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

Abstract

NEPC provides proteins containing a delta-like ligand 3 (DLL3) antigen-binding domain, multispecific antigen-binding constructs, and the like for novel therapies to treat cancers such as small cell carcinoma or small cell lung cancer. [Solution] The present invention describes a method of treatment or detection using a multispecific antigen-binding construct containing an antigen-binding region that binds to DLL3, such as a bispecific antibody, an anti-DLL3 antibody, an antigen-binding fragment thereof, or a multispecific antigen-binding construct, as well as related molecules, compositions, and methods.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application is U.S. Provisional Application No. 63 / 094,933, filed on October 22, 2020. and U.S. Provisional Application No. 63 / 094,934 filed on October 22, 2020 Priority is claimed, and each of these disclosures, by reference, is referred to herein in whole. It will be incorporated into it.

[0002] (Reference to electronically submitted sequence listings) This application is a distribution filed electronically via EFS-Web as an ASCII-formatted sequence listing. This sequence listing includes a column table, and the file name is "sequence listing JBI". It is "6411", created on October 7, 2021, and has a size of 275KB. The sequence listings submitted via EFS-Web are part of this specification and are referred to herein by reference. The entirety is incorporated herein.

[0003] (Field of invention) This application relates to Delta-like canonical Notch ligand 3. Proteins containing antigen-binding domains that bind to igand 3 (DLL3), and related compositions Regarding methods and procedures. [Background technology]

[0004] Prostate cancer is the second most common cancer and the sixth leading cause of cancer death in men. Yes, it accounts for 14% (903,500) of all new cancer cases in men worldwide and all other cancers. Metastatic prostate cancer accounts for 6% (258,400) of deaths. It is the second leading cause of death. The process from diagnosis to death of prostate cancer is influenced by the severity of the disease and hormone levels. Condition and presence or absence of detectable metastases: Localized disease, detectable metastases after radiotherapy or surgery The levels of prostate-specific antigen (PSA) continue to rise. Based on clinical metastasis during the non-castration or castration phase, a series of clinical stages It is classified as the best option. Surgery, radiation therapy, or a combination of both are used for localized disease. While it can be therapeutic for some, a significant proportion of these patients have PSA Elevated Bell's score is evidence of recurrent disease, which is also particularly in high-risk groups. This can lead to metastasis formation and a transition to the terminal stage of the disease.

[0005] Androgen depletion therapy (ADT) is the standard treatment. The generally predictable outcome is a decrease in PSA, a stable phase with no tumor growth, followed by a decrease in PSA. It is characterized by elevation and regrowth as a castration-resistant disease. For many years, ADT has been associated with metastatic prostate cancer. It was the standard treatment for patients with adenocarcinoma.

[0006] However, recent clinical data suggest that androgen depletion therapy is a progenitor of cell differentiation. Through Seth, neuroendocrine prostate cancer (NEPC) This suggests that it may lead to the emergence of androgen-independent tumor phenotypes. Delta-like canonical Notch ligand 3 (DLL3) is used at the RNA level and protein level. It has been shown to be abundant in NEPC tumors at both levels. Therefore, Strategies designed to target DLL3 have been shown in the NEPC / small cell carcinoma patient population. It may have clinical utility.

[0007] Small cell lung cancer accounts for approximately 20% of all lung cancers. In addition, because lymph node metastasis or distant metastasis has already occurred at the time of diagnosis, it progresses rapidly and requires surgical treatment. It is difficult to remove. This cancer shows a high response rate to anticancer drugs in the early stages. Therefore, chemotherapy is considered the first choice for treating cancer. However, The cancer quickly becomes resistant to chemotherapy, recurs, and the 3-year survival rate falls to less than 5%.

[0008] Therefore, new treatments for cancers such as NEPC, small cell carcinoma, or small cell lung cancer Treatment is needed.

[0009] In normal cells, DLL3 regulates notch signaling within the cell. In cells, DLL3 is expressed extracellularly, and in humans, for example, it consists of 618 amino acids and 6 EG molecules. It has eight extracellular domains, including an F-like repeat. Human DLL3 is found in cynomolgus monkeys and It is highly homologous to that of mouse / rat, with 96% and 83% of the amino acid sequence being identical, respectively. Although they share a common identity, they have less than 40% of the same identity with DLL1 and DLL4. L3 has low to undetectable expression in normal tissues, but in small cell lung cancer, prostate cancer, etc. It is highly expressed on the cell surface of glandular, large cell carcinoma, and neuroendocrine tumors, including bladder cancer. It is a target for T-cell redirection in the treatment of neuroendocrine cancers. [Overview of the project] [Means for solving the problem]

[0010] In a general embodiment, this disclosure relates to antigens that bind to delta-like protein 3 (DLL3). An isolated protein containing a binding region, wherein the antigen-binding region is as described in Sequence ID No. 263. Isolated proteins that bind to epitopes within residues 429-618 of human DLL3 To relate to.

[0011] In some embodiments, the isolated protein has the amino acid sequence of SEQ ID NO: 1 The heavy chain complementarity determining region of VH Heavy chain variable regions having HCDR1, HCDR2, and HCDR3 The le region (VH), and the light chain complementarity determination region of VL having the amino acid sequence of SEQ ID NO: 2. It has three regions (light chain variable regions, LCDR) 1, LCDR2, and LCDR3. The light chain variable region (VL), b) amino acid arrangement of SEQ ID NO: 3 VH having columns HCDR1, HCDR2, and HCDR3, and sequence numbers VL having the amino acid sequence of No. 4, and V having LCDR1, LCDR2, and LCDR3 L, c) VH having HCDR1, HCDR2, and HCDR3 of the VH of sequence number 5, d) VL of sequence number 6, LCDR1, LCDR2, and LCDR3, d) V of sequence number 7 H's HCDR1, HCDR2, and HCDR3, and VL's LCDR1 of sequence number 8, LCDR2 and LCDR3, e) HCDR1, HCDR2 and H of VH of SEQ ID NO: 9 CDR3, and LCDR1, LCDR2, and LCDR3 of VL of sequence number 10, f) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 11, and SEQ ID NO: 12 LCDR1, LCDR2, and LCDR3 of VL, or (g) HCD of VH of SEQ ID NO: 13 R1, HCDR2, and HCDR3, as well as LCDR1, LCDR of VL of Sequence ID No. 14 2, and antigen-binding regions including LCDR3 that compete for binding to the reference antibody and DLL3. Includes. Optionally, the reference antibody may be HCDR1, HCDR2, and H of SEQ ID NO: 3 VH. This includes CDR3, and LCDR1, LCDR2, and LCDR3 of the VL of sequence number 4.

[0012] The isolated proteins were selected as follows: a) Sequence IDs 15, 16, 17, and 33, respectively. , 34, 35, b) Sequence numbers 18, 19, 20, 36, 37, 38 respectively, c) each Sequence numbers 21, 22, 23, 39, 37, 40, d) and sequence numbers 24, 25, 2 respectively. 6, 41, 42, 43, e) Sequence numbers 18, 28, 29, 44, 45, 46, f respectively )Sequences 30, 31, 32, 47, 48, 49, g)Sequence 50, 51, 17, 33, 34, 35, h) Sequence numbers 52, 51, 17, 33, 34 respectively 35, i) Sequence numbers 53, 54, 20, 36, 37, 38 respectively, j) Sequence number (Numbers 55, 56, 23, 39, 37, 40, k) Sequence numbers 57, 58, 26, 41 respectively , 42, 43, l) respective sequence numbers 59, 60, 29, 44, 45, 46, or m) HCDR1, HCDR2, and HCD, respectively, with sequence numbers 61, 62, 32, 47, 48, and 49. Includes R3, LCDR1, LCDR2, and LCDR3. Optionally, isolated proteins The compounds are HCDR1 and HC, respectively, of sequence numbers 15, 16, 17, 33, 34, and 35. Includes DR2, HCDR3, LCDR1, LCDR2, and LCDR3. Optionally, D The antigen-binding regions that bind to LL3 are scFv, (scFv)2, Fv, Fab, and F(ab). ')2, Fd, dAb, or VHH. Optionally, an antigen-binding region that binds to DLL3. The region is Fab. Optionally, the antigen-binding region that binds to DLL3 is scFv. Optionally, scFv is VH, first linker (L1) from the N-terminus to the C-terminus. ), and VL(VH-L1-VL), or VL, L1, and VH(VL-L1-VH) Includes. Optionally, L1 contains a) approximately 5-50 amino acids, and b) approximately 5-40 amino acids. c) contains approximately 10-30 amino acids, d) contains approximately 10-20 amino acids. Optional selection: L1 corresponds to sequence numbers 27, 72, 73, 74, 75, 76, 79, 81, 82, 83, and 88. , 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 12 7, 128, 129, 130, 131, 132, 133, 134, 135, 136, 13 It contains amino acid sequences of 7, 138, or 139. Optionally, L1 is the same as sequence number 120. Contains amino acid sequence.

[0013] This disclosure also includes the VH versions of Sequence IDs 1, 3, 5, 7, 9, 11, or 13 and Sequence ID 2, The antigen-binding region that binds to DLL3 includes VLs 4, 6, 8, 10, 12, or 14. Provided. Optionally, the antigen-binding region that binds to DLL3 is a) VH and the corresponding part of SEQ ID NO: 1. VL of column number 2, a) VH of sequence number 3 and VL of sequence number 4, c) VH of sequence number 5 and d) VL of SEQ ID NO: 6, d) VH of SEQ ID NO: 7 and VL of SEQ ID NO: 8, e) V of SEQ ID NO: 9 H and VL of SEQ ID NO: 10, f) VH of SEQ ID NO: 11 and VL of SEQ ID NO: 12, and / Or g) Includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14. Optionally, linked to DLL3. The antigen-binding region that binds is at least 80% (e.g., at least 85%) of the VH region of SEQ ID NO: 3. %, at least 90%, at least 95%, at least 99%, or at least 100% ) VH and VL of sequence number 4 are identical and at least 80% (for example, at least 85%) , at least 90%, at least 95%, at least 99%, or at least 100%) It includes the same VL. Optionally, the antigen-binding region that binds to DLL3 is sequence number 63. Or a sequence of 64 amino acids and at least 80% (e.g., at least 85%, at least 9) 0%, at least 95%, at least 99%, or at least 100%) identical mesh Contains an acid sequence.

[0014] This disclosure relates to isolated tans that are monospecific proteins or multispecific antigen-binding constructs. The protein is provided. Optionally, the isolated protein is used in a multispecific antigen-binding construct. Yes. Optionally, a multispecific antigen-binding construct is a bispecific protein. By choice, the multispecific antigen-binding construct is a triple-specific protein. By choice, multispecific The isoantigen-binding construct includes an antigen-binding region that binds to an antigen on a lymphocyte. Optionally, Lymphocytes are T cells. By random selection, T cells are CD8 + These are T cells. Selected at will. Lymphocytes are natural killer (NK) cells. By random selection, In the hemispecific antigen-binding construct, the antigens on lymphocytes are CD3, CD3 epsilon (CD3 epsilon). 3 epsilon, CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, These are BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C. The antigen on the lymphocytes is selected at random: CD3ε.

[0015] In some embodiments, the multispecific antigen-binding construct is a) heavy chain complement of SEQ ID NO: 98 Sex determination region (HCDR) 1, HCDR2 (sequence number 99), HCDR3 (sequence number 100), Light chain complementarity determination region (LCDR)1 of sequence number 106, LCDR2 of sequence number 107, and a) LCDR3 of SEQ ID NO: 108, b) VH of SEQ ID NO: 84, and VL of SEQ ID NO: 85 , includes an antigen-binding region that binds to CD3ε. In some embodiments, multispecific antigen binding In the composite construct, the antigen-binding region that binds to CD3ε is HCDR1 of SEQ ID NO: 98, HCDR2 at column number 99, HCDR3 at sequence number 100, LCDR1 at sequence number 106, Includes LCDR2 of sequence number 107 and LCDR3 of sequence number 108. Several implementations Morphologically, in a multispecific antigen-binding construct, the antigen-binding region that binds to CD3ε is arranged VH of column number 84 and at least 80% (e.g., at least 85%, at least 90%) VH and distribution are identical (at least 95%, at least 99%, or at least 100%). VL of column number 85 and at least 80% (for example, at least 85%, at least 90%) Includes VLs that are identical (at least 95%, at least 99%, or at least 100%) .

[0016] In some embodiments, the multispecific antigen-binding construct is a) heavy chain complement of SEQ ID NO: 95 Sex determination region (HCDR) 1, HCDR2 (sequence number 96), HCDR3 (sequence number 97), distribution Light chain complementarity determination region (LCDR) 1 of sequence number 101, LCDR2 of sequence number 102, and b) containing LCDR3 of SEQ ID NO: 104, or VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80 It includes an antigen-binding region that binds to CD3ε. In some embodiments, it includes a multispecific antigen. The combined construct is HCDR1 of SEQ ID NO: 95, HCDR2 of SEQ ID NO: 96, and SEQ ID NO: 97. HCDR3, LCDR1 of sequence number 101, LCDR2 of sequence number 102, and sequence number Contains 104 LCDR3, including an antigen-binding region that binds to CD3ε. Several implementations In this state, the multispecific antigen-binding construct is at least 80% (e.g., VH of SEQ ID NO: 77) , at least 85%, at least 90%, at least 95%, at least 99%, or less At least 100%) VH and VL of sequence number 80 are identical and at least 80% (for example) , at least 85%, at least 90%, at least 95%, at least 99%, or less It contains an antigen-binding region that binds to CD3ε, including a VL that is identical (at least 100%).

[0017] This disclosure also includes an antigen-binding region that binds to delta-like protein 3 (DLL3), A separated multispecific antigen-binding construct, wherein the antigen-binding region that binds to DLL3 is a) Each of the following is sequence numbers 15, 16, 17, 33, 34, 35, and a) each of sequence numbers 18, 1 9, 20, 36, 37, and 38, c) Sequence IDs 21, 22, 23, 39, and 37 respectively , and 40, d) Sequence IDs 24, 25, 26, 41, 42, and 43 respectively, e) it f) Sequence numbers 18, 28, 29, 44, 45, and 46 respectively, f) Sequence numbers 30, 3 1, 32, 47, 48, and 49, g) Sequence IDs 50, 51, 17, 33, and 34 respectively , and 35, h) Sequence numbers 52, 51, 17, 33, 34, and 35 respectively, i) it (Sequences 53, 54, 20, 36, 37, 38, j) Sequences 55, 56, 23, 39, 37, and 40, k) Sequence IDs 57, 58, 26, 41, 42, 4 respectively 3, l) Sequence numbers 59, 60, 29, 44, 45, and 46 respectively, m) Sequence numbers HCDR1, HCDR2, HCDR3 numbers 61, 62, 32, 47, 48, and 49, LCDR1, LCDR2, and LCDR3, 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 SEQ ID NO: 6 VL, q) VH of SEQ ID NO: 7 and VL of SEQ ID NO: 8, r) VH of SEQ ID NO: 9 and SEQ ID NO: VL of 10, s) VH of SEQ ID NO: 11 and VL of SEQ ID NO: 12, or t) SEQ ID NO: 13 A multispecific antigen-binding construct is provided, containing VH and VL of SEQ ID NO: 14. The multispecific antigen-binding constructs are, respectively, SEQ ID NOs: 15, 16, 17, 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 Includes a binding domain that connects to DLL3.

[0018] In certain embodiments, the present disclosure relates to antigens that bind to delta-like protein 3 (DLL3). An isolated multispecific antigen-binding construct containing a translocation region, wherein the antigen binds to DLL3. The binding region is the heavy chain complementarity determination region (HCDR) 1 of the heavy chain variable region (VH) of SEQ ID NO: 3. Light chain complementarity determination of the light chain variable region (VL) of HCDR2, HCDR3, and SEQ ID NO: 4 Isolated multispecific antigens containing specific regions (LCDR)1, LCDR2, and LCDR3. Provides a combined structure.

[0019] This disclosure also includes an antigen-binding region that binds to delta-like protein 3 (DLL3), A separated multispecific antigen-binding construct, wherein the antigen-binding region that binds to DLL3 is sequence Including the heavy chain variable region (VH) of number 3 and the light chain variable region (VL) of sequence number 4, isolated The present invention provides a multispecific antigen-binding construct.

[0020] Optionally, the protein is conjugated to an isolated half-life extension region (moiety). The half-life extension portion is optionally selected from immunoglobulin (Ig) and Ig Fragments of the Ig steady-state region, fragments of the Ig steady-state region, Fc region, transferrin, albumin The domain is either albumin-binding or polyethylene glycol.Optionally, Ig stationary The fragment of the region includes the Fc region. Optionally, the antigen-binding region that binds to DLL3 is Ig It is conjugated to the N-terminus of a steady-state region or a fragment of an Ig steady-state region. Optionally, DLL The antigen-binding region that binds to 3 is conjugated at the C-terminus of the Ig constant region or a fragment of the Ig constant region. It is gated. Optionally, the antigen-binding region that binds to DLL3 is the second linker (L2 ) is conjugated to the Ig steady-state region or a fragment of the Ig steady-state region via ).Optionally, L2 corresponds to sequence numbers 27, 72, 73, 74, 75, 76, 79, 81, 82, 83, and 88. , 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 12 7, 128, 129, 130, 131, 132, 133, 134, 135, 136, 13 It contains amino acid sequences 7, 138, or 139. Optionally, it includes an Ig constant region or an Ig constant region. The region fragment is of the IgG1, IgG2, IgG3, or IgG4 isotype. (Optional) In the selection, the Ig constant region or fragment of the Ig constant region is the IgG1 isotype. (Optional selection) In the selection, the Ig constant region or a fragment of the Ig constant region is the Fcγ receptor of the protein (Fcγ receptor Contains at least one mutation resulting in reduced binding to tor (FcγR). Optional. Therefore, at least one mutation resulting in reduced binding to FcγR is F234A / L2 35A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234 A / L235A, S228P / F234A / L235A, N297A, V234A / G2 37A, K214T / E233P / L234V / L235A / G236 missing / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P33 1S, S267E / L328F, L234F / L235E / D265A, L234A / L 235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L Selected from the group consisting of 235A / G236 deletion / G237A / P238S, with residue numbers assigned. The EU index is followed. Optionally, a reduction in protein binding to FcγR is included. The variant is L234A_L235A_D265S.

[0021] This disclosure relates to an isolated protein comprising an antigen-binding region that binds to DLL3, and anti The original binding region is a) HCDR1 of sequence numbers 15, 16, 17, 33, 34, and 35, respectively. , HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, b) Sequence ID 1 a) VH of sequence number 2 and VL of sequence number 2, c) VH of sequence number 3 and VL of sequence number 4, d) sequence number Isolated protein containing scFv of number 63, and / or e) scFv of sequence number 64 Provides a protein. Optionally, isolated proteins bind to the antigen-binding region of DLL3. The antigen-binding region includes the following: a) SEQ ID NOs. 15, 16, 17, 33, 34, 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and b) containing VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2. Optionally, isolated The protein contains an antigen-binding region that binds to DLL3, and the antigen-binding region is a) each HCDR1, HCDR2, HCDR3 with sequence numbers 18, 19, 20, 36, 37, 38, LCDR1, LCDR2, and LCDR3, a) VH of SEQ ID NO: 5 and VL of SEQ ID NO: 6 , and / or including the scFv of SEQ ID NO: 65. Optionally, isolated proteins are: It includes an antigen-binding region that binds to DLL3, and the antigen-binding region is a) Sequence ID No. 21, 22, 23, 39, 37, 40 HCDR1, HCDR2, HCDR3, LCDR1, L CDR2 and LCDR3, a) VH of SEQ ID NO: 7 and VL of SEQ ID NO: 8, and / or Includes scFv of sequence number 66. Optionally, a single antigen-binding region that binds to DLL3 is included. The separated proteins, the antigen-binding regions are a) Sequence IDs 24, 25, and 26, respectively. , 41, 42, 43 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, b) VH of SEQ ID NO: 9 and VL of SEQ ID NO: 10, and / or SEQ ID NO: Contains 67 scFv. Optionally, isolated samples containing antigen-binding regions that bind to DLL3 are included. The protein has antigen-binding regions a) SEQ ID NOs. 27, 28, 29, and 44, respectively. 45, 46 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LC DR3, b) VH of SEQ ID NO: 11 and VL of SEQ ID NO: 12, and / or SEQ ID NO: 68 Contains scFv. Optionally, isolated proteins have an antigen-binding region that binds to DLL3. The antigen-binding region includes the following: a) Sequence IDs 30, 31, 32, 47, 48, and 49, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, b ) VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14, and / or scFv of SEQ ID NO: 69 include.

[0022] Selectively isolated proteins contain multiple antigen-binding regions that bind to CD3ε. It is a specific antigen-binding construct. Optionally, a multispecific antigen-binding construct is used, such as SEQ ID NO: 98. The heavy chain complementarity determination region (HCDR) 1 of , HCDR2 of SEQ ID NO: 99, and H of SEQ ID NO: 100 CDR3, Light Chain Complementarity Determination Region (LCDR) 1 of SEQ ID NO: 106, LC of SEQ ID NO: 107 DR2, and LCDR3 of SEQ ID NO: 108, and / or b) VH and distribution of SEQ ID NO: 84 Includes the VL at column number 85, and contains the antigen-binding region that binds to CD3ε. Optionally, multiple features are included. The isoantigen-binding construct is a) Heavy chain complementarity-determining region (HCDR) 1 of Sequence ID No. 95, sequence number HCDR2 of sequence number 96, HCDR3 of sequence number 97, and light chain complementarity determination region of sequence number 101. (LCDR)1, LCDR2 of sequence number 102, and LCDR3 of sequence number 104, and to / or b) an anti-CD3ε binding anti-CD3ε comprising VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80 Includes the original binding region.

[0023] This disclosure relates to a first antigen-binding region that binds to DLL3 and a second antigen-binding region that binds to lymphocyte antigens. Provides isolated anti-DLL3 / anti-CD3 proteins containing the antigen-binding region. Optional. Lymphocyte antigens are T cell antigens. Optionally, the T cell antigen is CD8 + T cell anti The origin is... Optionally, the lymphocyte antigen is the NK cell antigen. Optionally, the lymphocyte antigen... The originals are CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NK G2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or The antigen is NKG2C. Optionally, the lymphocyte antigen is CD3ε.

[0024] In the isolated anti-DLL3 / anti-CD3 protein, the binding to DLL3 was selected. The first antigen-binding region and / or the second antigen-binding region that binds to lymphocyte antigens are scF Includes v, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH Optionally, a first antigen-binding region that binds to DLL3 and / or a lymphocyte antigen. The second antigen-binding region includes Fab.Optionally, the first antigen-binding region that binds to DLL3 The binding region and / or the second antigen-binding region that binds to the lymphocyte antigen include scFv. Optional. In the selection, the first antigen-binding region that binds to DLL3 contains scFv and binds to lymphocyte antigens. The second antigen-binding region includes Fab.Optionally, the first antigen-binding region that binds to DLL3 The binding region includes Fab, and the second antigen-binding region that binds to lymphocyte antigens includes scFv. Optionally, scFv is VH, first linker (L1) from the N-terminus to the C-terminus. ), and VL(VH-L1-VL), or VL, L1, and VH(VL-L1-VH) Includes. Optionally, L1 contains a) approximately 5-50 amino acids, and b) approximately 5-40 amino acids. c) contains approximately 10-30 amino acids, d) contains approximately 10-20 amino acids. Optional selection: L1 corresponds to sequence numbers 27, 72, 73, 74, 75, 76, 79, 81, 82, 83, and 88. , 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 12 7, 128, 129, 130, 131, 132, 133, 134, 135, 136, 13 It contains amino acid sequences of 7, 138, or 139. Optionally, L1 is the same as sequence number 120. Contains amino acid sequence.

[0025] In the isolated anti-DLL3 / anti-CD3 protein, the binding to DLL3 was selected. The first antigen-binding region is found in sequence numbers 15, 18, 21, 24, 27, 30, 50, 52. HCDR1 of 53, 55, 57, 59, or 61, SEQ ID NOs: 16, 19, 22, 25, 2 HCDR2 of sequences 8, 31, 51, 54, 56, 58, 60, or 62, sequence numbers 17, 20 , HCDR3 of 23, 26, 29, 32, 17, 20, 23, 26, 29, or 32, distribution LCDR1 of column numbers 33, 36, 39, 41, 44, or 47, array numbers 34, 37, 4 LCDR2 of 2, 45, or 48, and SEQ ID NOs: 35, 38, 40, 43, 46, or 4 It contains 9 LCDR3. Optionally, the first antigen-binding region that binds to DLL3 is a. a. Sequence numbers 15, 16, 17, 33, 34, 35, and b. Sequence numbers 18, 19 respectively , 20, 36, 37, 38, c. Sequence numbers 21, 22, 23, 39, 37, 40 respectively d. Sequence numbers 24, 25, 26, 41, 42, 43 respectively, e. Sequence number 1 respectively 8, 28, 29, 44, 45, 46, f. Sequence numbers 30, 31, 32, 47, 4 respectively. 8, 49, g. Sequence numbers 50, 51, 17, 33, 34, 35, h. Column numbers 52, 51, 17, 33, 34, 35, i. Sequence numbers 53, 54, 20 respectively. 36, 37, 38, j. Sequence numbers 55, 56, 23, 39, 37, 40, k. Sequence IDs 57, 58, 26, 41, 42, 43, and 1, respectively, and sequence IDs 59 and 60, respectively. , 29, 44, 45, 46, or m. Sequence IDs 61, 62, 32, 47, 48 respectively. 49 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 This includes the following: Optionally, the first antigen-binding region that binds to DLL3 is sequence number 15. , 16, 17, 33, 34, 35 HCDR1, HCDR2, HCDR3, LCDR1, Includes LCDR2 and LCDR3.

[0026] In some embodiments, the first antigen-binding region that binds to DLL3 is a. Sequence ID No. 1 a. VH of sequence number 2 and VL of sequence number 2, b. VH of sequence number 3 and VL of sequence number 4, c. sequence number d. VH of sequence 5 and VL of sequence number 6, e. VH of sequence number 7 and VL of sequence number 8, e. VH of column number 9 and VL of sequence number 10, f. VH of sequence number 11 and V of sequence number 12 L, or g. Includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14. Optionally, DLL3 The first antigen-binding region that binds to contains the amino acid sequence of SEQ ID NO: 63 or 64. (Optional) In the selection, the first antigen-binding region that binds to DLL3 has the amino acid sequence of SEQ ID NO: 64 and is less 80% each (for example, at least 85%, at least 90%, at least 95%, less Both contain 99% or 100% identical amino acid sequences. Optionally, they bind to DLL3. The first antigen-binding region is at least 80% (e.g., at least 85%) of the VH of SEQ ID NO: 3. %, at least 90%, at least 95%, at least 99%, or at least 100% ) VH and VL of sequence number 4 are identical and at least 80% (for example, at least 85%) , at least 90%, at least 99%, at least 95%, or at least 100%) Includes the same VL. Optionally, a second antigen-binding region that binds to CD3 is the SEQ ID NO: HCDR1 of sequence 95 or 98, HCDR2 of sequence number 96 or 99, sequence number 97 or 10 HCDR3 of 0, LCDR1 of sequence number 101 or 106, and sequence number 102 or 107 Includes LCDR2 and LCDR3 of sequence number 104 or 108. Optionally, CD3 The second antigen-binding region that binds is HCDR1 in SEQ ID NO: 95 and HCDR2 in SEQ ID NO: 96. , HCDR3 with sequence number 97, LCDR1 with sequence number 101, LCDR with sequence number 102 2, and LCDR3 of sequence number 104. Optionally, the second that binds specifically to CD3. The antigen-binding regions of 2 are HCDR1 of SEQ ID NO: 98, HCDR2 of SEQ ID NO: 99, and SEQ ID NO: HCDR3 of 100, LCDR1 of sequence number 106, LCDR2 of sequence number 107, and Contains LCDR3 of SEQ ID NO: 108. Optionally, a second antigen-binding region that binds to CD3. This is VH of sequence number 77 and at least 80% (e.g., at least 85%, at least 9%). 0%, at least 95%, at least 99%, or at least 100%) identical VH and VL of sequence number 80 and at least 80% (e.g., at least 85%, at least 9%) 0%, at least 95%, at least 99%, or at least 100%) identical VL It includes. Optionally, a second antigen-binding region that specifically binds to CD3 is V of SEQ ID NO: 77 Includes H and VL of SEQ ID NO: 80.Optionally, a second antigen-binding region that binds to CD3 is , VH of sequence number 84 and at least 80% (e.g., at least 85%, at least 90%) %) identical VH and and VL of sequence number 85 and at least 80% (for example, at least 85%, at least 90%) VLs that are identical (at least 95%, at least 99%, or at least 100%) Includes. Optionally, the second antigen-binding region that binds to lymphocyte antigens is VH of SEQ ID NO: 84. and includes VL of sequence number 85.

[0027] In some embodiments, the first antigen-binding region that binds to DLL3 is the first immunoglobulin. Conjugated to a brin (Ig) steady-state region or a fragment of a first Ig steady-state region, / or the second antigen-binding region that binds to lymphocyte antigens is the second immunoglobulin (Ig) binding region. It is conjugated to the normal region or a fragment of the second Ig constant region. Optionally, it is isolated. The anti-DLL3 / anti-CD3 protein has a first antigen-binding domain that binds to DLL3 and a first Between the Ig constant region or the first Ig constant region, and the second antibody that binds to the lymphocyte antigen A second linker is placed between the original binding region and the second Ig steady-state region or a fragment of the second Ig steady-state region. -Includes (L2).Optionally, L2 includes sequence numbers 27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 1 23, 124, 125, 126, 127, 128, 129, 130, 131, 132, 1 Contains amino acid sequences 33, 134, 135, 136, 137, or 138. Optional. The fragment of the Ig steady-state region includes the Fc region. Optionally, the first Ig steady-state region or the first The fragment of the Ig constant region and the second Ig constant region or the fragment of the second Ig constant region are IgG1 The IgG2, IgG3, or IgG4 isotype is optional. A region or fragment of the first Ig steady-state region and a cross section of the second Ig steady-state region or the second Ig steady-state region. One is an IgG1 isotype. Optionally, the first Ig steady-state region or the first Ig constant region is selected. Fragments of the normal region and the second Ig steady-state region or fragments of the second Ig steady-state region are multivalent in FcγR Includes at least one mutation resulting in reduced binding of the hemospecific antigen-binding construct. Optional Selection results in reduced binding of multispecific antigen-binding constructs to FcγR, at least One mutation is F234A / L235A, L234A / L235A, L234A / L23 5A / D265S, V234A / G237A / P238S / H268A / V309L / A 330S / P331S, F234A / L235A, S228P / F234A / L235A , N297A, V234A / G237A, K214T / E233P / L234V / L23 5A / G236 deletion / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235 E / D265A, L234A / L235A / G237A / P238S / H268A / A3 30S / P331S, S228P / F234A / L235A / G237A / P238S, And from S228P / F234A / L235A / G236 (missing) / G237A / P238S A group is selected, and residue numbering follows the EU index. Optionally, FcγR is selected. Mutations that result in reduced binding of the multispecific antigen-binding construct are L234A_L235A_ This is D265S. Optionally, the protein has less of the CH3 domain in the Ig constant region. Both contain one mutation. Optionally, at least one mutation in the CH3 domain of the Ig constant region. One mutation is T350V, L351Y, F405A, Y407V, T366Y, T36 6W, F405W, T394W, T394S, Y407T, Y407A, T366S / L 368A / Y407V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y407V, T366L / K392M / T394W, L35 1Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K 409F, T366A / K409F, T350V / L351Y / F405A / Y407V and selected from the group consisting of T350V / T366L / K392L / T394W, residue The 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 is HC A first VH having DR1, HCDR2, and HCDR3, and LCDR1, LCDR 2, and a first VL having LCDR3, 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) Each contains the amino acid sequences of SEQ ID NOs. 61, 62, 32, 47, 48, and 49, respectively. , the first antigen-binding region that binds to DLL3. (2) A second antigen-binding region that binds to CD3ε, (a) HCDR1, HCDR2, and HCDR1 of sequence numbers 95, 96, and 97, respectively. A second VH having the DR3 amino acid sequence, and SEQ ID NOs. 101, 102, and A second VL having 104 LCDR1, LCDR2, and LCDR3 amino acid sequences, teeth (b) HCDR1, HCDR2, and H of sequence numbers 98, 99, and 100 respectively A second VH having the CDR3 amino acid sequence, and SEQ ID NOs. 106, 107, and A second VL having the LCDR1, LCDR2, and LCDR3 amino acid sequences of 108 This relates to a bispecific antigen-binding construct, which includes a second antigen-binding region.

[0029] In this specification, a bispecific antigen-binding construct is referred to as an "anti-DLL3 / anti-CD3 construct" or It is referred to as "anti-DLL3 / anti-CD3".

[0030] In some embodiments, isolated anti-DLL3 / anti-CD3 proteins are converted to DLL3. It includes a first antigen-binding region that binds and a second antigen-binding region that binds to CD3, a) DL The first antigen-binding regions that bind to L3 are, respectively, sequence numbers 15, 16, 17, 33, and 34. , 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR The second domain, which includes 3 and binds to lymphocyte antigens, is sequence numbers 95, 96, and 9, respectively. 7, 101, 102, 104 HCDR1, HCDR2, HCDR3, LCDR1, LC a) A first antigen-binding region comprising DR2 and LCDR3 and / or b) DLL3. This includes a Fab containing VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2, and a second bonded to CD3. The antigen-binding region comprises the scFv of SEQ ID NO: 105, and / or c) isolated anti-DL L3 / anti-CD3 proteins include HC1 (SEQ ID NO: 109), LC1 (SEQ ID NO: 110), and Contains HC1 of sequence number 112.

[0031] In some embodiments, isolated anti-DLL3 / anti-CD3 proteins are converted to DLL3. It includes a first antigen-binding region that binds and a second antigen-binding region that binds to CD3, a) DL The first antigen-binding regions that bind to L3 are, respectively, sequence numbers 15, 16, 17, 33, and 34. , 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR The second domains that include 3 and bind to CD3 are sequence numbers 95, 96, 97, and 1, respectively. 01, 102, 104 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 , and a) a first antigen-binding region comprising LCDR3 and / or b) DLL3, The Fab contains VH in column number 1 and VL in sequence number 2, and is a second antigen that binds to CD3. The binding region includes scFv of sequence number 119 and / or c) isolated anti-DLL3 / The anti-CD3 proteins are HC1 (SEQ ID NO: 109), LC1 (SEQ ID NO: 110), and sequence number Includes HC1 of number 113.

[0032] In some embodiments, isolated anti-DLL3 / anti-CD3 proteins are converted to DLL3. It includes a first antigen-binding region that binds and a second antigen-binding region that binds to CD3, a.DL The first antigen-binding regions that bind to L3 are, respectively, sequence numbers 15, 16, 17, 33, and 34. , 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR The second domains that include 3 and bind to CD3 are sequence numbers 98, 99, and 100, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR 106, 107, 108 2. The first antigen-binding region, which includes LCDR3 and / or binds to b.DLL3, The second antigen-binding region, which includes the scFv of SEQ ID NO: 63 and binds to CD3, is SEQ ID NO: 84 A Fab comprising VH of and VL of SEQ ID NO: 85, and / or c. isolated anti-DLL 3 / Anti-CD3 proteins include HC1 (SEQ ID NO: 111), HC2 (SEQ ID NO: 116), and Includes LC2 at column number 117.

[0033] In some embodiments, a first antigen-binding region that binds to DLL3 and a region that binds to CD3 An isolated anti-DLL3 / anti-CD3 protein comprising a second antigen-binding region, a The first antigen-binding regions that bind to .DLL3 are sequence numbers 15, 16, 17, and 33, respectively. , 34, 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and L The second domains containing CDR3 and binding to CD3 are sequence numbers 95, 96, and 9, respectively. 7, 101, 102, 104 HCDR1, HCDR2, HCDR3, LCDR1, LC A first antigen-binding region comprising DR2 and LCDR3 and / or binding to b.DLL3. It contains the scFv of SEQ ID NO: 63, and the second antigen-binding region that binds to CD3 is SEQ ID NO: A Fab comprising VH of 77 and VL of SEQ ID NO: 80, and / or c. isolated anti-D LL3 / anti-CD3 proteins include HC1 (sequence number 111), HC2 (sequence number 114), and It includes LC2 of sequence number 115.

[0034] In some embodiments, a first antigen-binding region that binds to DLL3 and a region that binds to CD3 An isolated anti-DLL3 / anti-CD3 protein comprising a second antigen-binding region, a The first antigen-binding regions that bind to .DLL3 are sequence numbers 15, 16, 17, and 33, respectively. , 34, 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and L The second domains containing CDR3 and binding to CD3 are sequence numbers 98, 99, and 1, respectively. 00, 106, 107, 108 HCDR1, HCDR2, HCDR3, LCDR1, L A first antigen-binding region comprising CDR2 and LCDR3 and / or binding to b.DLL3 The region contains the scFv of sequence number 64, and the second antigen-binding region that binds to lymphocyte antigens is, A Fab comprising VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85, and / or c. isolated The anti-DLL3 / anti-CD3 proteins identified are HC1 (SEQ ID NO: 71) and HC1 (SEQ ID NO: 118). Includes 2, and LC2 of sequence number 117.

[0035] In some embodiments, a first antigen-binding region that binds to DLL3 and a region that binds to CD3 An isolated anti-DLL3 / anti-CD3 protein comprising a second antigen-binding region, a The first antigen-binding regions that bind to .DLL3 are sequence numbers 15 and 16, respectively. , 17, 33, 34, 35 HCDR1, HCDR2, HCDR3, LCDDR1, LCD The second domain, which includes R2 and LCDR3 and binds to CD3, is sequence number 9, respectively. HCDR1, HCDR2, HCDR3, L for 8, 99, 100, 106, 107, 108 The first includes CDR1, LCDR2, and LCDR3 and / or is bound to b.DLL3 The antigen-binding region is at least 80% (e.g., at least 8) of the scFv of SEQ ID NO: 64. 5%, at least 90%, at least 95%, at least 99%, or 100%) are the same It contains scFv, and the second antigen-binding region that binds to CD3 is less than VH of SEQ ID NO: 84 At least 80% (for example, at least 85%, at least 90%, at least 95%, less VH is identical to VL of sequence number 85 by at least 99% or 100%. (For example, at least 85%, at least 90%, at least 95%, at least 99%) c. Contains a Fab containing a VL that is identical (or 100%) and / or isolated anti-DLL 3 / Anti-CD3 protein is at least 80% (e.g., less) of HC1 of SEQ ID NO: 71. (85%, at least 90%, at least 95%, at least 99%, or 100%) HC1 is one, and HC2 of sequence number 118 is at least 80% (for example, at least 85%) They are identical (at least 90%, at least 95%, at least 99%, or 100%). HC2 and SEQ ID NO: 117 and at least 80% (e.g., at least 85%, at least Includes LC2 which is identical by 90%, at least 95%, at least 99%, or 100%.

[0036] In some embodiments, a first antigen-binding region that binds to DLL3 and a region that binds to CD3 An isolated anti-DLL3 / anti-CD3 protein comprising a second antigen-binding region, a The first antigen-binding regions that bind to .DLL3 are sequence numbers 15, 16, 17, and 33, respectively. , 34, 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and L The second domains containing CDR3 and binding to CD3 are sequence numbers 98, 99, and 1, respectively. 00, 106, 107, 108 HCDR1, HCDR2, HCDR3, LCDR1, L A first antigen-binding region comprising CDR2 and LCDR3 and / or binding to b.DLL3 The region contains the scFv of sequence number 64, and the second antigen-binding region that binds to lymphocyte antigens is, A Fab comprising VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85, and / or c. isolated The anti-DLL3 / anti-CD3 proteins identified are HC1 (SEQ ID NO: 229) and H (SEQ ID NO: 230). Includes C2 and LC2 of sequence number 117.

[0037] In some embodiments, a first antigen-binding region that binds to DLL3 and a region that binds to CD3 An isolated anti-DLL3 / anti-CD3 protein comprising a second antigen-binding region, a The first antigen-binding regions that bind to .DLL3 are sequence numbers 15, 16, 17, and 33, respectively. , 34, 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and L The second domains containing CDR3 and binding to CD3 are sequence numbers 98, 99, and 1, respectively. 00, 106, 107, 108 HCDR1, HCDR2, HCDR3, LCDR1, L A first antigen-binding region comprising CDR2 and LCDR3 and / or binding to b.DLL3 The range is at least 80% (e.g., at least 85%, less) of scFv of sequence number 64. scFv that are identical (90%, at least 95%, at least 99%, or 100%) The second antigen-binding region, which includes a CD3-binding domain, is at least 80% of the VH of SEQ ID NO: 84. (For example, at least 85%, at least 90%, at least 95%, at least 99%) VH and VL of SEQ ID NO: 85 are identical (or 100%) and at least 80% (for example, less At least 85%, at least 90%, at least 95%, at least 99% or 100% ) Contains a Fab containing the same VL and / or c. Isolated anti-DLL3 / anti-CD3 The protein is at least 80% (e.g., at least 85%) of HC1 of sequence number 229. H is identical (at least 90%, at least 95%, at least 99%, or 100%). C1, HC2 of sequence number 230, and at least 80% (e.g., at least 85%, less HC2 and are identical (90%, at least 95%, at least 99%, or 100%) Sequence ID 117 and at least 80% (e.g., at least 85%, at least 90%, less It includes LC2 which is identical by at least 95%, and at least 99% or 100%.

[0038] This disclosure also includes an isolated antigen-binding region that binds to DLL3 of this disclosure, which is an immunocon. We provide jugate.

[0039] This disclosure also includes isolated proteins containing antigen-binding regions that bind to DLL3 of this disclosure. It provides an immune conjugate that includes [this].

[0040] This disclosure also includes an isolated, multispecific antigen-binding region that binds to DLL3 of this disclosure. We provide an immunoconjugate containing an antigen-binding construct.

[0041] This disclosure also includes a pharmaceutical composition comprising an isolated antigen-binding region that binds to DLL3 of this disclosure. To provide goods.

[0042] This disclosure also includes isolated proteins containing antigen-binding regions that bind to DLL3 of this disclosure. The present invention provides a pharmaceutical composition containing [a specific compound / substance].

[0043] This disclosure also includes an isolated, multispecific antigen-binding region that binds to DLL3 of this disclosure. The present invention provides a pharmaceutical composition containing an antigen-binding construct.

[0044] This disclosure also encodes an isolated antigen-binding region that binds to DLL3 of this disclosure. Provides separated polynucleotides.

[0045] This disclosure also includes isolated proteins containing antigen-binding regions that bind to DLL3 of this disclosure. Provides an isolated polynucleotide encoding [the specified character].

[0046] This disclosure also includes an isolated, multispecific antigen-binding region that binds to DLL3 of this disclosure. This provides isolated polynucleotides encoding antigen-binding constructs.

[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 this disclosure. .

[0049] This disclosure also relates to a method for treating DLL3-expressing cancer in a subject, wherein the therapeutic amount a protein containing an antigen-binding region that binds to DLL3, and an antigen-binding region that binds to DLL3. , a multispecific antigen-binding construct including an antigen-binding region that binds to DLL3, immunocontainer of the present disclosure Jugate, or the pharmaceutical composition of this disclosure, for a sufficient amount of time to treat DLL3-expressing cancer. The present invention provides a method that includes administering it to subjects who need it.

[0050] This disclosure also relates to a method for reducing the amount of DLL3-expressing tumor cells in a subject, DL A protein containing an antigen-binding region that binds to L3, and an antigen-binding region that binds to DLL3, DL A multispecific antigen-binding construct containing an antigen-binding region that binds to L3, an immunoconjugate of the present disclosure or the pharmaceutical composition of this disclosure for a sufficient amount of time to reduce the amount of DLL3-expressing tumor cells. The present invention provides a method that includes administering the drug to a subject over a certain period of time.

[0051] This disclosure also relates to a method for preventing the establishment of DLL3-expressing cancer in a subject, DLL A protein containing an antigen-binding region that binds to 3, and an antigen-binding region that binds to DLL3, DLL A multispecific antigen-binding construct containing an antigen-binding region that binds to 3, an immunoconjugate of the present disclosure. To administer the pharmaceutical composition of this disclosure to a subject in need thereof, and to achieve D in the subject This invention provides a method that includes preventing the establishment of LL3-expressing cancer.

[0052] This disclosure also relates to non-cancerous conditions in subjects at risk of developing DLL3-expressing cancer. A method for treating a sexual condition, comprising an antigen-binding region that binds to DLL3, and a region that binds to DLL3. Proteins containing antigen-binding regions, multispecific antigens containing antigen-binding regions that bind to DLL3. A conjugate, an immunoconjugate, or a pharmaceutical composition of the disclosure may be used as a conjugate, or if it is required The present invention provides a method that includes administering a drug to a subject to treat a non-cancerous condition.

[0053] This disclosure also relates to a method for treating prostate cancer in a subject, wherein the therapeutically effective dose is DL A protein containing an antigen-binding region that binds to L3, and an antigen-binding region that binds to DLL3, DL A multispecific antigen-binding construct containing an antigen-binding region that binds to L3, an immunoconjugate of the present disclosure The drug, or the pharmaceutical composition of the present disclosure, is administered for a sufficient amount of time to treat prostate cancer. The present invention provides a method that includes administering the substance to a subject who needs it.

[0054] This disclosure also relates to a method for treating small cell lung cancer in a subject, wherein the therapeutically effective dose is D A protein containing an antigen-binding region that binds to LL3, and an antigen-binding region that binds to DLL3, D A multispecific antigen-binding construct containing an antigen-binding domain that binds to LL3, the immunoconjugate of the present disclosure Gate, or the pharmaceutical composition of this disclosure, for a sufficient amount of time to treat small cell lung cancer The present invention provides a method that includes administering it to a subject who needs it.

[0055] This disclosure also relates to a method for detecting prostate cancer or small cell lung cancer in a subject, The disclosed immunoconjugate is administered to the target and the immunoconjugate to DLL3. This includes detecting binding and thereby detecting prostate cancer or small cell lung cancer. Provide a method.

[0056] This disclosure also provides an antigen-binding region that binds to DLL3, and an antigen-binding region that binds to DLL3. A multispecific antigen-binding construct containing a protein, an antigen-binding region that binds to DLL3, and this A kit is provided comprising an immunoconjugate of the Disclosure or a pharmaceutical composition of the Disclosure.

[0057] This disclosure also includes an anti-idiotyl that binds to the antigen-binding region of DLL3 of this disclosure. We provide antibodies.

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

[0059] The above is explained in more detail in the illustrative embodiments shown in the attached drawings. It will become clear in the future. [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] It shows the cytotoxicity against DLL3+ target cell lines in PBMC mediated by the bispecific anti-DLL3×CD3 antibody. Figure 8A shows the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by the bispecific anti-DLL3×CD3 antibody, and the E:T ratio is 10:1. Figure 8B shows the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by the bispecific anti-DLL3×CD3 antibody, and the E:T ratio is 5:1. Figure 8C shows the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by the bispecific anti-DLL3×CD3 antibody, and the E:T ratio is 1:1. <{ [Figure 8B] It shows the cytotoxicity against DLL3+ target cell lines in PBMC mediated by the bispecific anti-DLL3×CD3 antibody. Figure 8A shows the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by the bispecific anti-DLL3×CD3 antibody, and the E:T ratio is 10:1. Figure 8B shows the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by the bispecific anti-DLL3×CD3 antibody, and the E:T ratio is 5:1. Figure 8C shows the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by the bispecific anti-DLL3×CD3 antibody, and the E:T ratio is 1:1. [Figure 8C] It shows the cytotoxicity against DLL3+ target cell lines in PBMC mediated by the bispecific anti-DLL3×CD3 antibody. Figure 8A shows the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by the bispecific anti-DLL3×CD3 antibody, and the E:T ratio is 10:1. Figure 8B shows the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by the bispecific anti-DLL3×CD3 antibody, and the E:T ratio is 5:1. Figure 8C shows the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by the bispecific anti-DLL3×CD3 antibody, and the E:T ratio is 1:1. [Figure 9] It shows the proliferation of CD3+ T cells in response to the bispecific anti-DLL3×CD3 antibody in the total 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 in the background art and throughout this specification. This specification is disclosed, and the entire contents of each of these references are incorporated herein by reference. The discussion of documents, operations, materials, devices, articles, etc. included in this book provides the background for the present invention. This is a consideration. Such considerations are based on the fact that any or all of these things are disclosed or claimed. This does not constitute part of the prior art for any of the inventions being referred to.

[0061] Any method and materials similar to or equivalent to those described herein for the testing of the present invention Materials and methods that can be used to carry out this, but are illustrative examples, are described herein.

[0062] Unless otherwise specified, all technical and scientific terms used herein are defined in accordance with the present invention. It has the same meaning as generally understood by those skilled in the art to which it belongs. Otherwise, Certain terms used herein have the meanings set forth herein. All patents, published patent applications, and publications referenced in this specification are, by reference, as if they were true. The entirety is incorporated as described herein. This specification and the attached claims. When used within the scope of [this], the singular forms "a," "an," and "the" are used, especially when it is clear from the context. Unless otherwise noted, it should be kept in mind that it will include multiple referents. Therefore, for example, The term "a cell" includes combinations of two or more cells. .

[0063] If a list is presented, unless otherwise specified, each individual element of that list and its It should be understood that all combinations of the list are separate embodiments. For example, a list of embodiments presented as "A , B, or C" should be interpreted to include the embodiments "A", "B", "C" , "A or B", "A or C", "B or C", or "A, B, or C".

[0064] Unless otherwise specified, any numerical values such as concentrations or concentration ranges described herein should be understood to be modified by the term "about" in all cases. Therefore, the numerical values typically include ±10% of the recited value. For example, a dosage of 10 mg includes 9 mg to 11 mg. When used herein, the use of a numerical range includes all possible sub-ranges, including integers and fractional values within that range, and explicitly includes all individual numerical values within that range, unless explicitly specified otherwise in the context.

[0065] As used herein, the conjunctive term "and / or" between a plurality of recited 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 first element being applicable in the absence of the second element. The second option refers to the second element being applicable in the absence of the first element. The third option refers to the first and second elements being applicable together. Any one of these options is included in the meaning, and thus, when used herein, it is understood to meet the requirements of the term "and / or". The simultaneous applicability of two or more of the options is also included in the meaning, and thus, it is understood to meet the requirements of the term "and / or". ​​​​​​​​​​

[0066] Transition phrases: "comprising," "consisting essential" "ly of" and "consisting of" are generally accepted in patent terminology. It is intended to imply the meaning of "to be equipped / include (comp "Rising" is synonymous with "includes," "contains," or "characterizes," and is comprehensive or This is not restrictive and does not exclude other elements or methods not listed. (ii) "consisting of" is not specified in the claims. (iii) "Exclude any element, process or component that is not present, and (iii) "essentially from "The specified materials or processes, as well as the "basic and novel features" of the claimed invention" The scope of the patent claims is limited to "things that do not substantially affect" the term "equipment / includes" (and Embodiments described with the equivalent terms are also "consisting of" and "essentially from" It is provided as an embodiment that is described independently with the phrase "ru".

[0067] "Approximately" means that a particular value is within the acceptable margin of error as determined by those skilled in the art. This means that the value is measured or determined, i.e., the measurement system Partially dependent on the limitations of the system. In the context of a particular assay, result, or embodiment, Unless otherwise expressly stated in the examples or elsewhere in the specification, “about” means “about the technical field.” For implementation, within the range of one standard deviation or up to 5%, whichever is greater. It means something.

[0068] "Activation," "stimulation," "activated," or "stimulated" refers to the activation marker. This can lead to the expression of - cytokines, cytokine production, or mediation of target cell proliferation or cytotoxicity. This refers to the induction of a change in the biological state of a cell. Cells are activated by a primary stimulus signal. It is possible.

[0069] "Alternative scaffolds" are structured domains that associate with variable domains with high tolerance for three-dimensional structure. This refers to a single-chain protein framework containing A. The variable domain is part of the scaffold. To allow for polymorphisms to be introduced without compromising integrity, the variable domain is linked to the specific antigen. They can be genetically engineered and selected to bind together.

[0070] What is “antibody-dependent cytotoxicity”, “antibody-dependent cell-mediated cytotoxicity”, or “ADCC”? Antibody-coated target cells, natural killer cells (NK), monocytes, macromocytes, and macromocytes. Phages and effector cells with lytic activity such as neutrophils, and effector cells It induces cell death that depends on interactions mediated by the Fc gamma receptor (FcγR) expressed above. It refers to the mechanism by which something happens.

[0071] "Antibody-dependent cell phagocytosis" or "ADCP" refers to the action of macrophages or dendritic cells, etc. This refers to a mechanism by which antibody-coated target cells are eliminated through uptake by phagocytic cells.

[0072] An "antigen" is an antigen that can mediate an immune response through an antigen-binding domain or T cell receptor. Any molecule that can be bound (e.g., proteins, peptides, polysaccharides, glycoproteins, glycolipids) This refers to nucleic acids, parts thereof, or combinations thereof. An example of an immune response is the production of antibodies. This includes the activation of immune cells such as adult T cells, B cells, or NK cells. Antigens are tissue Samples, tumor samples, cells, or fluids, organisms, or proteins containing other biological components. / A biological sample such as antigen subunits, dead or inactivated whole cells, or lysates A gene that can be expressed from the sample, can be synthesized from the sample, or the sample It can be purified from.

[0073] The "antigen-binding region," "antigen-binding fragment," or "antigen-binding domain" each binds to the antigen. This refers to the portion of the full-length antibody that binds. The antigen-binding region is obtained from synthetic polypeptides or enzymatically. It may be a polypeptide, or a genetically modified polypeptide. The antigen-binding region is typical. It includes at least one or more portions of the VH region. The antigen-binding fragment is of the antibody A polyvalent molecule containing one, two, three, or more antigen-binding moieties and single-chain constructs. The VL and VH regions, or selected portions thereof, are linked by a synthetic linker or genetically modified. They are joined by a method to form a functional antigen-binding molecule. The antigen-binding fragment is also a single Antibody fragments consisting of monomeric variable antibody domains (VHH), also known as nanobodies. It may be a single-domain antibody (sdAb). The antigen-binding fragment Examples 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, e.g., Bird et al., Science 1988;242:423-426 and H See uston et al. PNAS 1988;85:5879-5883. i), dsFv, Fd (typically VH and CH1 domains), and dAb (typically VH domain) fragments; VH, VL, VHH, and V-NAR domains; single VH strand and Monovalent molecules containing a single VL chain; mini-bodies, dia-bodies, tria-bodies, tetra-bodies , and kappa bodies (e.g., Ill et al., Protein Eng 199) See 7;10:949-57); camel IgG; IgNAR; and one or Examples include two or more isolated CDRs or functional paratopes, and isolated CDRs or anti The primordial residue or polypeptide is the functional antibody fragment, the FR3-CDR3-FR4 portion. HCDR1, HCD, is the smallest recognition unit consisting of amino acid residues that mimic the CDR of antibodies. R2 and / or HCDR3 and LCDR1, LCDR2 and / or LCDR3, antigen It forms a multispecific antigen-binding construct that includes an alternative scaffold for binding, as well as an antigen-binding region. Therefore, they can be associated or linked together. Various types of antibody fragments can be, for example, H olliger and Hudson,Nat Biotechnol 2005;2 3:1126-1136, International Publication No. 2005040219, and the published U.S. Special Publication As described or generalized in Patent Application Publication Nos. 20050238646 and 20020161201 It is explained that antibody fragments can be obtained using conventional recombinant or protein engineering methods. This fragment can then be screened in the same way as an intact antibody for antigen binding or other functions. It is possible to do this. Various methods have been developed for the production of antibody fragments. Conventionally, These fragments were obtained from the proteolytic digestion of full-length antibodies (e.g., Morimoto et al., Journal of Biochemistry and Bioph. Medical Methods, 24:107-117 (1992), and Brenna See also n et al., Science, 229:81 (1985). However, these fragments are currently being produced directly by recombinant host cells. Alternatively, Fab'-SH fragments can be recovered directly from E. coli and chemically bonded. They can combine to form the F(ab')2 fragment (Carter et al., Bi o / Technology, 10:163-167 (1992). Another approach Furthermore, the F(ab')2 fragment can be directly isolated from recombinant host cell cultures. In the application method, the selected antibody is a single-chain Fv fragment (scFv). This is the case. International Publication No. 1993 / 16185, U.S. Patent No. 5,571,894, and the same. See Patent No. 5,587,458. Antibody fragments are also, for example, U.S. Patent No. 5,648. As described in issue 1,870, it may also be a "linear antibody." The body fragment may be monospecific or bispecific. Antigen-binding regions ((VH and VL) These are linked together via a synthetic linker to form various types of single-chain antibody designs. This is possible, and here, the VH / VL domains are separate VH domains and VL domains. When expressed in a single-strand form, single-strand Fv(scFv) forms pairs intramolecularly or intermolecularly. Alternatively, it may form a monovalent antigen-binding region such as a diabody. The antigen-binding region may also be bivalent. To genetically engineer heterosexual and multispecific antigen-binding constructs, use monospecific or multispecific methods. It may be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds. It is possible.

[0074] "Antibody" has a broad meaning, encompassing polyclonal antibodies, mouse antibodies, human antibodies, humanized antibodies, and textures. Monoclonal antibodies including lamonoclonal antibodies, antigen-binding region, bispecificity, triplicity Sex, tetraspecificity, and other multispecific antibodies, dimers, tetramers, or multimer antibodies, single-chain antibodies, etc. The main antibody, and any immunoglobulin molecule containing the required specific antigen-binding site. Contains immunoglobulin molecules with other modified configurations. "Full-length antibodies" are disulfide Two heavy chains (HC) and two light chains (C) are interconnected by bonding. It consists of hein (LC) and their polymers (e.g., IgM). Each heavy chain is heavy Chain variable region (VH), and heavy chain constant region (domains CH1, hinge, CH2, and CH3) It consists of a light chain variable region (VL) and a light chain constant region (light). It consists of a chain constant region (CL). The VH region and VL region are frame Framework regions (FRs) are scattered throughout the complementary determination region. It can be further classified into a highly variable region called the y determining region (CDR). Each VH and VL is in the following order from the amino terminus to the carboxyl terminus: FR1, CD Three CDRs arranged in R1, FR2, CDR2, FR3, CDR3, and FR4 It is composed of four FR segments. Immunoglobulins are amino acids in the heavy chain constant domain. Depending on the sequence, it is classified into five main classes: IgA, IgD, IgE, IgG, and IgM. It can be assigned. IgA and IgG are isotypes IgA1, IgA2, IgG1 These are further subdivided into IgG2, IgG3, and IgG4. Body light chains also fall into two distinct types based on the amino acid sequence of their constant domain. In other words, it can be assigned to either kappa (κ) or lambda (λ). The general principles of the molecular structure and various techniques for antibody production are, for example, those of Harlow and d Lane, ANTIBODIES: A LABORATORY MANUAL, Co ld Spring Harbor Laboratory Press,Cold S It was provided to Pring Harbor, NY, (1988).

[0075] The isolated proteins or constructs of this application may also include antibody derivatives. The term "antibody derivative" as used refers to a molecule containing a full-length antibody or its antigen-binding fragment. This refers to a molecule in which one or more amino acids are chemically modified or substituted. Chemical modifications that can be used in antibody derivatives include, for example, modifying the antibody to a second... For linking to the molecule, for example, alkylation, PEGylation, acylation, esterification, or Examples of modifications include amide formation. Exemplary modifications include PEGylation (e.g., cysteine ​​P). Examples include EG conversion, biotinylation, radiolabeling, and binding with a second reagent (e.g., a cytotoxic agent). It is possible.

[0076] The antibodies described herein are "amino acid sequence variants" that have modified antigen binding or biological activity. It contains "riant". An example of such amino acid modification is enhanced affinity for antibodies. Antibodies that have undergone modification (e.g., "affinity-mature" antibodies), and antibodies in which the Fc region has been altered (where present) For example, antibody dependent cellular cytotoxicity (AD) CC) and / or complement-dependent cytotoxicity (CDC) The antibody has been modified (increased or decreased) (e.g., International Publication No. 00 / 42072 (Pr esta, L.) and International Publication No. 99 / 51642 (Iduosogie et al. See also ( ). and / or antibodies with increased or decreased serum half-lives (e.g., country See International Publication No. 00 / 42072, Presta, L.) for example.

[0077] A "bispecific antigen-binding construct" or "bispecific construct" is a construct containing two different antigens or the same antigen. This refers to a construct that specifically binds to two different epitopes within the same antigen. The composite structure may be a protein, a protein complex, or an antibody. A bispecific antibody is, Other related antigens, e.g., human or monkey, e.g., cynomolgus monkey (Macaca cynomolgus) Other species such as cynomolgus (cyno) or chimpanzees (Pan troglodytes) They can exhibit cross-reactivity to antigens of the same origin (homologs), or two or It can bind to epitopes shared among three or more different antigens.

[0078] “Bispecific anti-DLL3 / anti-CD3 antibody”, “anti-DLL3×CD3”, “DLL3 / C "D3 antibody," "DLL3×CD3 antibody," "anti-DLL3 / anti-CD3 protein," etc. are D At least one binding domain that binds to LL3 and CD3 and specifically to DLL3 A construct comprising an in and at least one binding domain that specifically binds to CD3 or This refers to an antibody. The domains that specifically bind to DLL3 and CD3 are typically V H / V LThey are a pair. The bispecific anti-DLL3×CD3 antibody targets either DLL3 or CD3. It can be monovalent with respect to bonding.

[0079] The term "hypervariable region" as used herein refers to the atypical region of the antibody responsible for antigen binding. This refers to amino acid residues. The hypervariable region is generally derived from the "complementarity-determining region" or "CDR". Mino acid residues (residues 24-34 (L1), 50-56 (L2), and 8 in the light chain variable domain) 9-97 (L3) and 31-35 (H1) and 50-65 (H2) in the heavy chain variable domain and 95-102 (H3); (Kabat et al. (1991) Sequence s of Proteins of Immunological Interest, Fifth Edition,USDepartment of Health a nd Human Services,NIH Publication No.91- 3242), and / or residues derived from the "hypervariable loop" (residues 26- in the light chain variable domain) 32(L1), 50-52(L2), and 91-96(L3), as well as in the heavy chain variable domain 26-32 (H1), 53-55 (H2), and 96-101 (H3); Chothia (and Lesk, J.Mol.Biol.1987;196:901-917) Hmm. Typically, the numbering of amino acid residues in this region is done by Kabat et al. as described above. This is carried out by the method described herein. In this specification, "Kabat position" and "Ka Expressions such as "variable domain residue numbering in bat" and "by Kabat" are important. This refers to the numbering system for chain-variable domains or light-chain-variable domains. Using a numbering system, the actual linear amino acid sequence of the peptide is determined by the variable domain FR. Alternatively, it may contain fewer or additional amino acids to correspond to the shortening or insertion of CDRs. It is possible. For example, the heavy chain variable domain has a single amino acid insertion after residue 52 of CDR H2. The input (residue 52a according to Kabat) and the residue inserted after the heavy chain FR residue 82 (for example) , may include residues 82a, 82b, and 82c by Kabat. AT numbering assigns the sequence of a given antibody to the "standard" Kabat numbering assignment. This can be determined by aligning the columns within a homologous region.

[0080] A reference antibody is an antibody that binds to the same epitope as the reference antibody in a competitive assay. This refers to an antibody that blocks 50% or more of the binding to that antigen, while a reference antibody is a competitive antibody. In this study, the binding of the antibody to the antigen is blocked by 50% or more.

[0081] With respect to the method of the present invention, the term "administration" refers to the complex of the present invention, or its constituent products, composition By using the substance or drug, the syndrome, disorder, or disease described herein can be treated. It means a method of prevention, treatment, or remission, either therapeutically or preventively. Such methods include using an effective amount of the above antibody, its antigen-binding fragment, or their conjugates. , or formations, compositions, or drugs at different points in the course of treatment, or in combination The method of the present invention includes administering simultaneously in a formula. The method of the present invention encompasses all known therapeutic treatment regimens. It is understood to include.

[0082] The ability of a target antibody to "block" the binding of a target molecule to a natural target ligand is that the antibody can In assays using soluble or cell surface-bound target and ligand molecules, dose-dependent The binding of the target molecule to the ligand is detected in a way that is not present, and the target molecule is not present in the antibody. This means that the ligand is detectably bound to the present state.

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

[0084] "Complement-dependent cell damage" or "CDC" refers to the Fc effect of a protein bound to a target. The TARD domain binds to complement component C1q, activates it, and then activates the complement cascade. This refers to a mechanism that induces cell death by transforming the target cells into target cells. Complement activation also targets This can cause the deposition of complement components on the cell surface, which in turn can lead to the deposition of complement receptors (e.g., C) on leukocytes. The binding of R3) facilitates CDC.

[0085] The "complementarity-determining region" (CDR) is the region of the antibody that binds to the antigen. VH has three There are three types of CD-Rs (HCDR1, HCDR2, HCDR3), and VL has three CD-Rs (LC There are various types of CDRs, including DR1, LCDR2, and LCDR3. CDRs are described in various ways, such as the following: It can be defined using: Kabat (Wu et al. (1970) J Ex p Med 132:211-50, Kabat et al., Sequences of Proteins of Immunological Interest,5t h Ed.Public Health Service,National Inst. Itutes of Health, Bethesda, Md., 1991), Chot. hia(Chothia et al. (1987) J Mol Biol 196:9 01-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thor Various drawings such as those in the Nton J Bmol Biol 263:800-15, 1996. It can be defined using a copy. The correspondence between various descriptions and the numbering of the variable area is described. (For example, Lefranc et al. (2003) Dev Comp I mmunol 27:55-77, Honegger and Pluckthun(2 001), J Mol Biol 309:657-70;International ImMunoGeneTics (International ImMunoGeneTics, IMGT) database See the web resources, http: / / www.imgt_org. Use available programs such as abYsis provided by CL Business PLC And a CDR can be depicted. When used herein, "CDR" and "HC" are used. DR1", HCDR2, HCDR3, LCDR1, LCDR2, and The term "LCDR3" refers to the above Kabat unless otherwise expressly stated in the specification. , including CDRs as defined by the Chothia, IMGT, or AbM methods. For example, numbering systems including Kabat numbering and IMGT-specific numbering systems The correspondence between the stems is well known to those skilled in the art (for example, Kabat and Cho mentioned above). See also thia, Martin, and Lefranc mentioned above.

[0086] [Table 1]

[0087] "CD3" is part of the multimolecular T cell receptor (TCR) complex. Expressed on T cells, with two or four receptor chains: CD3 epsilon, CD3 delta, CD3 It consists of homodimers or heterodimers formed from the association of 3-zeta and CD3-gamma. This refers to an antigen. Human CD3 epsilon contains the amino acid sequence of SEQ ID NO: 253. All references to proteins, polypeptides, and protein fragments in this text refer to non-human proteins. Unless explicitly specified as being of species origin, each protein and polypeptide is a species-derived product. , or intended to refer to the human version of the protein fragment. Therefore, "CD3" These are designated as originating from non-human species, such as "mouse CD3" or "monkey CD3". Unless otherwise specified, it refers to human CD3.

[0088] Throughout this specification, the terms "CD3-specific," "specifically binds to CD3," or "anti" are used. "CD3 antibody" is CD3-ε extracellular domain (ECD) (sequence number CD3-ε polypeptide (SEQ ID NO. 253) containing an antibody that specifically binds to (SEQ ID NO. 254) This refers to an antibody that specifically binds to CD3. CD3-ε is a variant of CD3-γ, -δ, and -ζ, as well as T Together with the α / β and γ / δ heterodimers of the cell receptors, it forms the T cell receptor-CD3 complex. This complex is heavily involved in antigen binding, recognizing several intracellular signaling pathways. It plays a crucial role. The CD3 complex mediates signal transduction and promotes the activation and proliferation of T cells. CD3 is necessary for the immune response.

[0089] As used herein, "conjugate" refers to therapeutic peptides or proteins, or anti- One or more heterologous molecules, including but not limited to bodies, labels, or neurotoxic agents. This refers to a protein that is covalently bonded to another protein. When this occurs, it is also referred to as the fusion of two proteins. As an example, the antibody or antigen-binding fragment of this application is conjugated to another polypeptide, A fusion protein can be formed. In certain embodiments, the antibody or anti- The ability to fuse or conjugate a primordial binding fragment to another polypeptide via a linker. can.

[0090] "To decrease," "to decline," "to lower," "to reduce," or "to mitigate" Generally, the test molecule is reduced compared to the response mediated by the control or vehicle. This refers to the ability to mediate a response (i.e., a downstream effect). An example response is T cell expansion. Cell activation, or T cell-mediated tumor cell death, or the antigen or receptor of a protein. Binding to, binding to enhanced Fcγ, or enhanced ADCC, CDC and / or This is an enhanced Fc effector function such as ADCP. The decrease is compared to the test molecule and the control (or A statistically significant difference in the measured response between the vehicle and the measured response, or approximately 1.1, 1.2, 1.5 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 times, or that For example, 500, 600, 700, 800, 900, or 1000 times, or This includes all integers greater than or equal to 1 and any decimal points in between, e.g., 1.5, 1.6, 1. This may also be an increase in the measured response, such as an increase in 7.1.8, etc.

[0091] "Delta-like protein 3" or "DLL3" is also known as delta-like 3, delta 3, or shouji This refers to a known protein, also known as the fruit fly delta homolog 3. (Not specified) To the extent that it is used herein, DLL3 refers to human DLL3. All DLL3s Isoforms and variants are included in "DLL3". Various isoforms The minoic acid sequence is NCBI accession number NP_058637.1 (isoform 1). Precursor, 618 amino acids) and NP_982353.1 (Isoform 2 precursor, 58 It can be obtained from databases such as 7 amino acids. The amino acid distribution of full-length human DLL3 The sequence is shown in sequence number 255. The sequence of DLL3 is the DSL domain (residues 176-215). , EGF-1 domain (residues 216-249), EGF-2 domain (residues 274-31 0), EGF-3 domain (residues 312-351), EGF-4 domain (residues 353- 389), EGF-5 domain (residues 391-427), EGF-6 domain (residue 42 It includes residues 9-465) and the C-terminal domain (residues 466-618) (Figure 1). DLL3 The amino acid sequence of the DSL domain is shown in SEQ ID NO: 246. DLL3 EGF-1 The main amino acid sequence is shown in Sequence ID No. 247. DLL3 EGF-2 domain The amino acid sequence is shown in SEQ ID NO: 248. Amino acids of the DLL3 EGF-3 domain. The sequence is shown in Sequence ID No. 249. The amino acid sequence of the DLL3 EGF-4 domain is: The amino acid sequence of the DLL3 EGF-5 domain is shown in Sequence ID No. 250. The amino acid sequence of the DLL3 EGF-6 domain is shown in SEQ ID NO: 251. The amino acid sequence of the DLL3 EGF-6+ C-terminal domain is shown in SEQ ID NO: 263. It will be shown.

[0092] "Differentiation" is the process of reducing the capacity or proliferation of a cell, or restricting the cell's development. This refers to a method of moving something to a specific state.

[0093] "To code" or "to code" refers to a defined sequence of nucleotides (for example, (rRNA, tRNA, and mRNA) or having any of the defined amino acid sequences Templates for the synthesis of other polymers and macromolecules in biological processes. Nucleo in polynucleotides such as genes, cDNA, or mRNA that function as a gene. This refers to the unique characteristics of the specific sequence of a cydone, as well as the biological properties that arise therefrom. In cells or other biological systems, the transcription and translation of mRNA corresponding to genes are performed on proteins. When a gene, cDNA, or RNA produces a protein, that gene codes for a protein. The coding strand in which the rheotide sequence is identical to the mRNA sequence, and the transcription of the gene or cDNA. Any non-coding strand used as a plate is a protein of that gene or cDNA. It may be referred to as coding a quality or other product.

[0094] "Enhance," "Promote," "Increase," The terms "expand" or "improve" are generally used in contrast to or When compared to a response mediated by a vehicle, the test molecule produces a larger response (i.e., This refers to the ability to mediate downstream effects. Exemplary responses include T cell expansion, T cell activation, or T cell-mediated tumor cell death, or the binding and enhancement of proteins to their antigens or receptors. Binding to Fcγ, or enhanced ADCC, CDC and / or ADCP, etc. This is an enhanced Fc effector function. The enhancement occurs between the test molecule and the control (or vehicle). A statistically significant difference in the measured response, or 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, 5 00, 600, 700, 800, 900, or 1000 times, or more (1 All integers exceeding 1.5 and all decimal points in between, for example, 1.5, 1.6, 1.7, 1.8, etc. This may include an increase in the measured response, such as an increase in (including)

[0095] An "epitope" is a part of an antibody, or a part of an antigen to which an antibody-binding portion specifically binds. To refer to. An epitope is typically a chemically active (polar, nonpolar, or hydrophobic, etc.) part of a molecule. A surface group consisting of, for example, amino acids or polysaccharide side chains, with specific three-dimensional structural properties and specific It may have charge properties. Epitopes are continuous and / or form higher-order structural spatial units. It can be composed of discontinuous amino acids. In discontinuous epitopes, different parts of the linear sequence of the antigen The amino acids from the protein molecule are brought into very close proximity in three-dimensional space through the folding of the protein molecule. Yes. Antibodies, or "epitopes," differ depending on the methodology used to identify them. ru.

[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 within cells or in vitro. Therefore, expression products, such as proteins, are expressed by cells or in vitro. These may be intracellular, extracellular, or transmembrane proteins.

[0098] An "expression vector" is a vector containing polynucleotide sequences that can be used to express emotions. To direct the translation of the polypeptide to be translated, in a biological system or a reconstituted biological system This refers to the vectors that can be used.

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

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

[0101] "F(ab')2" or "F(ab')2 fragment" is a disulfide bridge within the hinge region. This refers to an antibody fragment containing two Fab fragments connected by [a specific mechanism / tool].

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

[0103] "Fv" or "Fv fragment" refers to the VH domain and VL domain derived from a single arm of the antibody. This refers to an antibody fragment composed of n. The Fv fragment is the constant region of the Fab(CH1 and CL) region. It lacks. VH and VL in the Fv fragment are joined together by non-covalent interactions. It is retained.

[0104] "Framework region" or "FR" residues are other than CDR as defined herein. These are the VH or VL residues.

[0105] A "full-length antibody" consists of two heavy chains (HC) linked together by disulfide bonds. It is composed of two light chains (LCs) and their polymers (e.g., IgM). The chain consists of a heavy chain variable domain (VH) and a heavy chain constant domain, and the heavy chain constant domain is It consists of subdomains CH1, hinge, CH2, and CH3. Each light chain is a light chain variable It consists of a main (VL) and a light chain constant domain (CL). VH and VL are frames A highly variable region called the Complementarity Determination Region (CDR), where work areas (FRs) are scattered. The region can be further subdivided. Each VH and VL is from the amino terminus to the carboxyl terminus. The following sequence is used for distribution: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. It consists of three CDR segments and four FR segments.

[0106] "Genetic modification" is the process by which a host cell expresses an introduced gene or sequence to produce a desired substance. Typically, it produces a protein or enzyme encoded by the introduced gene or sequence. To that end, "foreign" (i.e., exogenous or extracellular) genes, DNA, or RNA sequences This refers to introducing something into a host cell. The introduced gene or sequence is also called "cloned." They are sometimes called "foreign" genes or sequences, and include starters, terminaters, promoters, and sigmas. Chimeric antigen receptors such as other sequences used by the genetic mechanisms of the cell, secretion, or other cells. It may include a regulatory or control sequence that is operably ligated to the encoding polynucleotide. The gene or sequence may contain non-functional sequences that do not have known functions. The host cell that receives and expresses the DNA or RNA is "genetically engineered." DNA or RNA introduced into the principal cell may include cells of the same genus or species as the host cell. It may originate from the same source, or from a different genus or species.

[0107] "Different species" refers to two or more things that are not found in the same relationship to each other in the natural world. This refers to three or more polynucleotides or two or three or more polypeptides.

[0108] "Heterogeneous polynucleotides" refer to two or more neoantigens described herein that are coupled together. This refers to polynucleotides that do not exist in nature.

[0109] "Heterogeneous polypeptide" refers to two or more neoantigen polypeptides as described herein. This refers to polypeptides that do not exist in nature, including the 'd' component.

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

[0111] Human antibodies are optimized to produce the minimum possible immune response when administered to human subjects. This refers to antibodies that have been modified. The variable region of human antibodies is derived from the human immunoglobulin sequence. If the antibody contains a constant region or a portion of a constant region, that constant region is also human immunoglobulin Derived from the row. Human antibodies are derived from human germline immunoglobulins or rearranged in the variable region of human antibodies. When obtained from a system using the immunoglobulin gene, the sequence is of human origin. This includes a heavy chain variable region and a light chain variable region. Such exemplary systems are used to phages. A sprayed human immunoglobulin gene library and human immunoglobulin gene loci. These are transgenic non-human animals that possess "human antibodies," such as mice or rats. " is typically a system used to obtain human antibodies and human immunoglobulin gene loci. The difference lies in the intentional introduction of somatic mutations into the framework or CDR. By means of, or both of these, the amino acid content when compared to immunoglobulins expressed in humans It contains differences. Typically, "human antibodies" are human germline immunoglobulins or reorganized immunoglobulins. For the amino acid sequence encoded by the immunoglobulin gene, The sequence is at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, and 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% They are 98% or 99% identical. In some cases, "human antibodies" are, for example, Knappi k et al., (2000) J Mol Biol 296:57-86 Consensus framework sequences derived from human framework sequence analysis, or for example Shi et al., (2010) J Mol Biol 397:385-96 and The human immunoglobulin presented on phages, as described in International Publication No. 2009 / 085462. It may contain synthetic HCDR3 incorporated into the robulin gene library. Antibodies whose CDRs originate from non-human species are not included in the definition of "human antibodies."

[0112] "Humanized antibodies" are those in which at least one CDR is derived from a non-human species and at least one F The framework refers to antibodies derived from human immunoglobulin sequences. Humanized antibodies are frameworks. Because the framework can include substitutions, the framework expresses human immunoglobulins. It may not be an exact copy of the germline gene sequence of phosphorus or human immunoglobulin. .

[0113] "In combination with ~" means that two or more therapeutic agents are used together in a mixture form, or as a single agent. This means administering the drugs to the target simultaneously, or sequentially in any order, either as individual agents or as separate agents.

[0114] "Isolated" means that the molecule is not a component of the system in which it is produced, such as in recombinant cells. A homogeneous collection of molecules that have been substantially separated and / or purified (e.g., synthetic polynucleotides) Otides or polypeptides), and proteins subjected to at least one purification or isolation step. This refers to the substance. "Isolated" means substantially free from other cellular material and / or chemical substances. This refers to molecules that do not contain impurities, and indicates higher purity, for example, 80%, 81%, 82%, 83%, 84%, 8%. 5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9 Includes molecules isolated to a purity of 5%, 96%, 97%, 98%, 99%, or 100%. Yes. "Isolated" antibodies are those that have been isolated from components of their natural environment. In that embodiment, the antibody is subjected to electrophoresis (e.g., SDS-PAGE, isoelectric focusing). Electrophoresis (isoelectric focusing, IEF, capillary electrophoresis) or chromatography 95% or 99% as determined by (for example, ion exchange or reverse-phase HPLC) It is purified to a purity exceeding [a certain level]. For an overview of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B, 848:79-87( See (2007).

[0115] As used herein, "linker" refers to a chemical that covalently links two different entities. This refers to a linker or a single-chain peptide linker. The linker is the antibody of the present invention or its Any two of the following can be linked: fragments, fusion proteins, or conjugates. Carr, for example, VH and VL in scFv or monoclonal antibodies or their antigenicity The combined fragment can be linked to a second therapeutic molecule, such as a second antibody, via peptide bonds. 1 to 25 amino acids linked together, 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, 2 A single-chain peptide linker consisting of 4 or 25 amino acids can be used. In certain embodiments, the amino acids are selected from 20 natural amino acids. In other embodiments, one or more amino acids may be glycine, alanine, proline, etc. Selected from asparagine, glutamine, and lysine. Hydrocarbon linker, polyethylene Polyethylene glycol (PEG) linker, polypropylene glycol (Polypropylene glycol, PPG) linker, polysaccharide linker, polyester linker, P Hybrid linkers consisting of EG and embedded heterocycles, and chemical hydrocarbon chains, etc. A linker can also be used.

[0116] "To regulate" means to have a greater response compared to a response mediated by a control or vehicle. Enhanced or reduced test molecules that mediate a response of or less (i.e., downstream effects) It refers to one of the abilities that have been described.

[0117] A "monoclonal antibody" is an antibody in which the C-terminal lysine is removed from the antibody heavy chain, or the amino acids are removed. Isomerization or deamide, oxidation of methionine, or deamide of asparagine or glutamine The actual antibody molecule is identical except for possible, well-known changes such as post-translational modifications like amides. This refers to antibodies obtained from a qualitatively homogeneous population, that is, individual antibodies that make up the population. A bispecific monoclonal antibody binds to two different antigenic epitopes. Ronal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies It may be single-specific, or it may be multiple-specific, such as bi-specific, monovalent, divalent. It may be a single value or multiple values.

[0118] A "multispecific antigen-binding construct" or "multispecific molecule" is a construct that binds two or more different antigens or This refers to a construct that specifically binds to two or more different epitopes within the same antigen. The antigen-binding construct may be a protein, a protein complex, or an antibody. at least one other functional molecule (for example, a ligand for another antibody or receptor). It associates with or binds to peptides or proteins, thereby forming at least two different bonds. It contains an antibody or its antigen-binding fragment that forms a molecule that binds to a binding site or target molecule. The heavily specific molecule is associated with other related antigens, such as human or monkey antigens, such as cynomolgus monkey antigens (Macaca). fascicularis) (cynomolgus, cyno) or chimpanzee (Pan troglodyt) In some cases, cross-reactivity may occur with the same antigen (homolog) from other species, such as es. They may also bind to epitopes shared among two or more different antigens. Example Typical multispecific molecules include triplicate or bispecific antibodies and soluble receptor fragments. Alternatively, an antibody linked to a ligand may be used.

[0119] "Natural killer cells" and "NK cells" are used interchangeably and synonymously in this specification. When used herein, NK cells are CD16 + CD56 + and / or CD 57 + TCR - This refers to differentiated lymphocytes that exhibit a specific phenotype. NK cells are characterized by a specific cell-lytic enzyme. Activation of this process causes it to bind to and kill cells that cannot express their own MHC / HLA antigens. Ability to kill tumor cells or other disease cells that express ligands for NK cell activation receptors. They release protein molecules called cytokines that stimulate or inhibit the immune response. Characterized by its abilities.

[0120] The terms "operably linked" and similar phrases are used in reference to nucleic acids or amino acids. In combination, the functional relationships between nucleic acid sequences or amino acid sequences arranged in a mutually functional manner. Refers to a connection. For example, a promoter, enhancer element, or open motor that is operably connected. The leading frame, 5' and 3' UTR, and terminator sequence are nucleic acid molecules (e.g.) For example, it leads to the precise production of RNA, and in some cases, the production of polypeptides (that is, This results in the expression of an open reading frame. A functionally linked peptide. This is because the functional domains of the peptide are positioned at an appropriate distance from each other, and each domain is intended to perform its intended function. This refers to peptides that impart function.

[0121] The term "paratope" refers to an antigen that contains residues involved in antigen binding and interaction with the antigen. It refers to a region or area of ​​a body molecule. Paratopes are continuous and form higher-order structural spatial units. / or may consist of discontinuous amino acids. A given antibody paratope can be used in various experiments and calculations. Using calculation methods, it is possible to define and characterize in detail at various levels. Experimental methods Hydrogen / deuterium exchange mass spectrometry (H) This includes X-MS. Paratopes are defined differently depending on the mapping method used. Paratopes are amino acid residues that are directly involved in epitope binding (some of these are typical). (located within the CDR) and other amino acid residues that are not directly involved in binding, for example, specifically binding Amino acid residues that are effectively blocked by the antigen (i.e., amino acid residues that are specifically This may include the "solvent-excluded surface" and / or footprint of the bound antigen. .

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

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

[0124] A "medically acceptable carrier" or "excipient" is a non-toxic substance to the target and contains no active ingredient. This refers to components in external pharmaceutical compositions. Exemplary pharmaceutically acceptable carriers include buffers and stabilizers. , or a preservative.

[0125] "Polynucleotides" or "nucleic acids" are formed from a sugar-phosphate backbone or other equivalent covalent chemistry. Therefore, it refers to a synthetic molecule containing covalently bonded nucleotide chains. cDNA is a polynucleotide. This is a typical example of an ocide. Polynucleotides can be DNA or RNA molecules.

[0126] "Preventing," "preventing," "prevention," or "preventive methods" of a disease or disability are subject to the following rules: This means preventing failures from occurring.

[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 also These may each include an enhancer or repressor element that enhances or suppresses transcription.

[0129] In this specification, the terms "protein" and "polypeptide" used interchangeably refer to the respective peptides. One or two amino acid residues, linked by a tide bond. This refers to a molecule containing one or more polypeptides. The proteins may be monomers, or Even if a protein complex consists of two or more identical or different subunits Good. Small polypeptide molecules consisting of fewer than 50 amino acids can be called "peptides." Proteins are heterofusion proteins, glycoproteins, or phosphorylated, acetylated, and myrrhizated proteins. Stoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrulline Post-translational modifications such as polyglutamylation, ADP-ribosylation, pegylation, or biotinylation Therefore, it may be a modified protein. The protein can be recombinantly expressed.

[0130] A "recombinant" is a polynucleotide prepared, expressed, produced, or isolated by a recombinant means. This refers to ocides, polypeptides, vectors, viruses, and other macromolecules.

[0131] A "regulatory element" controls any cis or trans aspect of nucleic acid sequence expression. This refers to the action-oriented genetic element.

[0132] "Recurrent" means that the disease or signs and symptoms of the disease recur after a period of improvement following prior treatment with medication. It refers to resuming something.

[0133] "Intractable" refers to a disease that does not respond to treatment. Intractable diseases are considered before treatment or after treatment. Diseases that may be resistant to treatment at the outset, or are refractory, may become resistant to treatment during the course of treatment.

[0134] When used in relation to amino acid sequences, use the terms "sequence identity" or "sequence identity percentage (%)". The phrase "identity%" or "identity%" refers to the number of amino acid residues that make up the entire length of the amino acid sequence. The number of identical amino acids in two or more aligned amino acid sequences compared ( Record the "hits". Other alignments used for two or more sequences. In this terminology, the sequence is measured using a sequence comparison algorithm known in the art. In the case of manual alignment and visual inspection, the maximum agreement is compared and When entered, the percentage of amino acid residues that are the same (for example, the total amino acid sequence) Over the long term, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% A degree of identity (percentage, or 100%) can be determined. Therefore, in order to determine sequence identity The sequences being compared may differ due to amino acid substitutions, additions, or deletions. Suitable programs for aligning sequences are known to those skilled in the art. The percentage of column identity is, for example, CLUSTALW, Clustal Omega , programs such as FASTA or BLAST can be used, for example, NCBI BLAS T algorithm can be used for determination (Altschul SF, et al (199 7), 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 variable light chain region (VL) and at least one antibody fragment containing a variable heavy chain region (VH), where the VL and VH are continuously linked via a polypeptide linker and can be expressed as a single-chain polypeptide. Unless otherwise specified,[[ID=*12]] as used herein, scFv may have the VL and VH variable regions in either order, for example, with respect to the N-terminus and C-terminus of the polypeptide, scFv may contain VL- linker-VH or may contain VH-linker-VL.

[0136] "(scFv)2" or "tandem scFv" or "bis-scFv" fragment refers to a fusion protein comprising two variable light chain regions (VL) and two variable heavy chain regions (VH), where the two VLs and two VHs are continuously linked via a polypeptide linker and can be expressed as a single-chain polypeptide. The two VLs and two VHs are fused by a peptide linker to form a bivalent molecule VL A -linker-VH A -linker-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 bind," "specifically bind," "specifically bound," or "to bind" "A protein molecule is a parent of an antigen or an epitope within that antigen that is related to another antigen." This refers to binding with a higher affinity than typical affinity. Typically, protein molecules bind with approximately 1× 10 -7 M or less, for example, about 5 × 10 -8 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, or approximately 1 × 10 -12 More than M The equilibrium dissociation constant (K) below D ) binds to the antigen or an epitope within the antigen, typically K D teeth K for binding to nonspecific antigens (e.g., BSA, casein) D At least It's 100 times smaller.

[0138] "Subject" includes any human or non-human animal. "Non-human animal" includes any vertebrate Vertebrates, for example, non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, This includes mammals such as reptiles and non-mammals. The terms "subject" and "patient" are used in this context. It can be used interchangeably in the specification.

[0139] "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, and mature T lymphocytes. This includes T lymphocytes, quiescent T lymphocytes, or activated T lymphocytes. T cells are T helpers (T helper, Th) cells, for example, T helper 1 (Th1) or T helper 2 (Th2) It can be a cell. T cells are helper T cells (HTL, CD4+ T cells), CD4 + T thin cells, cytotoxic T cells (CTL, CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL CD8 + T cells), CD4 + CD8 + T cells, or any other subset of T cells It is possible. Also, "NKT cells" are included, which express semi-invariant αβT cell receptors. Furthermore, various molecular markers typically associated with NK cells, such as NK1.1, are also expressed. This refers to a special group of T cells. NKT cells include NK1.1 + and NK1.1 - ,Line up CD4 + CD4 - CD8 + , and CD8 - It contains cells. The TCR of NKT cells is It is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells produce cytokines that promote either inflammation or immune tolerance. Depending on their capabilities, they may have either protective or harmful effects. Also, "gamma delta T cells" (γδT cells) are also included, and these are small T cells that have a different TCR on their surface. This refers to a specific subset of TCRs, which are two sugar chains denoted as α- and β-TCR chains. Unlike most T cells, which are composed of protein chains, the TCR in γδT cells is γ It is composed of chains and delta chains. γδT cells play a role in immune surveillance and immunomodulation. It is possible to be an important source of IL-17, and a robust CD8 + Cytotoxic T cells It was found to induce a cellular response. It also includes "regulatory T cells" or "Tregs." This suppresses abnormal or excessive immune responses and plays a role in immune tolerance, affecting T cells. Tregs are typically Foxp3-positive CD4 + It is a T cell, and also IL-10 producing CD4 + This may also include T cells, specifically Foxp3-negative regulatory T cells.

[0140] In this specification, "therapeutic effective dose" or "effective dose" as used interchangeably refers to the required dosage. The effective therapeutic dose refers to the amount that is effective in obtaining the desired therapeutic outcome within a given period. Factors such as age, sex, and weight, as well as therapeutic agents that elicit the desired response in the individual This may vary depending on the ability of the combination of therapeutic drugs. Effective therapeutic drugs or combinations of therapeutic drugs Examples of matching indicators include, for example, improvement in the patient's health status, reduction in tumor volume, and tumor growth. This includes the cessation or slowing of growth, and / or the absence of metastasis of cancer cells to other parts of the body. ru.

[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 disorder such as cancer means: The following are exacerbations of symptoms characteristic of the treated disability that reduce the severity and / or duration of the disability. To suppress the progression of the disorder, or to limit or prevent the recurrence of the disorder in subjects who previously had the disorder. or limit or prevent the recurrence of symptoms in subjects who were previously symptomatic with respect to the disorder. This refers to achieving one or more of the following:

[0143] "Tumor cells" or "cancer cells" refer to cells identified in vivo, ex vivo, or through tissue culture. In which, cancerous, precancerous, or morphological changes with spontaneous or induced phenotypic changes This refers to convertive cells. These changes do not necessarily involve the uptake of new genetic material. No. Transformation involves infection with a transformed virus and the incorporation of new genomic nucleic acids, and the exogenous nucleus. It can also be generated by the uptake of acid, occurring spontaneously or after exposure to carcinogens. It can occur, and in some cases, this can lead to mutations in endogenous genes. Transformation / cancer is an infection. Morphological changes, cell immortalization, and abnormal proliferation control in vivo, in vivo, and ex vivo. Regarding lesion formation, proliferation, malignant lesions, regulation of tumor-specific marker levels, invasiveness, nude mammary glands This is exemplified by the proliferation of tumors in suitable animal hosts such as mice.

[0144] "Variant," "mutant," or "altered" means one or more modifications, for example , by substitution, insertion, or deletion of one or more reference polypeptides or reference polynuclei This refers to polypeptides or polynucleotides that are different from rheotides.

[0145] Throughout this specification, the numbering of amino acid residues in the constant region of antibodies is as specified herein. Unless otherwise explicitly stated, Kabat et al., Sequences of Proteins of Immunological Interest,5th E d.Public Health Service,National Institute EU Indicators of Health, Bethesda, MD. (1991) Follow Dex's instructions.

[0146] "VHH" refers to a single-domain antibody or nanobody composed exclusively of the antigen-binding region of the heavy chain. This refers to the CH1 domain of the light and heavy chains of the conventional Fab region. It lacks.

[0147] composition of a substance Antigen-binding region that binds to DLL3 This disclosure includes an antigen-binding region that binds to DLL3, and an antigen-binding region that binds to DLL3. Monospecific and multispecific antigen-binding constructs, encoding polynucleotides, vectors This document provides a ter, host cells, and methods for producing and using them. The antigen-binding region that binds to DLL3 exhibits improved properties in terms of improved thermal stability. The multispecific antigen-binding constructs disclosed herein mediate T cell-mediated cytotoxicity. Through this, it promotes the activation and proliferation of T cells, increases T cell cytokine release, and / or This may be particularly effective in demonstrating an increased antitumor effect.

[0148] This disclosure includes an isolated antigen-binding region that binds to delta-like protein 3 (DLL3). The protein has an antigen-binding region that binds to DLL3, as shown in SEQ ID NO: 263. Epitope in the EGF-6+ C-terminal domain of DLL3 (residues 429-618 of DLL3) Provides an isolated protein that binds to ) as shown in the examples. EGF-6 Multispecific antigenic ligations target epitopes within the domain or closer to the C-terminus of DLL3. The composite structure achieved a potent level of antitumor cytotoxicity.

[0149] In consideration of this disclosure, the antibody of this application can be bound using any known method in the art. The region within DLL3 can be identified, for example, using an ELISA assay. This allows for the identification of the domain within DLL3 to which the antibody binds. Domain mapping E In the LISA assay, the N-terminal DSL fusion domain (DL3W44, SEQ ID NO: 189) ), EGF-1+2 fusion (DL3W42, Sequence ID 187), EGF-2 (DL3W4 1. Sequence ID 186), EGF-3 (DL3W40, Sequence ID 185), EGF-4 (D L3W39 (Sequence ID 184), EGF-5 (DL3W38, Sequence ID 183), EGF -6 (DL3W37, Sequence ID 182), and EGF-6+ C-terminal domain fusion (DL Regarding the binding to recombinant DLL3 domain antigen across 3W36 (SEQ ID NO: 181), The DLL3 antibody was evaluated. MesoScale Discovery high binding plates were used. Plates were coated overnight with 20 nM antigen at 4°C. Plates were then coated with PB containing 0.1% Tween. Washed with S, then blocked with Starting Block solution for 30 minutes. Add the body and incubate at ambient temperature for 60 minutes, then remove any excess antibody from PBS (Gib). The antigen-binding antibody was removed by washing three times with co (#14190-136). Sulfotagged anti-human antibody (Meso Scale Discovery, R32AJ) The sample was detected at ambient temperature for 60 minutes, followed by another PBS wash. Signal acquisition was performed using 1 ×In the presence of MSD read buffer T (MSD, catalog number R92TC-1), MSD Performed with appropriate plate settings on Sector 600 Imager. Preferred domain We analyzed the data for the highest binding signal per domain, which indicates binding.

[0150] Using an H / D exchange assay, the residue in DLL3 to which the antibody binds can be determined. In the H / D exchange assay, recombinantly expressed soluble DLL3 is tested for the presence of antibodies or When incubated in deuterated water in the absence of the antibody for a predetermined time, the antibody... Deuterium incorporation occurs with unprotected, replaceable hydrogen atoms, followed by protein Protease digestion and peptide fragment analysis using LC-MS will be performed. H / D exchange The process can be carried out using known protocols. In some embodiments, By adding an entrenching buffer (e.g., 8M urea, 1M TCEP, pH 3.0) After quenching the H / D exchange mixture, the immobilized pepsin / FPXII was equilibrated at room temperature. The liquid is passed through column I (e.g., 600 μL / min). Then, the digestible fragments are placed in a reverse-phase trap column. Fill (e.g., 600 μL / min), desalt (e.g., 600 μL for 1 minute), Separate the samples (e.g., C18 column) and analyze them by mass spectrometry (e.g., capillary temperature). LT at 275℃, resolution 150,000, and mass range (m / z) 300~1,800 Q(TM) Orbitrap Fusion Lumos Mass Spectrometer (Thermo F (Use Isher Scientific)

[0151] In some embodiments, this application relates to an isolated antigen, such as an antibody, which includes an antigen-binding region. The protein has an antigen-binding region that binds to DLL3, and this specification describes the binding to DLL3. This document provides isolated proteins that compete with the reference antibody disclosed in the book. Several implementations In terms of morphology, the reference antibody is VH having HCDR1, HCDR2, and HCDR3, and Includes a VL having LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2 , HCDR3, LCDR1, LCDR2, and LCDR3 are, a. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 1, and SEQ ID NO: 2 LCDR1, LCDR2, and LCDR3 of the VL, b. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 3, and SEQ ID NO: 4 LCDR1, LCDR2, and LCDR3 of the VL, c. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 5, and SEQ ID NO: 6 LCDR1, LCDR2, and LCDR3 of the VL, d. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 7, and SEQ ID NO: 8 LCDR1, LCDR2, and LCDR3 of the VL, e. HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 9, and SEQ ID NO: 1 LCDR1, LCDR2, and LCDR3 of VL 0 f. HCDR1, HCDR2, and HCDR3 of VH in Sequence ID No. 11, and Sequence ID No. 12 VL LCDR1, LCDR2, and LCDR3, or g. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 13, and SEQ ID NO: These are the 14 VLs LCDR1, LCDR2, and LCDR3.

[0152] In certain such embodiments, the reference antibody is HCDR1,H of SEQ ID NO: 3 VH. CDR2 and HCDR3, and LCDR1, LCDR2, and L of VL of Sequence ID No. 4 Includes CDR3.

[0153] Regarding the binding of the test antibody that binds to soluble DLL3 (SEQ ID NO: 263) to the reference antibody of this application, The competition is that, considering this disclosure, assays can be performed in vitro using well-known methods. Yes, it is possible. For example, in the presence of an unlabeled reference antibody, DLL3, for example, the membrane proximal region of DLL3. The binding of labeled antibodies to the region can be evaluated by ELISA. Bioacore The competition can be demonstrated using analysis or flow cytometry. If the test antibody is soluble... The binding of the reference antibody to DLL3 is 85% or higher, for example, 90% or higher, When inhibiting by 95% or more, the test antibody competes with the reference antibody for binding to DLL3. do.

[0154] In some embodiments, this application relates to an antibody comprising an antigen-binding region that binds to DLL3. Which isolated protein has an antigen-binding region that binds to DLL3, HCDR1, VH having HCDR2 and HCDR3, and LCDR1, LCDR2, and LCD Includes VL having R3, HCDR1, HCDR2, HCDR3, LCDR1, LCDR 2, and LCDR3 are HCDR1, HCDR2, and HCDR3 of VH of Sequence ID No. 1. Furthermore, LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 2, or SEQ ID NO: 3 HCDR1, HCDR2, and HCDR3 of VH, and LCDR1 of VL of SEQ ID NO: 4 , LCDR2 and LCDR3, or HCDR1, HCDR2, and VH of sequence number 5. HCDR3, and LCDR1, LCDR2, and LCDR3 of VL of Sequence ID No. 6, or HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 7, and VL of SEQ ID NO: 8 LCDR1, LCDR2, and LCDR3, or HCDR1, HC of VH, sequence number 9 DR2 and HCDR3, and LCDR1, LCDR2, and L of VL of Sequence ID No. 10 CDR3, or HCDR1, HCDR2, and HCDR3 of VH of Sequence ID No. 11, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 12, or V in SEQ ID NO: 13 HCDR1, HCDR2, and HCDR3 of H, and LCDR1 of VL of SEQ ID NO: 14 The isolated proteins LCDR2 and LCDR3 are provided. Specific implementations In this state, the isolated protein contains an antigen-binding region that binds to DLL3. The antigen-binding regions that bind are HCDR1, HCDR2, and HCDR3 of the VH variant of SEQ ID NO: 3. , and also includes LCDR1, LCDR2, and LCDR3 of VL of Sequence ID No. 4.

[0155] In some embodiments, this application relates to an antibody comprising an antigen-binding region that binds to DLL3. Which isolated protein has an antigen-binding region that binds to DLL3? 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 HCDs having amino acid sequences 61, 62, 32, 47, 48, and 49, respectively. R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are isolated. It provides the protein.

[0156] In another embodiment, the present disclosure provides an isolated tangible substance comprising an antigen-binding region that binds to DLL3. These are proteins in which the antigen-binding region that binds to DLL3 is sequence numbers 15, 16, and 1, respectively. HCDR1, HCDR2, HCDR3, LCDR1, with sequence numbers 7, 33, 34, 35 This provides isolated proteins, including LCDR2 and LCDR3.

[0157] In another embodiment, the present disclosure provides an isolated tangible substance comprising an antigen-binding region that binds to DLL3. It is a protein whose antigen-binding region that binds to DLL3 is sequence numbers 1, 3, 5, 7, 9, 1. VH having an amino acid sequence of 1 or 13, and SEQ ID NOs: 2, 4, 6, 8, 10, 12, Alternatively, the present invention provides an isolated protein containing a VL having a 14-amino acid sequence.

[0158] This disclosure relates to an isolated protein comprising an antigen-binding region that binds to DLL3, D The antigen-binding region that binds to LL3 is The VH amino acid sequence of SEQ ID NO: 1 and the VL amino acid sequence of SEQ ID NO: 2 (Also known as VH and VL of Sequence 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 sequence number 13 and VL of sequence number 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 DLL3 containing an antigen-binding region. The protein has an antigen-binding region that binds to DLL3, and the VH and SEQ ID NOs of SEQ ID NOs are SEQ ID NOs. This provides isolated proteins containing 4 VLs.

[0160] This disclosure also relates to an isolated protein comprising an antigen-binding region that binds to DLL3. The antigen-binding region that binds to DLL3 is at least 80% (for example, VH of SEQ ID NO: 3) (At least 85%, at least 90%, at least 95%, or at least 99%) identical VH and VL of SEQ ID NO: 4 are at least 80% (for example, at least 85%, less) Isolated VL containing at least 90%, at least 95%, or at least 99% identical It provides the protein.

[0161] In some embodiments, the antigen-binding region that binds to DLL3 is, for example, SEQ ID NO: 3 VH and at least 80% (e.g., at least 85%, at least 90%, at least 9%) It contains VH and VL of SEQ ID NO: 4, which are identical in 5% or at least 99% of the total.

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

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

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

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

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

[0167] This disclosure relates to an isolated protein comprising an antigen-binding region that binds to DLL3, D The antigen-binding region that binds to LL3 is sequence number 63, 64, 65, 66, 67, 68, or This provides an isolated protein containing a sequence of 69 amino acids.

[0168] In certain embodiments, the disclosure provides an isolated DLL3 containing an antigen-binding region. It is an protein, and the antigen-binding region that binds to DLL3 has the amino acid sequence of SEQ ID NO: 63 It provides isolated proteins, including those listed.

[0169] In certain embodiments, the disclosure provides an isolated DLL3 containing an antigen-binding region. It is an protein, and the antigen-binding region that binds to DLL3 has the amino acid sequence of SEQ ID NO: 64 It provides isolated proteins, including those included.

[0170] This disclosure also relates to an isolated protein comprising an antigen-binding region that binds to DLL3. The antigen-binding region that binds to DLL3 is at least 80% different from the amino acid sequence of SEQ ID NO: 63. (For example, at least 85%, at least 90%, at least 95%, or at least 9 Provides isolated proteins containing identical amino acid sequences (9%).

[0171] This disclosure also relates to an isolated protein comprising an antigen-binding region that binds to DLL3. The antigen-binding region that binds to DLL3 is at least 80% different from the amino acid sequence of SEQ ID NO: 64. (For example, at least 85%, at least 90%, at least 95%, or at least 9 Provides isolated proteins containing identical amino acid sequences (9%).

[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 The VH domains and VL domains identified herein that bind to DLL3 or their All of the constituent components are either VH-linker-VL or VL-linker-VH oriented. The genes can be modified into the cFv format. VH identified herein Both VH-linker-VL-linker-VL-linker-VH, VH- Linker-VL-Linker-VH-Linker-VL, VH-Linker-VH-Linker- VL-linker-VL, VL-linker-VH-linker-VH-linker-VL, VL -Linker-VH-Linker-VL-Linker-VH, or VL-Linker-VL-Linker It can be used to generate sc(Fv)2 structures such as Kerr-VH-linker-VH. can.

[0182] VH and VL or their components identified herein are in scFv format It can be incorporated, and the coupling of the obtained scFv to DLL3 and its thermal stability are considered in this disclosure. It can be evaluated using known methods with due consideration. The coupling is ProteOn XPR3 6. Biacore3000 or KinExA equipment, ELISA, or equipment already known to those skilled in the art This can be evaluated using a competitive binding assay of knowledge. Binding is performed using purified scFv Alternatively, evaluation can be performed using E. coli supernatant or lysed cells containing expressed scFv. The affinity measurements of the test scFv to DLL3 can be obtained under different conditions (e.g., mol immersion). This may differ when measured under osmotic pressure (concentration, pH). Therefore, affinity and other binding parameters may vary. Meter (for example, K D , K on , K off The measurement of ) is typically performed under standardized conditions and standards This is done using a buffer solution. Thermal stability is good at high temperatures such as 50°C, 55°C, or 60°C. Heat the test scFv for a period of time such as minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes. This can be evaluated by measuring the binding of test scFv to DLL3. Unheated scFv When compared to the sample, scFv maintains the same level of coupling as DLL3, while having better thermal stability. It is said to exist.

[0183] In recombinant expression systems, the linker is a peptide linker, and any naturally occurring amino acid It may contain acids. Exemplary amino acids that may be present in the linker are Gly, Ser Pro, and T These are hr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker is , in order to maintain the desired activity such as binding to DLL3, the precise higher-order structures relative to each other It is necessary to have an appropriate length to connect VH and VL in a manner that forms such a connection. .

[0184] The linker can be approximately 5 to 50 amino acids long. In some embodiments, the linker - has an amino acid length of approximately 10 to 40. In some embodiments, the linker is approximately 1 The amino acid length is 0 to 35. In some embodiments, the linker is approximately 10 to 30. This is the length of the amino acids. In some embodiments, the linker is about 10 to 25 amino acids. It is long. In some embodiments, the linker is about 10 to 20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long. In some embodiments, the linker has a length of 6 amino acids. - is 7 amino acid length. In some embodiments, the linker is 8 amino acids It is long. In some embodiments, the linker is 9 amino acid long. In some embodiments, the linker is 10 amino acids long. The linker has an amino acid length of 11. In some embodiments, the linker has 12 amino acids. It is the amino acid length. In some embodiments, the linker is the length of 13 amino acids. In some embodiments, the linker has a length of 14 amino acids. The linker has a length of 15 amino acids. In some embodiments, the linker is 1 It has a length of 6 amino acids. In some embodiments, the linker has a length of 17 amino acids. Yes. In some embodiments, the linker is 18 amino acids long. In the application form, the linker has a length of 19 amino acids. In some embodiments, the linker - is 20 amino acid length. In some embodiments, the linker is 21 amino acids It is the length of the amino acid. In some embodiments, the linker is the length of 22 amino acids. In some embodiments, the linker has a length of 23 amino acids. In some embodiments, The linker has a length of 24 amino acids. In some embodiments, the linker has a length of 25 amino acids. The length is 1 amino acid. In some embodiments, the linker has a length of 26 amino acids. In some embodiments, the linker has a length of 27 amino acids. Morphologically, the linker has a length of 28 amino acids. In some embodiments, the linker It has a length of 29 amino acids. In some embodiments, the linker has a length of 30 amino acids. It is acidic. In some embodiments, the linker is 31 amino acids long. In one embodiment, the linker has a length of 32 amino acids. In some embodiments, The linker has a length of 33 amino acids. In some embodiments, the linker has 34 amino acids. This is the amino acid length. In some embodiments, the linker has a length of 35 amino acids. In some embodiments, the linker has a length of 36 amino acids. In this configuration, the linker has a length of 37 amino acids. In some embodiments, the linker is The length is 38 amino acids. In some embodiments, the linker is 39 amino acids. It is long. In some embodiments, the linker is 40 amino acids long. Used Examples of linkers obtained include a glycerin-rich linker, a glycerin and ser-containing linker, and a glycerin-rich linker. Linkers containing y and Ala, linkers containing Ala and Ser, and other flexible linkers That is the case.

[0185] Other linker sequences are derived from any immunoglobulin heavy or light chain isotype. The globulin hinge region may include parts of CL or CH1. Alternatively, polyethylene glycerin may be used. Polypropylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or polyethylene Various non-protein polymers including copolymers of polypropylene glycol and polypropylene glycol Remmers may be found to be used as linkers. Table 2 shows exemplary linkers that can be used. As shown. Additional linkers are described, for example, in International Publication No. 2019 / 060695. Yes, they are.

[0186] In some embodiments, scFv is VH, first ri, from the N-terminus to the C-terminus. Includes rker (L1) and VL (VH-L1-VL).

[0187] In some embodiments, scFv has VL, L1, and It includes VH(VL-L1-VH). In some embodiments, L1 is sequence number 120. , Sequence ID 27, Sequence ID 72, Sequence ID 73, Sequence ID 74, Sequence ID 75, Sequence ID 76, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 88, Array Number 90, Sequence ID 91, Sequence ID 92, Sequence ID 121, Sequence ID 122, Sequence ID 1 23, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 1 28, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 1 33, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 1 Contains amino acid sequence 38, or sequence number 139.

[0188] [Table 2]

[0189] In certain embodiments, L1 includes or is derived from the amino acid sequence of SEQ ID NO: 120. ru.

[0190] In some embodiments, scFv is the heavy chain complement of the heavy chain variable region (VH) of SEQ ID NO: 1. Sex-determining regions (HCDR) 1, HCDR2, and HCDR3, as well as the light chain variable of SEQ ID NO: 2 Light chain complementarity determination regions (LCDRs) 1, LCDR2, and LCDR3 of the region (VL), or HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 3, and VL of SEQ ID NO: 4 LCDR1, LCDR2, and LCDR3, or HCDR1, HC of VH, sequence number 5 DR2 and HCDR3, and LCDR1, LCDR2, and LCDR1 of VL in Sequence ID No. 6 DR3, or HCDR1, HCDR2, and HCDR3 of VH of sequence number 7, and sequence LCDR1, LCDR2, and LCDR3 of VL number 8, or HC of VH number 9. DR1, HCDR2, and HCDR3, as well as LCDR1, LCD of VL of Sequence ID No. 10 R2, and LCDR3, or HCDR1, HCDR2, and HCD of VH in sequence number 11. R3, and LCDR1, LCDR2, and LCDR3 of VL of sequence number 12, or sequence VH numbers 13, HCDR1, HCDR2, and HCDR3, and VL number 14. This includes LCDR1, LCDR2, and LCDR3. In a particular embodiment, scFv is HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 3, and VL of SEQ ID NO: 4 This includes LCDR1, LCDR2, and LCDR3.

[0191] In some embodiments, the scFv has HCDR1, HCDR2, and HCDR3. VH, and VL having LCDR1, LCDR2, and LCDR3, HCD R1, 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 certain embodiments, scFv is sequence numbers 15, 16, 17, 33, 34, respectively. 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 Includes.

[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 nothing.

[0198] In some embodiments, scFv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 12. nothing.

[0199] In some embodiments, scFv includes VH of SEQ ID NO: 1 and VL of SEQ ID NO: 14. nothing.

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

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

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

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

[0204] In some embodiments, scFv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 10. nothing.

[0205] In some embodiments, scFv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 12. nothing.

[0206] In some embodiments, scFv includes VH of SEQ ID NO: 3 and VL of SEQ ID NO: 14. nothing.

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

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

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

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

[0211] In some embodiments, scFv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 10 nothing.

[0212] In some embodiments, scFv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 12 nothing.

[0213] In some embodiments, scFv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 14 nothing.

[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 nothing.

[0219] In some embodiments, scFv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 12. nothing.

[0220] In some embodiments, scFv includes VH of SEQ ID NO: 7 and VL of SEQ ID NO: 14. nothing.

[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 nothing.

[0226] In some embodiments, scFv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 12 nothing.

[0227] In some embodiments, scFv includes VH of SEQ ID NO: 9 and VL of SEQ ID NO: 14 nothing.

[0228] In some embodiments, scFv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 2 nothing.

[0229] In some embodiments, scFv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 4 nothing.

[0230] In some embodiments, scFv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 6 nothing.

[0231] In some embodiments, scFv includes VH of SEQ ID NO: 11 and VL of SEQ ID NO: 8 nothing.

[0232] In some embodiments, scFv has VH of sequence number 11 and VL of sequence number 10 include.

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

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

[0235] In some embodiments, scFv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 2 nothing.

[0236] In some embodiments, scFv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 4 nothing.

[0237] In some embodiments, scFv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 6 nothing.

[0238] In some embodiments, scFv includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 8 nothing.

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

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

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

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

[0243] In some embodiments, scFv is at least 80% (e.g., VH of SEQ ID NO: 1) , at least 85%, at least 90%, at least 95%, or at least 99%) VH and VL of Sequence ID No. 2 are one and at least 80% (for example, at least 85%, less Includes VLs that are identical by at least 90%, at least 95%, or at least 99%.

[0244] In some embodiments, scFv is at least 80% (for example) of VH in Sequence ID No. 3. , at least 85%, at least 90%, at least 95%, or at least 99%) VH and VL of Sequence ID No. 4 are one and at least 80% (for example, at least 85%, less Includes VLs that are identical by at least 90%, at least 95%, or at least 99%.

[0245] In some embodiments, scFv is at least 80% (for example) of VH in SEQ ID NO: 5 , at least 85%, at least 90%, at least 95%, or at least 99%) VH and VL of Sequence ID No. 6 are one and at least 80% (for example, at least 85%, less Includes VLs that are identical by at least 90%, at least 95%, or at least 99%.

[0246] In some embodiments, scFv is at least 80% (for example) of VH in Sequence ID No. 7. , at least 85%, at least 90%, at least 95%, or at least 99%) VH and VL of Sequence ID No. 8 are one and at least 80% (for example, at least 85%, less Includes VLs that are identical by at least 90%, at least 95%, or at least 99%.

[0247] In some embodiments, scFv is at least 80% (for example) of VH in sequence number 9. , at least 85%, at least 90%, at least 95%, or at least 99%) VH and VL of Sequence ID No. 10 are one and at least 80% (for example, at least 85%) Includes VLs that are identical by at least 90%, at least 95%, or at least 99%.

[0248] In some embodiments, scFv is at least 80% (for example) of VH in SEQ ID NO: 11 (For example, at least 85%, at least 90%, at least 95%, or at least 99%) VH and VL of sequence number 12 are identical and at least 80% (for example, at least 85%) Includes VL that is identical (at least 90%, at least 95%, or at least 99%). .

[0249] In some embodiments, scFv is at least 80% (for example) of VH in SEQ ID NO: 13 (For example, at least 85%, at least 90%, at least 95%, or at least 99%) VH and VL of sequence number 14 are identical and at least 80% (for example, at least 85%) Includes VL that is identical (at least 90%, at least 95%, or at least 99%). .

[0250] In some embodiments, scFv is at least 80% (for example) of VH in Sequence ID No. 3. , at least 85%, at least 90%, at least 95%, or at least 99%) Includes VH, which is one, and VL, which is sequence number 4.

[0251] In some embodiments, scFv is less than VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4 at least 80% (for example, at least 85%, at least 90%, at least 95%, or Includes VLs that are identical (at least 99%).

[0252] In some embodiments, scFv is at least 95% identical to VH of Sequence ID No. 3. It contains VH and VL which is at least 95% identical to VL of SEQ ID NO: 4.

[0253] In some embodiments, scFv is at least 99% identical to VH of Sequence ID No. 3. It contains VH and VL which is at least 95% identical to VL of SEQ ID NO: 4.

[0254] In some embodiments, scFv is at least 99% identical to VH of Sequence ID No. 3. It contains VH and VL which is at least 99% identical to VL of sequence number 4.

[0255] In some embodiments, scFv is at least 95% identical to VH of Sequence ID No. 3. It contains VH and VL which is at least 99% identical to VL of sequence number 4.

[0256] In some embodiments, the linker is sequence numbers 63, 64, 65, 66, 67, 68 , or containing a sequence of 69 amino acids.

[0257] In some embodiments, scFv is sequence numbers 63, 64, 65, 66, 67, 68 , or 69 amino acid sequences and at least 80% (e.g., at least 85%, at least It contains amino acid sequences that are 90%, at least 95%, or at least 99% identical.

[0258] In some embodiments, scFv is at least 80 amino acids from the amino acid sequence of SEQ ID NO: 63. % (For example, at least 85%, at least 90%, at least 95%, or at least Contains amino acid sequences that are 99% identical.

[0259] In some embodiments, scFv is at least 80 amino acid sequences of SEQ ID NO: 64. % (For example, at least 85%, at least 90%, at least 95%, or at least Contains amino acid sequences that are 99% identical.

[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 or their components identified herein that bind to DLL3, Genetic engineering can be performed to Fab, F(ab')2, Fd, or Fv formats. Their binding to DLL3 and thermal stability were evaluated using the assay described herein. It can be valued.

[0263] In some embodiments, Fab, F(ab')2, Fd, or Fv is HCDR1, VH having HCDR2 and HCDR3, and LCDR1, LCDR2, and LCD Includes VL having R3, HCDR1, HCDR2, HCDR3, LCDR1, LCDR 2, and LCDR3 are HCDR1, HCDR2, and HCDR3 of VH of Sequence ID No. 1. Furthermore, LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 2, or SEQ ID NO: 3 HCDR1, HCDR2, and HCDR3 of VH, and LCDR1 of VL of SEQ ID NO: 4 , LCDR2 and LCDR3, or HCDR1, HCDR2, and VH of sequence number 5. HCDR3, and LCDR1, LCDR2, and LCDR3 of VL of Sequence ID No. 6, or HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 7, and VL of SEQ ID NO: 8 LCDR1, LCDR2, and LCDR3, or HCDR1, HC of VH, sequence number 9 DR2 and HCDR3, and LCDR1, LCDR2, and L of VL of Sequence ID No. 10 CDR3, or HCDR1, HCDR2, and HCDR3 of VH of Sequence ID No. 11, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 12, or V in SEQ ID NO: 13 HCDR1, HCDR2, and HCDR3 of H, and LCDR1 of VL of SEQ ID NO: 14 This includes LCDR2 and LCDR3.

[0264] In certain embodiments, Fab, F(ab')2, Fd, or Fv is VH of Sequence ID No. 3. HCDR1, HCDR2, and HCDR3, and LCDR1, L of VL of Sequence ID No. 4 Includes CDR2 and LCDR3.

[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 HCDR1 and HCDR2, respectively, with sequence numbers 61, 62, 32, 47, 48, and 49. This includes HCDR3, LCDR1, LCDR2, and LCDR3.

[0266] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. Includes VH and VL of sequence number 2.

[0267] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. Includes VH and VL of sequence number 4.

[0268] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. Includes VH and VL of sequence number 6.

[0269] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. Includes VH and VL of sequence number 8.

[0270] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. Includes VH and VL of sequence number 10.

[0271] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. Includes VH and VL of sequence number 12.

[0272] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. Includes VH and VL of sequence number 14.

[0273] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. Includes VH and VL of sequence number 2.

[0274] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. Includes VH and VL of sequence number 4.

[0275] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. Includes VH and VL of sequence number 6.

[0276] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. Includes VH and VL of sequence number 8.

[0277] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. Includes VH and VL of sequence number 10.

[0278] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. Includes VH and VL of sequence number 12.

[0279] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. Includes VH and VL of sequence number 14.

[0280] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 5. Includes VH and VL of sequence number 2.

[0281] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 5. Includes VH and VL of sequence number 4.

[0282] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 5. Includes VH and VL of sequence number 6.

[0283] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 5. Includes VH and VL of sequence number 8.

[0284] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 5. Includes VH and VL of sequence number 10.

[0285] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 5. Includes VH and VL of sequence number 12.

[0286] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 5. Includes VH and VL of sequence number 14.

[0287] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID 7. Includes VH and VL of sequence number 2.

[0288] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID 7. Includes VH and VL of sequence number 4.

[0289] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID 7. Includes VH and VL of sequence number 6.

[0290] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID 7. Includes VH and VL of sequence number 8.

[0291] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID 7. Includes VH and VL of sequence number 10.

[0292] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID 7. Includes VH and VL of sequence number 12.

[0293] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID 7. Includes VH and VL of sequence number 14.

[0294] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 9. Includes VH and VL of sequence number 2.

[0295] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 9. Includes VH and VL of sequence number 4.

[0296] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 9. Includes VH and VL of sequence number 6.

[0297] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 9. Includes VH and VL of sequence number 8.

[0298] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 9. Includes VH and VL of sequence number 10.

[0299] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 9. Includes VH and VL of sequence number 12.

[0300] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 9. Includes VH and VL of sequence number 14.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0315] In certain embodiments, Fab, F(ab')2, Fd, or Fv is VH of Sequence ID 1. and includes VL of sequence number 2.

[0316] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. VH and at least 80% (e.g., at least 85%, at least 90%, at least 9%) 5% or at least 99%) VH and VL of SEQ ID NO: 2 are identical and at least 80% (For example, at least 85%, at least 90%, at least 95%, or at least 9 9%) Includes VL which is identical.

[0317] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. VH and at least 80% (e.g., at least 85%, at least 90%, at least 9%) It contains VH and VL of SEQ ID NO: 2, which are identical in 5% or at least 99% of the total.

[0318] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. VH and VL of Sequence ID No. 2 are at least 80% (for example, at least 85%, at least Includes VLs that are 90%, at least 95%, or at least 99% identical.

[0319] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. VH is at least 95% identical to VH and VL of sequence number 2 is at least 95% identical to VH. Includes a certain VL.

[0320] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. VH is at least 99% identical to VH and VL of sequence number 2 is at least 95% identical to VH. Includes a certain VL.

[0321] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. VH is at least 99% identical to VH and VL of sequence number 2 is at least 99% identical to VH. Includes a certain VL.

[0322] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 1. VH is at least 99% identical to VH and VL of sequence number 2 is at least 95% identical to VH. Includes a certain VL.

[0323] In certain embodiments, Fab, F(ab')2, Fd, or Fv is VH of Sequence ID No. 3. and includes VL of sequence number 4.

[0324] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. VH and at least 80% (e.g., at least 85%, at least 90%, at least 9%) 5% or at least 99%) VH and VL of SEQ ID NO: 4 are identical and at least 80% (For example, at least 85%, at least 90%, at least 95%, or at least 9 9%) Includes VL which is identical.

[0325] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. VH and at least 80% (e.g., at least 85%, at least 90%, at least 9%) It contains VH and VL of SEQ ID NO: 4, which are identical in 5% or at least 99% of the total.

[0326] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. VH and VL of Sequence ID No. 4 are at least 80% (for example, at least 85%, at least Includes VLs that are 90%, at least 95%, or at least 99% identical.

[0327] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. VH is at least 95% identical to VH and VL of sequence number 4 is at least 95% identical to VH. Includes a certain VL.

[0328] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. VH is at least 99% identical to VH and VL of sequence number 4 is at least 95% identical to VH. Includes a certain VL.

[0329] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. VH is at least 99% identical to VH and VL of sequence number 4 is at least 99% identical to VH. Includes a certain VL.

[0330] In some embodiments, Fab, F(ab')2, Fd, or Fv is the same as in Sequence ID No. 3. VH is at least 99% identical to VH and VL of sequence number 4 is at least 95% identical to VH. Includes a certain VL.

[0331] VH and VL of Fab containing the antigen-binding region that binds to DLL3 are Fab-F, respectively. c HC (VH-CH1-Hinge-CH2-CH3) and Fab-Fc LC (VL-C In certain such embodiments, Fab -Fc HC is at least 80% (e.g., at least 85%) of sequence number 109. Contains amino acid sequences that are identical by at least 90%, at least 95%, or at least 99%. In certain embodiments, Fab-Fc HC is the same amino acid as SEQ ID NO: 109. Includes arrays.

[0332] In some embodiments, Fab-Fc LC is at least 80% of SEQ ID NO: 110 (For example, at least 85%, at least 90%, at least 95%, or at least 9 9%) Contains identical amino acid sequences. In certain embodiments, Fab-Fc LC contains Contains an amino acid sequence identical to that of column number 110.

[0333] As shown in the examples, the DLL3 is specifically coupled to be incorporated into the multispecific construct. A suitable antigen-binding region is Fab-Fc HC having the amino acid sequence of SEQ ID NO: 109 and It contains 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 bind to DLL3 are within the scope of this disclosure. For example, The riant retains improved functional properties compared to the parent antigen-binding region, or has As far as is possible, the antigen-binding region that binds to DLL3 should have 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 It may also contain 24, 25, 26, 27, 28, or 29 amino acid substitutions. In this embodiment, sequence identity is approximately 8 for the antigen-binding region that binds to DLL3 of the present disclosure. 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% It may also be the case. In some embodiments, diversity exists within the framework domain. In some embodiments, variants are generated by conservative substitution.

[0349] In some embodiments, an isolated protein containing an antigen-binding region that binds to DLL3 The quality is determined by the VH and VL of the antigen-binding region that binds to DLL3 as disclosed herein, respectively. At least 80% (for example, at least 85%, at least 90%, at least 95%) Or includes VH and VL that are identical (at least 99%).

[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 VH and VL are at least 80% identical to VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14. An antigen-binding region that binds to DLL3, including L, is also provided.

[0351] In some embodiments, the identity is 85%. The identity rate is 90%. In some embodiments, the identity rate is 91%. In terms of form, the identity is 91%. In some embodiments, the identity is 92%. In some embodiments, the identity is 93%. The percentage is 94%. In some embodiments, the percentage of identity is 94%. Morphologically, the identity is 95%. In some embodiments, the identity is 96%. In some embodiments, the identity is 97%. The accuracy rate is 98%. In some embodiments, the accuracy rate is 99%.

[0352] In some embodiments, the antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 1 and a small amount At least 80% (at least 85%, at least 90%, at least 95%, or less) VH and VL of Sequence ID No. 2 are identical (99%) and at least 80% (for example, less They are identical (85%, at least 90%, at least 95%, or at least 99%). Includes VL.

[0353] In some embodiments, the antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 3 and a small amount At least 80% (at least 85%, at least 90%, at least 95%, or less) VH and VL of sequence number 4 are identical (99%) and at least 80% (for example, less They are identical (85%, at least 90%, at least 95%, or at least 99%). Includes VL.

[0354] In some embodiments, the antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 5 and a small amount At least 80% (at least 85%, at least 90%, at least 95%, or less) VH and VL of sequence number 6 are identical (99%) and at least 80% (for example, less They are identical (85%, at least 90%, at least 95%, or at least 99%). Includes VL.

[0355] In some embodiments, the antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 7 and a small amount At least 80% (at least 85%, at least 90%, at least 95%, or less) VH and VL of sequence number 8 are identical (99%) and at least 80% (for example, less They are identical (85%, at least 90%, at least 95%, or at least 99%). Includes VL.

[0356] In some embodiments, the antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 9 and a small amount At least 80% (at least 85%, at least 90%, at least 95%, or less) VH and VL of sequence number 10 are identical (99%) and at least 80% (for example, less They are identical (at least 85%, at least 90%, at least 95%, or at least 99%) Includes VL.

[0357] In some embodiments, the antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 11 At least 80% (at least 85%, at least 90%, at least 95%, or less) VH is identical to VL of sequence number 12 (at least 99%) and at least 80% (for example, less (At least 85%, at least 90%, at least 95%, or at least 99%) identical Includes a certain VL.

[0358] In some embodiments, the antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 13 At least 80% (at least 85%, at least 90%, at least 95%, or less) VH is identical to VL of sequence number 14 (at least 99%) and at least 80% (for example, less (At least 85%, at least 90%, at least 95%, or at least 99%) identical Includes a certain VL.

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

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

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

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

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

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

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

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

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

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

[0369] In some embodiments, the antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 3 and a small amount It includes VH and VL (Sequence ID 4), which are 99% identical.

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

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

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

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

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

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

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

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

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

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

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

[0381] The identity percentage between the two sequences is introduced for optimal alignment of the two sequences. The number of gaps that need to be filled and the length of each gap are taken into consideration, and the same shared by the array It is a function of the number of positions (i.e., identity % = number of identical positions / total number of positions × 100) ru.

[0382] The percentage of identity between two amino acid sequences is calculated using the ALIGN program (version 2). 0) E. Meyers and W. Miller (Computer App) are incorporated into 0) Using the algorithm from Biosci4:11-17(1988), PAM120 Determined using a weighted residue table, gap length penalty 12, and gap penalty 4. It is possible. In addition, the percentage of identity between the two amino acid sequences is determined by the GCG software. GAP Programming (available at http: / / www.gcg.com) Needleman and Wunsch (J Mol Biol 4) are incorporated into the system. Using the algorithm 8:444-453 (1970), Blossum 62 Either the Trix or PAM250 matrix, and gap weightings of 16, 14, or 12. Determined using 10, 8, 6, or 4 and length weighting 1, 2, 3, 4, 5, or 6. It is also possible.

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

[0384] "Conservative modifications" can significantly affect the binding properties of antibodies, including those involving amino acid modifications. This refers to amino acid modifications that do not cause alteration. Conservative modifications include amino acid substitution, addition, and deletion. Includes substitutions. Conservative amino acid substitutions are when one amino acid is replaced by an amino acid residue that has a similar side chain. It is a substitution that can be replaced. Families of amino acid residues with similar side chains are clearly defined. It is defined as having acidic side chains (e.g., aspartic acid, glutamic acid) and basic side chains (e.g., (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucone) (e.g., syn, isoleucine, proline, phenylalanine, methionine), non-charged side chains (e.g.) For example, glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine (e.g., tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histamine) Thidine, tyrosine), aliphatic side chain (e.g., glycine, alanine, valine, leucine, i) (Seroisin, serine, threonine), amides (e.g., asparagine, glutamine), β Branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine) It contains amino acids (including methionine). Furthermore, any natural residue in the polypeptide is also Alanine scanning mutagenesis (MacLennan et al., (1988) A cta Physiol Scand Suppl 643:55-67;Sasaki Regarding et al., (1988) Adv Biophys 35:1-24) previously As described, it may be substituted with alanine. Amino acid substitutions for antibodies of this application This is done by known methods, such as PCR mutagenesis (U.S. Patent No. 4,683,195). It is possible. Alternatively, a variant library could be, for example, a random codon (NNK). ) or non-random codons (for example, 11 amino acids (Ala, Cys, Asp, Glu) DVK codes that code Gly, Lys, Asn, Arg, Ser, Tyr, Trp It can also be generated using (n). The resulting variant can be used in the assay described herein. Its characteristics can be tested using this method.

[0385] Method for producing antigen-binding fragments that bind to DLL3 The antigen-binding region that binds to DLL3 provided in this disclosure is fabricated using various techniques. To obtain, for example, using the Kohler-Milstein hybridoma method, DL VH / VL pairs that bind to L3 can be identified. In the hybridoma method, mice or It infects other host animals, such as hamsters, rats, or chickens, as well as humans and / or cats. Animals immunized with civet monkey DLL3, followed by immunization with myeloma cells using standard methods. The spleen cells are fused to form hybridoma cells. One immortalized hybrid Colonies derived from γ cells are evaluated based on their binding specificity, cross-reactivity, or lack thereof, and their relationship to the antigen. Antigen-binding regions that bind to DLL3 having desired properties such as compatibility and any desired functionality. Screening can be performed for the production of antibodies that include the region.

[0386] The antigen-binding region that binds to DLL3, which is generated by immunizing non-human animals, is used in humans. It can be transformed. Exemplary humanization techniques, including the selection of a human acceptor framework, include C DR transplantation (US Patent No. 5,225,539), SDR transplantation (US Patent No. 6,818,7 No. 49), Resurfacing (Padlan, (1991) Mol Immunol 2 8:489-499), Specificity-determining residue resurfacing (US Patent Application Publication No. 2010) (Patent No. 0261620), Human Framework compliant (US Patent No. 8,748,356), Alternatively, hyperhumanization (U.S. Patent No. 7,709,226) may be used. In these methods, C Based on the similarity of the lengths of DR, the identity of the canonical structure, or a combination thereof. Human frameworks that can be selected based on their overall homology to the parent framework. Then, the CDR or a subset of CDR residues of the parent antibody is transplanted.

[0387] The humanized antigen-binding region is described in International Publication No. 1090 / 007861 and No. 1992 / 22 Using techniques such as those described in issue 653, the modified framework support residues By incorporating and maintaining binding affinity (reverse mutation), or by having multiple CDRs By introducing variations, for example, by improving the affinity of the antigen-binding region, the desired antigen can be targeted. It can be further optimized to improve its selectivity or affinity.

[0388] Mice, rats, or Transgenic animals such as chickens are used to create antigen-binding fragments that bind to DLL3. These can be produced, for example, U.S. Patent No. 6,150,584, International Publication No. 1 No. 999 / 45962, No. 2002 / 066630, No. 2002 / 43478, This is described in issues 2002 / 043478 and 1990 / 04036. The endogenous immunoglobulin gene locus of an animal may be disrupted or deleted, homologous or Using non-homologous recombination, using transchromosomes, or minigenes Using this method, at least one complete or partial human immunoglobulin locus can be identified in animals. It can be inserted into the genome. Regeneron (http: / / _www_regene ron_com), Harbor Antibodies(http: / / _www_ harborantibodies_com), Open Monoclonal T echnology, Inc.(OMT)(http: / / _www_omtinc_n et), KyMab(http: / / _www_kymab_com), Trianni (http: / / _www.trianni_com), and Ablexis (http Companies such as : / / _www_ablexis_com use the above technologies to select There are efforts underway to provide human antibodies that target antigens. Several implementations In this study, Ablexis mice were immunized with soluble full-length DLL3 protein.

[0389] The antigen-binding region that binds to DLL3 is human immunoglobulin or a part thereof, for example, Fab , single-chain antibodies (scFv), or unpaired or synpaired antibody variable regions are expressed in the fur You can select from a library of genetically modified phage display devices. The antigen-binding region that binds to DLL3 is, for example, Shi et al., (2010)J As described in Mol Biol 397:385-96 and International Publication No. 09 / 085462). The antibody heavy chain is a fusion protein with the bacteriophage pIX coated protein that is included. It can also be isolated from phage display libraries expressing light chain variable regions. Screening for phage binding to human and / or cynomolgus monkey DLL3. The positive clones obtained are then further characterized, and Fab is isolated from the clone lysates. It may be 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 domains of the herein are carried out by recombinant proteins. This can be done using any suitable technique, such as generation. The immunogenic antigen is purified protein. In the form of a protein mixture containing whole cells or cell or tissue extracts, animals It may be administered, or the antigen may move from the nucleic acid encoding the antigen or a portion thereof. It may be newly formed (de novo) within the body of an object.

[0391] Fusion or conjugation of the half-life extension portion The antigen-binding region bound to DLL3 in this disclosure is fused or conjugated to the half-life extension portion. This can be done. Exceptional half-life extension portions include albumin, albumin variants, Albumin-binding proteins and / or domains, transferrin and their fragments and derivatives. This refers to an analogue, immunoglobulin (Ig), or a fragment thereof, such as an Fc region. (As mentioned above, this is related to the extension of the half-life.) The amino acid sequence of the portion is publicly known. Ig or its fragments are all isotypes, i.e. It includes IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE.

[0392] Additional half-life extensions that can be conjugated to the antigen-binding region bound to DLL3 in this disclosure In part, polyethylene glycol (PEG) molecules for the desired properties, for example, PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, e.g. For example, laurate ester, myristic acid ester, stearate ester, arachidic acid Esters, behenate esters, oleate esters, arachidonic acid esters, octane Acids, tetradecane dioic acid, octadecane dioic acid, docosanedioic acid, polylysine, octane, Examples include carbohydrates (dextran, cellulose, oligosaccharides, or polysaccharides). These parts This involves directly fusing the antigen-binding region that binds to DLL3 of the present disclosure with standard cloning and It can be produced by compound expression technology. Alternatively, known chemical coupling methods can be used to compound. The portion can be bound to the antigen-binding region of the DLL3 of this disclosure that is generated by substitution.

[0393] For example, a cysteine ​​residue is incorporated into the C-terminus of the antigen-binding region that binds to DLL3 of this disclosure. In the past, or through genetic manipulation, the residue position of cysteine ​​facing away from the DLL3 binding site By placing the pegyl group in place and bonding it to cysteine ​​using a well-known method, the present disclosure The pegyl portion can be conjugated to the antigen-binding region that binds to DLL3. can.

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

[0395] In some embodiments, the half-life extension portion is immunoglobulin (Ig), Ig fragments, Ig constant region, fragment of Ig constant region, Fc region, transferrin, albumin, albumin The mine-binding domain is or polyethylene glycol. In some embodiments, half The prolonged period is the steady-state Ig 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, the half-life extension portion may be fused or condimented. The antigen-binding region that binds to the fused DLL3 is evaluated for its pharmacokinetic properties. It is possible.

[0406] Fusion to immunoglobulin (Ig) constant region or fragment of the Ig constant region The antigen-binding region that binds to DLL3 in this disclosure is configured to bind to the Ig constant region or a fragment of the Ig constant region. It denjugates, and the Fc effector function is C1q binding, complement-dependent cell injury (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or cell surface receptor binding. Antibody-like properties including downregulation of receptors (e.g., B cell receptors, BCRs). It is possible to assign the Ig steady-state region or a fragment of the Ig steady-state region. It also functions as a half-life extension portion. Antigen binding to DLL3 of this disclosure The region can be genetically engineered using standard methods to create a conventional full-length antibody. A full-length antibody containing an antigen-binding region that binds to DLL3 is further described herein. It can be manipulated.

[0407] The constant region of the immunoglobulin heavy chain consists of subdomains CH1, hinge, CH2, and CH3. It is composed of residues numbered according to the EU index, and the CH1 domain remains in the heavy chain. The base extends from A118 to V215, and the CH2 domain residues extend from A231 to K340, CH3 The domain residues extend from G341 to K447. In some cases, G341 is the CH2 domain It is called a nucleotide residue. The hinge generally contains E216 and terminates at P230 in human IgG1. It is defined as follows: The Ig Fc region is at least CH2 and CH3 domain of the Ig steady state region. It includes and therefore extends at least approximately from A231 to K447 in the Ig heavy chain constant region. This includes the region in [location].

[0408] This application also applies to conjugates to immunoglobulin (Ig) constant regions or fragments of Ig constant regions. It provides an antigen-binding region that binds to the modified DLL3.

[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 steady-state region fragment consists of the CH2 domain and the CH3 domain. Includes.

[0415] In some embodiments, the Ig steady-state region fragment is at least a portion of the hinge, CH2 Includes domains and CH3 domains. Part of the hinge is one or more Ig hinges. This refers to the amino acid residue.

[0416] In some embodiments, the Ig steady-state region fragment consists of a hinge, a CH2 domain, and a CH3 domain. Includes the domain.

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

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

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

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

[0421] In some embodiments, L2 is represented by Sequence IDs 27, 72, 73, 74, 75, 76, 7 9, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 1 24, 125, 126, 127, 128, 129, 130, 131, 132, 133, 1 It contains the amino acid sequence 34, 135, 136, 137, 138, or 139.

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

[0423] The DLL3 of this disclosure is coupled to the Ig steady-state region or a fragment of the Ig steady-state region. The antigen-binding region can be evaluated for its functionality using several known assays. This can be done. The coupling to DLL3 can be evaluated using the method described herein. Changes induced by the Ig steady domain or fragments of the Ig steady region, such as the Fc region. The characteristics include FcγRI, FcγRII, FcγRIII, or FcRn receptors. In Fc receptor binding assays using the soluble form of the receptor, or, for example, ADCC Assay using a cell-based assay that measures CDC or ADCP. It is possible.

[0424] ADCC targets DLL3-expressing cells as target cells and NK cells as effector cells. It can be evaluated using an in vitro assay. Cell lysis is lysis. Release of labels (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) from cells. It can be detected by emission. In an exemplary assay, target cells are identified as one target cell It is used in a ratio to four effector cells. Target cells are pre-labeled with BATDA. Combine with effector cells and test antibody. Incubate the sample for 2 hours. Cell lysis is measured by measuring the amount of BATDA released into the supernatant. 0.67% Tr For the greatest cytotoxicity caused by iton X-100 (Sigma Aldrich), The data was normalized, and the spontaneous release of BATDA from target cells in the absence of any antibody was also measured. The minimum contrast is determined by the output.

[0425] ADCP uses monocyte-derived macrophages as effector cells, and GFP or other Any DLL3-expressing cell genetically engineered to express the labeling molecule is used as the target cell. It can be evaluated by using it. In an example assay, effector: label The target cell ratio can be, for example, 4:1. Effector cells are treated with the antibody of this application. The cells may be incubated with or without the solution for 4 hours. After the procedure, the cells can be detached using actase. Macrophages can be bound to fluorescent labels. This can be identified using combined anti-CD11b and anti-CD14 antibodies, and the proportion of phagocytic activity. The standard method is used for CD11 + CD14 + GFP fluorescence in macrophages It can be calculated based on percentages.

[0426] The CDC of cells, for example, Daudi cells, were supplemented with RPMI-B (1% BSA). RPMI) 1 x 10 5 Seed cells were seeded in individual cells / well (50 μL / well), and 50 μL of solution was added. Add the test protein to the wells at a final concentration of 0-100 μg / mL, and refuel the reaction mixture at room temperature for 15 minutes. Incubate for 1 minute, add 11 μL of pooled human serum to the well, and incubate the reaction mixture at 37°C. This can be measured by incubation for 45 minutes. The percentage of lysed cells (%) is: Using the standard method, the percentage of propidium iodide-stained cells in the FACS assay was detected. It may be released.

[0427] In some embodiments, the first antigen-binding region that binds to DLL3 is the first immunoglobulin. Fusing to a brin(Ig) steady-state region or a fragment of a first Ig steady-state region, and / or phosphorus The second antigen-binding region that binds to the Pacyl antigen is the second immunoglobulin (Ig) constant region. It is fused with a fragment of the second Ig constant region.

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

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

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

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

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

[0433] In some embodiments, the Ig steady-state region fragment consists of a hinge, a CH2 domain, and a CH3 domain. Includes the domain.

[0434] In some embodiments, the multispecific antigen-binding construct binds to DLL3 as a first antigen. Between the primordial binding region and the first Ig constant region or a fragment of the first Ig constant region, and against lymphocytes A second antigen-binding region that binds to the source and a second Ig constant region or a fragment of the second Ig constant region. In between, a second linker (L2) is further included.

[0435] In some embodiments, L2 is represented by Sequence IDs 27, 72, 73, 74, 75, 76, 7 9, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 1 24, 125, 126, 127, 128, 129, 130, 131, 132, 133, 1 It contains the amino acid sequence 34, 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, a first Ig steady-state region or a fragment of the first Ig steady-state region, and The 2nd Ig steady-state region or the fragment of the second Ig steady-state region is IgG1, IgG2, IgG3, or This is the IgG4 isotype.

[0438] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region and a second The Ig constant region or a fragment of the second Ig constant region is an IgG1 isotype.

[0439] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region and a second The Ig constant region or a fragment of the second Ig constant region is an IgG2 isotype.

[0440] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region and a second The Ig constant region or a fragment of the second Ig constant region is an IgG3 isotype.

[0441] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region and a second The Ig constant region or a fragment of the second Ig constant region is an IgG4 isotype.

[0442] In certain embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region and a second I The IgG steady-state region or the fragment of the second IgG steady-state region is an IgG1 isotype.

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

[0444] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region, and The Ig constant region of 2 or the fragment of the second Ig constant region is a multispecific antigen-binding structure to FcγR. It includes at least one mutation that results in reduced binding of structures.

[0445] In some embodiments, reduced binding of multispecific antigen-binding constructs to FcγR The resulting mutations are F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268 A / V309L / A330S / P331S, F234A / L235A, S228P / F2 34A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 Missing / A327G / P331A / D365E / L35 8M, H268Q / V309L / A330S / P331S, S267E / L328F, L 234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G23 7A / P238S, and S228P / F234A / L235A / G236 (missing) / G237 Selected from the group consisting of A / P238S, residue numbering follows the EU index. In this embodiment, the first Ig steady-state region or a fragment of the first Ig steady-state region and / or the second The Ig constant region or a fragment of the second Ig constant region has the following mutation: L234A_L23 Includes 5A_D265S.

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

[0447] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region, and The 2 Ig constant region or a fragment of the second Ig constant region affects the half-life of the multispecific antigen-binding construct. It includes at least one mutation that modifies [something].

[0448] In some embodiments, the multispecific antigen-binding construct is the CH3 of the first Ig constant region. At least one CH3 domain of the domain or fragment of the first Ig constant region Mutations and / or the CH3 domain or fragments of the second Ig constant region It contains at least one mutation in the CH3 domain.

[0449] In some embodiments, the CH3 domain of the first Ig steady region or the first Ig constant At least one mutation in the CH3 domain of the constitutive region fragment, and / or a second Ig determination. Small amount in the CH3 domain of the normal region or the CH3 domain of the fragment of the second Ig steady region At least one mutation is a U.S. Patent Application Publication No. 2012 / 0149876 or U.S. Patent Application Publication No. L351Y_ described in Application Publication No. 2013 / 0195849 (Zymeworks) F405A_Y407V / T394W, T366I_K392M_T394W / F405 A_Y407V, T366L_K392M_T394W / F405A_Y407V, L3 51Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_ K409F, Y407A / T366A_K409F, or T350V_L351Y_F4 From the group consisting of 05A_Y407V / T350V_T366L_K392L_T394W Selected.

[0450] In some embodiments, the CH3 domain of the first Ig steady region or the first Ig constant At least one mutation in the CH3 domain of the constitutive region fragment, and / or a second Ig determination. Small amount in the CH3 domain of the normal region or the CH3 domain of the fragment of the second Ig steady region At least one mutation is T366Y / described in International Publication No. 1996 / 027011. F405A, T366W / F405W, F405W / Y407A, T394W / Y407 T, T394S / Y407A, T366W / T394S, F405W / T394S, and Selected from the group consisting of T366W / T366S_L368A_Y407V.

[0451] In some embodiments, the protein or multispecific antigen-binding constructs of this application are AD One or more amino acids that reduce or eliminate the effector function of CC or CDC Modifications may include, for example, mutations that reduce or eliminate binding to the Fcγ receptor. The mutations are located at L234, L235, D270, N297, E318, K320, and K322. , P331, and P329, for example, one of L234A, L235A, and P331S, There may be two or three mutations, and the numbering of amino acid residues is as shown in Kabat EU Follow the index.

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

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

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

[0455] This disclosure also includes a multispecific antigen-binding structure comprising an antigen-binding region that binds to DLL3 of this disclosure. To provide a building.

[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 used for binding to DLL3. It is the value.

[0460] In some embodiments, the multispecific antigen-binding construct has two characteristics regarding binding to DLL3. It is the value.

[0461] This disclosure also relates to a first antigen-binding region that binds to DLL3 and a lymphocyte antigen (such as CD3). Provides an isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to ). ru.

[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 antigens are CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, These are BTNL8, PD-1, CD195, or NKG2C.

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

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

[0468] In some embodiments, a first antigen-binding region and / or lymphocytes that bind to DLL3. The second antigen-binding region, which binds to the antigen, contains Fab.

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

[0470] In some embodiments, a first antigen-binding region and / or lymphocytes that bind to DLL3. The second antigen-binding region, which binds to the antigen, contains VHH.

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

[0472] In some embodiments, a first antigen-binding region and / or lymphocytes that bind to DLL3. The second antigen-binding region, which binds to the antigen, contains Fd.

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

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

[0475] In certain embodiments, the multispecific antigen-binding construct is bispecific and binds to DLL3. The first antigen-binding region includes Fab, which binds to lymphocyte antigens (e.g., CD3). The antigen-binding region of 2 includes scFv.

[0476] In some embodiments, scFv is VH, first ri, from the N-terminus to the C-terminus. Inker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L Includes 1-VH).

[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 is represented by sequence numbers 27, 72, 73, 74, 75, 76, 7 9, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 1 24, 125, 126, 127, 128, 129, 130, 131, 132, 133, 1 It contains the amino acid sequence 34, 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 Sequence IDs 15, 18, 21, 24, 18, 30, 50, 52, 53, 55, 57, 59 , or HCDR1 of 61, SEQ ID NOs: 16, 19, 22, 25, 28, 31, 51, 54, HCDR2 of 56, 58, 60, or 62, SEQ ID NOs: 17, 20, 23, 26, 29, 3 HCDR3 of sequences 2, 17, 20, 23, 26, 29, or 32, sequence numbers 33, 36, 39 LCDR1 of 41, 44, or 47, L of sequence numbers 34, 37, 42, 45, or 48 Includes CDR2 and LCDR3 of sequence numbers 35, 38, 40, 43, 46, or 49.

[0484] In a particular embodiment, the first antigen-binding region that binds to DLL3 is, respectively, SEQ ID NO: 1 5, 16, 17, 33, 34 and 35 HCDR1, HCDR2, HCDR3, LCDR Includes 1, LCDR2, and LCDR3. In some embodiments, multispecific antigen binding. The construct mediates T cell-mediated cytotoxicity, promotes T cell activation and proliferation, and stimulates T cell cytotoxicity. It increases tokine 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 is used on the surface of CD8 T cells. 25, CD69, and CD71 expression are upregulated. In some embodiments, multiple specificity Sex antigen-binding constructs show increased tumor death.

[0485] In a particular embodiment, the first antigen-binding region that binds to DLL3 is, respectively, SEQ ID NO: 1 5, 16, 17, 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCD R1, LCDR2, and LCDR3, and optionally CD3, CD3 epsilon (C D3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, C D186, BTNL8, PD-1, CD195, or NKG2C, for example, CD3 It contains a second antigen-binding region that binds to the lymphocyte antigen.

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

[0487] In some embodiments, the first antigen-binding region that binds to DLL3 is V of SEQ ID NO: 3 H and at least 80% (at least 85%, at least 90%, at least 95%, or VH is identical to VL of SEQ ID NO: 4 by at least 99%, and VH is identical to VL of SEQ ID NO: 4 by at least 80% (for example, (At least 85%, at least 90%, at least 95%, or at least 99%) identical VL, and optionally CD3, CD3 epsilon (CD3ε), CD8, KI 2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, which bind to lymphocyte antigens such as CD3, It contains two antigen-binding regions. In some embodiments, isolated multispecific antigen-binding constructs The substance mediates T cell-mediated cytotoxicity, promoting T cell activation and proliferation, and T cell cytotoxicity It increases the release of and / or exhibits an increased antitumor effect. In some embodiments, The heavily specific antigen-binding constructs potently mediate the proliferation of cytotoxic CD8 T cells. In that embodiment, the multispecific antigen-binding construct is CD25 on the surface of CD8 T cells. Upregulating CD69 and CD71 expression. In some embodiments, multispecific antigens The combined structure shows increased tumor death.

[0488] In some embodiments, bispecific anti-DLL3×CD3 antibodies are T-cell cytotoxic antibodies. In Ssey, over 90% (e.g., 95%) of tumor lysis is achieved within 5 days.

[0489] In some embodiments, the first antigen-binding region that binds to DLL3 is V of SEQ ID NO: 3 H and at least 80% (at least 85%, at least 90%, at least 95%, or VH and VL of sequence number 4 are identical (at least 99%), and optionally CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG 2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, For example, it includes a second antigen-binding region that binds to the lymphocyte antigen, such as CD3.

[0490] In some embodiments, the first antigen-binding region that binds to DLL3 is V of SEQ ID NO: 3 H and VL of Sequence ID No. 4 and at least 80% (e.g., at least 85%, at least 9%) VLs that are identical by 0%, at least 95%, or at least 99%, and optionally, CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NK G2C includes a second antigen-binding region that binds to lymphocyte antigens, such as CD3.

[0491] In some embodiments, isolated multispecific antigen-binding constructs disclosed herein It mediates T cell-mediated cytotoxicity, promotes T cell activation and proliferation, and T cell cytotoxicity It may be particularly effective in increasing ion release and / or exhibiting increased antitumor efficacy.

[0492] In some embodiments, the first antigen-binding region that binds to DLL3 is V of SEQ ID NO: 3 Includes H and VL of sequence number 4.

[0493] In a particular embodiment, the first antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 3 And VL of sequence number 4, and optionally CD3, CD3 epsilon (CD3ε), CD 8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BT Lymphocyte antigens such as NL8, PD-1, CD195, or NKG2C, for example CD3, bind to NL8, PD-1, CD195, or NKG2C. It includes a second antigen-binding region.

[0494] In some embodiments, multispecific antigen-binding constructs mediate T cell-mediated cytotoxicity. In some embodiments, the multispecific antigen-binding construct is used to target cytotoxic CD8 T cells. It strongly mediates cell proliferation. In some embodiments, the multispecific antigen-binding construct is C Upregulates the expression of CD25, CD69, and CD71 on the surface of D8 T cells. In one embodiment, the multispecific antigen-binding construct exhibits increased tumor death. In this embodiment, the bispecific anti-DLL3×CD3 antibody was used in a T cell cytotoxic assay. And, within 5 days, over 90% (for example, 95%) of tumor lysis is achieved.

[0495] In some embodiments, the first antigen-binding region that binds to DLL3 is sequence number 63. It contains amino acid sequences of 64, 65, 66, 67, 68, or 69.

[0496] In some embodiments, the first antigen-binding region that binds to DLL3 is the one specified in SEQ ID NO: 63 It has an amino acid sequence.

[0497] In some embodiments, the first antigen-binding region that binds to DLL3 is the one specified in SEQ ID NO: 64 It has an amino acid sequence.

[0498] In some embodiments, the first antigen-binding region that binds to DLL3 is the one specified in SEQ ID NO: 65 It has an amino acid sequence.

[0499] In some embodiments, the first antigen-binding region that binds to DLL3 is the one specified in SEQ ID NO: 66 It has an amino acid sequence.

[0500] In some embodiments, the first antigen-binding region that binds to DLL3 is the same as in SEQ ID NO: 67 It has an amino acid sequence.

[0501] In some embodiments, the first antigen-binding region that binds to DLL3 is the same as in SEQ ID NO: 68 It has an amino acid sequence.

[0502] In some embodiments, the first antigen-binding region that binds to DLL3 is the same as the one in SEQ ID NO: 69 It has an amino acid sequence.

[0503] In some embodiments, the first antigen-binding region that binds to DLL3 is the one specified in SEQ ID NO: 63 The amino acid sequence and at least 80% (e.g., at least 85%, at least 90%, less) It contains amino acid sequences that are identical by at least 95%, or at least 99%.

[0504] In some embodiments, the first antigen-binding region that binds to DLL3 is the one specified in SEQ ID NO: 64 The amino acid sequence and at least 80% (e.g., at least 85%, at least 90%, less) It contains amino acid sequences that are identical by at least 95%, or at least 99%.

[0505] In certain embodiments, the first antigen-binding region that binds to DLL3 is sequence number 63 or 6 It contains a sequence of 4 amino acids.

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

[0507] In some embodiments, a second antigen-binding region binds to lymphocyte antigens (such as CD3). This is VH of sequence number 77 and at least 80% (at least 85%, at least 90%, less VH and VL of sequence number 80 are identical by at least 95%, or at least 99%. at least 80% (for example, at least 85%, at least 90%, at least 95%, or Includes VLs that are identical (at least 99%).

[0508] In some embodiments, the second antigen-binding region that binds to the lymphocyte antigen is represented by SEQ ID NO: 7 7 VH and at least 80% (for example, at least 85%, at least 90%, and at least It also includes VH and VL of SEQ ID NO: 80, which are 95% or at least 99% identical.

[0509] In some embodiments, the second antigen-binding region that binds to the lymphocyte antigen is represented by SEQ ID NO: 7 VH of 7 and VL of SEQ ID NO: 80 and at least 80% (e.g., at least 85%, less) Includes VL that is at least 90%, at least 95%, or at least 99% identical.

[0510] In a particular embodiment, the second antigen-binding region that binds to the lymphocyte antigen is the one specified in SEQ ID NO: 77 Includes VH and VL of sequence number 80.

[0511] In some embodiments, a second antigen-binding region binds to lymphocyte antigens (such as CD3). This is VH of sequence number 84 and at least 80% (at least 85%, at least 90%, less VH and VL of sequence number 85 are identical by at least 95%, or at least 99%. at least 80% (for example, at least 85%, at least 90%, at least 95%, or Includes VLs that are identical (at least 99%).

[0512] In some embodiments, the second antigen-binding region that binds to the lymphocyte antigen is represented by SEQ ID NO: 8 4 VH and at least 80% (for example, at least 85%, at least 90%, and at least It also includes VH and VL of SEQ ID NO: 85, which are 95% or at least 99% identical.

[0513] In some embodiments, the second antigen-binding region that binds to the lymphocyte antigen is represented by SEQ ID NO: 8 VH of 4 and VL of SEQ ID NO: 85 and at least 80% (e.g., at least 85%, less) Includes VL that is at least 90%, at least 95%, or at least 99% identical.

[0514] In a particular embodiment, the second antigen-binding region that binds to the lymphocyte antigen is the one specified in SEQ ID NO: 84 Includes VH and VL of sequence number 85.

[0515] In some embodiments, the second antigen-binding region that binds to the lymphocyte antigen is HCDR1 (sequence number 95), HCDR2 (sequence number 96), HCDR3 (sequence number 97), LCDR1 of sequence number 101, LCDR2 of sequence number 102, and LC of sequence number 104 Includes DR3, or VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80.

[0516] In some embodiments, the second antigen-binding region that binds to the lymphocyte antigen is HCDR1 (sequence number 98), HCDR2 (sequence number 99), HCDR3 (sequence number 100) LCDR1 of sequence number 106, LCDR2 of sequence number 107, and L of sequence number 108 Includes CDR3, or VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85.

[0517] In certain embodiments, the second antigen-binding region that binds to lymphocyte antigens (such as CD3) is HCDR1 (sequence number 95), HCDR2 (sequence number 96), HCDR3 (sequence number 97), LCDR1 at row number 101, LCDR2 at sequence number 102, and LCD at sequence number 104 Includes R3.

[0518] In a particular embodiment, the first antigen-binding region that binds to DLL3 is, respectively, SEQ ID NO: 1 5, 16, 17, 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCD The second component includes R1, LCDR2, and LCDR3, which bind to lymphocyte antigens (such as CD3). The antigen-binding regions are HCDR1 in SEQ ID NO: 95, HCDR2 in SEQ ID NO: 96, and SEQ ID NO: 9 7 HCDR3, Array 101 LCDR1, Array 102 LCDR2, and sequence Includes LCDR3 number 104.

[0519] In a particular embodiment, the first antigen-binding region that binds to DLL3 is, respectively, SEQ ID NO: 1 5, 16, 17, 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCD The second component includes R1, LCDR2, and LCDR3, which bind to lymphocyte antigens (such as CD3). The antigen-binding regions are HCDR1 in SEQ ID NO: 98, HCDR2 in SEQ ID NO: 99, and SEQ ID NO: 1 HCDR3 of 00, LCDR1 of sequence number 106, LCDR2 of sequence number 107, and Includes LCDR3 at column number 108.

[0520] In a particular embodiment, the first antigen-binding region that binds to DLL3 is, respectively, SEQ ID NO: 1 5, 16, 17, 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCD A second antigen-binding region comprising R1, LCDR2, and LCDR3 that binds to lymphocyte antigens. This includes VH of sequence number 77 and VL of sequence number 80. In some embodiments, multiplexing is used. Specific antigen-binding constructs mediate T cell-mediated cytotoxicity. In some embodiments, Multiple specific antigen-binding constructs potently mediate the proliferation of cytotoxic CD8 T cells. In several embodiments, the multispecific antigen-binding construct is CD25 on the surface of CD8 T cells. It upregulates the expression of CD69 and CD71. In some embodiments, it is multispecific anti- The original binding construct shows increased tumor death. In some embodiments, bispecific anti-DL L3×CD3 antibody showed over 90% effectiveness in T cell cytotoxicity assays by day 5 (for example, Achieves 95% tumor lysis.

[0521] In a particular embodiment, the first antigen-binding region that binds to DLL3 is, respectively, SEQ ID NO: 1 5, 16, 17, 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCD The second component includes R1, LCDR2, and LCDR3, which bind to lymphocyte antigens (such as CD3). The antigen-binding region includes VH in SEQ ID NO: 84 and VL in SEQ ID NO: 85. Several implementations Morphologically, multispecific antigen-binding constructs mediate T cell-mediated cytotoxicity and T cell activation. , promotes proliferation and expansion, and stimulates T cell cytokine release. It increases and / or shows increased antitumor efficacy. In some embodiments, multiple specificity Sex antigen-binding constructs potently mediate the proliferation of cytotoxic CD8 T cells. In terms of application, the multispecific antigen-binding construct is applied to CD25 and CD6 on the surface of CD8 T cells. 9, and upregulates CD71 expression. In some embodiments, a multispecific antigen binding structure The structures show increased tumor death. In some embodiments, bispecific anti-DLL3×C D3 showed that in T-cell toxicity assays, more than 90% (e.g., 95%) of tumors were eliminated by day 5. Achieve ulcer dissolution.

[0522] In a particular embodiment, the first antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 3 It includes the VL of sequence number 4 and a second antigen-binding region that binds to lymphocyte antigens (such as CD3). This includes HCDR1 (sequence number 95), HCDR2 (sequence number 96), and HCDR3 (sequence number 97). LCDR1 of sequence number 101, LCDR2 of sequence number 102, and L of sequence number 104 Includes CDR3.

[0523] In a particular embodiment, the first antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 3 The second antigen-binding region, which includes the VL of SEQ ID NO: 4 and binds to lymphocyte antigens, is SEQ ID NO: 8 Includes VH of 4 and VL of sequence number 85.

[0524] In a particular embodiment, the first antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 3 The second antigen-binding region, which includes the VL of SEQ ID NO: 4 and binds to lymphocyte antigens, is SEQ ID NO: 7 Includes VH of 7 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 modified to form a multispecific antibody. Furthermore, the antibody in question is also included within the scope of this application.

[0526] The antigen-binding fragments that bind to DLL3 are fully length fragments produced using Fab arm exchange. Genetic modification may be performed to create a highly specific antibody, and here, substitution is performed in vitro on a Fab arm. It is introduced into two monospecific bivalent antibodies within the Ig constant region CH3 domain to promote exchange. This method involves having a specific substitution in the CH3 domain that promotes the stability of the heterodimer. Thus, two monospecific bivalent antibodies are genetically engineered. These antibodies are located in the hinge region. Under sufficient reducing conditions for the cysteine ​​to isomerize the disulfide bond, The antibodies are incubated, and bispecific antibodies are generated by Fab arm exchange. The incubation conditions can be optimally returned to non-reducing conditions. Representative conditions that can be used The reducing agent is 2-mercaptoethylamine (2-MEA), dithio Dithiothreitol (DTT), dithioerythritol DTE), glutathione, tris(2-carboxyethyl)phosphine (tris(2-carboxy It consists of ethyl phosphine, TCEP, L-cysteine, and β-mercaptoethanol. Preferably, 2-mercaptoethylamine, dithiothreitol, and tris(2-) A reducing agent selected from the group consisting of (ruboxyethyl)phosphine. For example, at least Also at a temperature of 20°C, in the presence of at least 25 mM 2-MEA or at least 0.5 mM In the presence of dithiothreitol, at pH 5-8, for example, pH 7.0 or pH 7.4, At the very least, a 90-minute incubation period can be used.

[0527] Possible CH3 mutations to use include the knob-in-hole mutation (Genentech) and electrostatic mutation. Match mutations (Chugai, Amgen, NovoNordisk, Oncomed), Strand Exchange Engineer domain body ed Domain body, SEEDbody)(EMD Serono), Duo body(registered trademark) mutation (Genmab), and other asymmetric mutations (e.g., Zymew Examples of technologies include orks.

[0528] The nobu-in-hole mutation is disclosed, for example, in International Publication No. 1996 / 027011. Then, an amino acid with a small side chain (hole) is introduced into the first CH3 region, and the large side chain An amino acid containing a (knob) is introduced into the second CH3 region, and the first CH3 region and the second C This includes an interface mutation in the CH3 region that leads to preferential interaction with the H3 region. Exemplary CH3 region mutations that form knobs and holes include T366Y / F405A and T36 6W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y 407A, T366W / T394S, F405W / T394S, and T366W / T36 It is 6S_L368A_Y407V.

[0529] Heavy chain heterodimer formation is described in U.S. Patent Application Publication No. 2010 / 0015133, and the same patent application publication No. 20 Issues 09 / 0182127, 2010 / 028637, or 2011 / 0123 As described in No. 532, the positively charged residue on the first CH3 region and the second CH3 region By substituting the negatively charged residue above, the process is facilitated by utilizing electrostatic interactions. It is possible.

[0530] Other asymmetric mutations that can be used to promote heavy chain heterodimerization are specific to the United States. Approved application publication No. 2012 / 0149876 or No. 2013 / 0195849 (Zym L351Y_F405A_Y407V / T394W, T366I (as described in eworks) _K392M_T394W / F405A_Y407V, T366L_K392M_T39 4W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L 351Y_Y407A / T366V_K409F, Y407A / T366A_K409F , or T350V_L351Y_F405A_Y407V / T350V_T366L_K It is 392L_T394W.

[0531] SEED body mutations are described in U.S. Patent Application Publication No. 20070287170 as follows: By substituting selected IgG residues with IgA residues, heavy chain heterodimerization is promoted. To be accompanied by.

[0532] Other exemplary variants that may be used are from International Publication No. 2007 / 147901, International Publication No. Publication No. 2011 / 143545, International Publication No. 2013157954, International Publication No. 20130 R409D as described in Patent No. 96291 and U.S. Patent Application Publication No. 2018 / 0118849 _K370E / D399K_E357K, S354C_T366W / Y349C_T36 6S_L368A_Y407V, Y349C_T366W / S354C_T366S_L 368A_Y407V, T366K / L351D, L351K / Y349E, L351K / Y349D, L351K / L368E, L351Y_Y407A / T366A_K40 9F, L351Y_Y407A / T366V_K409F, K392D / D399K, K 392D / E356K, K253E_D282K_K322D / D239K_E240K These are _K292D, K392D_K409D / D356K_D399K.

[0533] The Duobody® mutation (Genmab) is, for example, registered in the United States under 9,150. Disclosed in Patent No. 663 and U.S. Patent Application Publication No. 2014 / 0303356, F4 05L / K409R, wild type / F405L_R409K, T350I_K370T_F4 05L / K409R, K370W / K409R, D399AFGHILMNRSTVWY / K409R, T366ADEFGHILMQVY / K409R, L368ADEGHN RSTVQ / K409AGRH, D399FHKRQ / K409AGRH, F405IK Examples of mutations include LSTVW / K409AGRH and Y407LWQ / K409AGRH. It can be done.

[0534] Additional bispecific or multispecific properties that can incorporate antigen-binding fragments that bind to DLL3. In terms of isomorphic structures, there is Dual Variable Domain Immunoglobulin. bulin, DVD) (International Publication No. 2009 / 134776; DVD is VH1-linker) A heavy chain having a -VH2-CH structure and a light chain having a VL1-linker-VL2-CL structure. It is a full-length antibody containing (and the linker is optional), with two different specificities Structures containing various dimerization domains for binding antibody arms, e.g., leucine zipper - or collagen dimerization domain (International Publication No. 2012 / 022811, U.S. Publication No. 5 (US Patent Nos. 932,448, U.S. Patent No. 6,833,441), conjugated together Two or more domain antibodies (dAbs), diabodies, camelid antibodies, and genes Modified heavy chain antibodies such as camelid antibodies, Dual Targeting (DT) - Ig(GSK / Domantis), Two-in-one Antibody(Gen entech), Cross-linked Mab(Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX / Pf izer), IgG-like Bispecific (InnClone / Eli L illy), Ts2Ab (MedImmune / AZ) and BsAb (Zymogene) tics), HERCULES (Biogen Idec), and TvAb (Roche) , ScFv / Fc Fusions (Academic Institution), S CORPION(Emergent BioSolutions / Trubion,Zy mogenetics / BMS)), Dual Affinity Retargeti ng Technology (Fc-DART) (MacroGenics) and Dua l(ScFv)2-Fab(National Research Center fo r Antibody Medicine--China), Dual-Action or This is Bis-Fab (Genentech), Dock-and-Lock (DNL) (I mmunoMedics), Bivalent Bispecific(Biotecn Examples include ol) and Fab-Fv (UCB-Celltech). ScFv antibody, Diabody-based antibodies and domain antibodies are bispecific T cell enforcers (Bisp ecific T Cell Engineer, BiTE) (Micromet), Tandem Diabody (Tan dem Diabody, Tandab) (Affimed), Dual Affinity Retargeting Technology (Dual Affini ty Retargeting Technology, DART) (MacroGenics), single-stranded diabo Academic, TCR-like antibody (AIT, ReceptorLogics), Human serum albumin ScFv fusion (Merrimack), and COMBODY (Ep igen Biotech), dual-target nanobody (Ablynx), dual-target heavy chain only This includes, but is not limited to, domain antibodies.

[0535] The antigen-binding region of DLL3 of this disclosure also comprises a polyparticle containing three polypeptide chains. Genetic engineering may be performed to create an isoantigen-binding construct. In such a design, at least one The antigen-binding region is in the form of an scFv. Examples of designs include the following: "1" indicates the first antigen-binding region, "2" indicates the second antigen-binding region, and "3" indicates the third (Indicates the antigen-binding region): Design 1: Chain A) scFv1-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-C H1-Hinge-CH2-CH3 Design 2: Chain A) scFv1-hinge-CH2-CH3, Chain B) VL2-CL, Chain C) V H2-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-C H1-Hinge-CH2-CH3 The CH3 operation is described in U.S. Patent Application Publication No. 2012 / 0149876 or U.S. Patent Application Publication L351Y_F405A as described in issue number 2013 / 0195849 (Zymeworks) _Y407V / T394W, T366I_K392M_T394W / F405A_Y40 7V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y 407A / T366A_K409F, L351Y_Y407A / T366V_K409F , Y407A / T366A_K409F, or T350V_L351Y_F405A_Y Variations such as 407V / T350V_T366L_K392L_T394W, etc., in designs 1-4. It can be incorporated.

[0536] In a particular embodiment, the design is as follows: Chain A)scFv1-Hinge-CH2-CH 3. Chain B) VL2-CL, Chain C) VH2-CH1-Hinge-CH2-CH3.

[0537] In some embodiments, isolated multispecific antigen-binding constructs bind to DLL3. The first antigen-binding region and the second antigen-binding region that binds to lymphocyte antigens (such as CD3) The first antigen-binding region that binds to DLL3 is included in SEQ ID NOs: 15, 18, 21, 24, 1 HCDR1 of sequence numbers 8, 30, 50, 52, 53, 55, 57, 59, or 61, sequence number 16 , 19, 22, 25, 28, 31, 51, 54, 56, 58, 60, or 62 HCDR 2. Sequence numbers 17, 20, 23, 26, 29, 32, 17, 20, 23, 26, 29, also 32 HCDR3, 33, 36, 39, 41, 44, or 47 LCDR1, LCDR2 of sequence numbers 34, 37, 42, 45, or 48, and sequence numbers 35, 38, 4 Includes LCDR3 of 0, 43, 46, or 49.

[0538] In some embodiments, isolated multispecific antigen-binding constructs bind to DLL3. The first antigen-binding region and the second antigen-binding region that binds to lymphocyte antigens (such as CD3) 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 HCDs having amino acid sequences 61, 62, 32, 47, 48, and 49, respectively. R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are isolated. It provides the protein.

[0539] In certain embodiments, the isolated multispecific antigen-binding construct binds to DLL3. A first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) The first antigen-binding regions that bind to DLL3 are, respectively, sequence numbers 15, 16, and 17. , 33, 34, 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and includes LCDR3. In some embodiments, isolated multispecific antigen-binding constructs It mediates T cell-mediated cytotoxicity, promotes T cell activation and proliferation, and T cell cytokines It increases release and / or exhibits an enhanced antitumor effect. In some embodiments, isolation The multispecific antigen-binding constructs potently mediate the proliferation of cytotoxic CD8 T cells. In some embodiments, isolated multispecific antigen-binding constructs are used on CD8 T cells. Upregulates surface expression of CD25, CD69, and CD71. In some embodiments, The isolated multispecific antigen-binding constructs show increased tumor death. Several implementations In this embodiment, the bispecific anti-DLL3×CD3 antibody was used in a T cell cytotoxic assay. Achieve over 90% (e.g., 95%) tumor lysis by [date].

[0540] In some embodiments, isolated multispecific antigen-binding constructs bind to DLL3. A first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) The first antigen-binding region, which includes the region and binds to DLL3, 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 sequence number 13 and VL of sequence number 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 Includes VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14.

[0541] In certain embodiments, the isolated multispecific antigen-binding construct binds to DLL3. A first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) The first antigen-binding region, which includes and binds to DLL3, is at least 80% of the VH of SEQ ID NO: 3. (at least 85%, at least 90%, at least 95%, or at least 99%) VH and VL of Sequence ID No. 4 are one and at least 80% (for example, at least 85%, less Includes VLs that are identical by at least 90%, at least 95%, or at least 99%.

[0542] In certain embodiments, the isolated multispecific antigen-binding construct binds to DLL3. A first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) The first antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 3 and V of SEQ ID NO: 4. L and at least 80% (for example, at least 85%, at least 90%, at least 95%) Includes VL that is identical by % or at least 99%. In some embodiments, as specified herein. The isolated multispecific antigen-binding constructs disclosed herein mediate T cell-mediated cytotoxicity. It promotes T cell activation and proliferation, increases T cell cytokine release, and / or increases It may be particularly effective in exhibiting antitumor efficacy. In certain embodiments, isolated multiple specific The sex antigen-binding construct consists of a first antigen-binding region that binds to DLL3 and a lymphocyte antigen (e.g.) It includes a second antigen-binding region that binds to CD3, and a first antigen-binding region that binds to DLL3. The region is VH of sequence number 3 and at least 80% (e.g., at least 85%, at least 9%). VH and VL of SEQ ID NO: 4 are identical (0%, at least 95%, or at least 99%). Includes.

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

[0544] In certain embodiments, the isolated multispecific antigen-binding construct binds to DLL3. A first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) The first antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 3 and V of SEQ ID NO: 4. L and at least 80% (for example, at least 85%, at least 90%, at least 95%) Includes VLs that are identical by % or at least 99%.

[0545] In certain embodiments, the isolated multispecific antigen-binding construct binds to DLL3. A first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) The first antigen-binding region, which includes and binds to DLL3, is at least 80% of the VH of SEQ ID NO: 3. (at least 85%, at least 90%, at least 95%, or at least 99%) VH and VL of Sequence ID No. 4 are one and at least 80% (for example, at least 85%, less Includes VLs that are identical by at least 90%, at least 95%, or at least 99%. In some embodiments, isolated multispecific antigen-binding constructs are used to induce T cell-mediated cytotoxicity. It mediates. In some embodiments, isolated multispecific antigen-binding constructs cause cell damage. It strongly mediates the proliferation of harmful CD8 T cells. In some embodiments, isolated multiplex The specific antigen-binding constructs are CD25, CD69, and CD71 on the surface of CD8 T cells. Upregulating expression. In some embodiments, isolated multispecific antigen-binding constructs , showing increased tumor death. In some embodiments, bispecific anti-DLL3×CD3 anti In T-cell cytotoxicity assays, more than 90% (e.g., 95%) of tumors were found within 5 days. Achieve ulcer dissolution.

[0546] In certain embodiments, the isolated multispecific antigen-binding construct binds to DLL3. A first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) The first antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 3 and V of SEQ ID NO: 4. Includes L.

[0547] In some embodiments, isolated multispecific antigen-binding constructs bind to DLL3. It includes a first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens, DLL3 The first antigen-binding region that binds to is sequence numbers 63, 64, 65, 66, 67, 68, or It contains a sequence of 69 amino acids.

[0548] In some embodiments, isolated multispecific antigen-binding constructs bind to DLL3. A first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) The first antigen-binding region, which includes the region and binds to DLL3, is the amino acid of SEQ ID NO: 63 or 64. Array and at least 80% (e.g., at least 85%, at least 90%, at least 9) Contains amino acid sequences that are identical by 5%, or at least 99%.

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

[0550] In certain embodiments, the isolated multispecific antigen-binding construct binds to DLL3. A first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) The second antigen-binding region, which binds to lymphocyte antigens, is the HCDR of SEQ ID NO: 95 or 98. 1. HCDR2 of sequence number 96 or 99, HCDR3 of sequence number 97 or 100, or LCDR1 of column number 101 or 106, LCDR2 of sequence number 102 or 107, and Includes LCDR3 with column numbers 103, 104, or 108.

[0551] In some embodiments, isolated multispecific antigen-binding constructs bind to DLL3. A first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) The second antigen-binding region, which includes the area and binds to lymphocyte antigens, HCDR1 (sequence number 95), HCDR2 (sequence number 96), HCDR3 (sequence number 97), LCDR1 of sequence number 101, LCDR2 of sequence number 102, and LC of sequence number 104 DR3, or HCDR1 (sequence number 98), HCDR2 (sequence number 99), HCDR3 (sequence number 100) LCDR1 of sequence number 106, LCDR2 of sequence number 107, and L of sequence number 108 Includes CDR3.

[0552] In certain embodiments, the isolated multispecific antigen-binding construct binds to DLL3. A first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens (e.g., CD3) The second antigen-binding region, which binds to lymphocyte antigens, is the sequence of HCDR1, sequence number 95. HCDR2 with sequence number 96, HCDR3 with sequence number 97, or LCDR1 with sequence number 101, Includes LCDR2 of sequence number 102 and LCDR3 of sequence number 104.

[0553] In some embodiments, isolated multispecific antigen-binding constructs bind to DLL3. It comprises a first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens, and The second antigen-binding region that binds to the antigen (e.g., CD3) is VH and sequence of SEQ ID NO: 77. Includes VL number 80.

[0554] In some embodiments, isolated multispecific antigen-binding constructs bind to DLL3. It comprises a first antigen-binding region and a second antigen-binding region that binds to lymphocyte antigens, and The second antigen-binding region that binds to the antigen is, HCDR1 (sequence number 98), HCDR2 (sequence number 99), HCDR3 (sequence number 100) LCDR1 of sequence number 106, LCDR2 of sequence number 107, and L of sequence number 108 Includes CDR3, or VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85.

[0555] This disclosure also relates to a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3. An isolated anti-DLL3 / anti-CD3 protein containing the primordial binding region, a. The first antigen-binding region that binds to DLL3 is sequence numbers 15, 16, and 17, respectively. 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 The second domain, which includes LCDR3 and binds to CD3, is sequence number 95, HCDR1, HCDR2, HCDR3, LCDR 96, 97, 101, 102, 104 1. Including LCDR2 and LCDR3, b. The first antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 1 and V of SEQ ID NO: 2 The second antigen-binding region, which contains L and binds to CD3, contains the scFv of SEQ ID NO: 105. and / or c. Isolated anti-DLL / anti-CD3 protein, HC1 of SEQ ID NO: 109, SEQ ID NO: Isolated anti-DLL3 / anti-CD3 containing LC1 of 110 and HC1 of sequence number 112. It provides protein.

[0556] This disclosure also relates to a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3. An isolated anti-DLL3 / anti-CD3 protein containing the primordial binding region, a. The first antigen-binding region that binds to DLL3 is sequence numbers 15, 16, and 17, respectively. 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 The second domain, which includes LCDR3 and binds to CD3, is sequence number 95, HCDR1, HCDR2, HCDR3, LCDR 96, 97, 101, 102, 104 1. Including LCDR2 and LCDR3, b. The first antigen-binding region that binds to DLL3 is VH of SEQ ID NO: 1 and V of SEQ ID NO: 2 The second antigen-binding region, which contains L and binds to CD3, contains the scFv of SEQ ID NO: 119. and / or c. Isolated anti-DLL3 / anti-CD3 protein, HC1 of SEQ ID NO. 109, sequence number Isolated anti-DLL3 / anti-CD compounds, including LC1 of code 110 and HC1 of sequence number 113. It provides 3 proteins.

[0557] This disclosure also relates to a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3. An isolated anti-DLL3 / anti-CD3 protein containing the primordial binding region, a. The first antigen-binding region that binds to DLL3 is sequence numbers 15, 16, and 17, respectively. 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 , and a second domain including LCDR3 that binds to CD3, each corresponding to sequence number 98, HCDR1, HCDR2, HCDR3, LCD for 99, 100, 106, 107, 108 Including R1, LCDR2, and LCDR3, and / or b. The first antigen-binding region that binds to DLL3 contains the scFv of SEQ ID NO: 63, and phosphorus The second antigen-binding region that binds to the Pacyl antigen is VH in SEQ ID NO: 84 and VL in SEQ ID NO: 85. Includes and / or c. Isolated anti-DLL3 / anti-CD3 protein, HC1 of SEQ ID NO: 111, sequence number Isolated anti-DLL3 / anti-CD2, including HC2 of code 116 and LC2 of sequence number 117. It provides 3 proteins.

[0558] This disclosure also relates to a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3. An isolated anti-DLL3 / anti-CD3 protein containing the primordial binding region, a. The first antigen-binding region that binds to DLL3 is sequence numbers 15, 16, and 17, respectively. 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 , and a second domain including LCDR3 that binds to CD3, each corresponding to sequence number 95, HCDR1, HCDR2, HCDR3, LCDR 96, 97, 101, 102, 104 1, including LCDR2 and LCDR3, and / or b. The first antigen-binding region that binds to DLL3 contains the scFv of SEQ ID NO: 63, and phosphorus The second antigen-binding region that binds to the pacyl antigen is VH in SEQ ID NO: 77 and VL in SEQ ID NO: 80. This includes and / or optionally, c. Isolated anti-DLL3 / anti-CD3 protein, HC1 of SEQ ID NO: 111, sequence number Isolated anti-DLL3 / anti-CD2, including HC2 of code 114 and LC2 of sequence number 115. It provides 3 proteins.

[0559] This disclosure also relates to a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3. An isolated anti-DLL3 / anti-CD3 protein containing the primordial binding region, a. The first antigen-binding region that binds to DLL3 is sequence numbers 15, 16, and 17, respectively. 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 , and a second domain including LCDR3 that binds to CD3, each corresponding to sequence number 98, HCDR1, HCDR2, HCDR3, LCD for 99, 100, 106, 107, 108 Includes R1, LCDR2, and LCDR3, and / or b. The first antigen-binding region that binds to DLL3 contains the scFv of SEQ ID NO: 64, and phosphorus The second antigen-binding region that binds to the Pacyl antigen is VH in SEQ ID NO: 84 and VL in SEQ ID NO: 85. Includes and / or c. Isolated anti-DLL3 / anti-CD3 protein, HC1 of SEQ ID NO: 71, SEQ ID NO: An isolated multispecific antigen-binding compound containing HC2 of 118 and LC2 of SEQ ID NO: 117 To provide a building.

[0560] This disclosure also relates to a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3. An isolated anti-DLL3 / anti-CD3 protein containing the primordial binding region, a. The first antigen-binding region that binds to DLL3 is sequence numbers 15, 16, and 17, respectively. 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 , and a second domain including LCDR3 that binds to CD3, each corresponding to sequence number 98, HCDR1, HCDR2, HCDR3, L for 99, 100, 106, 107, and 108 Includes CDR1, LCDR2, and LCDR3, and / or b. The first antigen-binding region that binds to DLL3 contains the scFv of SEQ ID NO: 64, and phosphorus The second antigen-binding region that binds to the Pacyl antigen is VH in SEQ ID NO: 84 and VL in SEQ ID NO: 85. Includes and / or c. Isolated anti-DLL3 / anti-CD3 protein, HC1 of sequence number 229, sequence number Isolated anti-DLL3 / anti-CD2, including HC2 of number 230 and LC2 of sequence number 117. It provides 3 proteins.

[0561] This disclosure also relates to a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3. An isolated anti-DLL3 / anti-CD3 protein containing the primordial binding region, a. The first antigen-binding region that binds to DLL3 is sequence numbers 15, 16, and 17, respectively. 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 , and a second domain including LCDR3 that binds to CD3, each corresponding to sequence number 98, HCDR1, HCDR2, HCDR3, L for 99, 100, 106, 107, and 108 Includes CDR1, LCDR2, and LCDR3, and / or b. The first antigen-binding region that binds to DLL3 is at least the scFv of SEQ ID NO: 64 80% (for example, at least 85%, at least 90%, at least 95%, at least) Contains scFv that is 99% or 100% identical, and a second antigen binding that binds to CD3. The region is at least 80% (for example, at least 85%, and at least) of VH of sequence number 84. VH and are identical (also 90%, at least 95%, at least 99%, or 100%) VL of sequence number 85 and at least 80% (e.g., at least 85%, at least 90%) Fab containing VL that is identical (at least 95%, at least 99%, or 100%) Includes and / or c. Isolated anti-DLL3 / anti-CD3 protein is less than HC1 of SEQ ID NO: 71 Also 80% (for example, at least 85%, at least 90%, at least 95%, and less HC1 is 99% or 100% identical to HC2 of sequence number 118, and at least 80% identical to HC1. (For example, at least 85%, at least 90%, at least 95%, at least 99%) (or 100%) identical to HC2 and SEQ ID NO: 117, at least 80% (for example, less (at least 85%, at least 90%, at least 95%, at least 99% or 100%) This provides isolated anti-DLL3 / anti-CD3 proteins containing the same LC2 protein.

[0562] This disclosure also relates to a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3. An isolated anti-DLL3 / anti-CD3 protein containing the primordial binding region, a. The first antigen-binding region that binds to DLL3 is sequence numbers 15, 16, and 17, respectively. 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 , and a second domain including LCDR3 that binds to CD3, each corresponding to sequence number 98, HCDR1, HCDR2, HCDR3, L for 99, 100, 106, 107, and 108 Includes CDR1, LCDR2, and LCDR3, and / or b. The first antigen-binding region that binds to DLL3 is at least the scFv of SEQ ID NO: 64 80% (for example, at least 85%, at least 90%, at least 95%, at least) Contains scFv that is 99% or 100% identical, and a second antigen binding that binds to CD3. The region is at least 80% (for example, at least 85%, and at least) of VH of sequence number 84. VH and are identical (also 90%, at least 95%, at least 99%, or 100%) VL of sequence number 85 and at least 80% (e.g., at least 85%, at least 90%) Fab containing VL that is identical (at least 95%, at least 99%, or 100%) Includes and / or c. Isolated anti-DLL3 / anti-CD3 protein is less than HC1 of SEQ ID NO: 229 80% each (for example, at least 85%, at least 90%, at least 95%, less HC1 is 99% or 100% identical to HC2 of sequence number 230, and at least 80 % (For example, at least 85%, at least 90%, at least 95%, at least 99 HC2 and SEQ ID NO: 117 are identical in % or 100% of the same amount (for example, a small amount of HC2 and SEQ ID NO: 117). At least 85%, at least 90%, at least 95%, at least 99% or 100% This provides isolated anti-DLL3 / anti-CD3 proteins containing the same LC2.

[0563] In certain embodiments, the disclosure relates to a first antigen-binding region that binds to DLL3 and to CD3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region, a) The first antigen-binding region that binds to DLL3 is sequence numbers 15, 16, and 17, respectively. 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 , and a second domain including LCDR3 that binds to CD3, each corresponding to sequence number 98, HCDR1, HCDR2, HCDR3, L for 99, 100, 106, 107, and 108 Includes CDR1, LCDR2, and LCDR3, and / or b) The first antigen-binding region that binds to DLL3 contains the scFv of SEQ ID NO: 64, and CD The second antigen-binding region that binds to 3 includes VH in SEQ ID NO: 77 and VL in SEQ ID NO: 80. The present invention provides isolated, multispecific antigen-binding constructs.

[0564] In some embodiments, isolated anti-DLL3 / anti-CD3 proteins are T cell-mediated It mediates sex cell damage. In some embodiments, isolated anti-DLL3 / anti-CD3 tannins The protein potently mediates the proliferation of cytotoxic CD8 T cells. In some embodiments, The isolated anti-DLL3 / anti-CD3 protein was found on CD25 on the surface of CD8 T cells. Upregulates CD69 and CD71 expression. In some embodiments, isolated anti-D LL3 / anti-CD3 protein shows increased tumor death. In some embodiments, single The detached anti-DLL3 / anti-CD3 protein was detected in T-cell cytotoxicity assays up to 5 days. This achieves over 90% (for example, 95%) tumor lysis.

[0565] This disclosure also relates to a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to CD3. An isolated anti-DLL3 / anti-CD3 protein containing the primordial binding region, a. The first antigen-binding region that binds to DLL3 is sequence numbers 15, 16, and 17, respectively. 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 , and a second domain including LCDR3 that binds to CD3, each corresponding to sequence number 98, HCDR1, HCDR2, HCDR3, L for 99, 100, 106, 107, and 108 Includes CDR1, LCDR2, and LCDR3, and / or b. The first antigen-binding region that binds to DLL3 is at least the scFv of SEQ ID NO: 64 80% (for example, at least 85%, at least 90%, at least 95%, at least) Contains scFv that is 99% or 100% identical, and a second antigen binding that binds to CD3. The region is at least 80% (for example, at least 85%, and at least) of VH of sequence number 84. VH and are identical (also 90%, at least 95%, at least 99%, or 100%) VL of sequence number 85 and at least 80% (e.g., at least 85%, at least 90%) Fab containing VL that is identical (at least 95%, at least 99%, or 100%) Includes and / or c. Isolated anti-DLL3 / anti-CD3 protein is less than HC1 of SEQ ID NO: 229 80% each (for example, at least 85%, at least 90%, at least 95%, less HC1 is 99% or 100% identical to HC2 of sequence number 230, and at least 80 % (For example, at least 85%, at least 90%, at least 95%, at least 99 HC2 and SEQ ID NO: 117 are identical in % or 100% of the same amount (for example, a small amount of HC2 and SEQ ID NO: 117). At least 85%, at least 90%, at least 95%, at least 99% or 100% This provides isolated anti-DLL3 / anti-CD3 proteins containing the same LC2.

[0566] In some embodiments, isolated anti-DLL3 / anti-CD3 proteins are T cell-mediated It mediates sex cell damage. In some embodiments, isolated anti-DLL3 / anti-CD3 tannins The protein potently mediates the proliferation of cytotoxic CD8 T cells. In some embodiments, The isolated anti-DLL3 / anti-CD3 protein was found on CD25 on the surface of CD8 T cells. Upregulates CD69 and CD71 expression. In some embodiments, isolated anti-D LL3 / anti-CD3 protein shows increased tumor death. In some embodiments, The released anti-DLL3 / anti-CD3 protein was detected in T-cell toxicity assays by 5 days. Achieves over 90% (for example, 95%) tumor lysis.

[0567] In certain embodiments, the disclosure relates to a first antigen-binding region that binds to DLL3 and to CD3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region, a. The first antigen-binding region that binds to DLL3 is sequence numbers 15, 16, and 17, respectively. 33, 34, and 35 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 , and a second domain including LCDR3 that binds to CD3, each corresponding to sequence number 98, HCDR1, HCDR2, HCDR3, L for 99, 100, 106, 107, and 108 Includes CDR1, LCDR2, and LCDR3, and / or b. The first antigen-binding region that binds to DLL3 contains the scFv of SEQ ID NO: 64, CD The second antigen-binding region that binds to 3 includes VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80. This provides isolated, multispecific antigen-binding constructs.

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

[0569] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 80 scFv of SEQ ID NO: 64. The second antigen-binding region, which contains the % identical scFv and binds to CD3, is the same as in SEQ ID NO: 77 VH is at least 80% identical to VH and VL of sequence number 80 is at least 80% identical to VH. This provides isolated, multispecific antigen-binding constructs containing VL.

[0570] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 85% of the scFv of SEQ ID NO: 64. The second antigen-binding region, which contains the same scFv and binds to CD3, is V in SEQ ID NO: 77 VH is at least 85% identical to H, and VL of sequence number 80 is at least 85% identical. This invention provides an isolated, multispecific antigen-binding construct containing a certain VL.

[0571] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 90% of the scFv of SEQ ID NO: 64. The second antigen-binding region, which contains the same scFv and binds to CD3, is V in SEQ ID NO: 77 VH is at least 90% identical to H, and VL of sequence number 80 is at least 90% identical. This invention provides an isolated, multispecific antigen-binding construct containing a certain VL.

[0572] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 95% compatible with the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 77 VH is at least 95% identical to H, and VL is at least 95% identical to Sequence ID No. 80. This invention provides an isolated, multispecific antigen-binding construct containing a certain VL.

[0573] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 99% compatible with the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 77 VH is at least 99% identical to H, and VL of sequence number 80 is at least 99% identical. This invention provides an isolated, multispecific antigen-binding construct containing a certain VL.

[0574] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 95% of the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 77 Isolated multiple markers containing VL which is at least 95% identical to VL of H and SEQ ID NO: 80 This provides an isoantigen binding construct.

[0575] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 99% compatible with the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 77 Isolated multiple specimens containing VL which is at least 99% identical to VL of H and SEQ ID NO: 80 This provides an isoantigen binding construct.

[0576] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 95% of the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 77 Isolated multiple markers containing VH, which is at least 95% identical to H, and VL of SEQ ID NO: 80. This provides an isoantigen binding construct.

[0577] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 99% compatible with the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 77 Isolated multiple markers containing VH, which is at least 99% identical to H, and VL of SEQ ID NO: 80. This provides an isoantigen binding construct.

[0578] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 contains the scFv of sequence number 64 and binds to CD3. The second antigen-binding region that binds is at least 95% identical to the VH of SEQ ID NO: 77. Isolated multispecific VL containing VL that is at least 95% identical to VL of SEQ ID NO: 80 To provide an antigen-binding construct.

[0579] In certain embodiments, the disclosure relates to a first antigen-binding region that binds to DLL3 and to CD3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region for binding, DL The first antigen-binding region that binds to L3 contains the scFv of SEQ ID NO: 64 and binds to CD3. The second antigen-binding region includes VH of SEQ ID NO: 77 and VL of SEQ ID NO: 80, which has been isolated. The present invention provides a multispecific antigen-binding construct.

[0580] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 80% of the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 84 VH is at least 80% identical to H, and VL of sequence number 85 is at least 80% identical. This invention provides an isolated, multispecific antigen-binding construct containing a certain VL.

[0581] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 85% of the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 84 VH is at least 85% identical to H, and VL of sequence number 85 is at least 85% identical. This invention provides an isolated, multispecific antigen-binding construct containing a certain VL.

[0582] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 90% of the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 84 VH is at least 90% identical to H, and VL of sequence number 85 is at least 90% identical. This invention provides an isolated, multispecific antigen-binding construct containing a certain VL.

[0583] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 95% of the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 84 VH is at least 95% identical to H, and VL is at least 95% identical to sequence number 85. This invention provides an isolated, multispecific antigen-binding construct containing a certain VL.

[0584] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 99% compatible with the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 84 VH is at least 99% identical to H, and VL is at least 99% identical to sequence number 85. This invention provides an isolated, multispecific antigen-binding construct containing a certain VL.

[0585] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 95% of the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 84 Isolated multiple markers containing VL which is at least 95% identical to VL of H and SEQ ID NO: 85 This provides an isoantigen binding construct.

[0586] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 99% compatible with the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 84 Isolated multiple specimens containing VL which is at least 99% identical to VL of H and SEQ ID NO: 85 This provides an isoantigen binding construct.

[0587] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 95% of the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 84 Isolated multiple chromium containing VH, which is at least 95% identical to H, and VL of SEQ ID NO: 85. This provides an isoantigen binding construct.

[0588] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 is at least 99% compatible with the scFv of SEQ ID NO: 64. It contains the same scFv, and the second antigen-binding region that binds to CD3 is V of SEQ ID NO: 84 Isolated multiple markers containing VH, which is at least 99% identical to H, and VL of SEQ ID NO: 85. This provides an isoantigen binding construct.

[0589] In some embodiments, the disclosure relates to a first antigen-binding region and CD that bind to DLL3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region that binds to 3, The first antigen-binding region that binds to DLL3 contains the scFv of sequence number 64 and binds to CD3. The second antigen-binding region that binds is at least 95% identical to the VH of SEQ ID NO: 84. Isolated multispecific VL containing VL that is at least 95% identical to VL of SEQ ID NO: 85 To provide an antigen-binding construct.

[0590] In certain embodiments, the disclosure relates to a first antigen-binding region that binds to DLL3 and CD3. An isolated multispecific antigen-binding construct comprising a second antigen-binding region for binding, DL The first antigen-binding region that binds to L3 contains the scFv of SEQ ID NO: 64 and binds to CD3. The second antigen-binding region includes VH of SEQ ID NO: 84 and VL of SEQ ID NO: 85, isolated The present invention provides a multispecific antigen-binding construct.

[0591] As shown in the examples, isolated multispecific antigen-binding constructs disclosed herein It mediates T cell-mediated cytotoxicity, promotes T cell activation and proliferation, and T cell cytotoxicity It may be particularly effective in increasing ion release and / or exhibiting increased antitumor efficacy. Therefore, in some embodiments, isolated multispecific antigens disclosed herein The composite construct mediates T cell-mediated cytotoxicity. In some embodiments, as described herein, The isolated, multispecific antigen-binding constructs shown enhance the proliferation of cytotoxic CD8 T cells. It mediates the force. In some embodiments, isolated multispecific antibodies disclosed herein The primordial construct enhances the expression of CD25, CD69, and CD71 on the surface of CD8 T cells. Upward control. In some embodiments, isolated multispecific antibodies disclosed herein The original binding construct shows increased tumor death. In some embodiments disclosed herein The bispecific anti-DLL3×CD3 antibody showed results in T-cell cytotoxicity assays up to 5 days. It achieves over 90% (for example, 95%) tumor lysis. Particularly remarkable is the highest tumor death rate. A multispecific antigen-binding construct exhibiting destructive properties is used to establish immune synapses between tumor cells and other cells. This is a location that is thought to impair the ability of multispecific antibodies to optimally position toxic T cells. The key fact is that it binds to the epitope on DLL3 that is closest to the cell membrane.

[0592] Isotype, allotype, and Fc gene manipulation Fragments of Ig constant regions, such as Ig constant regions or Fc regions, present in proteins of the present disclosure. This may be 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 isota It is Ip.

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

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

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

[0597] The Ig steady-state region or a fragment of the Ig steady-state region can be any allotype. It does not affect the properties of the Ig steady-state region, such as binding or Fc-mediated effector function. It is expected that the immunogenicity of therapeutic proteins containing the Ig constant region of the fragment will increase the risk of infusion reactions. This is associated with increased blood pressure and shortened treatment response time (Baert et al., (2003) (N Engl J Med 348:602-08). Therapy involving the Ig constant region of the fragment. The extent to which a protein induces an immune response in the host depends on the allotype of the Ig constant region. This can be determined to some extent (Stickler et al., (2011) Genes (and Immunity 12:213-21). The allotype of the Ig constant region is anti It is related to mutations in the amino acid sequence at specific locations within the body's constant region sequence. Table 3 shows selected results. The IgG1, IgG2, and IgG4 allotypes are shown.

[0598] [Table 3]

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

[0600] C-terminal lysine (CTL) is released from the Ig constant region into the bloodstream from endogenous sources. It can be removed by circulating carboxypeptidase (Cai et al., (2011) (Biotechnol Bioeng 108:404-412). In manufacture, U.S. Patent As described in Patent Publication No. 20140273092, extracellular Zn 2+ , EDTA, or E DTA-Fe 3+ By controlling the concentration, CTL removal can be controlled to below the maximum level. It is possible to measure the CTL content of a protein using known methods. Cut.

[0601] In some embodiments, an anti-Ig conjugate to the Ig constant region is bound to DLL3. The original binding fragment has a C-terminal lysine content of approximately 10% to approximately 90%. Several embodiments In this case, the C-terminal lysine content is approximately 20% to approximately 80%. In some embodiments, the C-terminal The terminal lysine content is approximately 40% to 70%. In some embodiments, C-terminal lysine content The amount is approximately 55% to approximately 70%. In some embodiments, the C-terminal lysine content is approximately 6 It is 0%.

[0602] Antigens that bind to the Ig constant region or DLL3 conjugated to a fragment of the Ig constant region. By performing Fc region mutations on the binding region, ADCC, ADCP, and / or ADCP This can modify effector functions such as and / or pharmacokinetic properties. c activation of FcγRs (FcγRI, FcγRIIa, FcγRIII), FcγRII This can be achieved by introducing mutations into Fc that control binding to b and / or FcRn. That is the case.

[0603] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is conjugated. The antigen-binding region that binds to DLL3 is at least one part of the Ig constant region or a fragment of the Ig constant region. It also includes one mutation.

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

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

[0606] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is conjugated. The antigen-binding region that binds to DLL3 is located in the Fc region, which regulates the binding of the antibody to FcRn. It contains at least one mutation.

[0607] Fc can be mutated to regulate its half-life (e.g., binding to FcRn). The positions are 250, 252, 253, 254, 256, 257, 307, and 376. Examples include 380, 428, 434, and 435. These can be performed individually or in combination. Examples of mutations obtained include mutations T250Q, M252Y, I253A, S254T, and T256. E, P257I, T307A, D376V, E380A, M428L, H433K, N4 These are 34S, N434A, N434H, N434F, H435A, and H435R. Exemplary mutations, either alone or in combination, that can be performed to increase the half-life of a substance are mutations. Different M428L / N434S, M252Y / S254T / T256E, T250Q / M42 These are 8L, N434A, and T307A / E380A / N434A. The half-life is reduced. Exemplary mutations that can occur, either alone or in combination, include mutations H435A and P257. I / N434H, D376V / N434H, M252Y / S254T / T256E / H4 These are 33K / N434F, T308P / N434A, and H435R.

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

[0609] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is conjugated. The antigen-binding region that binds to DLL3 is responsible for protein binding to the Fcγ receptor (FcγR). Enhances and / or C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity. Fc effect pedals that produce cytotoxicity (ADCC) and / or phagocytosis (ADCP) It includes at least one mutation in the Fc region that reduces function.

[0610] It reduces protein binding to activated FcγR, and subsequently reduces effector function. The Fc positions that can be mutated to achieve this are positions 214, 233, 234, 235, and 23 6, 237, 238, 265, 267, 268, 270, 295, 297, 309, 32 Examples include 7, 328, 329, 330, 331, and 365. They can be used individually or in combination. Exemplary mutations that can be added are IgG1, IgG2, IgG3, or IgG4 In this, mutations K214T, E233P, L234V, L234A, deletion of G236, V 234A, F234A, L235A, G237A, P238A, P238S, D265A , S267E, H268A, H268Q, Q268A, N297A, A327Q, P32 9A, D270A, Q295A, V309L, A327S, L328F, A330S, and P331S is an example of a combination mutation that results in reduced ADCC proteins. The difference lies in L234A / L235A in IgG1 and L234A / L23 in IgG1. 5A / D265S, V234A / G237A / P238S / H268A in IgG2 / V309L / A330S / P331S, F234A / L235A in IgG4, I S228P / F234A / L235A in gG4, and in all Ig isotypes N297A, V234A / G237A in IgG2, K214T / in IgG1 E233P / L234V / L235A / G236 (missing) / A327G / P331A / D36 5E / L358M, H268Q / V309L / A330S / P331S in IgG2 IgG1 S267E / L328F, IgG1 L234F / L235E / D265A, L234A / L235A / G237A / P238S / H in IgG1 268A / A330S / P331S, S228P / F234A / L23 in IgG4 5A / G237A / P238S, and S228P / F234A / L2 in IgG4 This is a mutation of 35A / G236 deletion / G237A / P238S. Additionally, residual IgG2-derived residues are present. Hybrids such as Fc having residues 117-260 and IgG4-derived residues 261-447 The IgG2 / 4 Fc domain may also be used.

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

[0612] By adding the well-known S228P mutation to an IgG4 antibody, the stability of IgG4 can be enhanced. Cut.

[0613] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is conjugated. The antigen-binding regions that bind to DLL3 are K214T, E233P, L234V, and L234 Missing A, G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297 A, A327Q, P329A, D270A, Q295A, V309L, A327S, L3 At least one selected from the group consisting of 28F, K322, A330S, and P331S. Contains one mutation.

[0614] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is conjugated. The antigen-binding region that binds to DLL3 contains the L234A / L235A / D265S mutation. In certain embodiments, the antigen-binding region that binds to DLL3 is L234A_L235A_ The IgG1 constant region or a fragment of the IgG1 constant region containing the D265S mutation is conjugated. Let it.

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

[0616] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is conjugated. The antigen-binding region that binds to DLL3 is responsible for protein binding to the Fcγ receptor (FcγR). Enhances and / or C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity. Fc effect pedals that produce cytotoxicity (ADCC) and / or phagocytosis (ADCP) It includes at least one mutation in the Fc region that enhances function.

[0617] Increases protein binding to activated FcγR and / or Fc effector function Fc positions that can be mutated to enhance this include positions 236, 239, 243, and 256. ,290,292,298,300,305,312,326,330,332,333 , 334, 345, 360, 339, 378, 396, or 430 (on the EU index) Examples include the numbering of residues that follows the rules. Exemplary mutations that can occur individually or in combination are: G236A, S239D, F243L, T256A, K290A, R292P, S298 A, Y300L, V305L, K326A, A330K, I332E, E333A, K3 These are 34A, A339T, and P396L. These are proteins with increased ADCC or ADCP levels. Exemplary combinations of mutations that result in S239D / I332E, S298A / E33 3A / K334A, F243L / R292P / Y300L, F243L / R292P / Y 300L / P396L, F243L / R292P / Y300L / V305I / P396L And G236A / S239D / I332E.

[0618] Fc locations that can mutate to enhance CDC include positions 267, 268, and 324. Examples include 326, 333, 345, and 430. These can be performed individually or in combination. Exemplary mutations include S267E, F1268F, S324T, K326A, K326W, E 333A, E345K, E345Q, E345R, E345Y, E430S, E430F And E430T. Exemplary combination mutations that result in increased CDC protein The difference is K326A / E333A, K326W / E333A, H268F / S324T, S 267E / H268F, S267E / S324T, and S267E / H268F / S32 It's 4T.

[0619] The specific mutations described herein are Ig mutations in SEQ ID NOs. 257, 258, and 259, respectively. These are mutations compared to the G1, IgG2, and IgG4 wild-type amino acid sequences.

[0620] Antibody binding to FcγR or FcRn is determined using flow cytometry, which analyzes each receptor. It can be evaluated in cells that have been genetically engineered to express it. In an exemplary binding assay... This is 2 x 10 in a 96-well plate. 5 Seed cells at a rate of 1 / well, BSA Stain Store in Buffer (BD Biosciences, San Jose, USA) at 4°C Block for 30 minutes. Place the cells on ice at 4°C for 1.5 hours with the test antibody. Incubate. After washing twice with BSA staining buffer, label the cells with R-PE-labeled anti-human I. gG secondary antibody (Jackson Immunoresearch Laboratori Incubate with (es) at 4°C for 45 minutes. Wash the cells twice with staining buffer. Next, a 1:200 dilution of DRAQ7 live / dead cell staining reagent (Cell Signal) is used. 150 μL of St containing (ing Technology, Danvers, USA) Resuspend in ain buffer. Observe the PE and DRAQ7 signals of the stained cells. Using the B2 and B4 channels respectively, Miltenyi MACSQuant flow - Detected by a cytometer (Miltenyi Biotec, Auburn, USA) Distribute the sample. Gate out live cells by DRAQ7 exclusion, and collect at least 10,000 live cells. For each event, the geometric mean fluorescence signal is determined. FlowJo software is used for the analysis. Use Tree Star. Data is obtained by comparing antibody concentration to average fluorescence signal. Plot the data logarithmically. Perform a nonlinear regression analysis.

[0621] Glycan gene manipulation Antigens that bind to the Ig constant region or DLL3 conjugated to a fragment of the Ig constant region. The ability of the binding region to mediate ADCC is due to oligosaccharides of the Ig constant region or fragments of the Ig constant region. The components can be enhanced by genetically modifying them. Human IgG1 or IgG3 , N-glycosylation occurs at Asn297. Here, the majority of the glycans are known It is in the form of a two-way branch G0, G0F, G1, G1F, G2, or G2F. Ig constant region-containing proteins that can be produced by CHO cells are typically at least It has a glycan fucose content of approximately 85%. It contains the Ig constant region or fragments of the Ig constant region. A branched-complex oligosaccharide bound to the antigen-binding domain that binds to jugated DLL3. When core fucose is removed, the antigen binding and CDC activity are improved without any changes. The ADCC of proteins is enhanced via FcγRIIIa binding. The quality is that it contains a relatively high level of defucosylated immunoglobulin with a bifurcated complex type of Fc oligosaccharide. This can be achieved using different methods that have been reported to lead to successful expression, including the following: ru: Control of osmotic pressure of cultures (Konno et al., Cytotechnology) 64(:249-65,2012), variant CHO strain Lec1 as host cell line Application of 3 (Shields et al., J Biol Chem 277:2673) 3-26740, 2002), Application of variant CHO strain EB66 as host cell line ( Olivier et al.,MAbs;2(4):405-415,2010;PM ID:20562582), rat hybridoma cell line YB2 / 0 as host cell line Application (Shinkawa et al., J Biol Chem 278:3466) -3473,2003), against the 1,6-fucosyltransferase (FUT8) gene This involves the introduction of specific small interfering RNAs (Mori et al., Biotechnol Bioeng 88:901-908,2004), or β-1,4-N-acetylg Glucosaminyltransferase III and Golgi α-mannosidase II or potent form. Co-expression of kifunensin, an alpha-mannosidase I inhibitor (Ferrara et al., J Biol Chem 281:5032-5036, 2006, Fe rrara et al.,Biotechnol Bioeng 93:851-86 1,2006;Xhou et al.,Biotechnol Bioeng 99: 652-65, 2008).

[0622] In some embodiments, a conjugate is added to the Ig steady-state region or a fragment of the Ig steady-state region in this disclosure. The antigen-binding region that binds to the modified DLL3 is approximately 1% to 15%, for example, approximately 15%. 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% It has a branched glycan structure with a fucose content of 2% or 1%. In the application method, DLL3 is connected to the Ig steady-state region or a fragment of the Ig steady-state region. The antigen-binding regions that bind are approximately 50%, 40%, 45%, 40%, 35%, 30%, and 25%. Alternatively, it has a glycan structure with a fucose content of 20%.

[0623] "Fucose content" refers to the amount of fucose monosaccharides in the sugar chain of Asn297. The relative amount of fucose is the ratio of the fucose-containing structure to the total sugar structure. These sugar structures This is done in several ways, for example, 1) as described in International Publication No. 2008 / 077546 , N-glycosidase F treated samples (e.g., complex, hybrid, and oligo-parallel 1) Use of MALDI-TOF (high mannose structure), 2) Enzyme of Asn297 glycan HPLC (UPLC) and / or HP with release, subsequent derivatization, and fluorescence detection. Detection / quantification by LC-MS (UPLC-MS), 3) First GlcNAc monosaccharide and the second The bond between the two GlcNAc monosaccharides is cleaved, and fucose is attached to the first GlcNAc. This involves processing Asn297 glycan with Endo S or other enzymes, or Analysis of intact proteins of natural or reduced mAbs without this treatment, 4) Enzyme digestion (e.g.) For example, the conversion of mAb constituent peptides by trypsin or endopeptidase (Lys-C) Digestion, followed by separation, detection and quantification by HPLC-MS (UPLC-MS), 5) As mAb tannins are induced by specific enzymatic deglycosylation with PNGase F at n 297. By isolating mAb oligosaccharides from the protein, they can be characterized and quantified. The oligosaccharides released were labeled with fluorophores, and the measured mass was compared to the theoretical mass. Matrix-assisted laser desorption ionization by comparison Ionization, MALDI) Mass spectrometry for detailed characterization of glycan structure, ion exchange Determination of the degree of sialylation by HPLC (GlycoSep C), normal-phase HPLC (Gly Separation and quantification of oligosaccharide types in accordance with hydrophilicity standards using coSep N), and high performance Capillary electrophoresis - Laser-induced fluorescence (high performance capillary electrophoresis) Separation and quantification of oligosaccharides by is-laser induced fluorescence (HPCE-LIF), This can be made possible by various supplementary techniques that allow for separation and identification.

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

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

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

[0627] This application also provides an anti-idylation that specifically binds to the antigen-binding region of DLL3 of the present disclosure. We provide otype antibodies.

[0628] Anti-idiotypic (Id) antibodies are antibodies whose antigenic determinants (e.g., parato) It is an antibody that recognizes the antigen (P or CDR). The Id antibody blocks the antigen. It may or may not be performed. The antigen blocking ID is the binding of free antigens in the sample. It can be used to detect regions (for example, antigen-binding regions that bind to DLL3 in this disclosure). Using an Id that does not block, the total antibodies in the sample (free antibodies, antigen-dependent antibodies) are used. It can detect substances that are partially bound or completely bound to the antigen. Id antibodies can be prepared by immunizing animals with an antibody that has been prepared as an anti-Id agent.

[0629] Furthermore, using an anti-Id antibody as an immunogen to induce an immune response in another animal, It is also possible to produce anti-anti-Id antibodies. Anti-anti-Id antibodies are produced from the original antigen that induced anti-Id. The binding region and the epitope may be identical. Therefore, the idiotype of the antigen-binding region is determined. By using antibodies against the group, other groups expressing antigen-binding regions with the same specificity can be created. It is possible to identify the loan. The anti-Id antibody is as described elsewhere in this specification. Varying by any suitable technique (thereby producing an anti-Id antibody variant), and / or can be induced.

[0630] Immunoconjugates The antigen-binding region that binds to DLL3 in this disclosure, and the antigen-binding region that binds to DLL3 A multispecific antigen-binding construct containing an antigen-binding region that binds to protein or DLL3 (Honmei These proteins (collectively referred to as DLL3-binding proteins in the detailed document) can be conjugated to heterologous molecules. ru.

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

[0632] This application also describes antigen binding cleavage to DLL3 conjugated with a detectable label. Provide a piece.

[0633] This application also describes antigen binding cleavage to DLL3 conjugated with a detectable label. It provides protein containing pieces.

[0634] This application also describes antigen binding cleavage to DLL3 conjugated with a detectable label. The present invention provides a multispecific antigen-binding construct containing a fragment.

[0635] This application also relates to antigen binding cleavage, which binds to DLL3 conjugated with a cytotoxic agent. Provide a piece.

[0636] This application also relates to an antigen-binding fragment that binds to DLL3 conjugated with a cytotoxic agent. It provides proteins, including [specific protein].

[0637] This application also relates to an antigen-binding fragment that binds to DLL3 conjugated with a cytotoxic agent. The present invention provides a multispecific antigen-binding construct that includes [the specified component].

[0638] Using the DL...

Claims

1. Isolated protein containing an antigen-binding region that binds to delta-like protein 3 (DLL3) The antigen-binding region is of a quality that binds to an epitope in the amino acid sequence of SEQ ID NO:

263. The isolated protein.

2. The aforementioned antigen-binding region is related to the heavy chain complementarity-determining region (HCDR) H for binding to DLL3. Heavy chain variable region (VH) including CDR1, HCDR2, and HCDR3, and light chain complementarity Decision region (LCDR) Light chain variable region (V) including LCDR1, LCDR2, and LCDR3 L) competes with the reference antibody, and the HCDR1, HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3 are a. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 1, and SEQ ID NO: 2 LCDR1, LCDR2, and LCDR3 of the VL, b. HCDR1, HCDR2, and HCDR3 of VH in Sequence ID 3, and Sequence ID 4 LCDR1, LCDR2, and LCDR3 of the VL, c. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 5, and SEQ ID NO: 6 LCDR1, LCDR2, and LCDR3 of the VL, d. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 7, and SEQ ID NO: 8 LCDR1, LCDR2, and LCDR3 of the VL, e. HCDR1, HCDR2, and HCDR3 of VH in Sequence ID 9, and Sequence ID 1 LCDR1, LCDR2, and LCDR3 of VL 0 f. HCDR1, HCDR2, and HCDR3 of VH in Sequence ID 11, and Sequence ID 12 VLs LCDR1, LCDR2, and LCDR3, or g. HCDR1, HCDR2, and HCDR3 of VH in Sequence ID No. 13, and Sequence ID No. 14 VLs LCDR1, LCDR2, and LCDR3 The isolated protein according to claim 1, having the amino acid sequence.

3. The antigen-binding region is a. Sequence numbers 15, 16, 17, 33, 34, 35, 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, f. Sequence numbers 30, 31, 32, 47, 48, 49, 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, 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. Sequence numbers 61, 62, 32, 47, 48, and 49, respectively. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and The isolated protein according to claim 1 or 2, comprising LCDR3.

4. The antigen-binding regions are, respectively, number 1, 3, 5, 7, 9, 11, or 13. At least 90% (for example, at least 90%, 91%, 92%, 93%, 94%, 95%) VH with identical amino acid sequences (96%, 97%, 98%, 99%, or 100%) , and at least 90% (for example, less) of sequence numbers 2, 4, 6, 8, 10, 12, or 14 (for example, less Even without it, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Claims 1 to 3 include a VL having an amino acid sequence that is 99% or 100% identical. The isolated protein described in either item.

5. The antigen-binding region is a. VH of sequence number 1 and VL of sequence number 2, b. VH of SEQ ID NO: 3 and VL of SEQ ID NO: 4 c. VH of sequence number 5 and VL of sequence number 6, d. VH of SEQ ID NO: 7 and VL of SEQ ID NO: 8 e. VH of sequence number 9 and VL of sequence number 10, f. VH of SEQ ID NO: 11 and VL of SEQ ID NO: 12, or g. VH of SEQ ID NO: 13 and VL of SEQ ID NO: 14 The isolated protein according to claim 4, comprising

6. The antigen-binding region is at least 80% (for example) of the amino acid sequence of SEQ ID NO: 63 or 64. If so, at least 85%, at least 90%, at least 91%, at least 92%, and less At least 93%, at least 94%, at least 95%, at least 96%, at least 9 7%, at least 98%, at least 99%, or 100%) identical amino acid sequences An isolated protein according to any one of claims 1 to 5, comprising scFv.

7. The isolation described in any one of claims 1 to 6, conjugated with a therapeutic agent or contrast agent. An immune conjugate containing modified proteins.

8. A multispecific antigen-binding construct comprising the protein described in any one of claims 1 to 6.

9. A second antibody that binds to antigens on lymphocytes such as T cells or natural killer (NK) cells. The multispecific antigen-binding construct according to claim 8, further comprising a protobinding region.

10. The antigens on the lymphocytes are CD3, CD3 epsilon (CD3ε), CD8, KI 2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, The multispecific antigen binding structure according to claim 9, which is PD-1, CD195, or NKG2C. building.

11. The second antigen-binding region binds to CD3ε, and to HCDR1, HCDR2, and H VH having CDR3, and VL having LCDR1, LCDR2, and LCDR3 Including the HCDR1, HCDR2, HCDR3, LCDR1 of the second antigen-binding region , LCDR2 and LCDR3 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 10, comprising the amino acid sequence.

12. The second antigen-binding region described above is a. The amino acid sequence of SEQ ID NO: 84 and at least 80% (e.g., at least 85%, less At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, VH and SEQ ID NO: 8 have amino acid sequences that are identical by at least 99% or 100%. 5 amino acid sequence and at least 80% (for example, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, At least 95%, at least 96%, at least 97%, at least 98%, VL having an amino acid sequence that is 99% or 100% identical, preferably SEQ ID NO: 8 VH containing the amino acid sequence of 4, and VL containing the amino acid sequence of SEQ ID NO: 85, teeth b. The amino acid sequence of SEQ ID NO: 77 and at least 80% (e.g., at least 85%, less) At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, VH and SEQ ID NO: 8 have amino acid sequences that are identical by at least 99% or 100%. 0 amino acid sequence and at least 80% (for example, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, At least 95%, at least 96%, at least 97%, at least 98%, The amino acids of VL and SEQ ID NO: 77 have amino acid sequences that are 99% or 100% identical. VH containing the sequence, and VL containing the amino acid sequence of SEQ ID NO:

80. A multispecific antigen-binding construct according to claim 11, comprising:

13. The antigen-binding regions that bind to DLL3 are, respectively, sequence numbers 15, 16, 17, and 33. HCDR1, HCDR2, HCDR3, LCDR1, L of amino acid sequences 34 and 35 It has CDR2 and LCDR3, and preferably the antigen-binding region that binds to DLL3 , Sequence ID 3 and at least 90% (for example, at least 90%, 91%, 92%, 93%) (94%, 95%, 96%, 97%, 98%, 99%, or 100%) Same amino acid composition VH having a column, and at least 90% (e.g., at least 90%, 91%) of sequence number 4. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ) Multiplex according to any one of claims 8 to 12, comprising VL having the same amino acid sequence. Specific antigen binding constructs.

14. The isolated protein according to any one of claims 1 to 6 or any of claims 8 to 13 A half-life extension portion fused to any one of the multispecific antigen-binding constructs described in item (moiety A fusion or conjugate comprising ) wherein the half-life extension portion is immunoglobulin (Ig), a fragment of Ig, a steady-state Ig region, a fragment of the steady-state Ig region, an Fc region, a transistor It is spherin, albumin, albumin-binding domain, or polyethylene glycol. , a fusion or conjugate.

15. The aforementioned half-life extension portions are T350V, L351Y, F405A, Y407V, T366 Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T3 66S / L368A / Y407V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y407V, T366L / K392M / T394 W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T3 66V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, T350V / T366L / K392L / T394W, and L234A / L2 Ig stationary region containing at least one mutation selected from the group consisting of 35A / D265S It includes fragments of the Ig constant region, such as the region, and the residue numbering follows the EU index. The fusion or conjugate according to claim 14.

16. A bispecific antigen-binding construct, (1) A first antigen-binding region that binds to DLL3, wherein the first antigen-binding region is A first VH having HCDR1, HCDR2, and HCDR3, and LCDR1, LC A first VL having DR2 and LCDR3, the HCDR1, the HCDR2, The HCDR3, the LCDR1, the LCDR2, and the LCDR3 are (a) Sequence numbers 15, 16, 17, 33, 34, 35, (b) Sequence numbers 18, 19, 20, 36, 37, 38, (c) Sequence numbers 21, 22, 23, 39, 37, 40 respectively, (d) Sequence numbers 24, 25, 26, 41, 42, 43, (e) Sequence numbers 18, 28, 29, 44, 45, 46, (f) Sequence numbers 30, 31, 32, 47, 48, 49, (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, (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) Sequence numbers 61, 62, 32, 47, 48, 49 respectively The first antigen-binding region includes the amino acid sequence, (2) A second antigen-binding region that binds to CD3ε, (a) HCDR1 and HCDR1 of the amino acid sequences of SEQ ID NOs. 95, 96, and 97, respectively. A second VH having R2 and HCDR3, and sequence numbers 101, 102, and A second V having the amino acids LCDR1, LCDR2, and LCDR3 in the sequence of 104 L, or (b) HCDR1, HC of the amino acid sequences of SEQ ID NOs. 98, 99, and 100, respectively. A second VH having DR2 and HCDR3, and sequence numbers 106 and 107, respectively. and a second V having LCDR1, LCDR2, and LCDR3 with a sequence of 108 amino acids L The second antigen-binding region, including A bispecific antigen-binding construct containing the above.

17. a. The first VH and the first VL are i. VH of sequence number 1 and VL of sequence number 2, ii. VH of sequence number 3 and VL of sequence number 4, respectively iii. VH of sequence number 5 and VL of sequence number 6, respectively iv. VH of sequence number 7 and VL of sequence number 8, respectively v. VH of sequence number 9 and VL of sequence number 10, respectively vi. VH of sequence number 11 and VL of sequence number 12, respectively, vii. VH of sequence number 13 and VL of sequence number 14 and at least 90% (for example, at least 90%, 91%, 92%, 93%, 94%, 9) They have identical amino acid sequences (5%, 96%, 97%, 98%, 99%, or 100%). 、 b. The second VH and the second VL are i. VH of sequence number 77 and VL of sequence number 80, respectively, ii. VH of sequence number 84 and VL of sequence number 85, respectively. and at least 90% (for example, at least 90%, 91%, 92%, 93%, 94%, 9) Having identical amino acid sequences (5%, 96%, 97%, 98%, 99%, or 100%) The bispecific antigen-binding construct according to claim 16.

18. The first antigen-binding region is the same as sequence numbers 15, 16, 17, 33, 34, and 35, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, which have amino acid sequences and a bispecific antigen-binding construct according to claim 16 or 17, comprising LCDR3.

19. The first VH is at least 90% (for example, at least 90%, 91%) of the sequence number 3. %) 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) The first VL contains the same amino acid sequence, and the first VL is at least 90% (e.g., the first VL is identical to SEQ ID NO: 4). For example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, The dual according to claim 18, comprising amino acid sequences that are 98%, 99%, or 100% identical. Specific antigen binding constructs.

20. The first antigen-binding region comprises the first scFv including the first VH and the first VL. or comprising a first Fab, wherein the second antigen-binding region comprises the second VH and the second V A second Fab or second scFv including L, as described in any one of claims 16 to 19 The listed bispecific antigen-binding construct.

21. The first antigen-binding region includes the first scFv, and the second antigen-binding region is The bispecific antigen-binding construct according to claim 20, comprising the second Fab.

22. A bispecific antibody comprising a first heavy chain and a second heavy chain, wherein the first heavy chain is the first The second heavy chain comprises VH, optionally further comprising the first VL, and the second V It includes H, and optionally further includes the second VL, and each of the first and second heavy chains is 1 The immunoglobulin (Ig) constant region further comprises one or more heterodimer mutations, A bispecific antigen-binding construct according to any one of claims 16 to 21.

23. (4) Amino acids selected from the group consisting of SEQ ID NOs: 111, 111, 71, and 229 The array is identical to at least 80%, for example, at least 85%, 90%, 95%, or 100%. A first heavy chain having a certain amino acid sequence, (5) Selected from the group consisting of sequence numbers 117, 115, 117, and 117 respectively The amino acid sequence and at least 80%, for example, at least 85%, 90%, 95%, or 10% Light chains having 0% identical amino acid sequences, and (6) Selected from the group consisting of sequence numbers 116, 114, 118, and 230, respectively The amino acid sequence and at least 80%, for example, at least 85%, 90%, 95%, or 10% The bispecificity according to claim 22, comprising a second heavy chain having an amino acid sequence that is 0% identical. Antigen-binding construct.

24. The protein according to any one of claims 1 to 6, or the protein according to any one of claims 8 to 13 The multispecific antigen-binding construct described above, the fusion or condyloma described in claim 14 or 15 A combate, or a bispecific antigen-binding construct according to any one of claims 16 to 23 Isolated nucleic acids.

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

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

27. The protein according to any one of claims 1 to 6, or the protein according to any one of claims 8 to 13 The multispecific antigen-binding construct described herein, the fusion according to any one of claims 14 to 25, is a conjugate, or a bispecific antigen-binding structure according to any one of claims 16 to 23. A method for producing a structure, comprising the protein, the multispecific antigen-binding structure, and the melt Under conditions that produce the combined or conjugated, or the aforementioned bispecific antigen-binding constructs, To culture the host cells described in item 26, and to recover them from the cells or cell culture. A method that includes doing something.

28. The protein according to any one of claims 1 to 6, the immunoconjugate according to claim 7 T, a multispecific antigen-binding construct according to any one of claims 8 to 13, claims 14 to 2 A fusion or conjugate according to any one of claims 5, or any one of claims 16 to 23. A bispecific antigen-binding construct according to claim 1, a nucleic acid according to claim 24, and the nucleic acid according to claim 25. A vector, or a host cell according to claim 26, and a pharmaceutically acceptable carrier, comprising A pharmaceutical composition.

29. A method for treating patients who require treatment for DLL3-expressing cancer, and treatment An effective amount of the pharmaceutical composition according to claim 28, sufficient to treat the DLL3-expressing cancer. This includes administering the substance to the subject over a period of time, preferably if the cancer is lung cancer (details omitted). Alveolar lung cancer, etc.), prostate cancer (neuroendocrine prostate cancer, or recurrent, refractory, malignant, or (e.g., castration-resistant prostate cancer), glioma, glioblastoma, melanoma, neuroendocrine pancreatic cancer Selected from the group consisting of hepatoblastoma and hepatocellular carcinoma, or any combination thereof. method.

30. A method for reducing the amount of DLL3-expressing tumor cells in a target, and a claim for a therapeutically effective dose. When the pharmaceutical composition described in item 28 is sufficient to reduce the amount of DLL3-expressing tumor cells This includes administering the drug to the subject over a period of time, preferably the subject having lung cancer (small cell lung cancer). Lung cancer, etc.), prostate cancer (neuroendocrine prostate cancer, or recurrent, refractory, malignant, or (e.g., castration-resistant prostate cancer), glioma, glioblastoma, melanoma, neuroendocrine pancreatic cancer, Cancer selected from the group consisting of hepatoblastoma, hepatocellular carcinoma, or any combination thereof. A method that requires treatment.

31. A method for treating non-cancerous conditions in subjects at risk of developing DLL3-expressing cancer. The pharmaceutical composition according to claim 28 in a therapeutically effective amount is administered to the subject, and the non-cancer This includes treating sexual conditions, preferably such that the non-cancerous condition is benign prostatic hyperplasia or benign prostate. High prostate-specific antigen (PS) levels in the absence of prostate hyperplasia (BPH) or diagnosed prostate cancer. A) A method that has a level.

32. A method for detecting the presence of neuroendocrine prostate cancer or small cell lung cancer in a subject, The immune conjugate described in claim 7 is suspected to have prostate cancer or small cell lung cancer. The objective is to administer the substance to the target and to enable the biological structure to which the immune conjugate is bound. A method including visualizing and thereby detecting the presence of prostate cancer or small cell lung cancer. Law.

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