiments, Ablexis mice were immunized with soluble full-length DLL3 protein.

[0389] Antigen-binding regions that bind to DLL3 can be selected from phage display libraries in which phages are genetically engineered to express human immunoglobulin or a portion thereof, such as Fab, single-chain antibodies (scFv), or unpaired or paired antibody variable regions. Antigen-binding regions that bind to DLL3 can be isolated from phage display libraries that express antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coated proteins, for example, as described in Shi et al., (2010) J Mol Biol 397:385-96 and International Publication No. 09 / 085462). The libraries may be screened for phage binding to human and / or cynomolgus monkey DLL3, positive clones obtained may be further characterized, Fab may be isolated from the clone lysates and converted to scFv or other components of the antigen-binding fragment.

[0390] The preparation of immunogenic antigens and the expression and generation of the antigen-binding regions of this disclosure can be carried out using any preferred technique, such as recombinant protein generation. The immunogenic antigen may be administered to an animal in the form of a purified protein or a protein mixture containing whole cells or cell or tissue extracts, or the antigen may be de novoly formed in the animal's body from nucleic acids encoding the antigen or a portion thereof.

[0391] Fusion or conjugation of the half-life extension portion The antigen-binding region that binds to DLL3 of this disclosure can be fused or conjugated to the half-life extension region. Exemplary half-life extension regions include albumin, albumin variants, albumin-binding proteins and / or domains, transferrin and its fragments and analogs, immunoglobulin (Ig) or its fragments, such as the Fc region. The amino acid sequences of the aforementioned half-life extension regions are known. Ig or its fragments include all isotypes, namely IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE.

[0392] Additional half-life extension moieties that can be conjugated to the antigen-binding domain of DLL3 of this disclosure include polyethylene glycol (PEG) molecules, e.g., PEG5000 or PEG20000, fatty acids and fatty acid esters of different chain lengths, e.g., laurate, myristic acid, stearate, arachidic acid, behenate, oleate, arachidonic acid, octanodioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, polylysine, octane, and carbohydrates (dextran, cellulose, oligosaccharides, or polysaccharides), for desired properties. These moieties can be directly fused with the antigen-binding domain of DLL3 of this disclosure and prepared by standard cloning and expression techniques. Alternatively, the moieties can be conjugated to the antigen-binding domain of recombinantly produced DLL3 of this disclosure using well-known chemical coupling methods.

[0393] For example, a pegyl moiety can be conjugated to the antigen-binding region of the DLL3 of this disclosure by incorporating a cysteine ​​residue into the C-terminus of the antigen-binding region that binds to the DLL3 of this disclosure, or by genetically manipulating the cysteine ​​to a residue position facing away from the DLL3-binding site, and then attaching a pegyl group to the cysteine ​​using a well-known method.

[0394] In some embodiments, the antigen-binding fragment that binds to DLL3 is fused to or conjugated to the half-life extension portion.

[0395] In some embodiments, the half-life extension portion is immunoglobulin (Ig), a fragment of Ig, the Ig constant region, a fragment of the Ig constant region, an Fc region, transferrin, albumin, an albumin-binding domain, or polyethylene glycol. In some embodiments, the half-life extension portion is the Ig constant region.

[0396] In some embodiments, the half-life extension portion is Ig.

[0397] In some embodiments, the half-life extension portion is a fragment of Ig.

[0398] In some embodiments, the half-life extension portion is the Ig steady-state region.

[0399] In some embodiments, the half-life extension portion is a fragment of the Ig steady-state region.

[0400] In some embodiments, the half-life extension portion is the Fc region.

[0401] In some embodiments, the half-life extension portion is albumin.

[0402] In some embodiments, the half-life extension portion is the albumin-binding domain.

[0403] In some embodiments, the half-life extension portion is transferrin.

[0404] In some embodiments, the half-life extension portion is polyethylene glycol.

[0405] Using known in vivo models, and taking this disclosure into consideration, antigen-binding regions that bind to DLL3 fused to or conjugated to the half-life extension portion can be evaluated for their pharmacokinetic properties.

[0406] Fusion to immunoglobulin (Ig) constant region or fragment of the Ig constant region. The antigen-binding region of DLL3 of this disclosure can be conjugated to an Ig constant region or a fragment of an Ig constant region to confer antibody-like properties, including Fc effector function (C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or downregulation of cell surface receptors (e.g., B cell receptors, BCRs). The Ig constant region or fragment of an Ig constant region can also function as a half-life extension portion, as discussed herein. The antigen-binding region of DLL3 of this disclosure can be genetically engineered using standard methods to produce a conventional full-length antibody. A full-length antibody containing an antigen-binding region that binds to DLL3 can be further genetically engineered as described herein.

[0407] The constant region of the immunoglobulin heavy chain consists of subdomains CH1, hinge, CH2, and CH3. Following EU index numbering, the CH1 domain in the heavy chain extends from residues A118 to V215, the CH2 domain from A231 to K340, and the CH3 domain from G341 to K447. In some examples, G341 is referred to as the CH2 domain residue. The hinge is generally defined as containing E216 and terminating at P230 in human IgG1. The Ig Fc region includes at least the CH2 and CH3 domains of the Ig constant region and therefore includes at least the region from approximately A231 to K447 of the Ig heavy chain constant region.

[0408] This application also provides an antigen-binding region that binds to a DLL3 conjugated to an immunoglobulin (Ig) constant region or a fragment of an Ig constant region.

[0409] In some embodiments, the Ig steady-state region is the heavy chain steady-state region.

[0410] In some embodiments, the Ig steady-state region is the light chain steady-state region.

[0411] In some embodiments, the Ig steady-state region fragment includes an Fc region.

[0412] In some embodiments, the Ig constant region fragment includes a CH2 domain.

[0413] In some embodiments, the Ig constant region fragment includes a CH3 domain.

[0414] In some embodiments, the Ig constant region fragment includes a CH2 domain and a CH3 domain.

[0415] In some embodiments, the Ig constant region fragment includes at least a portion of the hinge, the CH2 domain, and the CH3 domain. The portion of the hinge refers to one or more amino acid residues of the Ig hinge.

[0416] In some embodiments, the Ig constant region fragment includes a hinge, a CH2 domain, and a CH3 domain.

[0417] In certain embodiments, the Ig steady-state region fragment includes a hinge, a CH2 domain, and a CH3 domain.

[0418] In some embodiments, the antigen-binding region that binds to DLL3 is conjugated to the N-terminus of the Ig constant region or a fragment of the Ig constant region.

[0419] In some embodiments, the antigen-binding region that binds to DLL3 is conjugated to the C-terminus of the Ig constant region or a fragment of the Ig constant region.

[0420] In some embodiments, the antigen-binding region that binds to DLL3 is conjugated to the Ig constant region or a fragment of the Ig constant region via a second linker (L2).

[0421] In some embodiments, L2 includes the amino acid sequence of SEQ ID NOs: 27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139.

[0422] In certain embodiments, L2 includes the amino acid sequence of SEQ ID NO: 120.

[0423] Antigen-binding regions conjugated to the Ig constant domain or fragments of the Ig constant domain that bind to DLL3 of this disclosure can be evaluated for their functionality using several known assays. Binding to DLL3 can be evaluated using the methods described herein. Altered properties conferred by the Ig constant domain or fragments of the Ig constant domain, such as the Fc region, can be assayed in Fc receptor binding assays using soluble forms of receptors such as FcγRI, FcγRII, FcγRIII, or FcRn receptors, or using cell-based assays that measure ADCC, CDC, or ADCP, for example.

[0424] ADCC can be evaluated using an in vitro assay that uses DLL3-expressing cells as target cells and NK cells as effector cells. Cell lysis can be detected by the release of a label (e.g., radioactive substrate, fluorescent dye, or native intracellular protein) from the lysed cells. In an exemplary assay, target cells are used in a ratio of one target cell to four effector cells. Target cells are pre-labeled with BATDA and combined with effector cells and a test antibody. Cell lysis is measured by incubating the sample for 2 hours and measuring the BATDA released into the supernatant. Data are normalized to the maximum cytotoxicity with 0.67% Triton X-100 (Sigma Aldrich), and the minimum control is determined by the spontaneous release of BATDA from target cells in the absence of any antibody.

[0425] ADCP can be evaluated by using monocyte-derived macrophages as effector cells and any DLL3-expressing cells genetically engineered to express GFP or other labeled molecules as target cells. In an exemplary assay, the effector:target cell ratio may be, for example, 4:1. Effector cells may be incubated with target cells for 4 hours with or without the antibodies of this application. After incubation, the cells can be detached using actase. Macrophages can be identified using fluorescently labeled anti-CD11b and anti-CD14 antibodies, and the rate of phagocytosis can be determined using standard methods for CD11 + CD14 + This can be determined based on the GFP fluorescence percentage in macrophages.

[0426] For example, the CDC of cells is 1 × 10⁶ Daudi cells in RPMI-B (RPMI supplemented with 1% BSA). 5 The reaction can be measured by seeding cells per well (50 μL / well), adding 50 μL of test protein to the well at a final concentration of 0–100 μg / mL, incubating the reaction at room temperature for 15 minutes, adding 11 μL of pooled human serum to the well, and incubating the reaction at 37°C for 45 minutes. The percentage of lysed cells (%) can be detected as the percentage of propidium iodide-stained cells in the FACS assay using a standard method.

[0427] In some embodiments, a first antigen-binding region that binds to DLL3 is fused to a first immunoglobulin (Ig) constant region or a fragment of a first Ig constant region, and / or a second antigen-binding region that binds to a lymphocyte antigen is fused to a second immunoglobulin (Ig) constant region or a fragment of a second Ig constant region.

[0428] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include an Fc region.

[0429] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include a CH2 domain.

[0430] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include a CH3 domain.

[0431] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include a CH2 domain and a CH3 domain.

[0432] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include at least a portion of the hinge, a CH2 domain, and a CH3 domain.

[0433] In some embodiments, the Ig constant region fragment includes a hinge, a CH2 domain, and a CH3 domain.

[0434] In some embodiments, the multispecific antigen-binding construct further includes a second linker (L2) between a first antigen-binding region that binds to DLL3 and a first Ig constant region or a fragment of the first Ig constant region, and between a second antigen-binding region that binds to lymphocyte antigens and a second Ig constant region or a fragment of the second Ig constant region.

[0435] In some embodiments, L2 includes the amino acid sequence of SEQ ID NOs: 27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139.

[0436] In certain embodiments, L2 includes the amino acid sequence of SEQ ID NO: 120.

[0437] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region, and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG1, IgG2, IgG3, or IgG4 isotypes.

[0438] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG1 isotypes.

[0439] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG2 isotypes.

[0440] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG3 isotypes.

[0441] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG4 isotypes.

[0442] In certain embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG1 isotypes.

[0443] The first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region may be further genetically modified as described herein.

[0444] In some embodiments, the first Ig constant region or fragment of the first Ig constant region, and the second Ig constant region or fragment of the second Ig constant region, contain at least one mutation resulting in reduced binding of the multispecific antigen-binding construct to FcγR.

[0445] In some embodiments, at least one mutation resulting in reduced binding of the multispecific antigen-binding construct to FcγR is F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / The residues are selected from the group consisting of A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, and residue numbering follows the EU index. In certain embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and / or the second Ig steady-state region or a fragment of the second Ig steady-state region contain the following mutation: L234A_L235A_D265S.

[0446] In some embodiments, FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRIII, or any combination thereof.

[0447] In some embodiments, the first Ig constant region or fragment of the first Ig constant region, and the second Ig constant region or fragment of the second Ig constant region, include at least one mutation that modulates the half-life of the multispecific antigen-binding construct.

[0448] In some embodiments, the multispecific antigen-binding construct includes at least one mutation in the CH3 domain of a first Ig constant region or a fragment of the first Ig constant region and / or at least one mutation in the CH3 domain of a second Ig constant region or a fragment of the second Ig constant region.

[0449] In some embodiments, at least one mutation in the CH3 domain of the first Ig steady-state region or the CH3 domain of a fragment of the first Ig steady-state region, and / or at least one mutation in the CH3 domain of the second Ig steady-state region or the CH3 domain of a fragment of the second Ig steady-state region, is L351Y_F40 as described in U.S. Patent Application Publication 2012 / 0149876 or U.S. Patent Application Publication 2013 / 0195849 (Zymeworks). The following are selected from the group consisting of 5A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0450] In some embodiments, at least one mutation in the CH3 domain of the first Ig constant region or the CH3 domain of a fragment of the first Ig constant region, and / or at least one mutation in the CH3 domain of the second Ig constant region or the CH3 domain of a fragment of the second Ig constant region is selected from the group consisting of T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V as described in International Publication No. 1996 / 027011.

[0451] In some embodiments, the protein or multispecific antigen-binding constructs of this application may include one or more amino acid modifications that reduce or eliminate effector function such as ADCC or CDC, e.g., mutations that reduce or eliminate binding to the Fcγ receptor. Such mutations may be one, two, or three mutations at positions L234, L235, D270, N297, E318, K320, K322, P331, and P329, e.g., L234A, L235A, and P331S, where the amino acid residue numbering follows the EU index shown in Kabat.

[0452] Protein containing an antigen-binding region that binds to DLL3 in this disclosure The antigen-binding region of DLL3 in this disclosure can be genetically engineered using standard methods to create monospecific or multispecific antigen-binding constructs of various designs.

[0453] This disclosure also provides a single-specific protein comprising an antigen-binding region that binds to DLL3 of this disclosure.

[0454] In some embodiments, the single-specific protein is an antibody.

[0455] The disclosure also provides a multispecific antigen-binding construct comprising an antigen-binding region that binds to DLL3 of the disclosure.

[0456] In some embodiments, the multispecific antigen-binding construct is bispecific.

[0457] In some embodiments, the multispecific antigen-binding construct is triplicate.

[0458] In some embodiments, the multispecific antigen-binding construct is quadruplespecific.

[0459] In some embodiments, the multispecific antigen-binding construct is monovalent with respect to binding to DLL3.

[0460] In some embodiments, the multispecific antigen-binding construct is divalent with respect to binding to DLL3.

[0461] The disclosure also provides isolated, multispecific antigen-binding constructs comprising a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (such as CD3).

[0462] In some embodiments, the lymphocyte antigen is a T cell antigen.

[0463] In some embodiments, the T cell antigen is CD8 + It is a T cell antigen.

[0464] In some embodiments, the lymphocyte antigen is the NK cell antigen.

[0465] In some embodiments, the lymphocyte antigen is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C.

[0466] In some embodiments, the lymphocyte antigen is CD3ε.

[0467] In some embodiments, the first antigen-binding region that binds to DLL3 and / or the second antigen-binding region that binds to the lymphocyte antigen includes scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH.

[0468] In some embodiments, the first antigen-binding region that binds to DLL3 and / or the second antigen-binding region that binds to the lymphocyte antigen includes Fab.

[0469] In other embodiments, the first antigen-binding region that binds to DLL3 and / or the second antigen-binding region that binds to the lymphocyte antigen includes F(ab')2.

[0470] In some embodiments, the first antigen-binding region that binds to DLL3 and / or the second antigen-binding region that binds to the lymphocyte antigen includes VHH.

[0471] In some embodiments, the first antigen-binding region that binds to DLL3 and / or the second antigen-binding region that binds to the lymphocyte antigen includes Fv.

[0472] In some embodiments, the first antigen-binding region that binds to DLL3 and / or the second antigen-binding region that binds to the lymphocyte antigen includes Fd.

[0473] In some embodiments, the first antigen-binding region that binds to DLL3 and / or the second antigen-binding region that binds to the lymphocyte antigen includes scFv.

[0474] In certain embodiments, the multispecific antigen-binding construct is bispecific, with a first antigen-binding region that binds to DLL3 comprising scFv and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) comprising Fab.

[0475] In certain embodiments, the multispecific antigen-binding construct is bispecific, with a first antigen-binding region that binds to DLL3 comprising Fab, and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) comprising scFv.

[0476] In some embodiments, scFv includes VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH), from the N-terminus to the C-terminus.

[0477] In some embodiments, L1 contains approximately 5 to 50 amino acids.

[0478] In some embodiments, L1 contains approximately 5 to 40 amino acids.

[0479] In some embodiments, L1 contains approximately 10 to 30 amino acids.

[0480] In some embodiments, L1 contains approximately 10 to 20 amino acids.

[0481] In some embodiments, L1 includes the amino acid sequence of SEQ ID NOs: 27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139.

[0482] In certain embodiments, L1 includes the amino acid sequence of SEQ ID NO: 120.

[0483] In some embodiments, the extracellular antigen-binding region that binds to DLL3 is Includes HCDR1 of sequence numbers 15, 18, 21, 24, 18, 30, 50, 52, 53, 55, 57, 59, or 61; HCDR2 of sequence numbers 16, 19, 22, 25, 28, 31, 51, 54, 56, 58, 60, or 62; HCDR3 of sequence numbers 17, 20, 23, 26, 29, 32, 17, 20, 23, 26, 29, or 32; LCDR1 of sequence numbers 33, 36, 39, 41, 44, or 47; LCDR2 of sequence numbers 34, 37, 42, 45, or 48; and LCDR3 of sequence numbers 35, 38, 40, 43, 46, or 49.

[0484] In certain embodiments, the first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively. In some embodiments, the multispecific antigen-binding construct mediates T cell-mediated cytotoxicity, promotes T cell activation and proliferation, increases T cell cytokine release, and / or exhibits increased antitumor efficacy. In some embodiments, the multispecific antigen-binding construct potently mediates the proliferation of cytotoxic CD8 T cells. In some embodiments, the multispecific antigen-binding construct upregulates CD25, CD69, and CD71 expression on the surface of CD8 T cells. In some embodiments, the multispecific antigen-binding construct exhibits increased tumor death.

[0485] In certain embodiments, the first antigen-binding region that binds to DLL3 includes, respectively, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, and a second antigen-binding region that binds to lymphocyte antigens such as CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, for example, CD3.

[0486] In some embodiments, the antigen-binding region that binds to DLL3 includes, for example, the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 4, which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL.

[0487] In some embodiments, the first antigen-binding region that binds to DLL3 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 3 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 4, and optionally a second antigen-binding region that binds to a lymphocyte antigen such as CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, e.g., CD3. In some embodiments, the isolated multispecific antigen-binding construct mediates T cell-mediated cytotoxicity, promotes T cell activation and proliferation, increases T cell cytokine release, and / or exhibits enhanced antitumor effects. In some embodiments, the multispecific antigen-binding construct potently mediates the proliferation of cytotoxic CD8 T cells. In some embodiments, the multispecific antigen-binding construct upregulates the expression of CD25, CD69, and CD71 on the surface of CD8 T cells. In some embodiments, the multispecific antigen-binding construct exhibits increased tumor death.

[0488] In some embodiments, bispecific anti-DLL3×CD3 antibodies achieve more than 90% (e.g., 95%) tumor lysis by day 5 in T-cell cytotoxicity assays.

[0489] In some embodiments, the first antigen-binding region that binds to DLL3 includes VH and VL of SEQ ID NO: 4, which are at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 3, and optionally a second antigen-binding region that binds to a lymphocyte antigen such as CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, for example, CD3.

[0490] In some embodiments, the first antigen-binding region that binds to DLL3 includes a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 4, and optionally a second antigen-binding region that binds to a lymphocyte antigen such as CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, for example, CD3.

[0491] In some embodiments, the isolated multispecific antigen-binding constructs disclosed herein may be particularly effective in mediating T cell-mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release, and / or exhibiting enhanced antitumor efficacy.

[0492] In some embodiments, the first antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4.

[0493] In certain embodiments, the first antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, and optionally a second antigen-binding region that binds to lymphocyte antigens such as CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, for example, CD3.

[0494] In some embodiments, the multispecific antigen-binding construct mediates T cell-mediated cytotoxicity. In some embodiments, the multispecific antigen-binding construct potently mediates the proliferation of cytotoxic CD8 T cells. In some embodiments, the multispecific antigen-binding construct upregulates CD25, CD69, and CD71 expression on the surface of CD8 T cells. In some embodiments, the multispecific antigen-binding construct exhibits increased tumor death. In some embodiments, the bispecific anti-DLL3×CD3 antibody achieves over 90% (e.g., 95%) tumor lysis by day 5 in T cell cytotoxicity assays.

[0495] In some embodiments, the first antigen-binding region that binds to DLL3 includes the amino acid sequence of SEQ ID NOs. 63, 64, 65, 66, 67, 68, or 69.

[0496] In some embodiments, the first antigen-binding region that binds to DLL3 has the amino acid sequence of SEQ ID NO: 63.

[0497] In some embodiments, the first antigen-binding region that binds to DLL3 has the amino acid sequence of SEQ ID NO: 64.

[0498] In some embodiments, the first antigen-binding region that binds to DLL3 has the amino acid sequence of SEQ ID NO: 65.

[0499] In some embodiments, the first antigen-binding region that binds to DLL3 has the amino acid sequence of SEQ ID NO: 66.

[0500] In some embodiments, the first antigen-binding region that binds to DLL3 has the amino acid sequence of SEQ ID NO: 67.

[0501] In some embodiments, the first antigen-binding region that binds to DLL3 has the amino acid sequence of SEQ ID NO: 68.

[0502] In some embodiments, the first antigen-binding region that binds to DLL3 has the amino acid sequence of SEQ ID NO: 69.

[0503] In some embodiments, the first antigen-binding region that binds to DLL3 includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 63.

[0504] In some embodiments, the first antigen-binding region that binds to DLL3 includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 64.

[0505] In certain embodiments, the first antigen-binding region that binds to DLL3 includes the amino acid sequence of SEQ ID NO: 63 or 64.

[0506] The disclosure also provides a second antigen-binding region that binds to a lymphocyte antigen (such as CD3), wherein the antigen-binding region that binds to the lymphocyte includes the heavy chain variable region (VH) of SEQ ID NO: 77 and the light chain variable region (VL) of SEQ ID NO: 80, or the VH of SEQ ID NO: 84 and the VL of SEQ ID NO: 85.

[0507] In some embodiments, the second antigen-binding region that binds to a lymphocyte antigen (such as CD3) includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 77 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 80.

[0508] In some embodiments, the second antigen-binding region that binds to the lymphocyte antigen includes VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 77.

[0509] In some embodiments, the second antigen-binding region that binds to the lymphocyte antigen includes VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80, with VL being at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical.

[0510] In certain embodiments, the second antigen-binding region that binds to the lymphocyte antigen includes VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80.

[0511] In some embodiments, the second antigen-binding region that binds to a lymphocyte antigen (such as CD3) includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 84 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 85.

[0512] In some embodiments, the second antigen-binding region that binds to the lymphocyte antigen includes VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 84.

[0513] In some embodiments, the second antigen-binding region that binds to the lymphocyte antigen includes VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85, and VL is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH.

[0514] In certain embodiments, the second antigen-binding region that binds to the lymphocyte antigen includes VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85.

[0515] In some embodiments, the second antigen-binding region that binds to the lymphocyte antigen is Includes HCDR1 of sequence number 95, HCDR2 of sequence number 96, HCDR3 of sequence number 97, LCDR1 of sequence number 101, LCDR2 of sequence number 102, and LCDR3 of sequence number 104, or VH of sequence number 77 and VL of sequence number 80.

[0516] In some embodiments, the second antigen-binding region that binds to the lymphocyte antigen is Includes HCDR1 of sequence number 98, HCDR2 of sequence number 99, HCDR3 of sequence number 100, LCDR1 of sequence number 106, LCDR2 of sequence number 107, and LCDR3 of sequence number 108, or VH of sequence number 84 and VL of sequence number 85.

[0517] In certain embodiments, the second antigen-binding region that binds to lymphocyte antigens (such as CD3) includes HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 96, HCDR3 of SEQ ID NO: 97, LCDR1 of SEQ ID NO: 101, LCDR2 of SEQ ID NO: 102, and LCDR3 of SEQ ID NO: 104.

[0518] In a particular embodiment, the first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second antigen-binding region that binds to lymphocyte antigens (such as CD3) includes HCDR1 of SEQ ID NOs. 95, HCDR2 of SEQ ID NOs. 96, HCDR3 of SEQ ID NOs. 97, LCDR1 of SEQ ID NOs. 101, LCDR2 of SEQ ID NOs. 102, and LCDR3 of SEQ ID NOs. 104.

[0519] In a particular embodiment, the first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second antigen-binding region that binds to lymphocyte antigens (such as CD3) includes HCDR1 of SEQ ID NOs. 98, HCDR2 of SEQ ID NOs. 99, HCDR3 of SEQ ID NOs. 100, LCDR1 of SEQ ID NOs. 106, LCDR2 of SEQ ID NOs. 107, and LCDR3 of SEQ ID NOs. 108.

[0520] In certain embodiments, the first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second antigen-binding region that binds to lymphocyte antigens includes VH of SEQ ID NO. 77 and VL of SEQ ID NO. 80. In some embodiments, the multispecific antigen-binding construct mediates T cell-mediated cytotoxicity. In some embodiments, the multispecific antigen-binding construct potently mediates the proliferation of cytotoxic CD8 T cells. In some embodiments, the multispecific antigen-binding construct upregulates CD25, CD69, and CD71 expression on the surface of CD8 T cells. In some embodiments, the multispecific antigen-binding construct exhibits increased tumor death. In some embodiments, the bispecific anti-DLL3 × CD3 antibody achieves over 90% (e.g., 95%) tumor lysis by day 5 in T cell cytotoxicity assays.

[0521] In certain embodiments, the first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second antigen-binding region that binds to lymphocyte antigens (such as CD3) includes VH of SEQ ID NO. 84 and VL of SEQ ID NO. 85. In some embodiments, the multispecific antigen-binding construct mediates T cell-mediated cytotoxicity, promotes T cell activation, proliferation, and expansion, increases T cell cytokine release, and / or exhibits increased antitumor efficacy. In some embodiments, the multispecific antigen-binding construct potently mediates the proliferation of cytotoxic CD8 T cells. In some embodiments, the multispecific antigen-binding construct upregulates CD25, CD69, and CD71 expression on the surface of CD8 T cells. In some embodiments, the multispecific antigen-binding construct exhibits increased tumor death. In some embodiments, bispecific anti-DLL3×CD3 achieves over 90% (e.g., 95%) tumor lysis by day 5 in T-cell toxicity assays.

[0522] In certain embodiments, the first antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, and the second antigen-binding region that binds to lymphocyte antigens (such as CD3) includes HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 96, HCDR3 of SEQ ID NO: 97, LCDR1 of SEQ ID NO: 101, LCDR2 of SEQ ID NO: 102, and LCDR3 of SEQ ID NO: 104.

[0523] In certain embodiments, the first antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, and the second antigen-binding region that binds to lymphocyte antigens includes VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85.

[0524] In certain embodiments, the first antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, and the second antigen-binding region that binds to lymphocyte antigens includes VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80.

[0525] Preparation of multispecific antigen-binding constructs containing antigen-binding regions that bind to DLL3 The antigen-binding fragment that binds to DLL3 in this disclosure may be genetically engineered to form a multispecific antibody, which is also included within the scope of this application.

[0526] The antigen-binding fragment that binds to DLL3 may be genetically engineered into a full-length, multispecific antibody produced using Fab arm exchange, where the substitution is introduced into two monospecific bivalent antibodies within the Ig constant region CH3 domain that facilitate Fab arm exchange in vitro. In this method, two monospecific bivalent antibodies are genetically engineered to have a specific substitution in the CH3 domain that promotes heterodimer stability. These antibodies are incubated together under sufficiently reducing conditions for cysteine ​​in the hinge region to isomerize the disulfide bond, thereby generating a bispecific antibody via Fab arm exchange. The incubation conditions can, optimally, be returned to non-reducing conditions. Typical reducing agents that can be used include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation can be performed at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol at a pH of 5 to 8, for example, pH 7.0 or pH 7.4, for at least 90 minutes.

[0527] Possible CH3 mutations include techniques such as knob-in-hole mutations (Genentech), electrostatic match mutations (Chugai, Amgen, NovoNordisk, Oncomed), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Duobody® mutations (Genmab), and other asymmetric mutations (e.g., Zymeworks).

[0528] Knob-in-hole mutations, disclosed for example in International Publication No. 1996 / 027011, include interfacial mutations of the CH3 region in which an amino acid with a small side chain (hole) is introduced into the first CH3 region and an amino acid with a large side chain (knob) is introduced into the second CH3 region, resulting in a preferential interaction between the first and second CH3 regions. Exemplary CH3 region mutations that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0529] Heavy chain heterodimer formation can be facilitated by using electrostatic interactions by substituting a positively charged residue on the first CH3 region and a negatively charged residue on the second CH3 region, as described in U.S. Patent Application Publications 2010 / 0015133, 2009 / 0182127, 2010 / 028637, or 2011 / 0123532.

[0530] Other asymmetric mutations that can be used to promote heavy chain heterodimerization include L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T3 described in U.S. Patent Application Publication No. 2012 / 0149876 or 2013 / 0195849 (Zymeworks). These are 66L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0531] SEED body mutations, as described in U.S. Patent Application Publication No. 20070287170, involve the substitution of selected IgG residues with IgA residues to promote heavy chain heterodimerization.

[0532] Other exemplary variants that may be used include R409D_K370E / D399K_E357K, S354C_T366W / Y349C_T366S_L368A_Y407V, Y349C_T366W / S354C_T366W, and Y349C_T366W / S354C_T366S, as described in International Publication No. 2007 / 147901, International Publication No. 2011 / 143545, International Publication No. 2013157954, International Publication No. 2013096291, and U.S. Patent Application Publication No. 2018 / 0118849. S_L368A_Y407V, T366K / L351D, L351K / Y349E, L351K / Y349D, L351K / L368E, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, K392D / D399K, K392D / E356K, K253E_D282K_K322D / D239K_E240K_K292D, K392D_K409D / D356K_D399K.

[0533] Duobody® variants (Genmab) are disclosed, for example, in U.S. Patent No. 9,150,663 and U.S. Patent Application Publication No. 2014 / 0303356, and include variants such as F405L / K409R, wild-type / F405L_R409K, T350I_K370T_F405L / K409R, K370W / K409R, D399AFGHILMNRSTVWY / K409R, T366ADEFGHILMQVY / K409R, L368ADEGHNRSTVQ / K409AGRH, D399FHKRQ / K409AGRH, F405IKLSTVW / K409AGRH, and Y407LWQ / K409AGRH.

[0534] Additional bispecific or multispecific structures that can incorporate antigen-binding fragments that bind to DLL3 include: Dual Variable Domain Immunoglobulin (DVD) (International Publication No. 2009 / 134776; DVD is a full-length antibody comprising a heavy chain having a VH1-linker-VH2-CH structure and a light chain having a VL1-linker-VL2-CL structure, with the linker being optional); structures containing various dimerization domains for binding two antibody arms with different specificities, e.g., leucine zipper or collagen dimerization domain (International Publication No. 2012 / 022811, U.S. Patent No. 5,932,448, U.S. Patent No. 6,833,441); two or more domain antibodies (dAbs) conjugated together; diabodies; heavy-chain-only antibodies such as camelid antibodies and genetically modified camelid antibodies; Dual Targeting (DT)-Ig (GSK / Domantis); Two-in-one Antibody (Genentech), Cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX / Pfizer), IgG-like Bispecific (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche), ScFv / Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion,Examples include Zymogenetics / BMS, Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics), Dual (ScFv)2-Fab (National Research Center for Antibody Medicine--China), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), and Fab-Fv (UCB-Celltech). ScFv antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, bispecific T cell engagers (BiTE) (Micromet), tandem diabodies (Tandab) (Affimed), dual-affinity retargeting technology (DART) (MacroGenics), single-chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack), and COMBODY (Epigen Biotech), dual-target nanobodies (Ablynx), and dual-target heavy-chain-only domain antibodies.

[0535] The antigen-binding region that binds to DLL3 of this disclosure may also be genetically engineered into a multispecific antigen-binding construct comprising three polypeptide chains. In such a design, at least one antigen-binding region is in the form of an scFv. An exemplary design is shown below (where "1" indicates the first antigen-binding region, "2" indicates the second antigen-binding region, and "3" indicates the third antigen-binding region): Design 1: Chain A) scFv1-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Design 2: Chain A) scFv1-hinge-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Design 3: Chain A) scFv1-CH1-hinge-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Design 4: Chain A) CH2-CH3-scFv1, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 CH3 operation is described in U.S. Patent Publication No. 2012 / 0149876 or U.S. Patent Publication No. 2013 / 0195849 (Zymeworks) as L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F40 Variations such as 5A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W may be incorporated into designs 1-4.

[0536] In a particular embodiment, the design is as follows: Chain A) scFv1-hinge-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3.

[0537] In some embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (such as CD3), wherein the first antigen-binding region that binds to DLL3 is HCDR1 of SEQ ID NOs. 15, 18, 21, 24, 18, 30, 50, 52, 53, 55, 57, 59, or 61, SEQ ID NOs. 16, 19, 22, 25, 2 Includes HCDR2 of sequence numbers 8, 31, 51, 54, 56, 58, 60, or 62, HCDR3 of sequence numbers 17, 20, 23, 26, 29, 32, 17, 20, 23, 26, 29, or 32, LCDR1 of sequence numbers 33, 36, 39, 41, 44, or 47, LCDR2 of sequence numbers 34, 37, 42, 45, or 48, and LCDR3 of sequence numbers 35, 38, 40, 43, 46, or 49.

[0538] In some embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (such as CD3), wherein the first antigen-binding region that binds to DLL3 is Sequence numbers 15, 16, 17, 33, 34, 35, Sequence numbers 18, 19, 20, 36, 37, 38, respectively Sequence numbers 21, 22, 23, 39, 37, 40, Sequence numbers 24, 25, 26, 41, 42, 43, respectively Sequence numbers 18, 28, 29, 44, 45, 46, Sequence numbers 30, 31, 32, 47, 48, 49, The sequence numbers are 50, 51, 17, 33, 34, and 35 respectively. The sequence numbers are 52, 51, 17, 33, 34, and 35 respectively. Sequence numbers 53, 54, 20, 36, 37, 38, The sequence numbers are 55, 56, 23, 39, 37, and 40, respectively. Sequence numbers 57, 58, 26, 41, 42, 43, respectively Each of these corresponds to sequence numbers 59, 60, 29, 44, 45, 46, or The present invention provides isolated proteins, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 61, 62, 32, 47, 48, and 49, respectively.

[0539] In certain embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively. In some embodiments, the isolated multispecific antigen-binding construct mediates T cell-mediated cytotoxicity, promotes T cell activation and proliferation, increases T cell cytokine release, and / or exhibits enhanced antitumor effects. In some embodiments, the isolated multispecific antigen-binding construct potently mediates the proliferation of cytotoxic CD8 T cells. In some embodiments, the isolated multispecific antigen-binding construct upregulates CD25, CD69, and CD71 expression on the surface of CD8 T cells. In some embodiments, the isolated multispecific antigen-binding construct exhibits enhanced tumor death. In some embodiments, bispecific anti-DLL3×CD3 antibodies achieve over 90% (e.g., 95%) tumor lysis by day 5 in T-cell cytotoxic assays.

[0540] In some embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2 VH of SEQ ID NO: 1 and VL of SEQ ID NO: 4, VH of SEQ ID NO: 1 and VL of SEQ ID NO: 6 VH of SEQ ID NO: 1 and VL of SEQ ID NO: 8, VH of SEQ ID NO: 1 and VL of SEQ ID NO: 10, VH of SEQ ID NO: 1 and VL of SEQ ID NO: 12, VH of SEQ ID NO: 1 and VL of SEQ ID NO: 14, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 2, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 6 VH of SEQ ID NO: 3 and VL of SEQ ID NO: 8, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 10, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 12, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 14, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 2, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 6 VH of SEQ ID NO: 3 and VL of SEQ ID NO: 8, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 10, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 12, VH of SEQ ID NO: 3 and VL of SEQ ID NO: 14, VH of SEQ ID NO: 5 and VL of SEQ ID NO: 2 VH of SEQ ID NO: 5 and VL of SEQ ID NO: 4, VH of SEQ ID NO: 5 and VL of SEQ ID NO: 6, VH of SEQ ID NO: 5 and VL of SEQ ID NO: 8 VH of SEQ ID NO: 5 and VL of SEQ ID NO: 10 VH of SEQ ID NO: 5 and VL of SEQ ID NO: 12 VH of SEQ ID NO: 5 and VL of SEQ ID NO: 14 VH of sequence number 7 and VL of sequence number 2, VH of sequence number 7 and VL of sequence number 4, VH of sequence number 7 and VL of sequence number 6, VH of sequence number 7 and VL of sequence number 8, VH of sequence number 7 and VL of sequence number 10, VH of sequence number 7 and VL of sequence number 12, VH of sequence number 7 and VL of sequence number 14, VH of sequence number 9 and VL of sequence number 2, VH of sequence number 9 and VL of sequence number 4, VH of sequence number 9 and VL of sequence number 6, VH of sequence number 9 and VL of sequence number 8, VH of sequence number 9 and VL of sequence number 10, VH of sequence number 9 and VL of sequence number 12, VH of sequence number 9 and VL of sequence number 14, VH of sequence number 11 and VL of sequence number 2, VH of sequence number 11 and VL of sequence number 4, VH of sequence number 11 and VL of sequence number 6, VH of SEQ ID NO: 11 and VL of SEQ ID NO: 8, VH of sequence number 11 and VL of sequence number 10, VH of sequence number 11 and VL of sequence number 12, VH of sequence number 11 and VL of sequence number 14, VH of SEQ ID NO: 13 and VL of SEQ ID NO: 2 VH of SEQ ID NO: 13 and VL of SEQ ID NO: 4, VH of SEQ ID NO: 13 and VL of SEQ ID NO: 6, VH of SEQ ID NO: 13 and VL of SEQ ID NO: 8, VH of SEQ ID NO: 13 and VL of SEQ ID NO: 10, VH of SEQ ID NO: 13 and VL of SEQ ID NO: 12, or Includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14.

[0541] In certain embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding region that binds to DLL3 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 3 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 4.

[0542] In certain embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical. In some embodiments, the isolated multispecific antigen-binding constructs disclosed herein may be particularly effective in mediating T cell-mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release, and / or exhibiting enhanced antitumor efficacy. In certain embodiments, the isolated multispecific antigen-binding construct includes a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 3.

[0543] In certain embodiments, the isolated multispecific antigen-binding construct includes a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which are at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 3.

[0544] In certain embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical.

[0545] In certain embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding region that binds to DLL3 includes VH which is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 3 and VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VL of SEQ ID NO: 4. In some embodiments, the isolated multispecific antigen-binding construct mediates T cell-mediated cytotoxicity. In some embodiments, the isolated multispecific antigen-binding construct potently mediates the proliferation of cytotoxic CD8 T cells. In some embodiments, the isolated multispecific antigen-binding construct upregulates CD25, CD69, and CD71 expression on the surface of CD8 T cells. In some embodiments, the isolated multispecific antigen-binding construct exhibits increased tumor death. In some embodiments, bispecific anti-DLL3×CD3 antibodies achieve more than 90% (e.g., 95%) tumor lysis by day 5 in T-cell cytotoxicity assays.

[0546] In certain embodiments, the isolated multispecific antigen-binding construct includes a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4.

[0547] In some embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen, wherein the first antigen-binding region that binds to DLL3 comprises the amino acid sequence of SEQ ID NOs. 63, 64, 65, 66, 67, 68, or 69.

[0548] In some embodiments, the isolated multispecific antigen-binding construct includes a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding region that binds to DLL3 includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 63 or 64.

[0549] In certain embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding region that binds to DLL3 comprises the amino acid sequence of SEQ ID NO: 63 or 64.

[0550] In certain embodiments, the isolated multispecific antigen-binding construct includes a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the second antigen-binding region that binds to the lymphocyte antigen includes HCDR1 of SEQ ID NO: 95 or 98, HCDR2 of SEQ ID NO: 96 or 99, HCDR3 of SEQ ID NO: 97 or 100, or LCDR1 of SEQ ID NO: 101 or 106, LCDR2 of SEQ ID NO: 102 or 107, and LCDR3 of SEQ ID NO: 103, 104, or 108.

[0551] In some embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the second antigen-binding region that binds to the lymphocyte antigen is HCDR1 of sequence number 95, HCDR2 of sequence number 96, HCDR3 of sequence number 97, LCDR1 of sequence number 101, LCDR2 of sequence number 102, and LCDR3 of sequence number 104, or This includes HCDR1 (sequence number 98), HCDR2 (sequence number 99), HCDR3 (sequence number 100), LCDR1 (sequence number 106), LCDR2 (sequence number 107), and LCDR3 (sequence number 108).

[0552] In certain embodiments, the isolated multispecific antigen-binding construct includes a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3), wherein the second antigen-binding region that binds to the lymphocyte antigen includes HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 96, HCDR3 of SEQ ID NO: 97, or LCDR1 of SEQ ID NO: 101, LCDR2 of SEQ ID NO: 102, and LCDR3 of SEQ ID NO: 104.

[0553] In some embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen, wherein the second antigen-binding region that binds to a lymphocyte antigen (e.g., CD3) includes VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80.

[0554] In some embodiments, the isolated multispecific antigen-binding construct comprises a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to a lymphocyte antigen, wherein the second antigen-binding region that binds to a lymphocyte antigen is Includes HCDR1 of sequence number 98, HCDR2 of sequence number 99, HCDR3 of sequence number 100, LCDR1 of sequence number 106, LCDR2 of sequence number 107, and LCDR3 of sequence number 108, or VH of sequence number 84 and VL of sequence number 85.

[0555] This disclosure also relates to an isolated anti-DLL3 / anti-CD3 protein comprising a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to CD3, a. The first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 95, 96, 97, 101, 102, and 104, respectively. b. The first antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2, and the second antigen-binding region that binds to CD3 includes scFv of SEQ ID NO: 105, and / or c. The isolated anti-DLL3 / anti-CD3 protein provides isolated anti-DLL3 / anti-CD3 proteins, including HC1 of SEQ ID NO: 109, LC1 of SEQ ID NO: 110, and HC1 of SEQ ID NO: 112.

[0556] This disclosure also relates to an isolated anti-DLL3 / anti-CD3 protein comprising a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to CD3, a. The first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 95, 96, 97, 101, 102, and 104, respectively. b. The first antigen-binding region that binds to DLL3 includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2, and the second antigen-binding region that binds to CD3 includes scFv of SEQ ID NO: 119, and / or c. The isolated anti-DLL3 / anti-CD3 protein provides isolated anti-DLL3 / anti-CD3 proteins, including HC1 of SEQ ID NO: 109, LC1 of SEQ ID NO: 110, and HC1 of SEQ ID NO: 113.

[0557] This disclosure also relates to an isolated anti-DLL3 / anti-CD3 protein comprising a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to CD3, a. The first antigen-binding domain that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 98, 99, 100, 106, 107, and 108, respectively, and / or b. The first antigen-binding region that binds to DLL3 includes scFv of SEQ ID NO: 63, and the second antigen-binding region that binds to lymphocyte antigens includes VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85, and / or c. The isolated anti-DLL3 / anti-CD3 protein provides isolated anti-DLL3 / anti-CD3 proteins, including HC1 of SEQ ID NO: 111, HC2 of SEQ ID NO: 116, and LC2 of SEQ ID NO: 117.

[0558] This disclosure also relates to an isolated anti-DLL3 / anti-CD3 protein comprising a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to CD3, a. The first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 95, 96, 97, 101, 102, and 104, respectively, and / or b. The first antigen-binding region that binds to DLL3 includes scFv of SEQ ID NO: 63, and the second antigen-binding region that binds to lymphocyte antigens includes VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80, and / or optionally, c. The isolated anti-DLL3 / anti-CD3 protein provides isolated anti-DLL3 / anti-CD3 proteins, including HC1 of SEQ ID NO: 111, HC2 of SEQ ID NO: 114, and LC2 of SEQ ID NO: 115.

[0559] This disclosure also relates to an isolated anti-DLL3 / anti-CD3 protein comprising a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to CD3, a. The first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 98, 99, 100, 106, 107, and 108, respectively, and / or b. The first antigen-binding region that binds to DLL3 includes scFv of SEQ ID NO: 64, and the second antigen-binding region that binds to lymphocyte antigens includes VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85, and / or c. The isolated anti-DLL3 / anti-CD3 protein provides an isolated multispecific antigen-binding construct comprising HC1 of SEQ ID NO: 71, HC2 of SEQ ID NO: 118, and LC2 of SEQ ID NO: 117.

[0560] This disclosure also relates to an isolated anti-DLL3 / anti-CD3 protein comprising a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to CD3, a. The first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 98, 99, 100, 106, 107, and 108, respectively, and / or b. The first antigen-binding region that binds to DLL3 includes scFv of SEQ ID NO: 64, and the second antigen-binding region that binds to lymphocyte antigens includes VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85, and / or c. The isolated anti-DLL3 / anti-CD3 protein provides isolated anti-DLL3 / anti-CD3 proteins, including HC1 of SEQ ID NO: 229, HC2 of SEQ ID NO: 230, and LC2 of SEQ ID NO: 117.

[0561] This disclosure also relates to an isolated anti-DLL3 / anti-CD3 protein comprising a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to CD3, a. The first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 98, 99, 100, 106, 107, and 108, respectively, and / or b. A Fab comprising a first antigen-binding region that binds to DLL3, which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the scFv of SEQ ID NO: 64, and a second antigen-binding region that binds to CD3, which comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO: 84, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VL of SEQ ID NO: 85, and / or c. The isolated anti-DLL3 / anti-CD3 protein is provided, comprising HC1 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to HC1 of SEQ ID NO: 71, HC2 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to HC2 of SEQ ID NO: 118, and LC2 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 117.

[0562] This disclosure also relates to an isolated anti-DLL3 / anti-CD3 protein comprising a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to CD3, a. The first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 98, 99, 100, 106, 107, and 108, respectively, and / or b. A Fab comprising a first antigen-binding region that binds to DLL3, which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the scFv of SEQ ID NO: 64, and a second antigen-binding region that binds to CD3, which comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO: 84, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VL of SEQ ID NO: 85, and / or c. The isolated anti-DLL3 / anti-CD3 protein is provided, comprising HC1 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to HC1 of SEQ ID NO: 229, HC2 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to HC2 of SEQ ID NO: 230, and LC2 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 117.

[0563] In certain embodiments, the Disclosure relates to an isolated, multispecific antigen-binding construct comprising a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to CD3, a) The first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 98, 99, 100, 106, 107, and 108, respectively, and / or b) Provide an isolated multispecific antigen-binding construct in which a first antigen-binding region that binds to DLL3 comprises scFv of SEQ ID NO: 64, and a second antigen-binding region that binds to CD3 comprises VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80.

[0564] In some embodiments, isolated anti-DLL3 / anti-CD3 proteins mediate T cell-mediated cytotoxicity. In some embodiments, isolated anti-DLL3 / anti-CD3 proteins potently mediate the proliferation of cytotoxic CD8 T cells. In some embodiments, isolated anti-DLL3 / anti-CD3 proteins upregulate the expression of CD25, CD69, and CD71 on the surface of CD8 T cells. In some embodiments, isolated anti-DLL3 / anti-CD3 proteins exhibit increased tumor death. In some embodiments, isolated anti-DLL3 / anti-CD3 proteins achieve over 90% (e.g., 95%) tumor lysis by day 5 in T cell cytotoxicity assays.

[0565] This disclosure also relates to an isolated anti-DLL3 / anti-CD3 protein comprising a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to CD3, a. The first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 98, 99, 100, 106, 107, and 108, respectively, and / or b. A Fab comprising a first antigen-binding region that binds to DLL3, which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the scFv of SEQ ID NO: 64, and a second antigen-binding region that binds to CD3, which comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VH of SEQ ID NO: 84, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the VL of SEQ ID NO: 85, and / or c. The isolated anti-DLL3 / anti-CD3 protein is provided, comprising HC1 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to HC1 of SEQ ID NO: 229, HC2 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to HC2 of SEQ ID NO: 230, and LC2 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 117.

[0566] In some embodiments, isolated anti-DLL3 / anti-CD3 proteins mediate T cell-mediated cytotoxicity. In some embodiments, isolated anti-DLL3 / anti-CD3 proteins potently mediate the proliferation of cytotoxic CD8 T cells. In some embodiments, isolated anti-DLL3 / anti-CD3 proteins upregulate the expression of CD25, CD69, and CD71 on the surface of CD8 T cells. In some embodiments, isolated anti-DLL3 / anti-CD3 proteins exhibit increased tumor death. In some embodiments, isolated anti-DLL3 / anti-CD3 proteins achieve over 90% (e.g., 95%) tumor lysis by day 5 in T cell toxicity assays.

[0567] In certain embodiments, the Disclosure relates to an isolated, multispecific antigen-binding construct comprising a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to CD3, a. The first antigen-binding region that binds to DLL3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 15, 16, 17, 33, 34, and 35, respectively, and the second domain that binds to CD3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 98, 99, 100, 106, 107, and 108, respectively, and / or b. Provides an isolated multispecific antigen-binding construct in which a first antigen-binding region that binds to DLL3 includes scFv of SEQ ID NO: 64, and a second antigen-binding region that binds to CD3 includes VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80.

[0568] In some embodiments, the isolated multispecific antigen-binding construct contains lysine (e.g., K477) at the C-terminus of both Fc domains (i.e., HC1 and HC2 domains). Additional lysine may enhance the expression of the construct.

[0569] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 80% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 80% identical to the VH of SEQ ID NO: 77 and a VL that is at least 80% identical to the VL of SEQ ID NO: 80.

[0570] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 85% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 85% identical to the VH of SEQ ID NO: 77 and a VL that is at least 85% identical to the VL of SEQ ID NO: 80.

[0571] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 90% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 90% identical to the VH of SEQ ID NO: 77 and a VL that is at least 90% identical to the VL of SEQ ID NO: 80.

[0572] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 95% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 95% identical to the VH of SEQ ID NO: 77 and a VL that is at least 95% identical to the VL of SEQ ID NO: 80.

[0573] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 99% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 99% identical to the VH of SEQ ID NO: 77 and a VL that is at least 99% identical to the VL of SEQ ID NO: 80.

[0574] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 95% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VL that is at least 95% identical to the VH of SEQ ID NO: 77 and the VL of SEQ ID NO: 80.

[0575] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 99% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VL that is at least 99% identical to the VH of SEQ ID NO: 77 and the VL of SEQ ID NO: 80.

[0576] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 95% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 95% identical to the VH of SEQ ID NO: 77 and a VL of SEQ ID NO: 80.

[0577] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 99% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 99% identical to the VH of SEQ ID NO: 77 and a VL of SEQ ID NO: 80.

[0578] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises VH which is at least 95% identical to VH of SEQ ID NO: 77 and VL which is at least 95% identical to VL of SEQ ID NO: 80.

[0579] In certain embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80.

[0580] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 80% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 80% identical to the VH of SEQ ID NO: 84 and a VL that is at least 80% identical to the VL of SEQ ID NO: 85.

[0581] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 85% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 85% identical to the VH of SEQ ID NO: 84 and a VL that is at least 85% identical to the VL of SEQ ID NO: 85.

[0582] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 90% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 90% identical to the VH of SEQ ID NO: 84 and a VL that is at least 90% identical to the VL of SEQ ID NO: 85.

[0583] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 95% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 95% identical to the VH of SEQ ID NO: 84 and a VL that is at least 95% identical to the VL of SEQ ID NO: 85.

[0584] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 99% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 99% identical to the VH of SEQ ID NO: 84 and a VL that is at least 99% identical to the VL of SEQ ID NO: 85.

[0585] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 95% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VL that is at least 95% identical to the VH of SEQ ID NO: 84 and the VL of SEQ ID NO: 85.

[0586] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 99% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VL that is at least 99% identical to the VH of SEQ ID NO: 84 and the VL of SEQ ID NO: 85.

[0587] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv that is at least 95% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH that is at least 95% identical to the VH of SEQ ID NO: 84 and a VL of SEQ ID NO: 85.

[0588] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises an scFv which is at least 99% identical to the scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises a VH which is at least 99% identical to the VH of SEQ ID NO: 84 and a VL of SEQ ID NO: 85.

[0589] In some embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises VH which is at least 95% identical to VH of SEQ ID NO: 84 and VL which is at least 95% identical to VL of SEQ ID NO: 85.

[0590] In certain embodiments, the Disclosure provides an isolated multispecific antigen-binding construct comprising a first antigen-binding region bound to DLL3 and a second antigen-binding region bound to CD3, wherein the first antigen-binding region bound to DLL3 comprises scFv of SEQ ID NO: 64, and the second antigen-binding region bound to CD3 comprises VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85.

[0591] As shown in the examples, the isolated multispecific antigen-binding constructs disclosed herein may be particularly effective in mediating T cell-mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release, and / or exhibiting enhanced antitumor efficacy. Therefore, in some embodiments, the isolated multispecific antigen-binding constructs disclosed herein mediate T cell-mediated cytotoxicity. In some embodiments, the isolated multispecific antigen-binding constructs disclosed herein potently mediate the proliferation of cytotoxic CD8 T cells. In some embodiments, the isolated multispecific antigen-binding constructs disclosed herein upregulate the expression of CD25, CD69, and CD71 on the surface of CD8 T cells. In some embodiments, the isolated multispecific antigen-binding constructs disclosed herein exhibit enhanced tumor death. In some embodiments, the bispecific anti-DLL3×CD3 antibody disclosed herein achieves over 90% (e.g., 95%) tumor lysis by day 5 in T cell cytotoxicity assays. Particularly surprising is the fact that the multispecific antigen-binding construct exhibiting the greatest tumor-killing effect binds to an epitope on DLL3 closest to the cell membrane, a location thought to impair the multispecific antibody's ability to optimally position tumor cells and cytotoxic T cells to achieve immune synapses.

[0592] Isotype, allotype, and Fc gene manipulation The Ig constant region fragments present in the proteins of this disclosure, such as the Ig constant region or Fc region, may be of any allotype or isotype.

[0593] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is an IgG1 isotype.

[0594] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is an IgG2 isotype.

[0595] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is an IgG3 isotype.

[0596] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is an IgG4 isotype.

[0597] The Ig constant region or fragments of the Ig constant region can be any allotype. The allotype is not expected to affect the properties of the Ig constant region, such as binding or Fc-mediated effector function. The immunogenicity of therapeutic proteins containing fragments of the Ig constant region is associated with an increased risk of infusion reactions and a shortened therapeutic response (Baert et al., (2003) N Engl J Med 348:602-08). The extent to which therapeutic proteins containing fragments of the Ig constant region induce an immune response in the host may be determined to some extent by the allotype of the Ig constant region (Stickler et al., (2011) Genes and Immunity 12:213-21). The allotype of the Ig constant region is related to amino acid sequence mutations at specific positions in the antibody's constant region sequence. Table 3 shows the selected IgG1, IgG2, and IgG4 allotypes.

[0598] [Table 3]

[0599] In a particular embodiment, the allotype of the Ig constant region is huIgG1_G1m(17).

[0600] C-terminal lysine (CTL) can be removed from the Ig constant region by endogenous circulating carboxypeptidase in the bloodstream (Cai et al., (2011) Biotechnol Bioeng 108:404-412). During manufacturing, extracellular Zn is used as described in U.S. Patent Application Publication No. 20140273092. 2+ EDTA, or EDTA-Fe 3+ By controlling the concentration of [the substance], CTL removal can be controlled to below the maximum level. The CTL content of a protein can be measured using known methods.

[0601] In some embodiments, the antigen-binding fragment conjugated to the Ig constant region and bound to DLL3 has a C-terminal lysine content of about 10% to about 90%. In some embodiments, the C-terminal lysine content is about 20% to about 80%. In some embodiments, the C-terminal lysine content is about 40% to about 70%. In some embodiments, the C-terminal lysine content is about 55% to about 70%. In some embodiments, the C-terminal lysine content is about 60%.

[0602] By introducing mutations in the Fc region to the antigen-binding region that binds to the Ig constant region or DLL3 conjugated to a fragment of the Ig constant region, the effector functions and / or pharmacokinetic properties of ADCC, ADCP, and / or ADCP can be modified. This can be achieved by introducing mutations into Fc that control the binding of the mutated Fc to activated FcγRs (FcγRI, FcγRIIa, FcγRIII), FcγRIIb, and / or FcRn.

[0603] In some embodiments, the antigen-binding region that binds to DLL3 conjugated to the Ig constant region or a fragment of the Ig constant region includes at least one mutation in the Ig constant region or fragment of the Ig constant region.

[0604] In some embodiments, at least one mutation is located in the Fc region.

[0605] In some embodiments, the antigen-binding region that binds to the Ig constant region or DLL3 conjugated to a fragment of the Ig constant region contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations in the Fc region.

[0606] In some embodiments, the antigen-binding region that binds to the Ig constant region or a DLL3 conjugated to a fragment of the Ig constant region includes at least one mutation in the Fc region that modulates the binding of the antibody to FcRn.

[0607] Fc positions that can be mutated to regulate the half-life (e.g., binding to FcRn) include positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434, and 435. Exemplary mutations that can be performed individually or in combination are T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A, and H435R. Exemplary mutations, either alone or in combination, that can be performed to increase the half-life of an antibody are the mutations M428L / N434S, M252Y / S254T / T256E, T250Q / M428L, N434A, and T307A / E380A / N434A. Exemplary mutations, either alone or in combination, that can be performed to decrease the half-life are the mutations H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R.

[0608] In some embodiments, the antigen-binding region that binds to the Ig constant region or DLL3 conjugated to a fragment of the Ig constant region contains the M252Y / S254T / T256E mutation.

[0609] In some embodiments, an antigen-binding region that binds to the Ig constant region or DLL3 conjugated to a fragment of the Ig constant region includes at least one mutation in the Fc region that enhances protein binding to the Fcγ receptor (FcγR) and / or reduces Fc effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or phagocytosis (ADCP).

[0610] Fc sites that can be mutated to reduce protein binding to activated FcγR and subsequently reduce effector function include positions 214, 233, 234, 235, 236, 237, 238, 265, 267, 268, 270, 295, 297, 309, 327, 328, 329, 330, 331, and 365. Exemplary mutations that can be added individually or in combination include the K214T, E233P, L234V, L234A, G236 deletion, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S, and P331S mutations in IgG1, IgG2, IgG3, or IgG4. Exemplary mutation combinations that result in reduced ADCC protein include L234A / L235A in IgG1, L234A / L235A / D265S in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in all Ig isotypes, V234A / G237A in IgG2, and K214T / E233P / L234V / L235A / G in IgG1. These are mutations in IgG2: 236 deletion / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S. Alternatively, a hybrid IgG2 / 4 Fc domain, such as Fc having residues 117-260 derived from IgG2 and residues 261-447 derived from IgG4, may be used.

[0611] An example of a mutation that results in reduced CDC protein is the K322A mutation.

[0612] The well-known S228P mutation can be added to an IgG4 antibody to enhance the stability of IgG4.

[0613] In some embodiments, the antigen-binding region conjugated to the Ig constant region or a fragment of the Ig constant region, which binds to DLL3, contains at least one mutation selected from the group consisting of deletions of K214T, E233P, L234V, L234A, G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, K322, A330S, and P331S.

[0614] In some embodiments, the antigen-binding region that binds to DLL3 conjugated to an Ig constant region or a fragment of an Ig constant region contains the L234A / L235A / D265S mutation. In certain embodiments, the antigen-binding region that binds to DLL3 is conjugated to an IgG1 constant region or a fragment of an IgG1 constant region containing the L234A_L235A_D265S mutation.

[0615] In some embodiments, the antigen-binding region that binds to the Ig constant region or DLL3 conjugated to a fragment of the Ig constant region contains the L234A / L235A mutation.

[0616] In some embodiments, an antigen-binding region that binds to the Ig constant region or DLL3 conjugated to a fragment of the Ig constant region includes at least one mutation in the Fc region that enhances protein binding to the Fcγ receptor (FcγR) and / or enhances Fc effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or phagocytosis (ADCP).

[0617] Fc sites that can be mutated to increase protein binding to activated FcγR and / or enhance Fc effector function include positions 236, 239, 243, 256, 290, 292, 298, 300, 305, 312, 326, 330, 332, 333, 334, 345, 360, 339, 378, 396, or 430 (residue numbering according to the EU index). Exemplary mutations that can be performed individually or in combination include G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305L, K326A, A330K, I332E, E333A, K334A, A339T, and P396L. Exemplary mutation combinations that result in increased ADCC or ADCP proteins include S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E.

[0618] Fc positions that can be mutated to enhance CDC include positions 267, 268, 324, 326, 333, 345, and 430. Exemplary mutations that can occur individually or in combination are S267E, F1268F, S324T, K326A, K326W, E333A, E345K, E345Q, E345R, E345Y, E430S, E430F, and E430T. Exemplary combinations of mutations that result in proteins with increased CDC are K326A / E333A, K326W / E333A, H268F / S324T, S267E / H268F, S267E / S324T, and S267E / H268F / S324T.

[0619] The specific mutations described herein are those observed when compared to the wild-type amino acid sequences of IgG1, IgG2, and IgG4, respectively, of SEQ ID NOs. 257, 258, and 259.

[0620] The binding of antibodies to FcγR or FcRn can be evaluated using flow cytometry in genetically engineered cells expressing each receptor. In an exemplary binding assay, 2 × 10⁶ antibodies are used in a 96-well plate. 5 Cells are seeded at a rate of 1 / well and blocked in BSA Stain Buffer (BD Biosciences, San Jose, USA) at 4°C for 30 minutes. Cells are incubated with the test antibody on ice at 4°C for 1.5 hours. After washing twice with BSA stain buffer, cells are incubated with R-PE labeled anti-human IgG secondary antibody (Jackson Immunoresearch Laboratories) at 4°C for 45 minutes. Cells are washed twice with stain buffer and then resuspended in 150 μL of Stain Buffer containing a 1:200 dilution of DRAQ7 live / dead cell staining reagent (Cell Signaling Technology, Danvers, USA). PE and DRAQ7 signals from stained cells are detected using a Miltenyi MACSQuant flow cytometer (Miltenyi Biotec, Auburn, USA) with B2 and B4 channels, respectively. Live cells were gated out by DRAQ7 exclusion, and the geometric mean fluorescence signal was determined for at least 10,000 live cell events collected. FlowJo software (Tree Star) was used for the analysis. The data was plotted as the logarithm of antibody concentration against the mean fluorescence signal. Nonlinear regression analysis was performed.

[0621] Glycan gene manipulation The ability of an antigen-binding domain to mediate ADCC (Adverse Drug Continuity Comboconjugate) in an antigen-binding domain conjugated to DLL3 in the Ig constant region or a fragment of the Ig constant region can be enhanced by genetically engineering the oligosaccharide component of the Ig constant region or a fragment of the Ig constant region. Human IgG1 or IgG3 is N-glycosylated at Asn297, where the majority of the glycan is in the form of known bifurcated G0, G0F, G1, G1F, G2, or G2F. Ig constant region-containing proteins that can be produced by unengineered CHO cells typically have a glycan fucose content of at least about 85%. Removal of core fucose from a bifurcated complex oligosaccharide bound to an antigen-binding domain conjugated to DLL3 in the Ig constant region or a fragment of the Ig constant region enhances the protein's ADCC via improved FcγRIIIa binding without altering antigen binding or CDC activity.Such proteins can be achieved using different methods reported to lead to the successful expression of relatively high defucosylated immunoglobulins with branched complex-type Fc oligosaccharides, including: control of culture osmotic pressure (Konno et al., Cytotechnology 64(:249-65, 2012), application of variant CHO strain Lec13 as host cell line (Shields et al., J Biol Chem 277:26733-26740, 2002), application of variant CHO strain EB66 as host cell line (Olivier et al., MAbs;2(4):405-415, 2010;PMID:20562582), and application of rat hybridoma cell line YB2 / 0 as host cell line (Shinkawa et al., J Biol Chem (278:3466-3473, 2003), introduction of specific small interfering RNA to the 1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-908, 2004), or co-expression of β-1,4-N-acetylglucosaminyltransferase III and kifunensin, a potent inhibitor of Golgi α-mannosidase II or α-mannosidase I (Ferrara et al., J Biol Chem 281:5032-5036, 2006; Ferrara et al., Biotechnol Bioeng 93:851-861, 2006; Xhou et al., Biotechnol Bioeng 99:652-65, 2008).

[0622] In some embodiments, the antigen-binding region that binds to DLL3 conjugated to the Ig constant region or a fragment of the Ig constant region of the Disclosure has a branched glycan structure having a fucose content of about 1% to about 15%, for example, about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the antigen-binding region that binds to DLL3 conjugated to the Ig constant region or a fragment of the Ig constant region has a glycan structure having a fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, or 20%.

[0623] "Fucose content" refers to the amount of fucose monosaccharides within the sugar chain in Asn297. The relative amount of fucose is the ratio of the fucose-containing structure to the total sugar structure. These sugar structures can be analyzed by several methods, e.g., 1) using MALDI-TOF on N-glycosidase F-treated samples (e.g., complex, hybrid, and oligo- and high-mannose structures) as described in International Publication No. 2008 / 077546, 2) enzymatic release of Asn297 glycan, subsequent derivatization, and detection / quantification by HPLC (UPLC) and / or HPLC-MS (UPLC-MS) with fluorescence detection, 3) intact protein analysis of natural or reduced mAbs with or without treatment of Asn297 glycan with Endo S or other enzymes that cleave between the first and second GlcNAc monosaccharides, leaving fucose attached to the first GlcNAc, 4) digestion of mAbs into constituent peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C), followed by separation, detection and quantification by HPLC-MS (UPLC-MS), 5) Asn mAb oligosaccharides can be characterized and quantified by separating them from mAb proteins via specific enzymatic deglycosylation using PNGase F at position 297. The oligosaccharides thus released can be labeled with fluorophores and separated and identified by various supplemental techniques that enable detailed characterization of the glycan structure by matrix-assisted laser desorption ionization (MALDI) mass spectrometry by comparing the measured mass with the theoretical mass, determination of the degree of sialylation by ion-exchange HPLC (GlycoSep C), separation and quantification of oligosaccharide types according to hydrophilicity criteria by normal-phase HPLC (GlycoSep N), and separation and quantification of oligosaccharides by high-performance capillary electrophoresis-laser-induced fluorescence (HPCE-LIF).

[0624] When used herein, "low fucose" or "low fucose content" refers to an antigen-binding region that binds to the Ig constant region or a DLL3 conjugated to a fragment of the Ig constant region having a fucose content of approximately 1% to 15%.

[0625] When used herein, "normal fucose" or "normal fucose content" means that the antigen-binding region that binds to the Ig constant region or DLL3 conjugated to a fragment of the Ig constant region has a fucose content of more than approximately 50%, typically more than 80%, or more than 85%.

[0626] Anti-idiotype antibody The anti-idiotype antibody is an antibody that specifically binds to the antigen-binding region that binds to DLL3 of the present disclosure.

[0627] This application also provides an anti-idiotype antibody that specifically binds to the antigen-binding region of DLL3 of the present disclosure.

[0628] An anti-idiotypic (Id) antibody is an antibody that recognizes an antigenic determinant (e.g., a paratope or CDR). Id antibodies may or may not block the antigen. Antigen-blocking Id can be used to detect free antigen-binding regions in a sample (e.g., the antigen-binding region that binds to DLL3 in this disclosure). Non-blocking Id can be used to detect all antibodies in a sample (free, partially bound to the antigen, or fully bound to the antigen). Id antibodies can be prepared by immunizing animals with an antibody that has been prepared as an anti-Id.

[0629] Furthermore, so-called anti-anti-Id antibodies can be generated by using an anti-Id antibody as an immunogen to induce an immune response in yet another animal. The anti-anti-Id may have the same epitope as the original antigen-binding region that induced the anti-Id. Therefore, by using an antibody against the idiotype determinant of the antigen-binding region, it is possible to identify other clones expressing an antigen-binding region of the same specificity. Anti-Id antibodies can be modified (thereby producing anti-Id antibody variants) and / or induced by any preferred technique, such as those described elsewhere in this specification.

[0630] Immunoconjugates The antigen-binding domains that bind to DLL3, proteins containing the antigen-binding domains that bind to DLL3, or multispecific antigen-binding constructs containing the antigen-binding domains that bind to DLL3 (collectively referred to herein as DLL3-binding proteins) can be conjugated to heterologous molecules.

[0631] In some embodiments, the heterologous molecule is a detectable label or a cytotoxic agent.

[0632] This application also provides an antigen-binding fragment that binds to DLL3 conjugated with a detectable label.

[0633] This application also provides a protein comprising an antigen-binding fragment that binds to DLL3 conjugated with a detectable label.

[0634] This application also provides a multispecific antigen-binding construct comprising an antigen-binding fragment that binds to DLL3 conjugated with a detectable label.

[0635] This application also provides an antigen-binding fragment that binds to DLL3 conjugated with a cytotoxic agent.

[0636] This application also provides a protein comprising an antigen-binding fragment that binds to DLL3 conjugated with a cytotoxic agent.

[0637] This application also provides a multispecific antigen-binding construct comprising an antigen-binding fragment that binds to DLL3 conjugated with a cytotoxic agent.

[0638] The DLL3-binding proteins of this disclosure may be used to target therapeutic agents against DLL3-expressing cells, such as DLL3-expressing prostate cancer cells or small cell lung cancer cells. Alternatively, DLL3-expressing cells may be targeted using the DLL3-binding proteins of this disclosure conjugated to therapeutic agents intended to be internalized and then modify cellular function.

[0639] In some embodiments, the detectable label is also a cytotoxic agent.

[0640] The DLL3-binding protein of this disclosure, conjugated to a detectable label, can be used to evaluate DLL3 expression in a variety of samples.

[0641] The detectable label comprises a composition that, when conjugated to the DLL3-binding protein of this disclosure, makes the latter detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

[0642] Examples of detectable labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron density reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorophores, fluorescent quenchers, colored molecules, radioisotopes, scintillates, avidin, streptavidin, protein A, protein G, antibodies or their fragments, polyhistidine, Ni 2+ Examples include Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatases, peroxidases, luciferases, electron donors / receptors, acridinium esters, and colorimetric substrates.

[0643] A detectable label may spontaneously emit a signal, for example, when the detectable label is a radioactive isotope. In other cases, a detectable label emits a signal as a result of being stimulated by an external field.

[0644] Exemplary radioactive isotopes may be γ-emitting, Auger-emitting, β-emitting, α-emitting, or positron-emitting radioactive isotopes. Exemplary radioactive isotopes include: 3 H, 11 C, 13 C, 15 N, 18 F, 19 F, 55 Co, 57 Co, 60 Co, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 68 Ga, 72 As, 75 Br, 86 Y, 89 Zr, 90 Sr, 94m Tc, 99m Tc, 115 In, 123 1. 124 1. 125 I, 131 1. 211 At, 212 Bi, 213 Bi, 223 Ra, 226 Ra, 225 Ac, and 227 Ac is one example.

[0645] Exemplary metal atoms include metals with atomic numbers greater than 20, such as calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, bromine, krypton, rubidium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, antimony, tellurium, iodine, xenon, cesium, barium, lanthanum, hafnium, tantalum, tungsten, and rhenium atoms. These atoms are osmium, iridium, platinum, gold, mercury, thallium, lead, bismuth, francium, radium, actinium, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, or lawrencium.

[0646] In some embodiments, the metal atom may be an alkaline earth metal having an atomic number greater than 20.

[0647] In some embodiments, the metal atom may be a lanthanide.

[0648] In some embodiments, the metal atom may be an actinide.

[0649] In some embodiments, the metal atom may be a transition metal.

[0650] In some embodiments, the metal atom can be a base metal.

[0651] In some embodiments, the metal atom can be a gold atom, a bismuth atom, a tantalum atom, and a gadolinium atom.

[0652] In some embodiments, the metal atom can be a metal having an atomic number of 53 (i.e., iodine) to 83 (i.e., bismuth).

[0653] In some embodiments, the metal atom can be an atom suitable for magnetic resonance imaging.

[0654] The metal atom is Ba 2+ , Bi 3+ , Cs + , Ca 2+ , Cr 2+ , Cr 3+ , Cr 6+ , Co 2+ , Co 3+ , Cu + , Cu 2+ , Cu 3+ , Ga 3+ , Gd 3+ , Au + , Au 3+ , Fe 2+ , Fe 3+ , F 3+ , Pb 2+ , Mn 2+ , Mn 3+ , Mn 4+ , Mn 7+ , Hg 2+ , Ni 2+ , Ni 3+ , Ag + , Sr 2+ , Sn 2+ , Sn 4+ , and Zn 2+ which can be metal ions in the form of +1, +2, or +3 oxidation states. The metal atom can include a metal oxide, for example, iron oxide, manganese oxide, or gadolinium oxide.

[0655] Suitable dyes include, for example, any commercially available dye such as 5(6)-carboxyfluorescein, IRDye 680RD maleimide, or IRDye 800CW, or ruthenium polypyridyl dye.

[0656] Suitable fluorophores include fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDye (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594, Alexa647), near-infrared (NIR) (700-900 nm) fluorescent dyes, as well as carbocyanin and aminostyryl dyes.

[0657] The antigen-binding region of DLL3 conjugated with a detectable label can be used as a contrast agent.

[0658] Proteins containing an antigen-binding domain that binds to DLL3 conjugated with a detectable label can be used as contrast agents.

[0659] A multispecific antigen-binding construct containing an antigen-binding region that binds to DLL3 conjugated with a detectable label can be used as a contrast agent.

[0660] In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin or fragment thereof derived from bacteria, fungi, plants, or animals), or a radioisotope (i.e., a radioconjugate).

[0661] In some embodiments, the cytotoxic agent is daunomycin, doxorubicin, methotrexate, vindesine, bacterial toxins such as diphtheria toxin, lysine, geldanamycin, mytansinoids, or calicheamicin. The cytotoxic agent may induce its cytotoxic or cell proliferation inhibitory effect through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

[0662] In some embodiments, the cytotoxic agents are enzymatic toxins such as diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), bitter melon (momordica charantia) inhibitor, curcin, crotin, soapwort (sapaonaria officinalis) inhibitor, geronin, mitogellin, restrictosin, phenomycin, enomycin, and trichothecenes.

[0663] In some embodiments, the cytotoxic agent is 212 Bi, 131 I, 131 In, 90 Y, and 186 These are radioactive nuclides such as Re.

[0664] In some embodiments, the cytotoxic agent is drostatin or a peptide analog and derivative of drostatin, auristatin, or monomethyl auristatin phenylalanine. Exemplary molecules are disclosed in U.S. Patents 5,635,483 and 5,780,588. Drostatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) Antimicrob Agents and Chemother. 45(12):3580~3584) and possess anticancer and antifungal activity. The drostatin and auristatin drug sites can be bound to the antibodies of this application via the N (amino) or C (carboxyl) terminus of the peptide drug site (International Publication No. 02 / 088172), or via any genetically engineered cysteine ​​within the antibody.

[0665] The DLL3-binding protein of this disclosure can be conjugated to a detectable label using known methods.

[0666] In some embodiments, the detectable label forms a complex with a chelating agent.

[0667] In some embodiments, a detectable label is conjugated to the DLL3-binding protein of this disclosure via a linker.

[0668] A detectable label or cytotoxic moiety can be directly or indirectly linked to the DLL3-binding protein of this disclosure using known methods. Suitable linkers are known in the art and include, for example, prosthetic groups, non-phenol linkers (derivatives of N-succimidyl benzoate, dodecaborate), chelate moieties of both macrocyclic and acyclic chelating agents, such as derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), and diethylenetriaminepentaacetic acid. avid, derivatives of DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters ( Examples include disuccinimidyl sverat, aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bis-activated fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene), as well as other chelate moieties. Suitable peptide linkers are well known.

[0669] In some embodiments, the DLL3-binding protein of this disclosure is removed from the blood via renal clearance.

[0670] kit This application also provides a kit comprising an antigen-binding region that binds to DLL3.

[0671] This application also provides a kit comprising a protein containing an antigen-binding region that binds to DLL3.

[0672] This application also provides a kit comprising a multispecific antigen-binding construct that includes an antigen-binding region that binds to DLL3.

[0673] The kit can be used for therapeutic purposes and as a diagnostic kit.

[0674] The kit can be used to detect the presence of DLL3 in a sample.

[0675] In some embodiments, the kit comprises the DLL3-binding protein of the Disclosure and a reagent for detecting the DLL3-binding protein. The kit may also comprise one or more other elements, including instructions for use; other reagents, e.g., labels, therapeutic agents, or agents useful for chelation or other coupling methods, antibodies against labels or therapeutic agents, or radioprotective compositions; devices or other materials for preparing antibodies for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0676] In some embodiments, the kit includes an antigen-binding region that binds to DLL3 in a container, and instructions for using the kit.

[0677] In some embodiments, the kit includes a protein containing an antigen-binding region that binds to DLL3 in a container, and instructions for using the kit.

[0678] In some embodiments, the kit includes a multispecific antigen-binding construct containing an antigen-binding region that binds to DLL3 in a container, and instructions for using the kit.

[0679] In some embodiments, the antigen-binding region that binds to DLL3 in the kit is labeled.

[0680] In some embodiments, the protein containing the antigen-binding region that binds to DLL3 in the kit is labeled.

[0681] In some embodiments, a multispecific antigen-binding construct containing an antigen-binding region that binds to DLL3 in the kit is labeled.

[0682] How to detect DLL3 The present application also provides a method for detecting DLL3 in a sample, comprising: obtaining a sample; contacting the sample with an antigen-binding region that binds to DLL3 of the present disclosure; and detecting the bound DLL3 in the sample.

[0683] In some embodiments, the sample may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, synovial fluid, cells not associated with circulating cells or tissue (i.e., free cells), tissue (e.g., surgically excised tissue, biopsy material including fine-needle aspiration), tissue slides, etc.

[0684] The antigen-binding region that binds to DLL3 of this disclosure can be detected using known methods. Exemplary methods include directly labeling the antibody using fluorescent or chemiluminescent labeling, or radiolabeling, or conjugating the antibody of the present invention to an easily detectable moiety such as biotin, an enzyme, or an epitope tag. Exemplary labels and moieties include ruthenium, 111 In-DOTA, 111 These include in-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, fluoron dyes, oxazine dyes, phenanthidine dyes, rhodamine dyes, and Alexafluor® dyes.

[0685] The antigen-binding region of DLL3 of this disclosure can be used in a variety of assays for detecting DLL3 in a sample. Exemplary assays include Western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemical analysis, fluorescence-activated cell sorting (FACS), or ELISA assay.

[0686] Polynucleotides, host cells, and vectors The Disclosure also provides isolated polynucleotides encoding any of the following: a DLL3-binding protein of the Disclosure, which includes an antigen-binding region that binds to DLL3; a protein including an antigen-binding region that binds to DLL3; or a multispecific antigen-binding construct including an antigen-binding region that binds to DLL3.

[0687] In some embodiments, this application provides isolated polynucleotides encoding either the DLL3-binding protein or a fragment thereof disclosed herein.

[0688] In certain embodiments, the application provides an isolated polynucleotide encoding VH of SEQ ID NOs: 1, 3, 5, 7, 9, 11, or 13. In certain embodiments, the application provides an isolated polynucleotide encoding VL of SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14.

[0689] In certain embodiments, this application, VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2 VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4, VH of SEQ ID NO: 5 and VL of SEQ ID NO: 6, VH of sequence number 7 and VL of sequence number 8, VH of sequence number 9 and VL of sequence number 10, VH of SEQ ID NO: 11 and VL of SEQ ID NO: 12, or This provides isolated polynucleotides encoding VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14.

[0690] This application also provides isolated polynucleotides encoding polypeptides of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 63, 64, 65, 66, 67, 68, 69, 71, 77, 78, 80, 84, 85, 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 229, 190, 191, 192, 193, 194, 195, 196, 230, or 196.

[0691] This application also provides isolated polynucleotides encoding polypeptides of Sequence ID Nos. 86, 87, 89, 93, 94, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 202, 233, 234, 235, 236, 237, 238, 239, 256, 260, 261, 262, 264, 265, 266, 267, 268, 269, or 270.

[0692] In certain embodiments, the disclosure provides isolated polynucleotide sequences encoding the polynucleotide sequences of SEQ ID NOs. 71, 118, and 117.

[0693] In certain embodiments, the Disclosure provides isolated polynucleotide sequences encoding the polynucleotide sequences of SEQ ID NOs. 229, 230, and 117.

[0694] In certain embodiments, the Disclosure provides an isolated polynucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the polynucleotide of SEQ ID NO: 266, at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the polynucleotide of SEQ ID NO: 235, and at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the polynucleotide of SEQ ID NO: 236.

[0695] In certain embodiments, the disclosure provides an isolated polynucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%, or 100%) identical to the polynucleotide of SEQ ID NO: 266.

[0696] In certain embodiments, the disclosure provides an isolated polynucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%, or 100%) identical to the polynucleotide of SEQ ID NO: 235.

[0697] In certain embodiments, the Disclosure provides an isolated polynucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%, or 100%) identical to the polynucleotide of SEQ ID NO: 236.

[0698] In certain embodiments, the Disclosure provides an isolated polynucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the polynucleotide of SEQ ID NO: 239, at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the polynucleotide of SEQ ID NO: 237, and at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to the polynucleotide of SEQ ID NO: 238.

[0699] In certain embodiments, the Disclosure provides an isolated polynucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%, or 100%) identical to the polynucleotide of SEQ ID NO: 237.

[0700] In certain embodiments, the Disclosure provides an isolated polynucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%, or 100%) identical to the polynucleotide of SEQ ID NO: 238.

[0701] In certain embodiments, the disclosure provides an isolated polynucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%, or 100%) identical to the polynucleotide of SEQ ID NO: 239.

[0702] In certain embodiments, the Disclosure provides isolated polynucleotide sequences encoding the polynucleotide sequences of SEQ ID NOs. 64, 84, and 85. Some embodiments of the Disclosure also provide isolated or purified nucleic acids comprising a polynucleotide encoding the DLL3-binding protein of the Disclosure or a polynucleotide complementary to the polynucleotide that hybridizes under stringent conditions to the polynucleotide encoding the DLL3-binding protein of the Disclosure.

[0703] Polynucleotides that hybridize under stringent conditions may hybridize under high-stringency conditions. “High-stringency conditions” means that the polynucleotide hybridizes specifically to a target sequence (a nucleotide sequence of any of the nucleic acids described herein) in a detectable amount stronger than non-specific hybridization. High-stringency conditions include conditions that distinguish polynucleotides having precisely complementary sequences or containing only a few scattered mismatches from random sequences that end up having several sub-regions (e.g., 3–12 bases) that match the nucleotide sequence. Such complementary sub-regions melt more readily than full-length complements of 14–17 or more bases, and high-stringency hybridization makes them readily identifiable. Relatively high-stringency conditions include low-salt and / or high-temperature conditions, such as those provided by about 0.02–0.1 M NaCl or equivalent at a temperature of about 50–70°C. Such highly stringent conditions tolerate virtually no mismatch between the nucleotide sequence and the template or target chain, if any. It is generally understood that these conditions can be made even stringier by the addition of gradually increasing amounts of formamide.

[0704] The polynucleotide sequences of this disclosure can be operably ligated to one or more regulatory elements (e.g., promoters or enhancers) that express the nucleotide sequence in an intended host cell. The polynucleotides may be cDNAs. The promoters may be strong, weak, tissue-specific, inducible, or developmental stage-specific. Exemplary promoters that may be used include hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, and human muscle creatine. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the embodiments described. Examples of such viral promoters include the earliest promoter of cytomegalovirus (CMV), the early and late promoters of SV40, the promoter of mouse mammary tumor virus (MMTV), Moloney's leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and long terminal repeats (LTRs) of other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. Inducible promoters may be used, such as the metallothionein promoter, tetracycline-inducible promoter, doxycycline-inducible promoter, and promoters containing one or more interferon-stimulated response elements (ISREs), such as the protein kinase R2',5'-oligoadenylate synthase, the Mx gene, and ADAR1.

[0705] This application also provides vectors comprising the polynucleotides of this application. This disclosure also provides expression vectors comprising the polynucleotides of this application. Such vectors may be plasmid vectors, viral vectors, baculovirus expression vectors, transposon-based vectors, or any other vectors suitable for introducing the synthetic polynucleotides of the present invention into a given organism or genetic background by any means. The polynucleotides encoding the DLL3-binding protein of this disclosure can be operably ligated to regulatory sequences in the expression vector to ensure the expression of the DLL3-binding protein. Such regulatory elements may include transcription promoters, sequences encoding suitable mRNA-ribosome binding sites, and sequences that control the termination of transcription and translation. The expression vector may also include one or more non-transcription elements, e.g., origin of replication, suitable promoters and enhancers ligated to the gene to be expressed, other 5' or 3' adjacent non-transcription sequences, 5' or 3' untranslated sequences (e.g., essential ribosome binding sites), polyadenylation sites, splice donor and acceptor sites, or transcription termination sequences. A replication origin that confers the ability to replicate in the host can also be incorporated.

[0706] Expression vectors may contain naturally occurring or non-naturally occurring nucleotide linkages, or both. Non-naturally occurring or modified nucleotide linkages do not inhibit the transcription or replication of the vector.

[0707] Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the DLL3-binding protein of this disclosure, encoded by the incorporated polynucleotide. The transcriptional and translational regulatory sequences in the expression vector used to transform vertebrate cells can be provided by the viral source. Exemplary vectors can be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

[0708] The vectors of this disclosure may also contain one or more internal ribosome entry sites (IRESs). Including IRES sequences in the fusion vector may be beneficial for enhancing the expression of certain proteins. In some embodiments, the vector system may include one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. The components of the vector may be linked in close proximity, or arranged to provide optimal spacing for gene product expression (i.e., by introducing “spacer” nucleotides between ORFs), or otherwise positioned. Regulatory elements, such as IRES motifs, may also be arranged to provide optimal spacing for expression.

[0709] The vectors of this disclosure may be circular or linear. They may be prepared to contain a functional replication system in a prokaryotic or eukaryotic host cell. The replication system may be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papillomavirus, etc.

[0710] Recombinant expression vectors can be designed for transient expression, stable expression, or both. Furthermore, recombinant expression vectors can be constructed for constitutive or inducible expression.

[0711] Furthermore, recombinant expression vectors may be constructed to contain suicide genes. As used herein, the term “suicide gene” refers to a gene that causes cells expressing a suicide gene to die. A suicide gene may be a gene that confers sensitivity to a drug (e.g., a pharmacokinetic) to cells expressing the gene, causing the cells to die when they come into contact with or are exposed to the drug. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0712] The vector may also contain selection markers well known in the art. These selection markers include positive and negative selection markers. Marker genes include those for biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.) and complementation for providing protonutrient supply in nutritionally dependent hosts. Exemplary marker genes include antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene, histidinol resistance gene, histidinol × resistance gene), glutamine synthase genes, HSV-TK and HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase genes for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)), which have been developed and widely adopted. The nucleic acid sequence or cloning site encoding the selection marker may be upstream or downstream of the nucleic acid sequence or cloning site encoding the polypeptide of interest.

[0713] Examples of vectors that may be used include: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotes: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia), pEE6.4 (Lonza), and pEE12.4 (Lonza). Additional vectors include the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) can also be used. Exemplary plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Exemplary animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The expression vector may be a viral vector, such as a retroviral vector, such as a gamma retroviral vector.

[0714] In some embodiments, the vector includes a polynucleotide encoding VH of SEQ ID NOs. 1, 3, 5, 7, 9, 11, or 13. In certain embodiments, the vector includes a polynucleotide encoding VL of SEQ ID NOs. 2, 4, 6, 8, 10, 12, or 14.

[0715] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of SEQ ID NOs. 63, 64, 65, 66, 67, 68, or 69.

[0716] This application also provides host cells containing one or more vectors of this application. “Host cell” refers to a cell into which a vector has been introduced. The term “host cell” is understood to be intended to refer not only to a specific target cell, but also to the progeny of such cells, and to stable cell lines generated from a specific target cell. Such progeny may not be identical to the parent cell, because certain modifications may occur in subsequent generations due to either mutation or environmental influences, but they are still included within the scope of the term “host cell” as used herein. Such host cells may be eukaryotic cells, prokaryotic cells, plant cells, or archaeal cells. Examples of prokaryotic host cells include bacilli such as Escherichia coli and Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. Other microorganisms, such as yeast, are also useful for expression. Examples of suitable yeast host cells include Saccharomyces (e.g., S. cerevisi...

Claims

1. An isolated protein comprising an antigen-binding region that binds to delta-like protein 3 (DLL3), wherein the antigen-binding region is a. Sequence numbers 15, 16, 17, 33, 34, and 35, respectively b. Sequence numbers 18, 19, 20, 36, 37, and 38, respectively c. Sequence numbers 21, 22, 23, 39, 37, and 40, respectively d. Sequence numbers 24, 25, 26, 41, 42, and 43, respectively e. Sequence numbers 18, 28, 29, 44, 45, and 46, respectively f. Sequence numbers 30, 31, 32, 47, 48, and 49, respectively g. Sequence IDs 50, 51, 17, 33, 34, and 35, respectively h. Sequence numbers 52, 51, 17, 33, 34, and 35, respectively i. Sequence numbers 53, 54, 20, 36, 37, and 38, respectively j. Sequence numbers 55, 56, 23, 39, 37, and 40, respectively k. Sequence numbers 57, 58, 26, 41, 42, and 43, respectively l. Sequence numbers 59, 60, 29, 44, 45, and 46, respectively, or m. Sequence numbers 61, 62, 32, 47, 48, and 49 respectively An isolated protein containing the amino acid sequence of heavy chain complementarity-determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3.

2. The isolated protein according to claim 1, wherein the antigen-binding region comprises a heavy chain variable region (VH) having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13, and a light chain variable region (VL) having an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14.

3. The VH and VL of the antigen-binding region are a. Sequence ID 1 and Sequence ID 2, respectively b. Sequence ID 3 and Sequence ID 4, respectively c. Sequence ID 5 and Sequence ID 6, respectively d. Sequence ID 7 and Sequence ID 8, respectively e. Sequence ID 9 and Sequence ID 10, respectively f. Sequence ID 11 and Sequence ID 12, respectively, or g. Sequence ID 13 and Sequence ID 14, respectively The isolated protein according to claim 2, comprising the amino acid sequence.

4. The isolated protein according to any one of claims 1 to 3, wherein the antigen-binding region comprises an scFv having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 63 or 64.

5. An immunoconjugate comprising an isolated protein according to any one of claims 1 to 4, conjugated in a therapeutic agent or contrast agent.

6. A multispecific antigen-binding construct comprising an isolated protein according to any one of claims 1 to 4.

7. The multispecific antigen-binding construct according to claim 6, further comprising a second antigen-binding region for binding to an antigen on a lymphocyte.

8. The multispecific antigen-binding construct according to claim 7, wherein the antigen on the lymphocyte is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C.

9. The second antigen-binding region is bound to CD3ε and includes a second VH containing HCDR1, HCDR2, and HCDR3, and a second VL containing LCDR1, LCDR2, and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the second antigen-binding region are a. Sequence numbers 98, 99, 100, 106, 107, and 108, respectively, or b. Sequence numbers 95, 96, 97, 101, 102, and 104, respectively. A multispecific antigen-binding construct according to claim 8, comprising the amino acid sequence.

10. The VH and VL of the second antigen-binding region are a. Sequence ID 84 and Sequence ID 85, respectively, or b. Sequence ID 77 and Sequence ID 80, respectively The multispecific antigen-binding construct according to claim 9, comprising an amino acid sequence that is at least 90% identical to that of the other construct.

11. The VH and VL of the second antigen-binding region are a. Sequence ID 84 and Sequence ID 85, respectively, or b. Sequence ID 77 and Sequence ID 80, respectively A multispecific antigen-binding construct according to claim 10, comprising the amino acid sequence.

12. The multispecific antigen-binding construct according to any one of claims 6 to 11, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the antigen-binding region that binds to DLL3 each contain the amino acid sequences of SEQ ID NOs: 15, 16, 17, 33, 34, and 35, respectively.

13. A fusion or conjugate comprising a half-life extension portion fused to an isolated protein according to any one of claims 1 to 4 or a multispecific antigen-binding construct according to any one of claims 7 to 12, wherein the half-life extension portion is immunoglobulin (Ig), a fragment of Ig, an Ig constant region, a fragment of the Ig constant region, an Fc region, transferrin, albumin, an albumin-binding domain, or polyethylene glycol.

14. The aforementioned half-life extension portion is T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y407V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, The fusion or conjugate according to claim 13, comprising a fragment of the Ig constant region having at least one mutation selected from the group consisting of L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, T350V / T366L / K392L / T394W, and L234A / L235A / D265S, wherein the residue numbering follows the EU index.

15. A bispecific antigen-binding construct, (1) A first antigen-binding region that binds to DLL3, wherein the first antigen-binding region comprises a first VH including HCDR1, HCDR2, and HCDR3, and a first VL including LCDR1, LCDR2, and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are (a) Sequence numbers 15, 16, 17, 33, 34, and 35, respectively (b) Sequence numbers 18, 19, 20, 36, 37, and 38, respectively (c) Sequence IDs 21, 22, 23, 39, 37, and 40, respectively (d) Sequence numbers 24, 25, 26, 41, 42, and 43, respectively (e) Sequence numbers 18, 28, 29, 44, 45, and 46, respectively (f) Sequence numbers 30, 31, 32, 47, 48, and 49, respectively (g) Sequence IDs 50, 51, 17, 33, 34, and 35, respectively (h) Sequence numbers 52, 51, 17, 33, 34, and 35, respectively (i) Sequence numbers 53, 54, 20, 36, 37, and 38, respectively (j) Sequence IDs 55, 56, 23, 39, 37, and 40, respectively (k) Sequence IDs 57, 58, 26, 41, 42, and 43 respectively, (l) Sequence numbers 59, 60, 29, 44, 45, and 46, respectively, (m) Sequence numbers 61, 62, 32, 47, 48, and 49 respectively A first antigen-binding region comprising the amino acid sequence, and (2) A second antigen-binding region that binds to CD3ε, (a) A second VH containing HCDR1, HCDR2, and HCDR3 of the amino acid sequences of SEQ ID NOs. 95, 96, and 97, respectively, and a second VL containing LCDR1, LCDR2, and LCDR3 of the amino acids of the sequences of SEQ ID NOs. 101, 102, and 104, respectively, or (b) A second VH containing HCDR1, HCDR2, and HCDR3 of the amino acid sequences of SEQ ID NOs. 98, 99, and 100, respectively, and a second VL containing LCDR1, LCDR2, and LCDR3 of the amino acid sequences of SEQ ID NOs. 106, 107, and 108, respectively. The second antigen-binding region, including A bispecific antigen-binding construct containing the above.

16. a. The first VH and the first VL are i. Sequence ID 1 and Sequence ID 2, respectively ii. Sequence ID 3 and Sequence ID 4, respectively iii. Sequence ID 5 and Sequence ID 6, respectively iv. Sequence ID 7 and Sequence ID 8, respectively v. Sequence ID 9 and Sequence ID 10, respectively vi. Sequence ID 11 and Sequence ID 12, respectively, or vii. Sequence IDs 13 and 14 It contains an amino acid sequence that is at least 90% identical to, b. The second VH and the second VL are i. Sequence ID 77 and Sequence ID 80, respectively, or ii. Sequence ID 84 and Sequence ID 85, respectively The bispecific antigen-binding construct according to claim 15, comprising an amino acid sequence that is at least 90% identical to that of the other construct.

17. The bispecific antigen-binding construct according to claim 15 or 16, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first antigen-binding region each contain the amino acid sequences of SEQ ID NOs: 15, 16, 17, 33, 34, and 35, respectively.

18. The bispecific antigen-binding construct according to claim 17, wherein the first VH comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, and the first VL comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

4.

19. The bispecific antigen-binding construct according to any one of claims 15 to 18, wherein the first antigen-binding region comprises a first scFv or first Fab including the first VH and the first VL, and the second antigen-binding region comprises a second Fab or second scFv including the second VH and the second VL.

20. The bispecific antigen-binding construct according to claim 19, wherein the first antigen-binding region comprises the first scFv and the second antigen-binding region comprises the second Fab.

21. A bispecific antigen-binding construct according to any one of claims 15 to 20, comprising a first heavy chain and a second heavy chain, wherein the first heavy chain comprises the first VH, the second heavy chain comprises the second VH, and each of the first and second heavy chains further comprises an immunoglobulin (Ig) constant region comprising one or more heterodimer mutations.

22. (1) The first heavy chain contains an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 109, 111, 71, and 229. (2) A light chain containing an amino acid sequence in which the bispecific antigen-binding construct is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 115, and 117, and (3) The bispecific antigen-binding construct according to claim 21, comprising a second heavy chain having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 112, 113, 116, 114, 118, and 230.

23. Isolated nucleic acids encoding an isolated protein according to any one of claims 1 to 4, a multispecific antigen-binding construct according to any one of claims 6 to 12, a fusion or conjugate according to claim 13 or 14, or a bispecific antigen-binding construct according to any one of claims 15 to 22.

24. A vector comprising the nucleic acid described in claim 23.

25. A host cell comprising the nucleic acid described in claim 23 or the vector described in claim 24.

26. A method for producing an isolated protein according to any one of claims 1 to 4, a multispecific antigen-binding construct according to any one of claims 6 to 12, a fusion or conjugate according to claim 13 or 14, or a bispecific antigen-binding construct according to any one of claims 15 to 22, the method comprising culturing a host cell according to claim 25 under conditions that produce the protein, the multispecific antigen-binding construct, the fusion or conjugate, or the bispecific antigen-binding construct, and recovering the same from the host cell.

27. A pharmaceutical composition comprising an isolated protein according to any one of claims 1 to 4, an immunoconjugate according to claim 5, a multispecific antigen-binding construct according to any one of claims 6 to 12, a fusion or conjugate according to claim 13 or 14, a bispecific antigen-binding construct according to any one of claims 15 to 22, a nucleic acid according to claim 23, a vector according to claim 24, or a host cell according to claim 25, and a pharmaceutically acceptable carrier.

28. A pharmaceutical composition used in a method for treating DLL3-expressing cancer in a subject requiring treatment, wherein the pharmaceutical composition comprises an isolated protein according to any one of claims 1 to 4, an immune conjugate according to claim 5, a multispecific antigen-binding construct according to any one of claims 6 to 12, a fusion or conjugate according to claim 13 or 14, or a bispecific antigen-binding construct according to any one of claims 15 to 22, a nucleic acid according to claim 23, a vector according to claim 24, or a host cell according to claim 25.

29. The pharmaceutical composition according to claim 28, wherein the DLL3-expressing cancer is selected from the group consisting of lung cancer, prostate cancer, glioma, glioblastoma, melanoma, neuroendocrine pancreatic cancer, hepatoblastoma, and hepatocellular carcinoma, or any combination thereof.

30. A pharmaceutical composition used in a method for reducing the amount of DLL3-expressing tumor cells in a subject, wherein the pharmaceutical composition comprises an isolated protein according to any one of claims 1 to 4, an immunoconjugate according to claim 5, a multispecific antigen-binding construct according to any one of claims 6 to 12, a fusion or conjugate according to claim 13 or 14, or a bispecific antigen-binding construct according to any one of claims 15 to 22, a nucleic acid according to claim 23, a vector according to claim 24, or a host cell according to claim 25.

31. The pharmaceutical composition according to claim 30, wherein the subject requires treatment for a cancer selected from the group consisting of lung cancer, prostate cancer, glioma, glioblastoma, melanoma, neuroendocrine pancreatic cancer, hepatoblastoma, and hepatocellular carcinoma, or any combination thereof.

32. A pharmaceutical composition used in a method for treating a non-cancerous condition in a subject at risk of developing DLL3-expressing cancer, wherein the pharmaceutical composition comprises an isolated protein according to any one of claims 1 to 4, an immune conjugate according to claim 5, a multispecific antigen-binding construct according to any one of claims 6 to 12, a fusion or conjugate according to claim 13 or 14, or a bispecific antigen-binding construct according to any one of claims 15 to 22, a nucleic acid according to claim 23, a vector according to claim 24, or a host cell according to claim 25.

33. The pharmaceutical composition according to claim 32, wherein the non-cancerous state is a state having a high prostate-specific antigen (PSA) level in the absence of benign prostatic hyperplasia, benign prostatic hyperplasia (BPH), or diagnosed prostate cancer.

34. A pharmaceutical composition used in a method for detecting the presence of neuroendocrine prostate cancer or small cell lung cancer in a subject, wherein the pharmaceutical composition comprises an immunoconjugate as described in claim 5, and the method comprises administering the immunoconjugate to a subject suspected of having prostate cancer or small cell lung cancer, and visualizing the biological structure to which the immunoconjugate is bound, thereby detecting the presence of prostate cancer or small cell lung cancer.

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