An anti-DLL3 antibody, its preparation method, its drug conjugate, and its applications.

Abstract

This invention discloses an anti-DLL3 antibody, its preparation method, its drug conjugate, and its applications. The anti-DLL3 antibody of this invention exhibits excellent internalization activity, superior binding activity to human DLL3 protein, and strong affinity at the protein level. The antibody-drug conjugate targeting DLL3 of this invention possesses excellent drug-likeness, biological activity, and in vitro and in vivo antitumor activity, enabling the application of cytotoxic drugs in the treatment of patients with neuroendocrine tumors, including SCLC.

Description

[0001] This application claims priority to Chinese patent application CN2021108755368, filed July 30, 2021, and Chinese patent application CN2022102395912, filed March 11, 2022. The full text of the aforementioned patent applications is incorporated herein by reference. Technical Field

[0002] This invention belongs to the fields of biotechnology and medicine, specifically relating to an anti-DLL3 antibody, its preparation method, its drug conjugates, and its applications. Background Technology

[0003] Small cell lung cancer (SCLC) is the most malignant type of lung cancer, characterized by rapid progression, early metastasis, and high recurrence rate, accounting for approximately 15%-20% of newly diagnosed lung cancers. Its occurrence is closely related to long-term smoking. SCLC is mainly divided into limited-stage and extensive-stage patients, with extensive-stage patients being the majority, accounting for about 70% of SCLC cases. Currently, the main treatment for SCLC is chemotherapy, with first-line chemotherapy primarily based on platinum-based combination therapy. The most commonly used combination is etoposide + cisplatin or carboplatin (EP or CE regimen). Guidelines recommend that both limited-stage and extensive-stage SCLC patients should receive 4-6 cycles of cisplatin / carboplatin combination therapy. The response rate for first-line treatment in limited-stage SCLC is 70%-90%, while the response rate for extensive-stage SCLC is 50%-60%. Currently, within one year of first-line treatment, approximately 80% of limited-stage SCLC patients and almost all extensive-stage SCLC patients experience recurrence or progression. Therefore, the effectiveness of second-line treatment is crucial to the final survival of SCLC patients. Topotecan monotherapy, as a standard second-line treatment, has a response rate of approximately 22% and an overall survival of about 8 months. However, for patients with drug resistance and relapse, the response rate is only about 4%, with a median survival of only 5 months, indicating extremely limited clinical benefit and significant clinical unmet needs. In summary, SCLC faces numerous challenges in treatment due to its high relapse and drug resistance rates, limited second-line treatment options, and limited patient survival benefits. New treatment approaches are urgently needed to address these clinical unmet needs. Current research indicates that inhibition of the Notch pathway is highly correlated with the development and progression of SCLC, and its ligand DLL3 is considered one of the most promising targets for SCLC treatment.

[0004] DLL3 is an atypical Notch pathway ligand identified by high-throughput sequencing. This ligand is a single-pass transmembrane protein composed of 619 amino acids. Its complete structure includes one DSL domain, one intracellular domain, and six epidermal growth factor-like domains (EGF1, EGF2, EGF3, EGF4, EGF5, and EGF6), making it an important target for the development and progression of SCLC. Immunohistochemistry shows that this target is almost not expressed in normal tissues and is also absent in other tumors. However, it is specifically and abundantly expressed in neuroendocrine tumors. Approximately 80% of SCLC tumor tissues and cancer cells exhibit high levels of DLL3 on their surface, and about 85% of recurrent SCLC also highly express DLL3 protein, making it a constitutive receptor for SCLC. The fact that DLL3 is not expressed in normal tissues but specifically expressed in neuroendocrine tumors such as SCLC makes it a potential target for antibody or antibody-drug conjugate (ADC) development.

[0005] Several drugs targeting DLL3 are currently under development, primarily including bispecific antibodies, cell therapy, and ADCs (see CN104520324A for example). Rova-T, developed by AbbVie, was the first ADC targeting DLL3 and the first targeted therapy for SCLC in clinical trials. This drug utilizes DLL3 expressed on the surface of tumor cells to recognize them and deliver the cytotoxic drug PBD into the tumor cells, thus achieving targeted killing of tumor cells. However, in Phase 3 clinical trials, the drug's efficacy was insufficient due to the severe toxic side effects of the cytotoxic drug PBD, which was intolerable to patients. Development of Rova-T has since been terminated. Summary of the Invention

[0006] The technical problem this invention aims to solve is to address the shortcomings of existing anti-DLL3 antibodies and the current situation where no DLL3-targeting antibody-drug conjugates (ADCs) have been successfully developed clinically. This invention provides a novel anti-DLL3 antibody, a DLL3-targeting ADC, its intermediate, preparation method, and applications. Addressing the lack of DLL3-targeting antibody-drug conjugates in existing technologies, the antibody-drug conjugate of this invention can achieve therapeutic effects for patients with neuroendocrine-related tumors, including SCLC. This invention mainly solves the above-mentioned technical problems through the following technical means.

[0007] This invention provides an anti-DLL3 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein,

[0008] The VH contains the following complementarity-determining regions (CDRs) or mutations thereof: VH CDR1 as shown by the amino acid sequence of SEQ ID NO: 9, 19, 29, 39, 59, 69, 79, 89, 99 or 63; VH CDR2 as shown by the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 60, 70, 80, 90, 100, 110 or 83; and / or VH CDR3 as shown by the amino acid sequence of SEQ ID NO: 11, 21, 31, 41, 61, 71, 81, 91, 101, 111 or 93;

[0009] The VL contains the following CDRs or mutations thereof: VL CDR1 as shown by the amino acid sequence of SEQ ID NO: 12, 32, 42, 62, 72, 82, 92, 102, 112 or 103; VLCDR2 as shown by GAS, GAT, TTS, NAK, YTS, RAN, WAS, FTS or NAN; and / or VL CDR3 as shown by the amino acid sequence of SEQ ID NO: 14, 24, 34, 44, 64, 74, 84, 94, 104, 114 or 113;

[0010] The mutation is defined as an insertion, deletion, or substitution of 3, 2, or 1 amino acid in the amino acid sequence of the CDR.

[0011] In this application, "amino acid mutation" in phrases like "having an insertion, deletion, or substitution of 3, 2, or 1 amino acid" refers to a mutation in the sequence of a variant relative to the original amino acid sequence, including the insertion, deletion, or substitution of amino acids based on the original amino acid sequence. An exemplary interpretation is that mutations in CDRs can include mutations of 3, 2, or 1 amino acid. These CDRs can optionally select the same or different numbers of amino acid residues for mutation; for example, CDR1 could have a 1-amino acid mutation, while CDR2 and CDR3 could not have any amino acid mutations.

[0012] In this application, the mutations may include mutations known to those skilled in the art, such as mutations that may be performed on antibodies during the production or application of antibodies, such as mutations on potential post-translational modifications (PTMs) sites, especially in the CDR region, including mutations related to antibody aggregation, asparagine deamidation sites (NG, NS, NH, etc.), aspartic acid isomerization (DG, DP) sensitive sites, N-glycosylation (N-{P}S / T) sensitive sites, and oxidation sensitive sites.

[0013] Preferably, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are as shown in SEQ ID NO: 9, 10, and 11, respectively; or, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are as shown in SEQ ID NO: 19, 20, and 21, respectively; or, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are as shown in SEQ ID NO: 29, 30, and 31, respectively; or, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are as shown in SEQ ID NO: 39, 40, and 41, respectively; or, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are as shown in SEQ ID NO: 59, 60, and 61, respectively; or, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are as shown in SEQ ID NO: 19, 20, and 21, respectively; or, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are as shown in SEQ ID NO: 19, 20, and 21, respectively. The amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are shown in SEQ ID NO: 79, 80, and 81, respectively; or the amino acid sequences of VH CDR1, VH CDR2, and VHCDR3 contained in the VH are shown in SEQ ID NO: 89, 90, and 91, respectively; or the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are shown in SEQ ID NO: 99, 100, and 101, respectively; or the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in the VH are shown in SEQ ID NO: 89, 110, and 111, respectively; or the amino acid sequences of VHCDR1, VH CDR2, and VH CDR3 contained in the VH are shown in SEQ ID NO: 63, 83, and 93, respectively.

[0014] Preferably, the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are as shown in SEQ ID NO:12, GAS, and SEQ ID NO:14, respectively; or, the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are as shown in SEQ ID NO:12, GAS, and SEQ ID NO:24, respectively; or, the amino acid sequences of VL CDR1, VL CDR2, and VLCDR3 contained in the VL are as shown in SEQ ID NO:32, GAT, and SEQ ID NO:34, respectively; or, the amino acid sequences of VLCDR1, VL CDR2, and VL CDR3 contained in the VL are as shown in SEQ ID NO:42, TTS, and SEQ ID NO:44, respectively; or, the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are as shown in SEQ ID NO:62, NAK, and SEQ ID NO:64, respectively; or, the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are as shown in SEQ ID NO:62, NAK, and SEQ ID NO:64, respectively; or, the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are as shown in SEQ ID NO:62, NAK, and SEQ ID NO:64, respectively. The amino acid sequences of CDR1, VL CDR2, and VL CDR3 are shown in SEQ ID NO:72, NAK, and SEQ ID NO:74, respectively; or, the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are shown in SEQ ID NO:82, YTS, and SEQ ID NO:84, respectively; or, the amino acid sequences of VL CDR1, VL CDR2, and VLCDR3 contained in the VL are shown in SEQ ID NO:92, RAN, and SEQ ID NO:94, respectively; or, the amino acid sequences of VLCDR1, VL CDR2, and VL CDR3 contained in the VL are shown in SEQ ID NO:102, WAS, and SEQ ID NO:104, respectively; or, the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are shown in SEQ ID NO:112, FTS, and SEQ ID NO:114, respectively; or, the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in the VL are shown in SEQ ID NO:112, FTS, and SEQ ID NO:114, respectively; or, the amino acid sequences of VL CDR1, VL CDR2, and VLCDR3 contained in the VL are shown in SEQ ID NO:72, NAK, and SEQ ID NO:74, respectively. The amino acid sequences of CDR2 and VL CDR3 are shown in SEQ ID NO:103, NAN and 113, respectively.

[0015] In a preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in VH are shown in SEQ ID NO:9, 10, and 11, respectively, and the amino acid sequences of VL CDR1, VLCDR2, and VL CDR3 contained in VL are shown in SEQ ID NO:12, GAS, and SEQ ID NO:14, respectively. In another preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in VH are shown in SEQ ID NO:19, 20, and 21, respectively, and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in VL are shown in SEQ ID NO:12, GAS, and SEQ ID NO:24, respectively. In a preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in VH are shown in SEQ ID NO:29, 30, and 31, respectively, and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in VL are shown in SEQ ID NO:32, GAT, and SEQ ID NO:34, respectively. In another preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in VH are shown in SEQ ID NO:39, 40, and 41, respectively, and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in VL are shown in SEQ ID NO:42, TTS, and SEQ ID NO:44, respectively. In a preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VHCDR2, and VH CDR3 contained in VH are shown in SEQ ID NO:59, 60, and 61, respectively, and the amino acid sequences of VLCDR1, VL CDR2, and VL CDR3 contained in VL are shown in SEQ ID NO:62, NAK, and SEQ ID NO:64, respectively. In another preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in VH are shown in SEQ ID NO:69, 70, and 71, respectively, and the amino acid sequences of VL CDR1, VL CDR2, and VLCDR3 contained in VL are shown in SEQ ID NO:72, NAK, and SEQ ID NO:74, respectively.In a preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in VH are shown in SEQ ID NO:79, 80, and 81, respectively, and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in VL are shown in SEQ ID NO:82, YTS, and SEQ ID NO:84, respectively. In another preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in VH are shown in SEQ ID NO:89, 90, and 91, respectively, and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in VL are shown in SEQ ID NO:92, RAN, and SEQ ID NO:94, respectively. In a preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 contained in VH are shown in SEQ ID NO:99, 100, and 101, respectively, and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 contained in VL are shown in SEQ ID NO:102, WAS, and SEQ ID NO:104, respectively. In a preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VHCDR2, and VH CDR3 contained in VH are shown in SEQ ID NO:89, 110, and 111, respectively, and the amino acid sequences of VLCDR1, VL CDR2, and VL CDR3 contained in VL are shown in SEQ ID NO:112, FTS, and SEQ ID NO:114, respectively. In a preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 contained in VH are shown as SEQ ID NO:63, 83 and 93, respectively, and the amino acid sequences of VL CDR1, VL CDR2 and VLCDR3 contained in VL are shown as SEQ ID NO:103, NAN and 113, respectively.

[0016] Preferably, in the anti-DLL3 antibody, the heavy chain variable region (VH) further includes a heavy chain variable region framework region (VH FWR), wherein the VH FWR is the heavy chain variable region framework region of a human or mouse antibody. In a preferred embodiment of the invention, the VH may include an amino acid sequence such as SEQ ID NO: 15, 25, 35, 45, 65, 75, 85, 95, 105, 13 or 22 or a mutation thereof.

[0017] Preferably, in the anti-DLL3 antibody, the light chain variable region (VL) further includes a light chain variable region framework region (VL FWR); wherein the VL FWR is the light chain variable region framework region of a human or mouse antibody. In a preferred embodiment of the present invention, the VL may include an amino acid sequence such as SEQ ID NO: 16, 26, 36, 46, 66, 76, 86, 96, 106, 33 or 23 or a mutation thereof.

[0018] In one preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:15 or a mutation thereof, and the VL comprises an amino acid sequence as shown in SEQ ID NO:16 or a mutation thereof. In one preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:25 or a mutation thereof, and the VL comprises an amino acid sequence as shown in SEQ ID NO:26 or a mutation thereof. In one preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:35 or a mutation thereof, and the VL comprises an amino acid sequence as shown in SEQ ID NO:36 or a mutation thereof. In one preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:45 or a mutation thereof, and the VL comprises an amino acid sequence as shown in SEQ ID NO:46 or a mutation thereof. In one preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:65 or a mutation thereof, and the VL comprises an amino acid sequence as shown in SEQ ID NO:66 or a mutation thereof. In one preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:75 or a mutation thereof, and the VL comprises an amino acid sequence as shown in SEQ ID NO:76 or a mutation thereof. In one preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:85 or a mutation thereof, and the VL comprises an amino acid sequence as shown in SEQ ID NO:86 or a mutation thereof. In one preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:95 or a mutation thereof, and the VL comprises an amino acid sequence as shown in SEQ ID NO:96 or a mutation thereof. In one preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:105 or a mutation thereof, and the VL comprises an amino acid sequence as shown in SEQ ID NO:106 or a mutation thereof. In one preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:13 or a mutation thereof, and the VL comprises an amino acid sequence as shown in SEQ ID NO:33 or a mutation thereof. In one preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:22 or a mutation thereof, and the VL comprises an amino acid sequence as shown in SEQ ID NO:23 or a mutation thereof.

[0019] The above mutation is the deletion, substitution, or addition of one or more amino acid residues in the amino acid sequence of VH and / or VL, and the mutated amino acid sequence has at least 85% sequence identity with the amino acid sequence of VH and / or VL, and maintains or improves the binding of the antibody to DLL3; the at least 85% sequence identity is preferably at least 90% sequence identity, more preferably at least 95%, 96%, 97%, or 98% sequence identity, and most preferably at least 99% sequence identity.

[0020] In this application, the amino acid sequences of the CDRs listed above are all as shown in the Kabat definition rules (and are also shown in the Kabat definition rules in this application). However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Immunological Protein Sequences, 5th Edition, National Institutes of Health, Bethesda, Maryland (1991)) and the Chothia definition rules based on the location of structural loop regions (see J.Mol Biol 273:927-48, 1997). Those skilled in the art should understand that, unless otherwise specified, the terms “CDR” and “complementarity-determining region” for a given antibody or its region (e.g., a variable region) should be understood to encompass complementarity-determining regions defined as described above by the present invention. While the scope of protection claimed in this invention is based on the sequences shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0021] Preferably, the anti-DLL3 antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv (preferably scFv), bispecific antibody, multispecific antibody, heavy chain antibody, or single-domain antibody.

[0022] In a preferred embodiment, the anti-DLL3 antibody is a full-length antibody, comprising a heavy chain and a light chain; the heavy chain constant region included in the heavy chain is preferably a human or mouse antibody heavy chain constant region; the light chain constant region included in the light chain is preferably a human or mouse antibody light chain constant region. Preferably, the human light chain constant region is a human κ or λ light chain constant region; the human heavy chain constant region is human IgG1, IgG2, IgG3, or IgG4.

[0023] In a preferred embodiment, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 17, 27, 37, 47, 67, 77, 87, 97, 107, 43 or 73 or a mutation thereof, and / or the light chain comprises an amino acid sequence as shown in SEQ ID NO: 18, 28, 38, 48, 68, 78, 88, 98, 108, 53 or 109 or a mutation thereof.

[0024] In one preferred embodiment, the full-length antibody comprises, in the case of, the heavy chain as shown in SEQ ID NO:17 or a mutation thereof, and the light chain as shown in SEQ ID NO:18 or a mutation thereof. In one preferred embodiment, the heavy chain comprises, in the case of, the amino acid sequence shown in SEQ ID NO:27 or a mutation thereof, and the light chain comprises, in the case of, SEQ ID NO:28 or a mutation thereof. In one preferred embodiment, the heavy chain comprises, in the case of, the amino acid sequence shown in SEQ ID NO:37 or a mutation thereof, and the light chain comprises, in the case of, SEQ ID NO:38 or a mutation thereof. In one preferred embodiment, the heavy chain comprises, in the case of, the amino acid sequence shown in SEQ ID NO:47 or a mutation thereof, and the light chain comprises, in the case of, SEQ ID NO:48 or a mutation thereof. In one preferred embodiment, the heavy chain comprises, in the case of, the amino acid sequence shown in SEQ ID NO:67 or a mutation thereof, and the light chain comprises, in the case of, SEQ ID NO:68 or a mutation thereof. In one preferred embodiment, the heavy chain comprises, in the case of, the amino acid sequence shown in SEQ ID NO:77 or a mutation thereof, and the light chain comprises, in the case of, SEQ ID NO:78 or a mutation thereof. In one preferred embodiment, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:87 or a mutation thereof, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:88 or a mutation thereof. In one preferred embodiment, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:97 or a mutation thereof, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:98 or a mutation thereof. In one preferred embodiment, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:107 or a mutation thereof, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:108 or a mutation thereof. In one preferred embodiment, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:43 or a mutation thereof, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:53 or a mutation thereof. In one preferred embodiment, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:73 or a mutation thereof, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:109 or a mutation thereof.

[0025] The above-mentioned mutation is the deletion, substitution, or addition of one or more amino acid residues in the amino acid sequence of the heavy chain and / or light chain, and the mutated amino acid sequence has at least 85% sequence identity with the amino acid sequence of the heavy chain and / or light chain, and maintains or improves the binding of the antibody to DLL3; the at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95%, 96%, 97%, or 98% sequence identity; and most preferably at least 99% sequence identity.

[0026] In one aspect, the present invention also provides an anti-DLL3 antibody that competitively binds to the DLL3 protein, in contrast to the anti-DLL3 antibody described above.

[0027] In one aspect, the present invention also provides an isolated nucleic acid encoding the anti-DLL3 antibody as described above.

[0028] In one aspect, the present invention also provides a recombinant expression vector comprising the isolated nucleic acid as described above. Preferably, the expression vector comprises a eukaryotic cell expression vector and / or a prokaryotic cell expression vector.

[0029] In one aspect, the present invention also provides a transformant comprising the recombinant expression vector as described above. Preferably, the host cells of the transformant are prokaryotic and / or eukaryotic cells, wherein the prokaryotic cells are preferably E. coli cells such as TG1 or BL21 cells, and the eukaryotic cells are preferably HEK293 cells or CHO cells.

[0030] In one aspect, the present invention also provides an antibody-drug conjugate (ADC) with the general structural formula Ab-(L3-L2-L1-D). m ;

[0031] Among them, Ab is an anti-DLL3 antibody;

[0032] D is a cytotoxic drug.

[0033] m is 2 to 8;

[0034] The structure of L1 is shown in Formula I, II, III or IV, with its a end connected to the cytotoxic drug and its e end connected to the c end of L2.

[0035]

[0036] (L) pIn this context, L independently represents one or more of the following residues: phenylalanine residue, alanine residue, glycine residue, glutamic acid residue, aspartic acid residue, cysteine ​​residue, glutamic acid residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue, and valine residue; p is 2-4.

[0037] R 1 For one or more -NR 1-1 R 1-2 Substituted C1-C6 alkyl groups, with one or more R 1-3 S(O)2-substituted C1-C6 alkyl, C1-C6 alkyl, C3-C 10 cycloalkyl, C6-C 14 Aryl or 5-14 membered heteroaryl; the heteroatom in the 5-14 membered heteroaryl is selected from one or more of N, O and S, and the number of heteroatoms is 1, 2, 3 or 4; the R 1-1 R 1-2 and R 1-3 Each is independently a C1 to C6 alkyl group;

[0038] L2 is

[0039] Where n is independently 1 to 12, for example 8, 9, 10, 11 and 12, the c end is connected to L1 through a carbonyl group, and the f end is connected to the d end of L3.

[0040] L3 is The b end is connected to the Ab end, and the d end is connected to the f end of the L2 end.

[0041] In a preferred embodiment, the anti-DLL3 antibody binds to one of the following antigen-binding epitopes in the DLL3 protein: the DSL domain, the N-terminus, the EGF2 domain, and the EGF3-EGF6 domain.

[0042] In a preferred embodiment, the anti-DLL3 antibody is preferably the anti-DLL3 antibody as described above.

[0043] In a preferred embodiment, the anti-DLL3 antibody binds to the antigen-binding epitope of the EGF2 domain in the DLL3 protein.

[0044] In a preferred embodiment, the anti-DLL3 antibody comprises VH with an amino acid sequence as shown in SEQ ID NO:15 and VL with an amino acid sequence as shown in SEQ ID NO:16, or comprises VH with an amino acid sequence as shown in SEQ ID NO:25 and VL with an amino acid sequence as shown in SEQ ID NO:26.

[0045] In a preferred embodiment, the anti-DLL3 antibody is an anti-DLL3 antibody having a light chain with an amino acid sequence as shown in SEQ ID NO:1 and a heavy chain with an amino acid sequence as shown in SEQ ID NO:2, or an anti-DLL3 antibody having a light chain with an amino acid sequence as shown in SEQ ID NO:3 and a heavy chain with an amino acid sequence as shown in SEQ ID NO:4.

[0046] In a preferred embodiment, L is one or more of phenylalanine residue, alanine residue, glycine residue, isoleucine residue, leucine residue, proline residue and valine residue, preferably one or more of phenylalanine residue, alanine residue, glycine residue and valine residue.

[0047] In a preferred embodiment, L is a valine residue and / or an alanine residue, the plurality of residues refers to two or three, and p is 2. More preferably, (L) p for The g-terminus is connected to the c-terminus of L2 via a carbonyl group.

[0048] In a preferred embodiment, the R 1 For one or more -NR 1-1 R 1-2 Substituted C1-C6 alkyl groups, with one or more R 1-3 S(O)2-substituted C1-C6 alkyl, or C1-C6 alkyl, wherein R 1-1 R 1-2 and R 1-3 Each is independently a C1 to C4 alkyl group.

[0049] In a preferred embodiment, when the R 1 For one or more -NR 1-1 R 1-2 When the substituted C1-C6 alkyl group is used, the C1-C6 alkyl group is a C1-C4 alkyl group, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, preferably ethyl; the plurality of substituted alkyl groups is preferably two or three. More preferably, the R... 1-1 and R 1-2Each of the components is independently a C1-C4 alkyl group, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, more preferably methyl. Even more preferably, the -NR... 1-1 R 1-2 It is -N(CH3)2. More preferably, when the R... 1 For a -NR 1-1 R 1-2 When the C1-C6 alkyl group is substituted, the substance is replaced by a -NR 1-1 R 1-2 The substituted C1-C6 alkyl groups are

[0050] In a preferred embodiment, when the R 1 For one or more R 1-3 When S(O)2-substituted C1-C6 alkyl groups are used, the C1-C6 alkyl groups are C1-C4 alkyl groups, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and more preferably ethyl; the plurality of alkyl groups is preferably two or three. Preferably, the R... 1-3 It is a C1-C4 alkyl group, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, more preferably methyl. More preferably, when the R... 1 For being an R 1-3 When S(O)2- is substituted with a C1- to C6 alkyl group, the substance being replaced by an R 1-3 S(O)2-substituted C1- to C6 alkyl groups are

[0051] In a preferred embodiment, when the R 1 When the alkyl group is C1 to C6, the C1 to C6 alkyl group is C1 to C4 alkyl group, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and even more preferably methyl.

[0052] In a preferred embodiment, when the structure of L1 is as shown in Formula I, L2 is preferably... The L3 is preferably...

[0053] In a preferred embodiment, when the structure of L1 is as shown in Formula II, L2 is preferably... The L3 is preferably...

[0054] In a preferred embodiment of the present invention, when the structure of L1 is as shown in Formula III, L2 is preferably... The L3 is preferably...

[0055] In a preferred embodiment of the present invention, when the structure of L1 is as shown in Formula IV, L2 is preferably... The L3 is preferably...

[0056] In a preferred embodiment, the structure of L1 is preferably as shown in Formula I or III.

[0057] In a preferred embodiment, Formula III is preferably...

[0058]

[0059] In a preferred embodiment, Formula III is further preferably...

[0060]

[0061] In a preferred embodiment, the b-terminus of L3 is preferably linked to a thiol group on the antibody via a thioether bond. For example, The linkage form with the cysteine ​​residues in the antibody is as follows:

[0062] In a preferred embodiment, m is an integer from 2 to 8, such as 2, 3, 4, 5, 6, 7, 8, or a non-integer, such as 7.68, 7.53, 4.43, 7.12, 6.92, 7.43, 7.23, 6.83, 7.32, 7.56, 7.54, 7.47, 5.82, 6.78, 2.28, 6.32, 7.45, 7.65, 7.64, 7.36, 7.75, 7.80, 7.77, or 7.76.

[0063] Preferably, the antibody-drug conjugate is any of the following compounds:

[0064]

[0065]

[0066]

[0067]

[0068]

[0069] Wherein Ab is the aforementioned anti-DLL3 antibody, and m is 2 to 8, preferably 7.68, 7.53, 4.43, 7.12, 6.92, 7.43, 7.23, 6.83, 7.32, 7.56, 7.54, 7.47, 5.82, 6.78, 2.28, 6.32, 7.45, 7.65, 7.64, 7.36, 7.75, 7.80, 7.77, or 7.76.

[0070] In a preferred embodiment, the antibody-drug conjugate is any of the following compounds:

[0071]

[0072]

[0073]

[0074] The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:25 and the amino acid sequence VL as shown in SEQ ID NO:26; preferably, it contains a heavy chain as shown in SEQ ID NO:27 and a light chain as shown in SEQ ID NO:28.

[0075] In a preferred embodiment, the antibody-drug conjugate is any of the following compounds:

[0076]

[0077]

[0078]

[0079] The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:15 and the amino acid sequence VL as shown in SEQ ID NO:16; preferably, it contains a heavy chain as shown in SEQ ID NO:17 and a light chain as shown in SEQ ID NO:18.

[0080] In a preferred embodiment, the antibody-drug conjugate is a compound as shown below:

[0081] Ab is an anti-DLL3 antibody, which contains a heavy chain with an amino acid sequence as shown in SEQ ID NO:2 and a light chain with an amino acid sequence as shown in SEQ ID NO:1.

[0082] In a preferred embodiment, the antibody-drug conjugate is a compound as shown below:

[0083] Ab is an anti-DLL3 antibody, which contains a heavy chain with an amino acid sequence as shown in SEQ ID NO:4 and a light chain with an amino acid sequence as shown in SEQ ID NO:3.

[0084] In a preferred embodiment, the antibody-drug conjugate is a compound as shown below:

[0085] The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:22 and the amino acid sequence VL as shown in SEQ ID NO:23; preferably, it contains a heavy chain as shown in SEQ ID NO:73 and a light chain as shown in SEQ ID NO:109.

[0086] In a preferred embodiment, the antibody-drug conjugate is a compound as shown below:

[0087] The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:45 and the amino acid sequence VL as shown in SEQ ID NO:46; preferably, it contains a heavy chain as shown in SEQ ID NO:47 and a light chain as shown in SEQ ID NO:48.

[0088] In one aspect, the present invention also provides a chimeric antigen receptor comprising the anti-DLL3 antibody as described above.

[0089] In one aspect, the present invention also provides a genetically modified cell comprising the chimeric antigen receptor as described above. Preferably, the genetically modified cell is a eukaryotic cell, more preferably an isolated human cell; more preferably, an immune cell such as a T cell or an NK cell.

[0090] In one aspect, the present invention also provides a method for preparing an anti-DLL3 antibody, the method comprising the following steps: culturing a transformant as described above, and obtaining an anti-DLL3 antibody from the culture.

[0091] In one aspect, the present invention also provides a pharmaceutical composition comprising the anti-DLL3 antibody as described above, the antibody-drug conjugate as described above, the chimeric antigen receptor as described above, and / or the genetically modified cells as described above. Preferably, the pharmaceutical composition is in a liquid, gaseous, solid, or semi-solid dosage form, and / or, the pharmaceutical composition can be administered orally, by injection, nasal administration, transdermal administration, or mucosal administration. More preferably, the pharmaceutical composition further comprises a combination therapeutic agent, which includes a chemotherapeutic agent, a radiotherapy agent, an immunosuppressant, and / or a cytotoxic drug.

[0092] One aspect of the present invention also provides the use of the anti-DLL3 antibody, the antibody-drug conjugate, the chimeric antigen receptor, the genetically modified cell, and / or the pharmaceutical composition described above in the preparation of medicaments, kits, and / or delivery devices for treating and / or preventing diseases related to abnormal DLL3 expression; or the use of the anti-DLL3 antibody, the antibody-drug conjugate, the chimeric antigen receptor, the genetically modified cell, and / or the pharmaceutical composition described above in the treatment and / or prevention of diseases related to abnormal DLL3 expression. The DLL3 expression abnormality-related disease is preferably a tumor, the tumor is preferably cancer, the cancer is preferably an endocrine tumor such as neuroendocrine tumor, prostate cancer, pancreatic cancer, and colorectal cancer, and more preferably small cell lung cancer.

[0093] One aspect of the present invention also provides a kit comprising the anti-DLL3 antibody as described above, the antibody-drug conjugate as described above, the chimeric antigen receptor as described above, the genetically modified cells as described above, and / or the pharmaceutical composition as described above; and optionally, a specification.

[0094] In one aspect, the present invention also provides a drug delivery device comprising: (1) an infusion module for administering the drug composition as described above to a subject in need, and (2) an optional drug efficacy monitoring module.

[0095] In one aspect, the present invention also provides a method for detecting DLL3, comprising the step of using an anti-DLL3 antibody as described above. Preferably, the method is for non-diagnostic and / or therapeutic purposes.

[0096] In one aspect, the present invention also provides a method for diagnosing, preventing, and / or treating diseases related to abnormal DLL3 expression, comprising administering to a subject in need an anti-DLL3 antibody as described above, an antibody-drug conjugate as described above, genetically modified cells as described above, and / or a pharmaceutical composition as described above. The DLL3 expression abnormality-related disease is preferably a tumor, the tumor is preferably cancer, the cancer is preferably a neuroendocrine tumor, and more preferably small cell lung cancer.

[0097] The present invention also provides an antibody group (including molecules comprising or composed of antibody fragments or variants), wherein the group members correspond to one, two, three, four, five, or more different antibodies of the present invention [e.g., complete antibodies, Fab, F(ab)2 fragments, and scFv, etc.].

[0098] In one aspect, this invention also provides an antibody-drug conjugate with the general structural formula Ab-(L5-L4-L3-L2-L1-D). m ;

[0099] Wherein, Ab is an anti-DLL3 antibody; the anti-DLL3 antibody binds to the DSL domain and / or N-terminus of the DLL3 protein;

[0100] D is a cytotoxic drug;

[0101] m is 2 to 8;

[0102] The structure of L1 is shown in Formula I, II, III or IV, with its a end connected to the cytotoxic drug and its e end connected to the c end of L2.

[0103]

[0104] (L) p In this context, L independently represents one or more of the following residues: phenylalanine residue, alanine residue, glycine residue, glutamic acid residue, aspartic acid residue, cysteine ​​residue, glutamic acid residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue, and valine residue; p is 2-4.

[0105] R 1 For one or more -NR 1-1 R 1-2 Substituted C1-C6 alkyl groups, with one or more R 1-3 S(O)2-substituted C1-C6 alkyl, C1-C6 alkyl, C3-C 10 cycloalkyl, C6-C 14Aryl or 5-14 membered heteroaryl; the heteroatom in the 5-14 membered heteroaryl is selected from one or more of N, O and S, and the number of heteroatoms is 1, 2, 3 or 4; the R 1-1 R 1-2 and R 1-3 Each is independently a C1 to C6 alkyl group;

[0106] L2 is Where n is independently 1 to 12, the c end is connected to L1 through a carbonyl group, and the f end is connected to the d end of L3.

[0107] L3 is Wherein, end b is connected to Ab, and end d is connected to end f of L2;

[0108] The L4 is absent, or is selected from cuttable connectors, non-cuttable connectors, hydrophilic connectors, pre-charged connectors, and dicarboxylic acid-based connectors;

[0109] The L5 is absent, or is a compound represented by the following formula V:

[0110]

[0111] Wherein, X1 is selected from hydrogen atom, halogen, hydroxyl, cyano, alkyl, alkoxy and cycloalkyl;

[0112] X2 is selected from alkyl, cycloalkyl and heterocyclic groups; m is 0-5; S is a sulfur atom.

[0113] In a preferred embodiment, D is a cytotoxic drug containing a hydroxyl, thiol, or amino group, such as a microtubule inhibitor and / or a topoisomerase inhibitor.

[0114] In a preferred embodiment, L is one or more of phenylalanine residue, alanine residue, glycine residue, isoleucine residue, leucine residue, proline residue and valine residue, preferably one or more of phenylalanine residue, alanine residue, glycine residue and valine residue; more preferably, L is valine residue and / or alanine residue, the plurality of residues is two or three, and p is 2.

[0115] In a preferred embodiment, the R 1 For one or more -NR 1-1 R 1-2 Substituted C1-C6 alkyl groups, with one or more R 1-3 S(O)2-substituted C1-C6 alkyl, or C1-C6 alkyl, wherein R 1-1 R 1-2 and R1-3 Each is independently a C1 to C4 alkyl group.

[0116] In a preferred embodiment, when the structure of L1 is as shown in Formula I, L2 is...

[0117] In a preferred embodiment, when the structure of L1 is as shown in Formula II, the L2 is...

[0118] In a preferred embodiment, when the structure of L1 is as shown in Formula III, the L2 is...

[0119] In a preferred embodiment, when the structure of L1 is as shown in Formula IV, the L2 is...

[0120] In a preferred embodiment, n is independently 8, 9, 10, 11, and 12.

[0121] In a preferred embodiment, m is an integer or non-integer of 2 to 8.

[0122] In a preferred embodiment, L3 is

[0123] In a preferred embodiment, L4 is selected from N-succinimide-4-(2-pyridyldithio)valerate (SPP), N-succinimide-(4-iodoacetyl)aminobenzoate (SIAB), N-succinimide-4-(maleimidemethyl)cyclohexanecarboxylate (SMCC), 6-maleimide-hexanoyl (MC), maleimide-propionyl (MP), valine-citrulline (VC), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and MC-VC-PAB.

[0124] In a preferred embodiment, in L5, when X1 is a hydrogen atom, X2 is an alkyl group, and m is 1, the compound represented by formula V is S-(3-carbonylpropyl) thioacetate.

[0125] In a preferred embodiment, D is one or more of maytansine derivatives, sea haretoxin 10 derivatives, doxorubicin analogs, or camptothecin analogs.

[0126] The maytansin derivatives include, for example, DM1, DM3 and DM4.

[0127] The sea haretoxin 10 derivatives include, for example, MMAE and MMAF.

[0128] Doxorubicin analogues include, for example, doxorubicin and camsirubicin.

[0129] More preferably, D is a camptothecin analogue, and even more preferably a compound with the structure shown in formula Va or Vb:

[0130]

[0131] R 2 and R 5 Each can be independently H, C1-C6 alkyl, or halogen;

[0132] R 3 and R 6 Each can be independently H, C1-C6 alkyl, or halogen;

[0133] R 4 and R 7 Each is independently a C1-C6 alkyl group.

[0134] In a preferred embodiment, the (L) p for The g-terminus is connected to the c-terminus of L2 via a carbonyl group.

[0135] In a preferred embodiment, Formula III is:

[0136]

[0137] In a preferred embodiment, the antibody-drug conjugate is any of the following compounds:

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144] The anti-DLL3 antibody is an anti-DLL3 antibody having a light chain with an amino acid sequence as shown in SEQ ID NO:1 and a heavy chain with an amino acid sequence as shown in SEQ ID NO:2, or an anti-DLL3 antibody having a light chain with an amino acid sequence as shown in SEQ ID NO:3 and a heavy chain with an amino acid sequence as shown in SEQ ID NO:4.

[0145] The value of m is 7.23 or 7.32.

[0146] Preferably, the antibody-drug conjugate is any of the following compounds:

[0147] Ab is an anti-DLL3 antibody, which contains a heavy chain with an amino acid sequence as shown in SEQ ID NO:2 and a light chain with an amino acid sequence as shown in SEQ ID NO:1.

[0148] Ab is an anti-DLL3 antibody, which contains a heavy chain with an amino acid sequence as shown in SEQ ID NO:4 and a light chain with an amino acid sequence as shown in SEQ ID NO:3.

[0149] In one aspect, the present invention also provides a method for preparing the antibody-drug conjugate as described herein, the method comprising the following steps: adding an excess of the linker-drug conjugate dissolved in DMSO dropwise into a buffer containing anti-DLL3 antibody to obtain the antibody-drug conjugate.

[0150] In another aspect, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate as described herein.

[0151] Preferably, the pharmaceutical composition is a liquid, gaseous, solid, or semi-solid dosage form, and / or the pharmaceutical composition can be administered orally, by injection, nasal administration, transdermal administration, or mucosal administration.

[0152] More preferably, the pharmaceutical composition further comprises a combination therapy agent, which includes a chemotherapeutic agent, a radiotherapy agent, an immunosuppressant, and / or a cytotoxic drug.

[0153] In another aspect, the present invention also provides the use of antibody-drug conjugates and / or pharmaceutical compositions as described herein in the preparation of medicaments, kits and / or delivery devices for treating and / or preventing diseases related to abnormal DLL3 expression;

[0154] The diseases associated with abnormal DLL3 expression are preferably tumors, the tumors are preferably cancers, the cancers are preferably neuroendocrine tumors, and more preferably small cell lung cancer.

[0155] In one aspect, the present invention also provides a kit comprising an antibody-drug conjugate as described in the present invention and / or a pharmaceutical composition as described in the present invention; and optionally, a specification.

[0156] In one aspect, the present invention also provides a drug delivery device, characterized in that the drug delivery device comprises: (1) an infusion module for administering an antibody-drug conjugate and / or a drug composition as described in the present invention to a subject in need, and (2) an optional drug efficacy monitoring module.

[0157] Another aspect of the present invention provides a method for detecting DLL3, characterized in that it includes the step of detection using an antibody-drug conjugate as described in the present invention. Preferably, the method for detecting DLL3 is for non-diagnostic and / or therapeutic purposes.

[0158] In one aspect, the present invention also provides a method for diagnosing, preventing, and / or treating diseases related to abnormal DLL3 expression, comprising administering to a subject in need an antibody-drug conjugate as described in the present invention and / or a pharmaceutical composition as described in the present invention.

[0159] In this invention, the conditions and operations of the coupling reaction can be the conventional conditions and operations of the coupling reaction in the art.

[0160] In this invention, m represents the molar ratio of cytotoxic drug molecules to Ab (also known as DAR, i.e., drug-antibody conjugation ratio). m can be an integer or a decimal. Preferably, it is understood as the average molar ratio of drug molecules to monoclonal antibody molecules in the antibody-drug conjugate obtained after conjugating monoclonal antibody molecules with cytotoxic drugs. It can generally be determined by hydrophobic-interaction chromatography (HIC), polyacrylamide-SDS gel electrophoresis (SDS-PAGE), liquid chromatography-mass spectrometry (LC-MS), etc.

[0161] In this invention, the term "C1-C6 alkyl" alone or in combination refers to a saturated straight-chain or branched alkyl group containing 1 to 6, particularly 1 to 4, carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and "C1-C6 alkyl" is preferably methyl or ethyl.

[0162] The antibodies of the present invention can be prepared using techniques well known in the field, such as hybridoma methods, recombinant DNA technology, phage display technology, synthesis technology, or combinations thereof, or other techniques known in the field.

[0163] The terms “selectivity” or “specificity” as used herein refer to the fact that the disclosed antagonists do not exhibit significant binding to substances other than DLL3, except in those special cases where the antagonist is supplemented to have a different specificity than the DLL3-specific binding portion (e.g., a bispecific or bifunctional molecule, wherein the molecule is designed to bind or perform two functions, at least one of which is a specific binding to DLL3).

[0164] As used herein, "antibody molecule" or "antibody" refers to immunoglobulin molecules and the immunoactive portion of immunoglobulin molecules, i.e., molecules containing antigen-binding sites that specifically bind to immune antigens. Therefore, the term antibody encompasses not only complete antibody molecules but also fragments of said antibodies and variants (including derivatives) of said antibodies and antibody fragments. The term antibody molecule as used herein includes, but is not limited to, single-chain Fv (scFv), Fab fragments, Fab' fragments, F(ab')2, disulfide-linked Fv (sdFv), Fv, and complete or full-length antibodies. The term "single-chain Fv" or "scFv" refers to a polypeptide containing a VL domain of an antibody linked to the VH domain of the antibody. Antibodies that specifically bind DLL3 can cross-react with other antigens. Preferably, antibodies that specifically bind DLL3 do not cross-react with other antigens. Antibodies that specifically bind DLL3 can be identified, for example, by immunoassay or other methods known to those skilled in the art. A “complete antibody” or “full-length antibody” refers to a protein comprising two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, the protein comprising: (1) a heavy chain variable region (hereinafter abbreviated as “VH”) and a heavy chain constant region containing three domains CH1, CH2, and CH3, with respect to the heavy chain; and (2) a light chain variable region (hereinafter abbreviated as “VL”) and a light chain constant region containing one domain CL, with respect to the light chain. The antibodies of the present invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab′) fragments, anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies of the present invention), and epitope-binding fragments of any of the above antibodies. The immunoglobulin molecules of the present invention can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulins. The antibodies of the present invention can be monoclonal or polyclonal antibodies, preferably mouse anti-human monoclonal antibodies. The antibodies of the present invention can be hyperhumanized antibodies or biclonal antibodies.

[0165] In this application, the term "heavy chain antibody" refers to an antibody containing only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as HCAbs.

[0166] "Single-domain antibody," also known as "nanobody," refers to the VHH structure cloned from heavy chain antibodies, which is the smallest known unit that can bind to a target antigen.

[0167] The amino acid sequences described in this invention as having "90%, 95%, 98%, or 99% or more homology" are obtained by inserting, deleting, or substituting the amino acid sequences shown in the aforementioned sequence listing. Substitution may include, for example, computer structural simulation analysis of the sequence, analysis of potential post-translational modifications (PTMs) sites, particularly in the CDR region, including analysis and substitution of sites such as antibody aggregation, asparagine deamidation (NG, NS, NH, etc.), aspartic acid isomerization (DG, DP) sensitive sites, N-glycosylation (N-{P}S / T) sensitive sites, and oxidation sensitive sites.

[0168] "KD" refers to the dissociation constant derived from the ratio of Kd (the dissociation rate of the specific bound molecule-target protein interaction) to Ka (the binding rate of the specific bound molecule-target protein interaction) (or Kd / Ka, expressed as molar concentration (M)). KD values ​​can be determined using methods well-established in the art. A preferred method for determining the KD of bound molecules is through the use of surface plasmon resonance, such as a biosensor system like the Biacore™ (GE Healthcare Life Sciences) system.

[0169] The term "treatment" or its equivalents, when used, for example, in the context of cancer, refers to a procedure or process used to reduce or eliminate the number of cancer cells in a patient's body or to alleviate the symptoms of cancer. "Treatment" for cancer or other proliferative disorders does not necessarily mean that cancer cells or other disorders will actually be eliminated, that the number of cells or disorders will actually be reduced, or that the symptoms of cancer or other disorders will actually be alleviated. Often, methods for treating cancer are undertaken even with only a low probability of success, but are still considered to induce an overall beneficial effect, taking into account the patient's medical history and estimated survival expectations.

[0170] The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium capable of delivering an effective amount of the active substance of this invention without interfering with the biological activity of the active substance and without toxic side effects on the host or patient. Representative carriers include water, oil, vegetables and minerals, ointment bases, lotion bases, and ointment bases. These bases include suspending agents, thickeners, transdermal penetration enhancers, etc. Their formulations are well known to those skilled in the art of cosmetics or topical pharmaceuticals.

[0171] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0172] The reagents and raw materials used in this invention are all commercially available.

[0173] The positive and progressive effects of this invention are as follows: the anti-DLL3 antibody of this invention has excellent internalization activity, good binding activity to human DLL3 protein, and strong affinity at the protein level. The antibody-drug conjugate of this invention has excellent drug-likeness, biological activity, and in vitro and in vivo antitumor activity, which enables the application of cytotoxic drugs in the treatment of patients with neuroendocrine tumors, including SCLC. Attached Figure Description

[0174] Figure 1 This is a spectrum of the pV81 vector.

[0175] Figure 2 The results of antibody internalization in Example 4 are shown.

[0176] Figure 3 The dose-response curve of the binding activity of the chimeric antibody to hDLL3 is shown.

[0177] Figure 4 The dose-response curves show the binding activity of the chimeric antibody with hDLL1 (left) and hDLL4 (right).

[0178] Figure 5 The dose-response curves of the binding activity of the chimeric antibody with mouse (left) and monkey DLL3 (right) are shown.

[0179] Figure 6 The dose-response plot of cytotoxic activity of different ADC candidates on DLL3 target cells after 6 days of treatment is shown.

[0180] Figure 7 This study demonstrates the in vivo antitumor activity of ADC drugs in the NCI-H82 model. Detailed Implementation

[0181] Table a. Explanation of Abbreviations

[0182] DMEM basal culture medium HAT Screening culture medium PEI Polyetherimide MOI Multiple infection GFP Green fluorescent protein SPF Specific pathogen-free animals PBST Phosphate buffer solution containing Tween HRP Horseradish peroxidase TMB 3,3',5,5'-Tetramethylbenzidine PBS Phosphate buffer solution APC Allophycocyanin

[0183] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0184] Example 1. Preparation of monoclonal antibodies FDA027 and FDA031 specifically targeting hDLL3

[0185] In this invention, monoclonal antibodies FDA027 and FDA031 with high affinity and specific targeting of hDLL3 were selected. FDA027 has a light chain amino acid sequence as shown in SEQ ID NO:1 and a heavy chain amino acid sequence as shown in SEQ ID NO:2, and FDA031 has a light chain amino acid sequence as shown in SEQ ID NO:3 and a heavy chain amino acid sequence as shown in SEQ ID NO:4.

[0186] The FDA027 light and heavy chain nucleotide sequences were obtained through whole-genome synthesis (Suzhou Genewiz), and were separately constructed into the pV81 vector by double digestion with EcoRI (purchased from NEB, R3104S) and Hind III (purchased from NEB, R3101S). Figure 1The cells were ligated and transformed into Trans 1-T1 competent cells (purchased from TransGen, CD501). Clones were selected and sequenced for confirmation. Positive clones were cultured and amplified, and plasmids were extracted in medium quantities to obtain the antibody light chain eukaryotic expression plasmid FDA027-L / pV81 and the antibody heavy chain eukaryotic expression plasmid FDA027-H / pV81, with a light chain-to-heavy chain eukaryotic expression plasmid mass ratio of 1.5:1. These plasmids were then transformed into CHO cells adapted for suspension growth (purchased from ATCC) by electroporation. Cells were seeded at 2000-5000 cells / well in 96-well plates and cultured for 3 weeks. Expression levels were measured using HTRF (homogeneous time-resolved fluorescence) according to the kit instructions (purchased from CisBio, 62HFCPEG). Cells with the highest expression levels were selected. Cell pools were expanded into 125ml shake flasks (culture volume 30ml), incubated at 37℃, 5.0% CO2, and shaking at 130 rpm for 3 days. After 3 days, the cells were expanded into 1000ml shake flasks (culture volume 300ml), incubated at 37℃, 5.0% CO2, and shaking at 130 rpm. Starting on the fourth day, 5-8% of the initial culture volume of feed medium was added every other day. Culture was continued for 10-12 days. The harvested culture was centrifuged at 9500 rpm for 15 min to remove the cell pellet. The supernatant was collected and filtered through a 0.22μm filter. The purified sample was then purified using a MabSelect affinity chromatography column (purchased from GE) to obtain high-purity anti-DLL3 antibody FDA027. FDA031 was prepared using the same method as FDA027.

[0187] Example 2. Preparation of human hDLL3 overexpression vector and stable cell line

[0188] The human DLL3 expression plasmid pCMV3-DLL3-t1 (purchased from Beijing Yiqiao, HG20010-UT) was transformed into E. coli competent cells Trans1-T1 (purchased from TransGen, CD501) for plasmid amplification. The transformation procedure followed the instructions for use with competent cells. Single clones were picked from the transformation plate and amplified overnight in culture medium. After centrifugation at 6000 rpm for 20 min, the bacterial culture was collected for plasmid extraction. Plasmid DNA was extracted using a plasmid medium-volume extraction kit (purchased from MN, product number: DP117) following the manufacturer's instructions.

[0189] HEK293 cells in good growth phase (purchased from the Chinese Academy of Sciences Cell Bank) were selected and diluted to 5 × 10⁶ cells / mL with 10% fetal bovine serum in fresh DMEM medium. 5Cells were seeded at a density of 1 cell / ml, with 2 ml per well in 6-well culture plates and cultured in an incubator (37°C, 5% CO2). The following day, plasmid pCMV3-DLL3-t1 was transfected into HEK293 cells using lipofectamin 3000 (Thermo, L3000-008), following the instructions of the lipofectamin 3000 transfection kit. 48 hours after transfection, cells were seeded at a density of 1 cell per well in 96-well culture plates and cultured in fresh medium containing 200 μg / ml hygromycin B (Thermo, 10687010). The medium was replaced with fresh medium containing hygromycin B every 3 to 4 days to obtain stable monoclonal cells expressing hDLL3. HEK293 cells expressing hDLL3 were screened by flow cytometry. The detection antibody, goat anti-hDLL3-PE (purchased from R&D, FAB4315P), was diluted with PBS to 10 μg / ml and added to a 5×10⁻⁶ volumetric cytokine solution. 5 The cells were incubated at 4°C for 1 hour. Then, 1 ml of PBS containing 2% fetal bovine serum was added to resuspend the cells. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded. This process was repeated twice. The cells were then resuspended in 1 ml of PBS and analyzed using CytoFLEX (Beckman). A positive DLL3 expression rate greater than 85% was considered a positive clone, indicating successful construction of the HEK293-DLL3 stable cell line.

[0190] HEK293 overexpressing cell lines containing different hDLL3 domains were stably transfected using the same method described above. These cell lines were: an hDLL3 overexpressing cell line without the N-terminus (the sequence of which is shown in SEQ ID NO:5); an hDLL3 overexpressing cell line without the N-terminus and DSL domains (the sequence of which is shown in SEQ ID NO:6); an hDLL3 overexpressing cell line without the N-terminus, DSL, and EGF1 domains (the sequence of which is shown in SEQ ID NO:7); and an hDLL3 overexpressing cell line without the N-terminus, DSL, EGF1, and EGF2 domains (the sequence of which is shown in SEQ ID NO:8).

[0191] SEQ ID NO:5

[0192] GKPIPNPLLGLDSTSGARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGGGSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA

[0193] SEQ ID NO:6

[0194] GKPIPNPLLGLDSTSG APLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGGGSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA

[0195] SEQ ID NO:7

[0196] GKPIPNPLLGLDSTSGGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGG GAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGGGSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA

[0197] SEQ ID NO:8

[0198] GKPIPNPLLGLDSTSG SGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRER ADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGGGSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA

[0199] Note: Underlined text indicates V5 tags, and italicized text indicates intracellular sequences.

[0200] Example 3. Preparation and screening of hybridoma monoclonal antibodies

[0201] 3-1. Immunization and serum titer testing

[0202] Six 6-week-old SPF-grade female Balb / C healthy mice (purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd.) were randomly divided into two groups, A and B. The mice were immunized with recombinant DLL3 protein (purchased from ACRO, DL3-H5255) every two weeks. The initial immunization used Freund's complete adjuvant, and subsequent immunizations used Freund's incomplete adjuvant. Blood samples were collected from the orbital sinus of the mice on days 35 and 49, and the serum titers of the immune serum were measured using standard methods including ELISA and flow cytometry (FACS).

[0203] ELISA detection method: Dilute DLL3 protein with PBS to a concentration of 1 μg / ml, add 100 μl / well to a 96-well microplate and incubate overnight at 4°C. The next day, discard the supernatant and add 200 μl of PBS solution containing 2% milk to each well for blocking. After 2 hours, discard the blocking solution and add 200 μl of PBST (1‰ Tween 20) to wash the wells 3 times. Then, pre-dilute the serum to be tested 100 times as the starting concentration, and perform 11 3-fold serial dilutions. Add 100 μl of each concentration to the microplate and incubate at room temperature for 1 hour. Discard the supernatant and add 200 μl of PBST (1‰ Tween 20) to wash 5 times. Add 100 μl of HRP-labeled goat anti-mouse secondary antibody (Jackson, 1:10000) and incubate at room temperature for 1 hour. Discard the supernatant and add 200 μl of PBST (1‰ Tween 20) to each well and wash 7 times. Add 100 μL of TMB chromogenic solution to each well and incubate for 10 min. After stopping the reaction by adding 2 M HCl, read the absorbance value at 450 nm using a microplate reader (purchased from Biotek, Elx808).

[0204] FACS testing method: Add 3×10⁻⁶ to each well of a 96-well V-type microplate (purchased from Axygen, wipp02280). 5 HEK293-DLL3 stable transgenic cells or SHP-77 endogenously expressed cells (purchased from ATCC) were centrifuged at 1500 rpm for 1 min, and the supernatant was discarded. Immune serum was diluted 1:50 with PBS, and then serially diluted 4-fold to obtain 8 concentrations. 50 μL / well was added to each well of a microplate and incubated on ice for 30 min. Cells were resuspended in 150 μL of PBS in each well and centrifuged at 1500 rpm for 1 min. This process was repeated twice. 50 μL of APC-labeled goat anti-mouse IgG (Jackson, catalog number 115-605-164, diluted 1:800 with PBS) was added to each well and incubated on ice for 30 min. Cells were resuspended in 150 μL of PBS in each well and centrifuged at 1500 rpm for 1 min. This process was repeated twice. Detection was performed using CytoFLEX (purchased from Beckman). After testing, mice with the highest titer and stable titers in two consecutive immune serum fusions were selected for fusion. Three days before fusion, mice were given a shock immunization by intraperitoneal injection of DLL3 protein.

[0205] 3-2. Hybridoma fusion screening and cloning

[0206] Spleens from Balb / C mice subjected to shock immunization were used to prepare cell suspensions. Spleen cells were then fused with SP2 / 0 cells (purchased from the Chinese Academy of Sciences Cell Bank, TCM42) using an electrofusion method. After fusion, hybridoma cells were added at a concentration of 2 × 10⁻⁶ cells / mL. 4Cells were seeded into 96-well plates and screened using HAT medium. After 7 days, the plate head was collected for ELISA testing, as described in Example 3-1. ELISA-positive cells were then analyzed using FACS to detect DLL3-overexpressing 293 cell lines and SHP-77 endogenous cell lines. Cells with positive binding were subjected to limiting dilutions, repeated twice. The diluted clones were then confirmed as positive clones using the ELISA and FACS methods described in Example 3-1. Hybridomas of positive clones detected after two limiting dilutions were amplified for antibody production, purification, and sequencing of antibody expression genes.

[0207] 3-3. Hybridoma antibody production and purification

[0208] To generate mg of antibody for functional characterization, selected hybridoma cells were cultured at 2.5 × 10⁻⁶ mg / mL. 5 The cells were seeded at a density of 1 / mL into cell culture flasks containing 250 mL of serum-free hybridoma medium (purchased from Yuanpei Biotechnology, H630KJ), and cultured at 37°C with shaking at 130 rpm. Once the cell viability dropped to approximately 30%, the cell supernatant was harvested by centrifugation. Mouse monoclonal antibodies were purified using Protein G media and then dialyzed into PBS pH 7.2 buffer. The antibody concentration and purity were determined by measuring the absorbance using a micro-spectrophotometer (purchased from Hangzhou Aosheng Instruments, Nano-300).

[0209] Example 4. Analysis of antibody endocytosis activity

[0210] The endocytic activity of the hybridoma antibody obtained in Example 3-3 was detected using the FACS detection method described in Example 3-1 and SHP-77 cells. The detection method is briefly described below: six aliquots (5 × 10⁶ cells each) were dispensed. 5100 SHP-77 cells were placed in a 96-well V-type microplate and centrifuged at 1500 rpm for 1 min, then the supernatant was discarded. 200 μL of saturated 1 μg / ml antibody to be tested was added to each well, and the cells were incubated on ice for 30 min. Then, 150 μL of PBS was added to each well, and the cells were centrifuged at 1500 rpm for 1 min, and the supernatant was discarded. This process was repeated 4 times. Resuspend the cells in 200 μl of PBS. Take one sample from each well and place it in a 37°C cell culture incubator for 5 hours, 3 hours, 1 hour, 0.5 hours, and 15 minutes, respectively. Place the remaining sample on ice. After the incubation time, place all cell samples at 4°C, centrifuge at 1500 rpm for 1 minute, discard the supernatant, and add 50 μl of APC-labeled goat anti-mouse IgG (Jackson, catalog number 109-605-098, diluted 1:800 in PBS) to each well. Incubate on ice for 30 minutes. Then, resuspend the cells in 150 μl of PBS to each well, centrifuge at 1500 rpm for 1 minute, discard the supernatant, and repeat this process 4 times for washing. Finally, resuspend the cells in 100 μl of PBS to each well and perform detection using CytoFLEX (Beckman). The fluorescence values ​​are shown in Table 1 below. Figure 2 The internalization results of the antibody obtained in Example 3-3 are presented, and the results show that the antibody obtained in Example 3-3 has good internalization activity.

[0211] Table 1. Fluorescence values ​​of antibody internalization detected by flow cytometry

[0212]

[0213] Example 5. Obtaining the gene sequence of hybridoma antibody expression

[0214] Collect 2×10 per clone 5 Hybridoma cells (from Examples 3-2) were used for RNA extraction. Cells were centrifuged at 300g (speed unit in this example, same below) for 5 min, and the supernatant was discarded. 250 μL of Trizol (purchased from TAKARA, T9108) lysis buffer was added to lyse the hybridoma cells. Then, 50 μL of chloroform was added, and the mixture was vortexed to form an emulsion. The mixture was allowed to stand at room temperature for 5 min, then centrifuged at 12000g, 4°C for 15 min. The supernatant was transferred to a new RNase-free 1.5 ml centrifuge tube, and 125 μL of isopropanol was added. The mixture was allowed to stand at room temperature for 10 min. Centrifugation at 12000g, 4°C for 10 min revealed a precipitate. The supernatant was discarded, and 250 μL of 75% ethanol was added. The mixture was gently inverted to mix, and centrifuged at 12000g, 4°C for 10 min. The supernatant was discarded, and the precipitate was dried at room temperature for 10 min. Then, 20 μL of RNase-free water was added to dissolve the precipitate for 30 min. After dissolution, the mixture was mixed with a pipette, and the concentration was determined. PrimeScript was used based on RNA concentration.TM The RNA of the cloned sample was reverse transcribed into cDNA using the RT Master Mix kit (TAKARA, RR036A). Antibody light and heavy chain specific primers were synthesized, and the antibody light and heavy chain sequences were obtained by PCR. The amplified fragments were then gel-cleaved using a Universal DNA purification and recovery kit (Tiangen Biotech, DP214-03), and subsequently ligated with the pMD18T vector (TAKARA, 6011) at 4°C for 30 min. Add 5 μL of the ligation product to 50 μL of LTG-1 (Lucigen) competent cells, mix gently, and heat-shock at 42°C for 90 s. After heat-shock, incubate on ice for 3 min. Add 1 mL of antibiotic-free 2YT liquid medium (purchased from Sangon Biotech, A507019-0250), and incubate at 37°C and 220 rpm for 1 h on a shaker. Then, take 100 μL of the bacterial culture and spread it evenly on Amp-resistant (purchased from Sangon Biotech, A600469-0005) 2YT solid plates and incubate upside down at 37°C for 14-16 h. Select single clones and send them to Sangon Biotech for sequencing. After obtaining the sequencing results, analyze the sequencing results using Vector NTI and Vbase2 software to obtain the amino acid sequences of antibodies with good binding and endocytic activities. The results are shown in Table 2.

[0215] Table 2. Amino acid sequence numbers of CDRs and variable regions of the light and heavy chains of the obtained antibodies (using Kabat encoding)

[0216]

[0217]

[0218] Example 6. Construction and preparation of chimeric antibodies and detection of binding activity

[0219] The hybridoma clone heavy chain variable region sequence with good binding and endocytic activity obtained in Example 5 above was synthesized and cloned into the pFUSEss-CHIg-hG1 vector (purchased from InvivoGen, catalog number pfusess-hchg1) containing the amino acid sequence of the constant region of the human antibody IgG1 heavy chain via homologous recombination to obtain a chimeric antibody heavy chain expression vector. The cloned light chain variable region sequence was cloned into the pFUSE2ss-CLIg-hk vector (purchased from InvivoGen, catalog number pfuse2ss-hclk) containing the amino acid sequence CL of the constant region of the human antibody Kappa light chain using the same method to obtain a chimeric antibody light chain expression vector. The amino acid sequence numbers corresponding to the full length of the light and heavy chains of the constructed chimeric antibodies are shown in Table 3-1. 293F cells in good logarithmic growth phase were collected, seeded into 250 mL cell culture flasks, and cultured in 50 mL of medium. 25 μg of each light and heavy chain expression plasmid were co-transfected with PEI. Cell supernatant was collected on day 7 post-transfection culture, centrifuged, and filtered through a 0.45 μM filter. Antibody was purified using Protein A media and then dialyzed to displace it into PBS pH 7.2 buffer. The absorbance was measured using a micro-spectrophotometer (Hangzhou Ausen Instruments, Nano-300) to determine the antibody concentration. The binding activity of the obtained chimeric antibody to human DLL3 protein was detected by ELISA (results are shown in Figure 1). Figure 3 (As shown in Table 3-2), the ELISA detection method is as described in Example 3-1. Figure 3 It can be seen that the obtained chimeric antibodies all have good binding activity to human DLL3 protein.

[0220] Table 3-1. Full-length amino acid sequence numbers of the light and heavy chains of chimeric antibodies.

[0221] chimeric antibodies Heavy chain total length Light chain full length ch1A5 SEQ ID NO:17 SEQ ID NO:18 ch33F11 SEQ ID NO:27 SEQ ID NO:28 ch23G2 SEQ ID NO:37 SEQ ID NO:38 ch32E8 SEQ ID NO:47 SEQ ID NO:48 ch13C2 SEQ ID NO:57 SEQ ID NO:58 ch19C7 SEQ ID NO:67 SEQ ID NO:68 ch26D9 SEQ ID NO:77 SEQ ID NO:78 ch10B1 SEQ ID NO:87 SEQ ID NO:88 ch36H10 SEQ ID NO:97 SEQ ID NO:98 ch4H7 SEQ ID NO:107 SEQ ID NO:108 ch6A2 SEQ ID NO:43 SEQ ID NO:53 ch15G2 SEQ ID NO:73 SEQ ID NO:109

[0222] Table 3-2. Binding activity of chimeric antibodies to human DLL3 protein

[0223] chimeric antibodies EC50 (ng / mL) FDA027 10.84* FDA031 23.35 ch32E8 8.828 ch1A5 9.213 ch33F11 9.546 ch23G2 9.406 ch4H7 11.48 ch19C7 8.658 ch6A2 13.13 ch36H10 15.88 ch10B1 16.77 ch26D9 11.78 ch13C2 394.8 ch15G2 8.526

[0224] Example 7. Chimeric Antibody Competition Analysis and Binding Epitope Analysis

[0225] The chimeric antibodies obtained in Example 6 were labeled with Biotin, and the competitive binding activity of the antibodies was detected by ELISA. The detection method is briefly described as follows: Dilute DLL3 protein to 1 μg / ml with PBS, plate it in a 384-well microplate (purchased from Corning, 3700) and incubate overnight at 40°C. The next day, discard the supernatant from the wells, block with 80 μl of 3% milk (dissolved in PBS) for 2 hours, and wash three times with 80 μl of PBST (1‰ Tween 20). Then, pre-dilute the unlabeled chimeric antibody to 100 μg / ml, and then serially dilute it 3-fold to 11 concentration points. Add 25 μl of each antibody to the blocked microplate and incubate at room temperature for 1 hour. After that, discard the supernatant from the wells, wash five times with 80 μl of PBST (1‰ Tween 20), add 25 μl of Biotin-labeled chimeric antibody and incubate at room temperature for 1 hour. Discard the supernatant again, and wash five times with 80 μl of PBST (1‰ Tween 20). Finally, the sample was incubated with HRP-labeled streptavidin secondary antibody at a dilution of 1:5000 (purchased from GenScript, M00091) at room temperature for 1 hour. The supernatant was discarded, and the sample was washed 7 times with 80 μl of PBST (1‰ Tween 20). 25 μl of TMB chromogenic buffer was added for 10 min of color development. The reaction was terminated by adding 2M HCl, and the absorbance value at 450 nm was read using a microplate reader (purchased from Biotek, Elx808).

[0226] To determine the specific binding region of the antibody, the binding ability of the chimeric antibody to HEK293 overexpression cell lines containing different hDLL3 domains (corresponding sequences: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8) as described in Example 2 was detected by FACS to determine the antibody binding region. In short: 5 × 10 5Cells overexpressing DSL, EGF1, EGF2, and EGF3 were placed in 96-well V-type microplates and centrifuged at 1500 rpm for 1 min, discarding the supernatant. 200 μL of saturated 1 μg / ml antibody was added to each well, and the plates were incubated on ice for 30 min. Then, 150 μL of PBS was added to each well, and the plates were centrifuged at 1500 rpm for 1 min, discarding the supernatant. The washing process was repeated four times. Finally, 50 μL of APC-labeled goat anti-human IgG (Jackson, PBS 1:800 dilution) was added to each well, and the plates were incubated on ice for 30 min. After washing the plate four times with PBS, 100 μL of PBS was added to each well to resuspend the cells. CytoFLEX assay (Beckman) was performed. The results showed that antibodies ch1A5, ch33F11, and ch32E8 bound to the EGF2 domain of DLL3; antibodies FDA031, ch13C2, and ch15G2 bound to the N-terminus of DLL3; FDA027 bound to the DSL domain of DLL3; and the remaining antibodies bound to the EGF3-EGF6 domain of DLL3. Therefore, the binding epitope of ch15G2 was the N-terminal domain; the binding epitopes of ch32E8, ch1A5, and ch33F11 were the EGF2 domain; and the binding epitopes of ch36H10, ch6A2, ch19C7, ch26D9, ch23G2, ch10B1, and ch4H7 were the EGF3-EGF6 domain.

[0227] Example 8. Cross-reactivity of antibodies with human DLL1, DLL4 proteins and mouse and monkey DLL3 cells

[0228] The chimeric antibody obtained in Example 6 was tested for its binding ability to human DLL1 (purchased from Acro, DL1-H52H8) and DLL4 (purchased from Acro, DL4-H5227) proteins by ELISA. For the ELISA assay, DLL1 and DLL4 proteins were diluted with PBS to a final concentration of 1 μg / ml, and then plated into 384-well microplates (purchased from Corning, 3700) and incubated overnight at 4°C. The next day, the supernatant was discarded, and 80 μl of the supernatant was used. Blocking was performed with 3% milk (dissolved in PBS) for 2 hours. The wells were washed three times with 80 μl of PBST (1‰ Tween 20). Then, the chimeric antibody, FDA027, and FDA031 were diluted to 20 μg / ml, and serially diluted 3-fold to 11 concentration points. 25 μl of each solution was added to each well of the blocked plate. The plate was incubated at room temperature for 1 hour. The supernatant was discarded, and the plate was washed five times with 80 μl of PBST (1‰ Tween 20). 25 μl of HRP-labeled goat anti-human secondary antibody (Jackson, 1:10000) was added to each well, and the plate was incubated at room temperature for 1 hour. The supernatant was discarded, and the plate was washed seven times with 80 μl of PBST (1‰ Tween 20). 25 μl of TMB chromogenic buffer was added for 10 min of color development. The reaction was terminated with 2M HCl. The absorbance at 450 nm was read using a microplate reader (Biotek, Elx808). Results (see...) Figure 4 The results showed that none of the 12 candidate antibodies had significant cross-reactivity with hDLL1 and hDLL4 proteins. Among them, the 36H10 and 4H7 antibodies showed weak cross-reactivity with hDLL1 at high concentrations (20 μg / mL), while the 6A2 antibody showed some cross-reactivity with hDLL4 at high concentrations (20 μg / mL).

[0229] To detect the cross-reactivity of antibodies against mouse and monkey DLL3, this study constructed overexpression cell lines using the mouse DLL3 expression plasmid pCMV3-mDLL3 (purchased from Beijing Yiqiao, MG58052-UT) and the monkey DLL3 expression plasmid pCMV3-rheDLL3 (purchased from Beijing Yiqiao, CG90919-UT). The construction method of the overexpression cells is as described in Examples 2-3. The binding ability of the chimeric antibody to these two cell lines was detected by flow cytometry to determine the species cross-reactivity of the antibody. The constructed 5×10⁶ cells were then used as overexpression cells. 5Mouse-DLL3 and monkey-DLL3 cells were seeded into 96-well V-type microplates (purchased from Axygen, wipp02280), centrifuged at 1500 rpm for 1 min, and the supernatant was discarded. Chimeric antibodies, FDA027, and FDA031 were pre-diluted to 50 μg / ml, and serially diluted 4-fold to 8 concentration points. Different concentrations of the antibody to be tested were added to each well (200 μL), and the plates were incubated on ice for 30 min. Then, 150 μL of PBS was added to each well, centrifuged at 1500 rpm for 1 min, and the supernatant was discarded. The plates were washed 4 times. APC-labeled goat anti-human IgG (Jackson, PBS 1:800 dilution) was added to each well (50 μL), and the plates were incubated on ice for 30 min. After washing 4 times with PBS, 100 μL of PBS was added to each well to resuspend the cells. Detection was performed using CytoFLEX (Beckman). The results (e.g., ...) were obtained. Figure 5 As shown in the figure, except for ch32E8, ch13C2 and ch15G2 which do not recognize monkey and mouse DLL3, the other 9 antibodies can bind to mouse and monkey DLL3.

[0230] Example 9. Affinity detection of antibody binding to human DLL3 protein

[0231] To detect the affinity of the antibody for human DLL3 protein, this study used the BLI method to detect the binding kinetics of immobilized antibody and free DLL3. The detection method followed the instrument's (supplier: Fortebio, model: Octet 96e) instructions. In short, the AMC sensor was first equilibrated for 60 seconds with Loading Buffer / Sample Dilution Buffer (1×PBS, pH 7.4, with 0.1% BSA and 0.02% Tween-20) to obtain Baseline 1. The antibody to be tested was diluted to a concentration of 10 μg / ml with Loading Buffer and bound to the equilibrated sensor. The bound sensor was then reequilibrated with Loading Buffer to obtain Baseline 2. The antibody-loaded sensor was then placed in human DLL3, His tag (supplier: Acrobiosystems, catalog number: DL3-H52H4), diluted to 100 to 3.13 nM with sample dilution buffer, and bound for 90 seconds to obtain the antibody-protein binding curve. The antigen-bound sensor was then placed back into Sample Dilution Buffer for 180 seconds to dissociate, yielding dissociation curves. The k-on and k-off values ​​of antibody-protein binding were calculated from the binding and dissociation curves, and the KD value was also calculated. The results (see Table 4) show that these chimeric antibodies exhibit strong affinity at the protein level.

[0232] Table 4. Affinity of antibody to human DLL3 protein

[0233] Antibody name KD(M) kon(1 / Ms) Koff(1 / s) ch1A5 5.97E-11 1.24E+06 7.40E-05 ch33F11 5.21E-11 1.21E+06 6.31E-05 ch23G2 1.58E-11 1.76E+06 2.79E-05 ch13C2 5.37E-09 2.23E+05 1.20E-03 ch32E8 9.00E-11 1.49E+06 1.34E-04 ch19C7 7.39E-10 1.30E+06 9.62E-04 ch26D9 3.81E-11 1.73E+06 6.58E-05 ch10B1 2.04E-10 1.37E+06 2.79E-04 ch36H10 9.91E-11 1.69E+06 1.68E-04 ch4H7 1.04E-10 1.07E+06 1.11E-04 ch6A2 4.66E-10 1.47E+06 6.84E-04 ch15G2 7.02E-11 1.72E+06 1.21E-04

[0234] Example 10. Preparation of linker drug conjugates

[0235] The structures of the linker drug conjugates LE00 and LE01-LE24 used in this invention are shown in Table 5. LE00 (GGFG-Dxd) was synthesized according to the method reported in WO2015146132A, and LE01-LE24 were synthesized according to the method reported in WO2020259258A1.

[0236] Table 5. Structure of Linker-Based Drug Conjugates

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243] Example 11. Preparation of ADC by conjugation of antibody and linker drug conjugate

[0244] The different anti-DLL3 antibodies obtained in Examples 1 and 6 were replaced with 50 mM MPB / 1.0 mM EDTA buffer (pH 7.0) using a G25 desalting column. 12 equivalents of TECP were added, and the mixture was stirred at 37°C for 2 hours to fully open the disulfide bonds between the antibody chains. The pH of the reduced antibody solution was then adjusted to 6.0 using phosphoric acid, and the water bath temperature was lowered to 25°C for the coupling reaction. The linker-drug conjugate prepared according to the method in Example 10 was dissolved in DMSO. 12 equivalents of the linker-drug conjugate were then added dropwise to the reduced anti-DLL3 antibody solution. All samples were prepared according to the highest DAR (i.e., over-coupling). The precipitation during each coupling reaction was observed, and DMSO was added to a final concentration of 10% (v / v). The mixture was stirred at 25°C for 0.5 hours. After the reaction was complete, the samples were filtered through a 0.22 μm membrane. Uncoupled small molecules were purified using a tangential flow ultrafiltration system. The buffer solution was 50 mM PB / 1.0 mM EDTA (pH 6.0). After purification, 6% sucrose was added to the final concentration and the solution was stored at -20°C. The absorbance was measured at 280 nm and 370 nm using UV spectroscopy, and the DAR value was calculated. The results are shown in Table 6. The results showed that antibodies ch1A5, ch33F11, ch32E8, ch13C2, FDA027, FDA031, ch15G2, and ch4H7 could be normally conjugated with LE14; ch33F11 could be normally conjugated with LE01-LE11, LE23, and LE24; and ch1A5 could be normally conjugated with LE00, LE12, LE13, LE15, LE16, LE17, LE18, LE19, LE20, LE21, and LE22. The DAR value and free Dxd content of the conjugated ADCs met the expected requirements. When antibodies ch19C7, ch23G2, ch6A2, and ch26D9 were conjugated with LE14, their ADC samples showed obvious flocculent precipitation during centrifugation and concentration, resulting in a small amount collected, indicating that their conjugation for drug formation was not feasible. The DAR values ​​of the ADC samples obtained by conjugating antibodies ch36H10 and ch10B1 were 1.89 and 1.90, respectively, both with high impurity content. In particular, the small molecule residue of ch36H10 was as high as 49.35%, indicating that the conjugation failed.

[0245] Table 6. Drugability Evaluation of ADC Samples Prepared with Different Antibodies Conjugated to LE14

[0246]

[0247] Example 12. Evaluation of in vitro cytotoxic activity of ADC samples

[0248] HEK293 cells, stably transfected with high DLL3 expression, were selected as the cell line for in vitro activity assays to observe the dose-response effect of different antibody-drug conjugates on cell killing. 2000 cells per well were seeded in 96-well cell culture plates and cultured for 20–24 hours. The antibody-drug conjugates prepared according to Example 11 were diluted to nine concentration gradients (80, 20, 5, 1.25, 0.3125, 0.0781, 0.0195, 0.00488, and 0.000488 μg / ml). 100 μl of each diluted solution was added to the cell-seeded culture plates, and the plates were incubated at 37°C in a 5% CO2 incubator for 144 hours. Luminescent Cell Viability Assay Reagent (50 μl / well), mixed by shaking at 500 rpm for 10 minutes at room temperature, data were read using a SpectraMax L microplate reader (OD570 nm, 2-second intervals). The viability of the untreated whole-cell group was used as 100% for data processing, and the IC50 was calculated. Results are shown below. Figure 6 See Table 7.

[0249] Table 7. IC50 of candidate antibodies in in vitro killing of DLL3 / HEK293 cells by ADC 50 Summary of values ​​and maximum kill rate

[0250] Sample Name <![CDATA[IC 50 (μg / mL)]]> Maximum lethality (%) ch1A5-LE00 0.321 96.3 ch33F11-LE01 0.432 95.4 ch33F11-LE02 0.564 97.3 ch33F11-LE03 0.375 95.2 ch33F11-LE04 0.453 94.8 ch33F11-LE05 0.532 96.2 ch33F11-LE06 0.383 95.7 ch33F11-LE07 0.432 95.2 ch33F11-LE08 0.634 96.8 ch33F11-LE09 0.563 94.3 ch33F11-LE10 0.276 96.7 ch33F11-LE11 0.364 93.1 ch1A5-LE12 0.412 94.8 ch1A5-LE13 0.337 96.3 ch1A5-LE15 0.432 95.2 ch1A5-LE16 0.386 96.5 ch1A5-LE17 0.254 93.5 ch1A5-LE18 0.227 95.6 ch1A5-LE19 0.521 97.2 ch1A5-LE20 0.632 95.4 ch1A5-LE21 0.534 92.7 ch1A5-LE22 0.432 97.1 ch33F11-LE23 0.369 93.6 ch33F11-LE24 0.463 96.5 ch36H10-LE14 0.695 96.9 ch32E8-LE14 0.225 96.8 ch1A5-LE14 0.277 96.6 ch19C7-LE14 8.69 88.4 ch23G2-LE14 0.303 96.8 ch33F11-LE14 0.377 98.3 ch6A2-LE14 0.992 96.2 ch10B1-LE14 0.46 97.6 ch4H7-LE14 0.718 96.9 ch13C2-LE14 2.72 97.9 ch15G2-LE14 0.385 90.5 ch26D9-LE14 0.563 95.6 FDA031-LE14 0.389 95.2 FDA027-LE14 <![CDATA[0.087 1 ]]> <![CDATA[95.1 2 ]]>

[0251] 1 and 2 All values ​​are averages.

[0252] Table 7 shows that, except for ch19C7-LE14 and ch13C2-LE14, which exhibit relatively weak in vitro cytotoxic activity, all the ADCs disclosed in this invention have significant in vitro cytotoxic effects on DLL3-positive cells. FDA027-LE14 shows the strongest in vitro cytotoxic activity and also has the best in vitro IC50. The IC90 values ​​of each ADC disclosed in this invention are not significantly different from those of FDA027-LE14.

[0253] Example 13. In vivo antitumor activity of ADC drugs in the NCI-H82 model

[0254] Female Balb / c nude mice aged 6-8 weeks (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were subcutaneously injected with 5×10⁻⁶ Matrigel solution dissolved in 200 μL of Matrigel in their right back. 6 Human small cell lung cancer cells (NCI-H82) were used until the tumor grew to an average volume of 140 mm. 3 At approximately 10:00 AM, mice were randomly divided into 15 groups based on tumor size and body weight. Groups 1-13 each contained 8 animals: a solvent blank control group (ch1A5-LE14), two dosage groups of conjugates (ch33F11-LE14, ch23G2LE14, ch32E8-LE14, FDA031-LE14, and FDA027-LE14, respectively, administered intravenously once weekly for a total of 3 times); and groups 14-15 each contained 6 animals: a control group (Lurbinectedin) and a control group (topotecan hydrochloride), administered intravenously once weekly for a total of 3 times. The Lubbinectedin group received the Lubbinectedin, and the topotecan hydrochloride group received the topotecan, administered intraperitoneally twice weekly, on days 1 and 2 consecutively, for a total of 3 times. Animal body weight and tumor volume were measured three times weekly, and animal survival status was observed during the experiment. Results are as follows: Figure 7 As shown in Table 8, the average tumor volume of mice in the solvent blank control group (Group 01) was 2773 mm² on day 21 after the end of drug administration. 3 In the FDA027-LE14 (2.5 mg / kg, group 12) treatment group, the mean tumor volume on day 21 after the end of administration was 486 mm. 3 In the FDA027-LE14 (5 mg / kg, group 13) treatment group, the mean tumor volume on day 21 after the end of administration was 305 mm. 3 The mean tumor volume in the ch1A5-LE14 treatment group (Group 02) at 2.5 mg / kg was 607 mm² on day 21 after the end of administration. 3 The mean tumor volume in the ch1A5-LE14 treatment group (Group 03) at 5 mg / kg was 245 mm on day 21 after the end of administration. 3 The mean tumor volume in the ch33F11-LE14 treatment group (Group 04) at 2.5 mg / kg was 715 mm² on day 21 after the end of administration. 3 The mean tumor volume in the ch33F11-LE14 treatment group (Group 05) at 5 mg / kg was 293 mm on day 21 after the end of administration. 3 The mean tumor volume in the ch23G2-LE14 treatment group (Group 06) at 2.5 mg / kg was 1322 mm² on day 21 after the end of administration. 3The mean tumor volume in the ch23G2-LE14 treatment group (Group 07) at 5 mg / kg was 441 mm² on day 21 after the end of administration. 3 The mean tumor volume in the ch32E8-LE14 treatment group (Group 08) at 2.5 mg / kg was 945 mm² on day 21 after the end of administration. 3 In the ch32E8-LE14 treatment group (Group 09) with 5 mg / kg of the test drug, the mean tumor volume was 433 mm on day 21 after the end of administration. 3 In the FDA031-LE14 (2.5 mg / kg, group 10) treatment group, the mean tumor volume on day 21 after the end of administration was 564 mm. 3 In the FDA031-LE14 (5 mg / kg, group 11) treatment group, the mean tumor volume was 245 mm on day 21 after the end of treatment. 3 The control group (0.18 mg / kg) of Lubbinectedin (purchased from Zhongke Chuangyue Pharmaceutical Co., Ltd., batch number 000039-87-CE013BA-a1) had an average tumor volume of 2268 mmHg on day 21 after the end of treatment. 3 In the control group (1.8 mg / kg topotecan hydrochloride, purchased from Saen Chemical Technology (Shanghai) Co., Ltd., batch number R3URR4E), the mean tumor volume on day 21 after the end of administration was 1553 mmHg. 3 The experimental results showed that the above treatment groups (especially ch1A5-LE14) all had good in vivo anti-tumor activity. At the same time, except for the control group with topotecan hydrochloride, there were no deaths or weight loss in any of the experimental mice, indicating that the tested drugs had good safety.

[0255] Table 8. In vivo antitumor effects of ADC drugs in the NCI-H82 model

[0256]

[0257] Example 14. In vivo antitumor activity of ADC drugs in the NCI-H209 model

[0258] Female Balb / c nude mice aged 6-8 weeks were selected and subcutaneously injected into the right back with 1×10⁻⁶ of Matrigel dissolved in 200 μL. 7 Human small cell lung cancer cells (NCI-H209) were used until the tumor grew to an average volume of 150 mm. 3At approximately 10:00 AM, mice were randomly divided into 7 groups of 6 animals each, based on tumor size and body weight. The groups consisted of a solvent blank control group, three dosage groups for ch1A5-LE14 and FDA027-LE14 conjugates (1.5 mg / kg, 2.5 mg / kg, and 5.0 mg / kg respectively), administered as a single dose. Animal body weight and tumor volume were measured three times weekly, and animal survival status was observed throughout the experiment. The results are shown in Table 9 below. The average tumor volume of mice in the solvent blank control group (Group 1) was 2006 mm² at the end of the administration period. 3 In the 1.5 mg / kg FDA027-LE14 treatment group (Group 5), the mean tumor volume was 0 mm on day 21 after the end of administration. 3 In the 2.5 mg / kg FDA027-LE14 treatment group (Group 6), the mean tumor volume was 0 mm on day 21 after the end of administration. 3 In the 5 mg / kg FDA027-LE14 treatment group (Group 7), the mean tumor volume was 0 mm on day 21 after the end of administration. 3 In the 1.5 mg / kg ch1A5-LE14 treatment group (Group 2), the mean tumor volume was 0 mm on day 21 after the end of administration. 3 In the 2.5 mg / kg ch1A5-LE14 treatment group (Group 3), the mean tumor volume was 0 mm on day 21 after the end of administration. 3 The mean tumor volume in the 5 mg / kg ch1A5-LE14 treatment group (Group 4) was 0 mm on day 21 after the end of administration. 3 The experimental results showed that FDA027-LE14 and ch1A5-LE14 had good in vivo antitumor activity, and no mice died or lost weight, indicating that the tested drugs had good safety.

[0259] Table 9. In vivo antitumor activity of ADC drugs in the NCI-H209 model

[0260]

[0261] Example 15. Toxicity evaluation of ADC drugs in mice

[0262] KM strain mice were selected for toxicity evaluation. Mice were randomly divided into four groups of 10 mice each (half male and half female) based on body weight. Each group received a single intravenous injection of 600 μL of the ch1A5-LE14 conjugate at doses of 200 mg / kg, 400 mg / kg, 500 mg / kg, and 640 mg / kg. Toxicity reactions caused by the ch1A5-LE14 conjugate were observed for 14 days post-administration. Results showed that in the 200 mg / kg group, only one animal experienced a decrease in body weight on day 3, followed by recovery and weight gain. Other animals showed an increasing trend throughout the experiment. Clinical observation revealed no abnormalities in the 200 mg / kg group. Mice in the other groups developed ruffled fur on the day of administration, which quickly returned to normal. At the end of the 14-day observation period, all animals were alive and well without any signs of mortality or death. These results indicate that the conjugate of this invention is well tolerated in mice, with a maximum tolerated dose of 640 mg / kg. In another study evaluating the toxicity of the ch1A5-LE14 conjugate in KM mice, intraperitoneal injection doses of 410 mg / kg, 512 mg / kg, 640 mg / kg, 800 mg / kg, and 1000 mg / kg were administered. Symptoms observed in each group included ruffled fur, arched back, reduced activity, curling up, diarrhea, soft stools, and perianal soiling. Five mice (out of 10 mice per group) died in the 1000 mg / kg group, and one mouse in the 800 mg / kg group experienced a weight loss exceeding 30% three times consecutively during the 14-day observation period. This mouse was euthanized according to animal welfare principles. No deaths were observed in other groups. These results further demonstrate that the conjugate of this invention exhibits very good tolerability in mice.

[0263] Example 16. Toxicity evaluation of ADC drugs in rats

[0264] The tolerable dose of ch1A5-LE14 in rats was evaluated by intravenous injection of the ch1A5-LE14 conjugate of this invention into SD rats. Rats were randomly divided into three groups (n=8 males and 50 females per group) based on their body weight. One group received 100 mg / kg of the conjugate once a week for three weeks, while the other group received a single dose of 300 mg / kg. A blank control group was also established, with an administration volume of 10 μL / g based on rat body weight. A 12-day observation period followed the last administration. No animals in any group experienced near-death or death during the entire experiment. In the high-dose (300 mg / kg) group, all animals developed ruffled fur, while no abnormalities were observed in the 100 mg / kg group. The high-dose (300 mg / kg) group experienced a slight decrease in body weight, but all surviving animals showed a recovery trend in body weight after day 7. At the end of the observation period, all animals were euthanized and gross examinations were performed, revealing no abnormalities in the organs. The in vivo toxicity study in rats further demonstrates that the conjugate of this invention also exhibits a very good tolerable dose in rats.

[0265] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An anti-DLL3 antibody, characterized in that, It comprises a heavy chain variable region VH and a light chain variable region VL; wherein, The amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 contained in the VH are shown in SEQ ID NO:9, 10 and 11, respectively, and the amino acid sequences of VL CDR1, VL CDR2 and VL CDR3 contained in the VL are shown in SEQ ID NO:12, GAS and SEQ ID NO:14, respectively.

2. The anti-DLL3 antibody as described in claim 1, characterized in that, The heavy chain variable region VH further includes a heavy chain variable region framework region VH FWR, and / or the light chain variable region VL further includes a light chain variable region framework region VL FWR; wherein, the VHFWR is the heavy chain variable region framework region of a human or mouse antibody, and the VL FWR is the light chain variable region framework region of a human or mouse antibody.

3. The anti-DLL3 antibody as described in claim 2, characterized in that, The VH comprises an amino acid sequence as shown in SEQ ID NO:15; and / or, the VL comprises an amino acid sequence as shown in SEQ ID NO:

16.

4. The anti-DLL3 antibody as described in claim 3, characterized in that, The amino acid sequence of VH is shown in SEQ ID NO:15, and the amino acid sequence of VL is shown in SEQ ID NO:

16.

5. The anti-DLL3 antibody according to any one of claims 1-4, wherein it is a full-length antibody, Fab, Fab', F(ab')2 or Fv, and / or, it is a monoclonal antibody.

6. The anti-DLL3 antibody as described in claim 5, characterized in that, The Fv is scFv.

7. The anti-DLL3 antibody as described in claim 5, wherein it is a full-length antibody, and the full-length antibody comprises a heavy chain and a light chain; wherein, The heavy chain comprises an amino acid sequence as shown in SEQ ID NO:17, and / or the light chain comprises an amino acid sequence as shown in SEQ ID NO:

18.

8. The anti-DLL3 antibody as described in claim 7, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO:17, and the amino acid sequence of the light chain is shown in SEQ ID NO:

18.

9. An isolated nucleic acid encoding an anti-DLL3 antibody as described in any one of claims 1-8.

10. A recombinant expression vector comprising the isolated nucleic acid as described in claim 9.

11. The recombinant expression vector as described in claim 10, characterized in that, The recombinant expression vector includes a eukaryotic cell expression vector and / or a prokaryotic cell expression vector.

12. A transformant comprising the recombinant expression vector as described in claim 10 or 11.

13. The transformant as described in claim 12, characterized in that, The host cells of the transformant are prokaryotic cells and / or eukaryotic cells.

14. The transformant as described in claim 13, characterized in that, The prokaryotic cells are E. coli cells, and the eukaryotic cells are HEK293 cells or CHO cells.

15. The transformant as described in claim 14, characterized in that, The prokaryotic cells are TG1 or BL21 cells.

16. An antibody-drug conjugate having the general structural formula Ab-(L3-L2-L1-D). m ; in, Ab is the anti-DLL3 antibody as described in any one of claims 1-8; D is a cytotoxic drug. m is 2 to 8; The structure of L1 is shown in Formula I, II, III or IV, with its a end connected to the cytotoxic drug and its e end connected to the c end of L2. (L) p In this context, L independently represents one or more of the following residues: phenylalanine residue, alanine residue, glycine residue, glutamic acid residue, aspartic acid residue, cysteine ​​residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue, and valine residue; p is 2-4. R 1 For one or more -NR 1-1 R 1-2 Substituted C1-C6 alkyl groups, with one or more R 1-3 S(O)2-substituted C1-C6 alkyl, C1-C6 alkyl, C3-C 10 cycloalkyl, C6-C 14 Aryl or 5-14 membered heteroaryl; the heteroatom in the 5-14 membered heteroaryl is selected from one or more of N, O and S, and the number of heteroatoms is 1, 2, 3 or 4; the R 1-1 R 1-2 and R 1-3 Each is independently a C1 to C6 alkyl group; L2 is Where n is independently 1 to 12, the c end is connected to L1 through a carbonyl group, and the f end is connected to the d end of L3. L3 is The b end is connected to the Ab end, and the d end is connected to the f end of the L2 end.

17. The antibody-drug conjugate as described in claim 16, characterized in that, The anti-DLL3 antibody binds to the antigen-binding epitope of the EGF2 domain in the DLL3 protein; And / or, the L is one or more of phenylalanine residue, alanine residue, glycine residue, isoleucine residue, leucine residue, proline residue and valine residue; And / or, the R 1 For one or more -NR 1-1 R 1-2 Substituted C1-C6 alkyl groups, with one or more R 1-3 S(O)2-substituted C1-C6 alkyl, or C1-C6 alkyl, wherein R 1-1 R 1-2 and R 1-3 Each is independently a C1 to C4 alkyl group; And / or, when the structure of L1 is as shown in Formula I, the L2 is And / or, when the structure of L1 is as shown in Formula II, the L2 is And / or, when the structure of L1 is as shown in Formula III, the L2 is And / or, when the structure of L1 is as shown in Formula IV, the L2 is And / or, the n is independently 8, 9, 10, 11, and 12; And / or, m is an integer or non-integer of 2 to 8; And / or, the L3 is 18. The antibody-drug conjugate as described in claim 17, characterized in that, The L is one or more of phenylalanine residues, alanine residues, glycine residues, and valine residues.

19. The antibody-drug conjugate as described in claim 18, characterized in that, The "multiple" mentioned refers to two or three types.

20. The antibody-drug conjugate as described in claim 18, characterized in that, The L is a valine residue and / or an alanine residue, and the p is 2.

21. The antibody-drug conjugate according to any one of claims 16-20, characterized in that, The (L) mentioned above p for The g-terminus is connected to the c-terminus of L2 via a carbonyl group; And / or, Formula III is 22. The antibody-drug conjugate of claim 16, wherein the antibody-drug conjugate is any of the following compounds: The value of m is 7.68, 7.53, 4.43, 7.12, 6.92, 7.43, 7.23, 6.83, 7.32, 7.56, 7.54, 7.47, 5.82, 6.78, 2.28, 6.32, 7.45, 7.65, 7.64, 7.36, 7.75, 7.80, 7.77, or 7.

76.

23. The antibody-drug conjugate as described in claim 22, characterized in that, The antibody-drug conjugate is any of the following compounds: The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:15 and the amino acid sequence VL as shown in SEQ ID NO:

16. The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:15 and the amino acid sequence VL as shown in SEQ ID NO:

16. The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:15 and the amino acid sequence VL as shown in SEQ ID NO:

16. The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:15 and the amino acid sequence VL as shown in SEQ ID NO:

16. The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:15 and the amino acid sequence VL as shown in SEQ ID NO:

16. The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:15 and the amino acid sequence VL as shown in SEQ ID NO:

16. The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:15 and the amino acid sequence VL as shown in SEQ ID NO:

16. The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:15 and the amino acid sequence VL as shown in SEQ ID NO:

16. The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:15 and the amino acid sequence VL as shown in SEQ ID NO:

16. The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:15 and the amino acid sequence VL as shown in SEQ ID NO:

16. The Ab is an anti-DLL3 antibody, which contains the amino acid sequence VH as shown in SEQ ID NO:15 and the amino acid sequence VL as shown in SEQ ID NO:

16. Ab is an anti-DLL3 antibody, which contains VH with an amino acid sequence as shown in SEQ ID NO:15 and VL with an amino acid sequence as shown in SEQ ID NO:

16.

24. A chimeric antigen receptor comprising an anti-DLL3 antibody as described in any one of claims 1-8.

25. A genetically modified cell, characterized in that, It contains the anti-DLL3 antibody as described in any one of claims 1-8.

26. The gene-modified cell as described in claim 25, characterized in that, The genetically modified cells are eukaryotic cells.

27. The gene-modified cell as described in claim 26, characterized in that, The genetically modified cells are isolated human cells.

28. The gene-modified cell as described in claim 27, characterized in that, The genetically modified cells are immune cells.

29. The gene-modified cell as described in claim 28, characterized in that, The genetically modified cells are T cells or NK cells.

30. A method for preparing an anti-DLL3 antibody, the method comprising the following steps: Cultivate the transformant as described in any one of claims 12-15 to obtain an anti-DLL3 antibody from the culture.

31. A pharmaceutical composition comprising an anti-DLL3 antibody as described in any one of claims 1-8, an antibody-drug conjugate as described in any one of claims 16-23, a chimeric antigen receptor as described in claim 24, and / or a genetically modified cell as described in any one of claims 25-29.

32. The pharmaceutical composition according to claim 31, characterized in that, The pharmaceutical composition is in the form of a liquid, gas, solid, or semi-solid dosage form, and / or the pharmaceutical composition can be administered orally, by injection, transdermal, or via mucosal administration.

33. The pharmaceutical composition according to claim 32, characterized in that, The pharmaceutical composition can be administered via the nose.

34. The pharmaceutical composition according to any one of claims 31-33, characterized in that, The pharmaceutical composition further comprises a combination therapy agent, which includes a chemotherapeutic agent, a radiotherapy agent, an immunosuppressant, and / or a cytotoxic agent.

35. The use of the anti-DLL3 antibody as described in any one of claims 1-8, the antibody-drug conjugate as described in any one of claims 16-23, the chimeric antigen receptor as described in claim 24, the genetically modified cell as described in any one of claims 25-29, and / or the pharmaceutical composition as described in any one of claims 31-34 in the preparation of medicaments, kits, and / or delivery devices for treating and / or preventing neuroendocrine tumors with abnormal DLL3 expression.

36. The application as described in claim 35, characterized in that, Neuroendocrine tumors are a type of small cell lung cancer.

37. A kit comprising an anti-DLL3 antibody as claimed in any one of claims 1-8, an antibody-drug conjugate as claimed in any one of claims 16-11, a chimeric antigen receptor as claimed in claim 24, genetically modified cells as claimed in any one of claims 25-29, and / or a pharmaceutical composition as claimed in any one of claims 31-34; and optionally, a specification.

38. A drug delivery device, characterized in that, The drug delivery device comprises: (1) an infusion module for administering the drug composition of any one of claims 31-34 to a subject in need, and (2) an optional drug efficacy monitoring module.

39. A method for detecting DLL3, characterized in that, It includes the step of using an anti-DLL3 antibody as described in any one of claims 1-8, wherein the method is for non-diagnostic and / or therapeutic purposes.

Images