CN116271079A

CN116271079A - anti-DLL 3 antibody, preparation method thereof, drug conjugate and application thereof

Info

Publication number: CN116271079A
Application number: CN202210911943.4A
Authority: CN
Inventors: 郭青松; 沈毅珺; 杨彤; 高贝; 吴芳; 孟李凯; 王宝霞; 张文伯
Original assignee: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Current assignee: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Priority date: 2021-07-30
Filing date: 2022-07-29
Publication date: 2023-06-23

Abstract

The invention discloses an anti-DLL 3 antibody, a preparation method thereof, a drug conjugate and application thereof. The anti-DLL 3 antibody has good internalization activity, better binding activity with human DLL3 protein and stronger affinity at protein level; the DLL 3-targeted antibody coupled drug has good drug property, biological activity and in-vivo and in-vitro anti-tumor activity, and can realize the application of cytotoxic drugs in treating tumor patients with neuroendocrine characteristics including SCLC.

Description

anti-DLL 3 antibody, preparation method thereof, drug conjugate and application thereof

The present application claims priority from chinese patent application CN2021108755368, whose application date is 2021, 7, 30, and chinese patent application CN2022102395912, whose application date is 2022, 3, 11. The present application refers to the entirety of the above-mentioned patent application.

Technical Field

The invention belongs to the field of biotechnology and medicine, and particularly relates to an anti-DLL 3 antibody, a preparation method thereof, a drug conjugate and application thereof.

Background

Small Cell Lung Cancer (SCLC) is the highest malignant type of lung cancer, and has the characteristics of rapid progress, early metastasis, easy recurrence and the like, and is about 15% -20% of new lung cancer, and the occurrence of the small cell lung cancer is closely related to long-term smoking. SCLC is largely divided into limited patients and extensive patients, with extensive patients being the major factor accounting for about 70% of SCLC. At present, treatment of SCLC is mainly chemotherapy, and first-line chemotherapy is mainly combination therapy based on platinum drugs, and most commonly etoposide+cisplatin or carboplatin (EP or CE regimen). Guidelines suggest that a combination of cisplatin/carboplatin treatment should be administered for 4-6 courses of treatment, either in the restricted or in the extensive phase, wherein the response rate for first-line treatment of SCLC in the restricted phase is 70% -90% and the response rate for SCLC in the extensive phase is 50% -60%. Current SCLC recurs or progresses within 1 year of first-line treatment with about 80% of patients with restricted periods and almost all patients with extensive periods, and it can be seen that the effect of second-line treatment is related to the final survival of SCLC patients. Topotecan single as standard therapy for second line treatment with a patient response rate of about 22% and a total patient survival of about 8 months; for drug-resistant recurrent patients, the response rate is only about 4%, the median survival is only 5 months, the clinical benefit is extremely limited, and the clinical benefit is greatly unsatisfied. In view of the above, SCLC still faces many challenges in terms of treatment due to the high recurrence rate and drug resistance rate, and limited selection of two-line therapeutic drugs, limited patient survival benefit, and there is a strong need for new therapeutic approaches to change existing clinical unmet. Several studies now suggest that Notch pathway inhibition is highly correlated with the development of SCLC, and that ligand DLL3 is considered one of the most potential targets for treatment of SCLC.

DLL3 is an atypical Notch pathway ligand identified by high-throughput sequencing, which is a single transmembrane protein consisting of 619 amino acids, the complete structure comprising 1 DSL domain, 1 intracellular domain and 6 epidermal growth factor-like domains (i.e. domains EGF1, EGF2, EGF3, EGF4, EGF5 and EGF 6), which are important targets for SCLC development and progression. Immunohistochemistry showed that this target was hardly expressed in normal tissues and not in other tumors, whereas it was expressed in large amounts specifically in neuroendocrine tumors, with high levels of DLL3 present on the tumor tissue and cancer cell surface of approximately 80% of SCLCs, and approximately 85% of recurrent SCLCs also expressed DLL3 proteins, a constitutive expression receptor for SCLCs. DLL3 is not expressed in normal tissues, but specifically expressed in neuroendocrine tumors such as SCLC, making it one of the targets for antibody or antibody drug conjugate (antibody drug conjugate, ADC) development.

There are a number of drugs currently being developed that target DLL3 targets, mainly bispecific antibodies, cytotherapeutic and ADC drugs (see for example CN104520324 a). The Rova-T is the first ADC drug targeting the DLL3 target point developed by ibovine and is also the first targeting therapeutic drug of SCLC in clinical research, the drug utilizes DLL3 expressed on the surface of tumor cells to identify the tumor cells and convey cytotoxic drug PBD into the tumor cells so as to achieve the effect of directionally killing the tumor cells, but in clinical phase 3 research, the drug is intolerable to patients due to too great toxic and side effect of the cytotoxic drug PBD, thereby resulting in insufficient effectiveness, and the development is stopped at present.

Disclosure of Invention

Aiming at the defects of the anti-DLL 3 antibody in the prior art and the current situation that the ADC drug targeting the DLL3 has not been successfully developed clinically, the invention provides a brand-new anti-DLL 3 antibody, the ADC targeting the DLL3, an intermediate, a preparation method and application thereof. Aiming at the defect of lack of the antibody drug conjugate for targeting DLL3 in the prior art, the antibody drug conjugate can realize the effect of treating tumor patients with neuroendocrine characteristics including SCLC. The invention mainly solves the technical problems through the following technical means.

The present invention provides an anti-DLL 3 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein,,

the VH comprises the following Complementarity Determining Regions (CDRs) or mutations thereof: VH CDR1 as shown in the amino acid sequence of SEQ ID NO 9, 19, 29, 39, 59, 69, 79, 89, 99 or 63, VH CDR2 as shown in 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 in the amino acid sequence of SEQ ID NO 11, 21, 31, 41, 61, 71, 81, 91, 101, 111 or 93;

the VL comprises the following CDRs or mutations thereof: VL CDR1 as shown in the amino acid sequence of SEQ ID No. 12, 32, 42, 62, 72, 82, 92, 102, 112 or 103, VL CDR2 as shown in GAS, GAT, TTS, NAK, YTS, RAN, WAS, FTS or NAN, and/or VL CDR3 as shown in the amino acid sequence of SEQ ID No. 14, 24, 34, 44, 64, 74, 84, 94, 104, 114 or 113;

Wherein the mutation is a 3, 2 or 1 amino acid insertion, deletion or substitution in the amino acid sequence of the CDR.

In this application, an "amino acid mutation" in "an analogous" insertion, deletion or substitution of 3, 2 or 1 amino acids "refers to a mutation in which an amino acid is present in the sequence of the variant as compared to the original amino acid sequence, including an insertion, deletion or substitution of an amino acid that occurs on the basis of the original amino acid sequence. An exemplary interpretation is that mutations to a CDR may comprise 3, 2 or 1 amino acid mutations, and optionally the same or different numbers of amino acid residues may be selected between these CDRs for mutation, e.g., 1 amino acid mutation to CDR1, and no amino acid mutation to CDR2 and CDR 3.

In this application, the mutations may include mutations which are currently known to those skilled in the art, for example, during the production or use of the antibody, and may be performed on the antibody, for example, mutations at sites which may exist, particularly post-transcriptional modification of the CDR regions (post-translational modifications, PTMs), including aggregation, deamidation sensitivity (asparagine deamidation, site (NG, NS, NH, etc.), aspartic acid isomerism (DG, DP) sensitivity, N glycosylation (N- { P } S/T) sensitivity, oxidation sensitivity, and the like.

Preferably, the VH comprises the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 shown in SEQ ID NOs 9, 10 and 11 respectively; or, the VH comprises the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 shown in SEQ ID NO 19, 20 and 21 respectively; or, the VH comprises the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 shown in SEQ ID NO. 29, 30 and 31 respectively; or, the VH comprises the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 shown in SEQ ID NO 39, 40 and 41 respectively; or, the VH comprises the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 shown as SEQ ID NO 59, 60 and 61 respectively; or, the VH comprises the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 shown as SEQ ID NO 69, 70 and 71 respectively; or, the VH comprises the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 shown as SEQ ID NO 79, 80 and 81 respectively; or, the VH comprises the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 shown as SEQ ID NO 89, 90 and 91 respectively; or, the VH comprises the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 shown as SEQ ID NO 99, 100 and 101 respectively; or, the VH comprises the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 shown as SEQ ID NO 89, 110 and 111 respectively; or, the VH comprises the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 shown in SEQ ID NO. 63, 83 and 93 respectively.

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

In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 of the anti-DLL 3 antibody are shown in SEQ ID NOs 9, 10 and 11, respectively, and the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 of the VL antibody are shown in SEQ ID NOs 12, GAS and 14, respectively. In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 are shown in SEQ ID NO. 19, 20 and 21, respectively, and the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 are shown in SEQ ID NO. 12, GAS and SEQ ID NO. 24, respectively. In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 are shown in SEQ ID NO. 29, 30 and 31, respectively, and the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 are shown in SEQ ID NO. 32, GAT and SEQ ID NO. 34, respectively. In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 are shown in SEQ ID NO 39, 40 and 41, respectively, and the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 are shown in SEQ ID NO 42, TTS and SEQ ID NO 44, respectively. In a preferred embodiment, the amino acid sequences of the VH comprising VH CDR1, VH CDR2 and VH CDR3 are shown in SEQ ID NO 59, 60 and 61, respectively, and the amino acid sequences of the VL comprising VL CDR1, VL CDR2 and VL CDR3 are shown in SEQ ID NO 62, NAK and SEQ ID NO 64, respectively. In a preferred embodiment, the amino acid sequences of the VH comprising VH CDR1, VH CDR2 and VH CDR3 are shown in SEQ ID NO:69, 70 and 71, respectively, and the amino acid sequences of the VL comprising VL CDR1, VL CDR2 and VL CDR3 are shown in SEQ ID NO:72, NAK and SEQ ID NO:74, respectively. In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 are shown in SEQ ID NO. 79, 80 and 81, respectively, and the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 are shown in SEQ ID NO. 82, YTS and SEQ ID NO. 84, respectively. In a preferred embodiment, the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 comprised by the VH are shown in SEQ ID NOs 89, 90 and 91, respectively, and the amino acid sequences of VL CDR1, VL CDR2 and VL CDR3 comprised by the VL are shown in SEQ ID NOs 92, RAN and 94, respectively. In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 are shown as SEQ ID NO 99, 100 and 101, respectively, and the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 are shown as SEQ ID NO 102, WAS and SEQ ID NO 104, respectively, in the anti-DLL 3 antibody. In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 are shown in SEQ ID NO 89, 110 and 111, respectively, and the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 are shown in SEQ ID NO 112, FTS and SEQ ID NO 114, respectively. In a preferred embodiment, the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 of the anti-DLL 3 antibody are shown in SEQ ID nos. 63, 83 and 93, respectively, and the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 of the VL antibody are shown in SEQ ID nos. 103, NAN and 113, respectively.

Preferably, in the anti-DLL 3 antibody, the heavy chain variable region (VH) further comprises a heavy chain variable region framework region (VH FWR), wherein the VH FWR is a heavy chain variable region framework region of a human or murine antibody. In a preferred embodiment of the invention, the VH may comprise an amino acid sequence as shown in

SEQ ID NO

15, 25, 35, 45, 65, 75, 85, 95, 105, 13 or 22 or a mutation thereof.

Preferably, in the anti-DLL 3 antibody, the light chain variable region (VL) further comprises a light chain variable region framework region (VL FWR); wherein the VL FWR is a human or murine antibody light chain variable region framework region. In a preferred embodiment of the invention, the VL may comprise an amino acid sequence as shown in SEQ ID NO. 16, 26, 36, 46, 66, 76, 86, 96, 106, 33 or 23 or a mutation thereof.

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

The mutation described above is a deletion, substitution or addition of one or more amino acid residues in the amino acid sequence of the VH and/or VL, and the mutated amino acid sequence has at least 85% sequence identity to the amino acid sequence of the VH and/or VL and maintains or improves binding of the antibody to DLL 3; the at least 85% sequence identity is preferably at least 90% sequence identity, more preferably at least 95%, 96%, 97%, 98% sequence identity, most preferably at least 99% sequence identity.

In the present application, the amino acid sequences of the CDRs listed above are all shown according to the kabat definition rules (in the present application also shown according to the kabat definition rules). However, it is well known to those skilled in the art that CDRs of antibodies can be defined in the art by a variety of methods, such as Kabat definition rules based on sequence variability (see, kabat et al, protein sequences for immunology, fifth edition, national institutes of health, besseda, malyland (1991)) and Chothia definition rules based on structural loop region position (see, jmol Biol 273:927-48,1997). It will be appreciated by those skilled in the art that unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are to be understood as encompassing complementarity determining regions defined in any of the above known schemes as described by the present invention. Although the scope of the claimed invention is based on the sequences shown by the kabat definition rules, amino acid sequences corresponding to the definition rules according to other CDRs should also fall within the scope of the claimed invention.

Preferably, the anti-DLL 3 antibody is a full length antibody, fab ', F (ab') 2, fv is preferably an scFv, a bispecific antibody, a multispecific antibody, a heavy chain antibody or a single domain antibody.

In a preferred embodiment, the anti-DLL 3 antibody is a full length antibody, which comprises a heavy chain and a light chain; the heavy chain constant region included in the heavy chain is preferably a human or mouse-derived antibody heavy chain constant region; light chain constant regions included in the light chain the antibody light chain constant regions are preferably human or mouse-derived antibody light chain constant regions. Preferably, the human light chain constant region is a human kappa or lambda light chain constant region; the human heavy chain constant region is human IgG1, igG2, igG3, or IgG4.

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.

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

The mutation described above is a deletion, substitution or addition of one or more amino acid residues in the amino acid sequence of the heavy and/or light chain, and the mutated amino acid sequence has at least 85% sequence identity to the amino acid sequence of the heavy and/or light chain and maintains or improves the binding of the antibody to DLL 3; the at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95%, 96%, 97% or 98% sequence identity; most preferably at least 99% sequence identity.

In one aspect, the invention also provides an anti-DLL 3 antibody that competitively binds to DLL3 protein with an anti-DLL 3 antibody as described above.

In a further aspect the invention provides an isolated nucleic acid encoding an anti-DLL 3 antibody as described above.

In a further aspect the invention provides a recombinant expression vector comprising an isolated nucleic acid as described above. Preferably, the expression vector comprises a eukaryotic expression vector and/or a prokaryotic expression vector.

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

In one aspect, the invention also provides an Antibody Drug Conjugate (ADC) having the structural formula Ab- (L) ₃ -L ₂ -L ₁ -D) _m ；

Wherein Ab is an anti-DLL 3 antibody;

d is a cytotoxic drug

m is 2-8;

L ₁ the structure of which is shown as formula I, II, III or IV, the a end of which is connected with the cytotoxic drug, the e end of which is connected with the L ₂ C end of (2) is connected;

(L) _p wherein L is independently one or more of a phenylalanine residue, an alanine residue, a glycine residue, a glutamic acid residue, an aspartic acid residue, a cysteine residue, a glutamic acid residue, a histidine residue, an isoleucine residue, a leucine residue, a lysine residue, a methionine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, a tyrosine residue, and a valine residue; p is 2-4;

R ¹ is one or more of-NR ^1-1 R ^1-2 Substituted C ₁ ～C ₆ Alkyl, substituted by one or more R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ Alkyl, C ₁ ～C ₆ Alkyl, C ₃ ～C ₁₀ Cycloalkyl, C ₆ ～C ₁₄ Aryl or 5-14 membered heteroaryl; the hetero atom in the 5-14 membered heteroaryl is selected from one or more of N, O and S, and the number of the hetero atom is 1, 2, 3 or 4; said R is ^1-1 、R ^1-2 And R is ^1-3 Each independently is C ₁ ～C ₆ An alkyl group;

L ₂ is that

Wherein n is independently 1 to 12, e.g., 8, 9, 10, 11 and 12, c-terminal is bonded to L by carbonyl ₁ The f end is connected with the L ₃ Is connected with the d end of the (C);

L ₃ is that

Wherein the b-terminal is connected with the Ab and the d-terminal is connected with the L ₂ Is connected to the f terminal of (c).

In a certain preferred embodiment, the anti-DLL 3 antibody binds to one of the following antigen binding epitopes in the DLL3 protein: DSL domain, N-terminal, EGF2 domain and EGF3-EGF6 domain.

In a certain preferred embodiment, the anti-DLL 3 antibody is preferably an anti-DLL 3 antibody as described above.

In a certain preferred embodiment, the anti-DLL 3 antibody binds to an antigen binding epitope of the EGF2 domain in the DLL3 protein.

In a preferred embodiment, the anti-DLL 3 antibody comprises a VH having the amino acid sequence shown in SEQ ID NO. 15 and a VL having the amino acid sequence shown in SEQ ID NO. 16, or comprises a VH having the amino acid sequence shown in SEQ ID NO. 25 and a VL having the amino acid sequence shown in SEQ ID NO. 26.

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

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

In a preferred embodiment, L is a valine residue and/or an alanine residue, the plurality of L is two or three, and the p is 2. More preferably, said (L) _p Is that

Wherein the g-terminus is bound to the L via a carbonyl group ₂ Is connected to the c terminal of (c).

In a preferred embodiment, said R ¹ Is one or more of-NR ^1-1 R ^1-2 Substituted C ₁ ～C ₆ Alkyl, substituted by one or more R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ Alkyl, or C ₁ ～C ₆ Alkyl, said R ^1-1 、R ^1-2 And R is ^1-3 Each independently is C ₁ ～C ₄ An alkyl group.

In a preferred embodiment, when R is ¹ Is one or more of-NR ^1-1 R ^1-2 Substituted C ₁ ～C ₆ In the case of alkyl, said C ₁ ～C ₆ Alkyl is C ₁ ～C ₄ Alkyl, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably ethyl; the plurality is preferably two or three. Preferably, said R ^1-1 And R is ^1-2 Each independently is C ₁ ～C ₄ Alkyl is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, more preferably methyl. More preferably, said-NR ^1-1 R ^1-2 is-N (CH) ₃ ) ₂ . Further preferably, when R is ¹ Is covered by a-NR ^1-1 R ^1-2 Substituted C ₁ ～C ₆ In the case of alkyl, said radical is substituted by one-NR ^1-1 R ^1-2 Substituted C ₁ ～C ₆ Alkyl is

In a preferred embodiment, when R is ¹ To be covered by one or more R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ In the case of alkyl, said C ₁ ～C ₆ Alkyl is C ₁ ～C ₄ Alkyl, preferably methylA group, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl group, more preferably ethyl; the plurality is preferably two or three. Preferably, said R ^1-3 Is C ₁ ～C ₄ Alkyl is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, more preferably methyl. More preferably, when said R ¹ Is covered by one R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ In the case of alkyl, said radical is substituted by one R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ Alkyl is

In a preferred embodiment, when R is ¹ Is C ₁ ～C ₆ In the case of alkyl, said C ₁ ～C ₆ Alkyl is C ₁ ～C ₄ Alkyl is more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and still more preferably methyl.

In a preferred embodiment, when L ₁ When the structure of the formula I is shown in the specification, L ₂ Preferably is

The L is ₃ Preferably +.>

In a preferred embodiment, when L ₁ When the structure of (C) is shown as formula II, L ₂ Preferably is

The L is ₃ PreferablyIs that

In a preferred embodiment of the invention, when L ₁ When the structure of (C) is shown as formula III, L ₂ Preferably is

The L is ₃ Preferably +. >

In a preferred embodiment of the invention, when L ₁ When the structure of (C) is shown as formula IV, the L ₂ Preferably is

The L is ₃ Preferably +.>

In a preferred embodiment, L ₁ The structure of (a) is preferably as shown in formula I or III.

In a preferred embodiment, the compound of formula III is preferably

In a preferred embodiment, the formula III is further preferred

In a preferred embodiment, said L ₃ Preferably linked to the thiol group of said antibody as a thioether bond. To be used for

For example, a->

The form of linkage to the cysteine residues in the antibodies is

In a preferred embodiment, m is an integer from 2 to 8, e.g., 2, 3, 4, 5, 6, 7, 8, or a non-integer, e.g.: 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.

Preferably, the antibody drug conjugate is any one of the compounds shown as follows:

where Ab is the anti-DLL 3 antibody described above, m is from 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.

In a preferred embodiment, the antibody drug conjugate is any one of the compounds shown below:

wherein Ab is an anti-DLL 3 antibody comprising a VH having an amino acid sequence shown as SEQ ID NO. 25 and a VL having an amino acid sequence shown as SEQ ID NO. 26; preferably comprises a heavy chain having an amino acid sequence shown in SEQ ID NO. 27 and a light chain having an amino acid sequence shown in SEQ ID NO. 28.

wherein Ab is an anti-DLL 3 antibody comprising a VH having an amino acid sequence shown as SEQ ID NO. 15 and a VL having an amino acid sequence shown as SEQ ID NO. 16; preferably comprises a heavy chain having an amino acid sequence shown in SEQ ID NO. 17 and a light chain having an amino acid sequence shown in SEQ ID NO. 18.

In a preferred embodiment, the antibody drug conjugate is a compound as shown below:

wherein Ab is an anti-DLL 3 antibody comprising a heavy chain having an amino acid sequence shown in SEQ ID NO. 2 and a light chain having an amino acid sequence shown in SEQ ID NO. 1.

wherein Ab is an anti-DLL 3 antibody comprising a heavy chain having an amino acid sequence shown in SEQ ID NO. 4 and a light chain having an amino acid sequence shown in SEQ ID NO. 3.

wherein Ab is an anti-DLL 3 antibody comprising a VH having an amino acid sequence shown as SEQ ID NO. 22 and a VL having an amino acid sequence shown as SEQ ID NO. 23; preferably comprises a heavy chain having an amino acid sequence shown in SEQ ID NO. 73 and a light chain having an amino acid sequence shown in SEQ ID NO. 109.

wherein Ab is an anti-DLL 3 antibody comprising a VH having the amino acid sequence shown in SEQ ID NO. 45 and a VL having the amino acid sequence shown in SEQ ID NO. 46; preferably comprises a heavy chain having an amino acid sequence shown in SEQ ID NO. 47 and a light chain having an amino acid sequence shown in SEQ ID NO. 48.

In a further aspect the invention provides a chimeric antigen receptor comprising an anti-DLL 3 antibody as described above.

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

In one aspect, the invention also provides a method for preparing an anti-DLL 3 antibody, the method comprising the steps of: the transformant as described above was cultured, and an anti-DLL 3 antibody was obtained from the culture.

In a further aspect the invention provides a pharmaceutical composition comprising an anti-DLL 3 antibody as described above, an antibody drug conjugate as described above, a chimeric antigen receptor as described above, and/or a genetically modified cell as described above. Preferably, the pharmaceutical composition is in liquid, gaseous, solid and semi-solid dosage forms, and/or the pharmaceutical composition may be administered orally, by injection, nasally, transdermally or mucosally. More preferably, the pharmaceutical composition further comprises a combination therapeutic agent comprising a chemotherapeutic agent, a radiation therapeutic agent, an immunosuppressant, and/or a cytotoxic drug.

In a further aspect the invention provides the use of an anti-DLL 3 antibody as described above, an antibody drug conjugate as described above, a chimeric antigen receptor as described above, a genetically modified cell as described above, and/or a pharmaceutical composition as described above for the preparation of a medicament, kit and/or administration device for the treatment and/or prevention of a disease associated with abnormal DLL3 expression; or an anti-DLL 3 antibody as described above, an antibody drug conjugate as described above, a chimeric antigen receptor as described above, a genetically modified cell as described above, and/or a pharmaceutical composition as described above for use in the treatment and/or prevention of a disease associated with abnormal DLL3 expression. The DLL3 expression abnormality related disease is preferably a tumor, the tumor is preferably a cancer, the cancer is preferably an endocrine tumor such as neuroendocrine tumor, prostate cancer, pancreatic cancer and colorectal cancer, and the like, and more preferably small cell lung cancer.

In a further aspect the invention provides a kit comprising an anti-DLL 3 antibody as described above, an antibody drug conjugate as described above, a chimeric antigen receptor as described above, a genetically modified cell as described above, and/or a pharmaceutical composition as described above; and optionally, instructions.

In one aspect, the invention provides a drug delivery device comprising: (1) An infusion module for administering a pharmaceutical composition as described above to a subject in need thereof, and (2) optionally a efficacy monitoring module.

In one aspect the invention also provides a method of detecting DLL3 comprising the step of detecting using an anti-DLL 3 antibody as described above. Preferably, the method is for non-diagnostic and/or therapeutic purposes.

In a further aspect the invention provides a method of diagnosing, preventing and/or treating a disease associated with abnormal DLL3 expression comprising administering to a subject in need thereof an anti-DLL 3 antibody as described above, an antibody drug conjugate as described above, a genetically modified cell 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 a cancer, the cancer is preferably a neuroendocrine tumor, and more preferably a small cell lung cancer.

The invention also provides a panel of antibodies (including molecules comprising or consisting of antibody fragments or variants), wherein the members of the panel correspond to one, two, three, four, five, or more different antibodies of the invention [ e.g., complete antibodies, fab, F (ab) ] ₂ Fragments and scFv and the like]。

In one aspect, the invention also provides an antibody drug conjugate, which has a structural formula of Ab- (L) ₅ -L ₄ -L ₃ -L ₂ -L ₁ -D) _m ；

Wherein Ab is an anti-DLL 3 antibody; the anti-DLL 3 antibody binds to a DSL domain and/or the N-terminus in a DLL3 protein;

d is a cytotoxic drug;

m is 2-8;

R ¹ is one or more of-NR ^1-1 R ^1-2 Substituted C ₁ ～C ₆ Alkyl, substituted by one or more R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ Alkyl, C ₁ ～C ₆ Alkyl, C ₃ ～C ₁₀ Cycloalkyl, C ₆ ～C ₁₄ Aryl or 5-14 membered heteroaryl;the hetero atom in the 5-14 membered heteroaryl is selected from one or more of N, O and S, and the number of the hetero atom is 1, 2, 3 or 4; said R is ^1-1 、R ^1-2 And R is ^1-3 Each independently is C ₁ ～C ₆ An alkyl group;

L ₂ is that

Wherein n is independently 1 to 12, c-terminal is bound to L via a carbonyl group ₁ The f end is connected with the L ₃ Is connected with the d end of the (C);

L ₃ is that

Wherein the b-terminal is connected with the Ab and the d-terminal is connected with the L ₂ Is connected with the f end of the (E);

the L is ₄ Absent, or selected from cleavable linkers, non-cleavable linkers, hydrophilic linkers, pre-charged linkers and dicarboxylic acid-based linkers;

the L is ₅ Absent, or a compound of formula V:

wherein X is ₁ Selected from the group consisting of hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy, and cycloalkyl;

X ₂ selected from alkyl, cycloalkyl and heterocyclyl; m is 0-5; s is a sulfur atom.

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

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

In a preferred embodiment, when L ₁ When the structure of the formula I is shown in the specification, L ₂ Is that

In a preferred embodiment, when L ₁ When the structure of (C) is shown as formula II, L ₂ Is that

In a preferred embodiment, when L ₁ When the structure of (C) is shown as formula III, L ₂ Is that

In a preferred embodiment, when L ₁ When the structure of (C) is shown as formula IV, the L ₂ Is that

In a certain preferred embodiment, said n is independently 8, 9, 10, 11 and 12.

In a preferred embodiment, m is an integer or non-integer from 2 to 8.

In a preferred embodiment, said L ₃ Is that

In a preferred embodiment, the L ₄ Selected from the group consisting of N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), 6-Maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (VC), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB) and MC-VC-PAB.

In a preferred embodiment, the L ₅ In (1), when X ₁ Is a hydrogen atom, X ₂ When the alkyl group and m are 1, the compound shown in the formula V is S- (3-carbonyl propyl) thioacetate.

In a preferred embodiment, D is one or more of a maytansine derivative, a dolastatin 10 derivative, an doxorubicin analog or a camptothecin analog.

Such as DM1, DM3 and DM4.

Such as MMAE and MMAF.

Such as doxorubicin and cambriubicin.

More preferably, D is a camptothecin analog, further preferably a compound having the structure shown as formula Va or Vb:

R ² and R is ⁵ H, C each independently of the other ₁ -C ₆ Alkyl or halogen;

R ³ and R is ⁶ H, C each independently of the other ₁ -C ₆ Alkyl or halogen;

R ⁴ and R is ⁷ Each independently is C ₁ -C ₆ An alkyl group.

In a preferred embodiment, the said (L) _p Is that

In a preferred embodiment, the compound of formula III

the anti-DLL 3 antibody is an anti-DLL 3 antibody with a light chain with an amino acid sequence shown as SEQ ID NO. 1 and a heavy chain with an amino acid sequence shown as SEQ ID NO. 2, or an anti-DLL 3 antibody with a light chain with an amino acid sequence shown as SEQ ID NO. 3 and a heavy chain with an amino acid sequence shown as SEQ ID NO. 4;

And m is 7.23 or 7.32.

wherein Ab is an anti-DLL 3 antibody comprising a heavy chain having an amino acid sequence shown in SEQ ID NO. 2 and a light chain or +.>

In a certain aspect, the invention also provides a preparation method of the antibody drug conjugate, which comprises the following steps: and (3) dropwise adding the excessive connecting group-drug conjugate dissolved in DMSO into a buffer solution containing the anti-DLL 3 antibody to obtain the antibody drug conjugate.

In a further aspect the invention provides a pharmaceutical composition comprising an antibody drug conjugate according to the invention.

Preferably, the pharmaceutical composition is in a liquid, gaseous, solid or semi-solid dosage form, and/or the pharmaceutical composition may be administered orally, by injection, nasally, transdermally or mucosally.

More preferably, the pharmaceutical composition further comprises a combination therapeutic agent comprising a chemotherapeutic agent, a radiation therapeutic agent, an immunosuppressant, and/or a cytotoxic drug.

In one aspect, the invention also provides the use of an antibody drug conjugate and/or a pharmaceutical composition according to the invention for the preparation of a medicament, kit and/or administration device for the treatment and/or prevention of diseases associated with abnormal DLL3 expression;

the DLL3 expression abnormality related disease is preferably a tumor, the tumor is preferably a cancer, the cancer is preferably a neuroendocrine tumor, and more preferably a small cell lung cancer.

In a further aspect the invention provides a kit comprising an antibody drug conjugate according to the invention and/or a pharmaceutical composition according to the invention; and optionally, instructions.

In one aspect, the invention provides a drug delivery device comprising: (1) An infusion module for administering an antibody drug conjugate according to the invention and/or a pharmaceutical composition according to the invention to a subject in need thereof, and (2) optionally a efficacy monitoring module.

In a further aspect the invention provides a method of detecting DLL3, which comprises the step of detecting using an antibody drug conjugate according to the invention. Preferably, the method of detecting DLL3 is for non-diagnostic and/or therapeutic purposes.

In a further aspect the invention provides a method of diagnosing, preventing and/or treating a disease associated with abnormal DLL3 expression comprising administering to a subject in need thereof an antibody drug conjugate according to the invention and/or a pharmaceutical composition according to the invention.

In the present invention, the conditions and operations of the coupling reaction may be those conventional in the art.

In the present invention, m represents the molar ratio of cytotoxic drug molecule to Ab (also known as DAR, i.e. drug antibody coupling ratio), m may be an integer or a fraction, preferably understood as: the average molar ratio of the drug molecules in the antibody drug conjugate obtained after coupling the monoclonal antibody molecules with the cytotoxic drugs can be generally determined by means of Hydrophobic chromatography (Hydrophobic-Interaction Chromatography, HIC), polyacrylamide-SDS gel electrophoresis (SDS-PAGE), liquid phase mass spectrometry (liquid chromatograph-mass spectrometer, LC-MS) and the like.

In the present invention, the term "C ₁ ～C ₆ Alkyl "alone or in combination denotes a saturated, straight-chain or branched alkyl radical having from 1 to 6, in particular from 1 to 4, carbon atoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl," C ₁ ～C ₆ The alkyl group "is preferably methyl or ethyl.

Antibodies of the invention may be prepared using techniques well known in the art, such as hybridoma methods, recombinant DNA techniques, phage display techniques, synthetic techniques, or combinations thereof, or other techniques known in the art.

The term "selective" or "specific" as used herein refers to the fact that: the disclosed antagonists do not show significant binding to substances other than DLL3, except in those particular cases: wherein the supplemental antagonist imparts an additional specificity (e.g., a bispecific or bifunctional molecule wherein the molecule is designed to bind or perform both functions, at least one of which is specifically binding to DLL 3) to a moiety other than the DLL3 specific binding moiety.

An "antibody molecule" or "antibody" as described herein refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen. Thus, the term antibody encompasses not only intact antibody molecules, but also fragments of such antibodies as well as variants (including derivatives) of such antibodies and antibody fragments. The term antibody molecule as described herein includes, for example, but is not limited to, single chain Fv (scFv), fab fragments, fab 'fragments, F (ab') 2, disulfide-linked Fv (sdFv), fv, and whole or full length antibodies. The term "single chain Fv" or "scFv" refers to a polypeptide comprising the VL domain of an antibody linked to the VH domain of an antibody. Antibodies that immunospecifically bind DLL3 can cross-react with other antigens. Preferably, antibodies that immunospecifically bind DLL3 do not cross react with other antigens. Antibodies that immunospecifically bind DLL3 can be identified, for example, by immunoassays or other methods known to those skilled in the art. "intact antibody" or "full length antibody" refers to a protein comprising two heavy chains (H) and two light chains (L), said heavy and light chains being linked to each other by disulfide bonds, said protein comprising: (1) In the case of heavy chains, comprising a heavy chain variable region (abbreviated herein as "VH") and a heavy chain constant region comprising three domains CH1, CH2, CH 3; and (2) in the case of a light chain, a light chain variable region (abbreviated herein as "VL") and a light chain constant region comprising one domain CL. Antibodies of the 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 invention), and epitope-binding fragments of any of the antibodies described above. The immunoglobulin molecules of the invention may be of any type (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG1, igG2, igG3, igG4, igA1 and IgA 2) or subclass of immunoglobulin. The antibody of the present invention may be a monoclonal antibody, preferably a murine anti-human monoclonal antibody, or a polyclonal antibody. The antibodies of the invention may be super-humanized antibodies or diabodies.

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

"Single-domain antibody", also known as "nanobody", refers to a VHH structure cloned from a heavy chain antibody, which is the smallest unit known to bind an antigen of interest.

The amino acid sequence with the homology of 90%, 95%, 98% or more than 99% is obtained by inserting, deleting or replacing the amino acid sequence shown in the sequence table, wherein the replacement can be as follows: for example, the sequences are subjected to in silico analysis, and analysis and substitution of possible post-transcriptional modification (PTMS) sites, particularly CDR regions, including aggregation, deamidation sensitivity (asparagine deamidation, site (NG, NS, NH, etc.), aspartate isomerism (DG, DP) sensitivity, N glycosylation (N- { P } S/T) sensitivity, oxidation sensitivity, etc. of antibodies.

"KD" refers to the dissociation constant obtained from the ratio (or Kd/Ka, expressed in molar concentration (M)) of Kd (the rate of dissociation of a particular binding molecule-target protein interaction) to Ka (the rate of binding of a particular binding molecule-target protein interaction). KD values can be determined using methods well established in the art. A preferred method of determining the KD of a binding molecule is by using surface plasmon resonance, e.g., a biosensor system, such as the Biacore TM (GE Healthcare Life Sciences) system.

The term "treatment" or its equivalent, when used in reference to, for example, cancer, refers to a procedure or process used to reduce or eliminate the number of cancer cells in a patient or to alleviate symptoms of cancer. "treating" of cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will actually be eliminated, that the number of cells or disorders will actually be reduced or that the symptoms of the cancer or other disorder will actually be reduced. In general, methods of treating cancer are performed even with low likelihood of success, but are still considered to induce an overall beneficial course of action, given the patient's medical history and estimated survival expectancy.

The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium representative of a carrier capable of delivering an effective amount of the active agents of the present invention, which does not interfere with the biological activity of the active agents and which does not have toxic or side effects to the host or patient, including water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, and the like. Such matrices include suspending agents, viscosity enhancers, transdermal enhancers, and the like. Their formulations are well known to those skilled in the cosmetic or topical pharmaceutical arts.

The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that: the anti-DLL 3 antibody has good internalization activity, better binding activity with human DLL3 protein and stronger affinity at protein level. The antibody-conjugated drug has good drug property, biological activity and in-vitro and in-vivo anti-tumor activity, and can realize the application of cytotoxic drugs in treating tumor patients with neuroendocrine characteristics including SCLC.

Drawings

FIG. 1 is a map of the pV81 vector.

FIG. 2 shows the internalization results of the antibodies of example 4.

Figure 3 shows the dose-response curve of the binding activity of chimeric antibodies to hlll 3.

Figure 4 shows the dose-response curves of the binding activity of chimeric antibodies to hDLL1 (left), hDLL4 (right).

Fig. 5 shows dose-response curves of binding activities of chimeric antibodies to mice (left side), monkey DLL3 (right side).

Figure 6 shows graphs of cytotoxic activity of different ADC candidates for 6 days of DLL3 target cell treatment.

FIG. 7 shows the in vivo anti-tumor status of ADC drugs in NCI-H82 model.

Detailed Description

Table a. Abbreviation description

DMEM	Basal medium
		HAT	Screening media
PEI	Polyether imide
		MOI	Multiplicity of infection
GFP	Green fluorescent protein
		SPF	Animals without specific pathogen
PBST	Tween-containing phosphate buffer salt solution
		HRP	Horseradish peroxidase
TMB	3,3', 5' -tetramethylbenzidine
		PBS	Phosphate buffered saline solution
APC	Allophycocyanin

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 preparation of monoclonal antibodies FDA027 and FDA031 specifically targeting hDLL3

In the invention, monoclonal antibodies FDA027 and FDA031 with high affinity and specificity targeting hDLL3 are selected, wherein FDA027 has a light chain amino acid sequence shown as SEQ ID NO. 1 and a heavy chain amino acid sequence shown as SEQ ID NO. 2, and FDA031 has a light chain amino acid sequence shown as SEQ ID NO. 3 and a heavy chain amino acid sequence shown as SEQ ID NO. 4.

FDA027 light and heavy chain nucleotide sequences were obtained by total gene synthesis (Suzhou Jin Weizhi), double-digested single constructs of EcoR I (purchased from NEB, R3104S) and Hind III (purchased from NEB, R3101S) into the pV81 vector (see FIG. 1), transformation into Trans 1-T1 competent cells (purchased from full gold, CD 501) by ligation, sequencing confirmation, amplification-positive clones were picked up from them, plasmid medium extraction was performed to obtain antibody light chain eukaryotic expression plasmid FDA027-L/pV81 and antibody heavy chain eukaryotic expression plasmid FDA027-H/pV81, the mass ratio of light and heavy chain eukaryotic expression plasmid was 1.5:1, transformation into suspension-grown adapted CHO cells (purchased from ATCC) by electric shock, inoculation of the shock-adapted cells into 96 well plates at 2000-5000 cells/well plates, shaking-up to a maximum expression of 37% CO (37 ml) in shake flask (37% by shake flask) according to the kit (purchased from CisO, 62 HFEG) after 3 weeks culture ₂ Shake culturing at 130r/min, spreading into 1000ml shake flask (culture volume 300 ml) after 3 days, 37deg.C, 5.0% CO ₂ Shake culturing at 130r/min, feeding culture medium with 5-8% of initial culture volume every other day, culturing until 10-12 days, centrifuging the obtained solution at 9500r/min for 15min, removing cell precipitate, collecting supernatant, filtering with 0.22 μm filter membrane, purifying the treated sample with MabSelect affinity chromatography column (purchased from GE company), and finally obtaining high purity anti-DLL 3 antibody FDA027.FDA031 is prepared by the same procedure as FDA027.

Example 2 preparation of human hDLL3 overexpression vector and stably transformed cell line

Human DLL3 expression plasmid pCMV3-DLL3-T1 (purchased from Beijing Yiqiao, HG 20010-UT) was used to transform E.coli competent cells Trans1-T1 (purchased from full gold, CD 501) for amplification of the transfected expression plasmid. The transformation process was referred to the instructions for use of competent cells. The single clone was picked from the transformation plate and amplified in the medium overnight, centrifuged at 6000r/min for 20min, the bacterial liquid was collected for plasmid extraction, and plasmid DNA was extracted according to the procedure of the specification using a plasmid medium extraction kit (purchased from MN, product number: DP 117).

HEK293 cells (purchased from a cell bank of the department of Chinese sciences) with good growth state in logarithmic growth phase are selected, 10% fetal bovine serum is added to fresh culture medium DMEM to dilute the cells to 5×10 ⁵ The cells/ml density, 2 ml/well, were inoculated into 6-well plates, and placed in an incubator (37 ℃ C., 5% CO) ₂ ) Culturing. The following day, transfection reagent lipofectamine 3000 (purchased from Thermo, L3000-008) transfected plasmid pCMV3-DLL3-t1 to HEK293 cells, the procedure was performed according to the instructions of lipofectamine 3000 transfection kit. 48hr after transfection, cells were seeded at a 1-per-well seeding density in 96-well plates and cultured with fresh medium containing 200 μg/ml hygromycin B (available from Thermo, 10687010), and the fresh medium containing hygromycin B was changed every 3 to 4 days to obtain stably transfected monoclonal cells stably expressing hDLL 3. Flow cytometry detection screening hDLL3 expressing HEK293 cells, PBS dilution detection antibody goat anti-hDLL3-PE (purchased from R)&D, FAB 4315P) to 10. Mu.g/ml, 100. Mu.l to 5X 10 are added ⁵ The cells to be tested were incubated at 4℃for 1hr. Then, 1ml of PBS containing 2% fetal bovine serum was added to resuspend the cells, and the supernatant was discarded after centrifugation at 1500r/min for 5min, and the procedure was repeated twice. 1ml PBS was added to resuspend the cells, and the cells were tested with CytoFLEX (purchased from Beckman), and the positive clones with more than 85% positive expression rate of human DLL3 were successfully constructed with HEK293-DLL3 stable cell lines.

HEK293 overexpressing cell lines containing different hDLL3 domains, i.e., hDLL3 (the sequence of which is shown in SEQ ID NO: 5) overexpressing cell lines containing NO N-terminal, hDLL3 (the sequence of which is shown in SEQ ID NO: 6) overexpressing cell lines containing NO N-terminal, DSL and EGF1 domains, hDLL3 (the sequence of which is shown in SEQ ID NO: 7) overexpressing cell lines containing NO N-terminal, DSL and EGF1 and EGF2 domains, respectively, were stably transfected by the same method as described above.

SEQ ID NO:5

GKPIPNPLLGLDSTSGARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGGGSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA

SEQ ID NO:6

GKPIPNPLLGLDSTSGAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGGGSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA

SEQ ID NO:7

GKPIPNPLLGLDSTSGGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGGGSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA

SEQ ID NO:8

GKPIPNPLLGLDSTSGSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLSGGGGSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA

Note that: underlined is the V5 tag, italics is the intracellular sequence

Example 3 preparation of hybridoma monoclonal antibodies and screening of antibodies

3-1 immunization and serum Titer detection

6 female healthy mice of SPF grade Balb/C6 weeks old (purchased from Shanghai Ji Hui laboratory animal feeding Co., ltd.) were selected, randomly divided into A, B two groups, and the DLL3 protein (purchased from ACRO, DL 3-H5255) was subjected to immunization recombination once at two weeks intervals, the first immunization was performed using Freund's complete adjuvant, and the subsequent immunization was performed using Freund's incomplete adjuvant. Mice were bled orbital at day 35, day 49 and their immune serum titers were detected by ELISA and flow cytometry (FACS) conventional methods.

ELISA detection method: the DLL3 protein was diluted to a concentration of 1. Mu.g/ml with PBS, 100. Mu.l/well was added to 96-well ELISA plate at 4℃overnight, the plate supernatant was discarded the next day, 200. Mu.l of PBS solution containing 2% milk was added to each well for blocking, blocking solution was discarded after 2hr and 200ul of PBST (1%Tween 20) was added to wash the plate wells 3 times, after which the serum to be examined was pre-diluted 100-fold as the initial concentration, 3-fold gradient diluted to 11 concentrations, 100ul of each well was added to the ELISA plate, plate supernatant was discarded after 1hr reaction at room temperature, 200ul of PBST (1%Tween 20) was added to wash 5 times, 100ul of HRP-labeled goat anti-mouse antibody (Jackson, 1:10000) was added, and after 1hr incubation at room temperature, the supernatant was discarded, 200ul of PBST (1%Tween 20) was added to wash 7 times. 100ul of TMB chromogenic solution was added to each well and developed for 10min, and after termination of the reaction by addition of 2M HCl, the microplate reader read the absorbance at 450nm (available from biotek, elx 808).

FACS detection method: a96 well V-shaped microplate (available from axygen, wipp 02280) was added 3X 10 per well ⁵ HEK293-DLL3 stably or endogenously expressed cells SHP-77 (purchased from ATCC), centrifuged at 1500r/min for 1min and the supernatant discarded. After diluting immune serum with PBS at 1:50, diluting 8 concentrations in 4-fold gradient, adding 50 mu L/well into a microplate, incubating on ice for 30min, adding 150 mu L of PBS per well to resuspend cells, centrifuging at 1500r/min for 1min, and repeating the operation for 2 times. APC-labeled goat anti-mouse IgG (Jackson, cat. No. 115-605-164, PBS 1:800 dilution) was added and incubated on ice for 30min at 50. Mu.L per well. Cells were resuspended in 150. Mu.L PBS per well, centrifuged at 1500r/min for 1min, and the procedure repeated 2 times for detection with CytoFLEX (available from Beckman). After detection, select the mice with highest titers and smooth serial two immune serum titers, and perform one by intraperitoneal injection of DLL3 protein 3 days before fusion Secondary impact immunization.

3-2 hybridoma fusion screening and cloning

Preparing cell suspension from spleen of Balb/C mouse with impact immunity, fusing spleen cells with SP2/0 cells (purchased from cell bank of Chinese sciences, TCM 42) by electrofusion method, and mixing hybridoma cells according to 2×10 ⁴ Each cell was plated into 96-well plates and screened with HAT medium, and after 7 days the supernatant of the plates was assayed by ELISA, as described in example 3-1, and ELISA positive cells were assayed by FACS assay for DLL3 overexpressing 293 cell line and SHP-77 endogenous cell line, respectively. Cell-binding positive clones were subjected to limiting dilution and repeated 2 times, and the diluted clones were examined to confirm positive clones by ELISA and FACS methods of example 3-1. The positive cloned hybridomas detected after 2 limiting dilutions were amplified for antibody production purification and sequencing of antibody expression genes.

3-3 purification of hybridoma antibody production

To generate mg amounts of antibody for functional characterization, selected hybridoma cells were grown at 2.5X10 ⁵ The density of/mL was inoculated into a cell culture flask containing 250mL of hybridoma serum-free medium (purchased from source culture organism, H630 KJ), shake flask culture was performed at 37℃for 130r/min, and cell supernatant was harvested by centrifugation after the cell viability was reduced to about 30%. The mouse monoclonal antibody was purified using Protein G medium and the antibody was replaced by dialysis into PBS ph7.2 buffer. Antibody concentration and purity were determined by measuring absorbance with a micro-spectrophotometer (available from Hangzhou o Cheng Yiqi, nano-300).

Example 4 analysis of endocytic Activity of antibodies

Endocytosis of the hybridoma antibodies obtained in example 3-3 was detected using SHP-77 cells by the FACS detection method described in example 3-1. The detection method is briefly described as follows, 6 parts of 5×10 are packaged ⁵ The SHP-77 cells were centrifuged at 1500r/min for 1min in 96-well V-shaped microplates and the supernatant was discarded. Saturated 1. Mu.g/ml of antibody to be detected was added, 200. Mu.L of antibody was added to each well, and incubated on ice for 30min. Then 150. Mu.L of PBS was added to each well, centrifuged at 1500r/min for 1min, the supernatant was discarded, and the procedure was repeated 4 times. Resuspension with 200 μl PBSTaking out 1 part, placing into 37 deg.C cell incubator, standing for 5hr, 3hr, 1hr, 0.5hr, and 15min respectively, placing the rest last cell on ice, standing for incubation time, centrifuging at 4deg.C for 1min at 1500r/min, discarding supernatant, adding APC labeled goat anti-mouse IgG (Jackson, cat No. 109-605-098, PBS 1:800 for dilution), adding 50 μl per well, incubating on ice for 30min, adding 150 μl PBS per well for resuspension cell, centrifuging at 1500r/min for 1min, discarding supernatant, and repeating the operation for 4 times. Finally, 100. Mu.L PBS was added to each well to resuspend the cells, and the cells were assayed by CytoFLEX (available from Beckman), and the assay fluorescence statistics are shown in Table 1 below, and FIG. 2 shows the internalization results of the antibodies obtained in 3-3 of example 3, which indicate that the antibodies obtained in 3-3 of example 3 have good internalization activity.

TABLE 1 flow cytometry detection of fluorescence values for antibody internalization

Example 5 acquisition of hybridoma antibody expression Gene sequences

2X 10 collections per clone ⁵ Each hybridoma cell (from example 3-2) was used for RNA extraction, 300g (in this example, rotation speed unit, the same applies below) was centrifuged for 5min, the supernatant was discarded, 250uL of Trizol (from TAKARA, T9108) lysate was added to lyse the hybridoma cells, then 50uL of chloroform was added to vortex and mix well to emulsion, standing still for 5min at room temperature, 12000g was centrifuged for 15min at 4 ℃, the supernatant was sucked into a 1.5ml centrifuge tube of new Rnase-free, and 125 uL of isopropanol was added to mix well and then standing still for 10min at room temperature. 12000g, centrifuging at 4deg.C for 10min to obtain precipitate, discarding supernatant, adding 250 μl of 75% ethanol, mixing by gently upside down, centrifuging at 4deg.C for 10min to obtain supernatant, and drying at room temperature for 10min. Then, 20. Mu.l of RNase-free water was added to the mixture to dissolve the precipitate for 30 minutes, and the mixture was mixed with a pipette after dissolving the precipitate, and the concentration was measured. PrimeScript was used depending on RNA concentration ^TM The RT Master Mix kit (purchased from TAKARA, RR 036A) reverse transcribes the RNA of the clone to be tested into cDNA. Synthesizing antibody light chain and heavy chain specific primers, and obtaining the antibody by a PCR method The amplified fragments were gel recovered using Universal DNA purification recovery kit (purchased from kava biochemistry, DP 214-03) and then ligated with pMD18T vector (purchased from TAKARA, 6011) at 4 ℃ for 30min. mu.L of the ligation product was added to 50uLTG-1 (Lucigen Co.) competent cells, gently mixed, heat-shocked at 42℃for 90s, incubated on ice for 3min after heat-shocking, 1mL of a non-resistant 2YT liquid medium (from Bio-technology, A507019-0250) was added, and after 1h of shaking bed at 37℃for 220r/min, 100. Mu.L of the bacterial liquid was uniformly spread on a 2YT solid plate with Amp resistance (from bio-technology, A600469-0005) and cultured upside down at 37℃for 14-16h. The monoclonal is picked and sent to the engineering for sequencing. After the sequencing result is obtained, the sequencing result is analyzed by Vector NTI and Vbase2 software, so that the amino acid sequence of the antibody with better binding activity and endocytic activity is obtained, and the obtained result is shown in Table 2.

TABLE 2 amino acid sequence numbering of the CDRs and variable regions of the resulting antibodies (encoded by kabat)

EXAMPLE 6 construction and preparation of chimeric antibodies and detection of binding Activity

The hybridoma clone heavy chain variable region sequence with better binding activity and endocytosis activity obtained in example 5 above was subjected to gene synthesis and cloned into pfuses-CHIg-hG 1 vector (purchased from invitrogen, trade name pFUSEss-hchg 1) containing the amino acid sequence of the heavy chain constant region of the human antibody IgG1 by means of homologous recombination to obtain chimeric antibody heavy chain expression vector. The cloned light chain variable region sequences were cloned into pFUSE2ss-CLIg-hk vectors (available from InvivoGen under the trade designation pFUSE2 ss-hclk) containing the amino acid sequence CL of the human antibody Kappa light chain constant region, respectively, by homologous recombination using the same method, to obtain chimeric antibody light chain expression vectors. The amino acid sequence numbers corresponding to the full length of the heavy and light chains of the chimeric antibody thus constructed are shown in Table 3-1. 293F cells of a well-grown logarithmic growth phase were collected, inoculated into 250mL cell culture flasks and cultured in 50mL medium, and PEI co-transfected with 25. Mu.g each of the light and heavy chain expression plasmids. Cell supernatants from day 7 post-transfection cultures were collected, centrifuged and filtered using a 0.45 μm filter, protein a medium purified for antibodies and the antibodies were replaced by dialysis into PBS ph7.2 buffer. The antibody concentration was determined by measuring absorbance with a micro-spectrophotometer (available from Hangzhou O Cheng Yiqi, nano-300). The binding activity of the obtained chimeric antibody to human DLL3 protein was detected by ELISA (results are shown in FIG. 3 and Table 3-2), and ELISA detection method is described in example 3-1. As can be seen from FIG. 3, the obtained chimeric antibodies all have better binding activity to human DLL3 protein.

TABLE 3-1 chimeric antibody light and heavy chain full-length amino acid sequence numbering

Chimeric antibodies	Heavy chain full length	Full length of light chain
			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

TABLE 3-2 binding Activity of chimeric antibodies to human DLL3 protein

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

Example 7 chimeric antibody Competition assay and binding epitope assay

The chimeric antibodies obtained in example 6 were labeled with Biotin, and the activity of competing the antibodies for binding was detected by ELISA. Briefly, the assay was performed by diluting the DLL3 protein to 1. Mu.g/ml in PBS, spreading the solution over a 384-well ELISA plate (from Corning, 3700) for 40 days, discarding the plate well supernatant the next day, blocking with 80. Mu.l of 3% milk (dissolved in PBS) for 2hr, washing with 80. Mu.l of PBST (1. Mu.m Tween 20) for 3 times, pre-diluting the unlabeled chimeric antibody to 100. Mu.g/ml, respectively, then diluting the solution by a 3-fold gradient for 11 concentration spots, adding 25. Mu.l of each well to the blocked ELISA plate, reacting at room temperature for 1hr, discarding the plate well supernatant, washing with 80. Mu.l of PBST (1. Mu.m Tween 20) for 5 times, adding 25. Mu.l of Biotin-labeled chimeric antibody, incubating at room temperature for 1hr, discarding the supernatant again, washing with 80. Mu.l of PBST (1. Mu.m Tween 20) for 5 times. Finally, HRP-labeled streptavidin secondary antibody 1:5000 Incubation (from gold, M00091) at room temperature for 1hr, discarding the supernatant and washing 7 times with 80 μl of PBST (1%o Tween 20). After the reaction was stopped by adding 25. Mu.l of TMB chromogenic solution and allowing to develop for 10min and adding 2M HCl, the absorbance at 450nm was read by an ELISA reader (available from biotek, elx 808).

To determine the region to which the antibody specifically binds, HEK293 overexpressing 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 were examined by FACS for the binding capacity of the chimeric antibody to these four cells to determine the binding region of the antibody. Briefly: will be 5X 10 ⁵ The over-expressed cells of DSL, EGF1, EGF2 and EGF3 were centrifuged at 1500r/min for 1min in 96-well V-shaped microplates and the supernatant was discarded. Saturated 1. Mu.g/ml of antibody to be detected was added, 200. Mu.L per well and incubated on ice for 30min. Then 150. Mu.L PBS was added to each well, centrifuged at 1500r/min for 1min, the supernatant was discarded, and the plate was washed 4 times. APC-labeled goat anti-human IgG (Jackson, PBS 1:800 dilution) was added and incubated on ice for 30min at 50. Mu.L per well. After washing the plates 4 times with PBS, 100. Mu.L of PBS was added to each well to resuspend the cells, and the assay was performed with CytoFLEX (available from Beckman) and the results showed that: antibodies ch1A5, ch33F11, and ch32E8 bind to the EGF2 domain of DLL3, FDA031, ch13C2, and ch15G2 antibodies bind to the N-terminus of DLL3, FDA027 binds to the DSL domain of DLL3, and the remaining antibodies all bind to the EGF3 to EGF6 domains of DLL3, giving a binding epitope for ch15G2 as the N-terminal domain; the binding epitope of ch32E8, ch1A5 and ch33F11 is the EGF2 domain; the binding epitopes of ch36H10, ch6A2, ch19C7, ch26D9, ch23G2, ch10B1 and ch4H7 are EGF3-EGF6 domains.

Example 8 Cross-reactivity of antibodies to human DLL1, DLL4 proteins and murine and monkey DLL3 cells

The chimeric antibody obtained in example 6 was tested for its binding capacity to human DLL1 (purchased from Acro, DL1-H52H 8), DLL4 (purchased from Acro, DL 4-H5227) protein by ELISA, by ELISA test methods, respectively diluting DLL1 and DLL4 proteins with PBS to a final concentration of 1. Mu.g/ml, respectively spreading onto 384-well ELISA plates (purchased from corning, 3700) at 4℃overnight, discarding the plate well supernatant the next day, blocking 2hr with 80. Mu.l of 3% milk (dissolved in PBS), washing the plate well 3 times with 80. Mu.l of PBST (1. Mu.l of Tween 20), then diluting the chimeric antibody to be tested, FDA027 and FDA031 to 20. Mu.g/ml, 3-fold gradient dilution of 11 concentration spots, 25. Mu.l each well to the enzyme plate which has been blocked, room temperature reacting for 1hr, washing 5 mu.l of PBST (1. Mu.m) with 80. Mu.l of Tween20, washing 5 mu.l of PBST (1. Mu.m.m) each well with 7, and 7. Mu.l of anti-human Tween (7: 7). After addition of 25. Mu.l of TMB chromogenic solution for 10min and termination of the reaction with 2M HCl, the microplate reader reads the absorbance at 450nm (from biotek, elx 808) and the results (see FIG. 4) show that there is no significant cross-over between the 12 candidate antibodies and both hDLL1 and hDLL4 proteins, with the 36H10, 4H7 antibodies having a weaker cross-reaction with hDLL1 at high concentration (20. Mu.g/mL); the 6A2 antibody had some cross-reactivity with hDLL4 at high concentrations (20. Mu.g/mL).

To test the cross-reactivity of antibodies to murine and monkey DLL3, the present study used murine DLL3 expression plasmid pCMV3-mDLL3 (purchased from beijing yiqiao, MG 58052-UT) and monkey DLL3 expression plasmid pCMV3-rheDLL3 (purchased from beijing yiqiao, CG 90919-UT) to construct overexpressed cell lines. The construction of the overexpressing cells was as described in examples 2-3. The binding capacity of the chimeric antibody to these two cells was examined by flow cytometry, and the species cross-reactivity of the antibody was determined. The 5×10 of the construction ⁵ The Mouse DLL3 cell Mouse-DLL3 and the monkey DLL3 cell Rhesus-DLL3 were inoculated into 96-well V-type microplates (available from Axygen, wipp 02280), respectively, and centrifuged at 1500r/min for 1min, and the supernatants were discarded. The chimeric antibody, FDA027 and FDA031 were pre-diluted to 50. Mu.g/ml, diluted 4-fold in a gradient to 8 concentration points, and the antibodies to be detected were added at different concentrations, 200. Mu.L per well, and incubated on ice for 30min. Then 150. Mu.L PBS was added to each well, centrifuged at 1500r/min for 1min, the supernatant was discarded, and the plate was washed 4 times. APC-labeled goat anti-human IgG (Jackson, PBS 1:800 dilution) was added and incubated on ice for 30min at 50. Mu.L per well. After washing the plate 4 times with PBS, 100. Mu.L of PBS was added to each well to resuspend the cells, and the detection was performed with CytoFLEX (Beckman), and the obtained results (shown in FIG. 5) show that the remaining 9 antibodies were able to bind to mouse and monkey DLL3 except that ch32E8, ch13C2 and ch15G2 did not recognize monkey and mouse DLL 3.

Example 9 affinity detection of antibody binding to human DLL3 protein

To examine the affinity of antibodies for human DLL3 protein binding, the present study examined the binding kinetics of immobilized antibodies to free DLL3 using BLI, which was performed with reference to the instructions of the instrument (vendor: fortebio, apparatus model: octet 96 e), briefly, the AMC sensor was equilibrated for 60s with Loading Buffer/Sample dilution Buffer (1 XPBS, pH7.4, with 0.1%BSA and 0.02%Tween-20), yielding Baseline 1. And diluting the antibody to be detected to a concentration of 10ug/ml by using a Loading Buffer, combining the diluted antibody with the balanced sensor, and re-balancing the combined sensor by using the Loading Buffer again to obtain Baseline 2. The antibody-loaded sensor was then placed in human DLL3, his tag (suppliers: acrobiosystems, cat# DL3-H52H 4) diluted to 100 to 3.13nM with sample dilution for 90s to give an antibody-protein binding curve. The antigen-bound sensor was then again placed in Sample Dilution Buffer for dissociation 180s, resulting in a dissociation curve. The k-on, k-off values of antibody binding to protein were calculated by binding and dissociation curves, respectively, and KD values were calculated, and the results obtained (see Table 4) are shown. As a result, these chimeric antibodies were found to have strong affinity at the protein level.

TABLE 4 affinity of antibodies to human DLL3 protein binding

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

Example 10 preparation of linker drug conjugates

The structures of the linker drug conjugates LE00 and LE01-LE24 used in the invention are shown in Table 5, wherein LE00 (GGFG-Dxd) is synthesized by the method reported in WO2015146132A, and LE01-LE24 is synthesized by the method reported in WO2020259258A 1.

TABLE 5 linker drug conjugate structures

Example 11 preparation of ADC by conjugation of antibody to linker drug conjugate

The different anti-DLL 3 antibodies obtained in examples 1 and 6 above were each replaced to 50mM PB/1.0mM EDTA buffer (pH 7.0) using a G25 desalting column, 12 equivalents TECP were added, and stirred at 37 ℃ for 2 hr to allow the inter-chain disulfide bond of the antibody to be completely opened, followed by adjusting the pH of the reduced antibody solution to 6.0 using phosphoric acid, and reducing the water bath temperature to 25 ℃ for the coupling reaction. The linker drug conjugate prepared according to example 10 was dissolved in DMSO, 12 equivalents of the linker-drug conjugate were extracted therefrom and added drop-wise to the reduced anti-DLL 3 antibody solution, the samples were prepared according to the highest DAR (i.e., excessive conjugation), the occurrence of precipitation at each coupling reaction was observed, DMSO was added to a final concentration of 10% (v/v), the reaction was stirred at 25 ℃ for 0.5 hr, and after the reaction was completed, the samples were filtered using a 0.22 μm membrane. Uncoupled small molecules were removed by purification using a tangential flow ultrafiltration system with buffer 50mM PB/1.0mM EDTA solution (pH 6.0), and after purification 6% sucrose was added and stored in a-20deg.C freezer. The absorbance values were measured at 280nm and 370nm by UV method, respectively, and DAR values were calculated, and the results are shown in Table 6. The results show that the antibodies of ch1A5, ch33F11, ch32E8, ch13C2, FDA027, FDA031, ch15G2 and ch4H7 can be coupled with LE14 normally, the antibodies of ch33F11, LE01-LE11, LE23 and LE24 can be coupled normally, the antibodies of ch1A5, LE00, LE12, LE13, LE15, LE16, LE17, LE18, LE19, LE20, LE21 and LE22 can be coupled normally, and DAR values, free Dxd content and the like of the coupled ADC meet the expected requirements. The ADC samples of the four antibodies of ch19C7, ch23G2, ch6A2 and ch26D9 have obvious flocculent precipitation phenomenon during centrifugal concentration when being coupled with LE14, so that the collection amount is small, which indicates that the coupling is not feasible, the DAR values of the ADC samples obtained by coupling the two antibodies of ch36H10 and ch10B1 are respectively 1.89 and 1.90, the impurity content of the two antibodies is very high, and particularly the small molecule residue of the ch36H10 is up to 49.35%, which indicates that the coupling is failed.

TABLE 6 evaluation of the drug resistance of ADC samples prepared by different antibody-conjugated LE14

Example 12 evaluation of in vitro cytotoxic Activity of ADC samples

HEK293 cells which are stably transfected by DLL3 and high in expression are selected as cell strains for detecting the in vitro activity of experiments, and the quantitative effect of different antibody drug conjugates on cell killing is observed. 2000 cells per well are inoculated in a 96-well cell culture plate and cultured for 20 to 24 hours; antibody drug conjugates prepared according to the method of example 11 were formulated into 9 concentration gradient test solutions of 80, 20, 5, 1.25, 0.3125, 0.0781, 0.0195, 0.00488 and 0.000488. Mu.g/ml, 100. Mu.l/well of diluted test solution was added to a cell-inoculated culture plate and placed at 37℃in 5% CO ₂ Culturing in incubator for 144 hr, adding

Luminescent Cell Viability Assay Reagent (50. Mu.l/well), shaking at 500rpm at room temperature for 10 minutes, mixing, data processing and IC50 calculation were performed with 100% viability of the whole cell group without drug after SpectraMaxL microplate reader reading (OD 570nm,2s interval reading), and the results are shown in FIG. 6 and Table 7./>

TABLE 7 in vitro killing of ADCs of candidate antibodies against DLL3/HEK293 cells IC ₅₀ Statistical summary of values and maximum kill rates

Sample name	IC ₅₀ (μg/mL)	Maximum kill rate (%)
			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	0.087 ¹	95.1 ²

¹ And ² : all are averages.

The experimental results in Table 7 show that besides relatively weak in-vitro cytotoxic activities of ch19C7-LE14 and ch13C2-LE14, the ADC disclosed by the invention has obvious in-vitro cell killing effect on DLL3 positive cells, relatively strongest in-vitro cytotoxic activity of FDA027-LE14 and optimal in-vitro activity IC50 of FDA027-LE 14. The IC90 value of each ADC disclosed by the invention is not greatly different from that of FDA027-LE 14.

EXAMPLE 13 in vivo anti-tumor Activity of ADC drugs in NCI-H82 model

Female Balb/c nude mice (available from Shanghai Ling Biotech Co., ltd.) 6-8 weeks old were selected for 5X 10 subcutaneous injection in 200ul Matrigel on the right back ⁶ Human small cell lung cancer cell (NCI-H82) for tumor growth to average volume of 140mm ³ When the mice were left and right, the mice were randomly divided into 15 groups according to tumor size and mouse weight, 8 animals in each of 1-13 groups were respectively a vehicle blank group, and each of the two dose groups of ch1A5-LE14, ch33F11-LE14, ch23G2LE14, ch32E8-LE14, FDA031-LE14 and FDA027-LE14 conjugates was respectively 2.5mg/kg and 5.0mg/kg, and were intravenously administered once a week for 3 times. The number of animals in each of 14-15 groups is 6, namely, the Lurbinectein and topotecan hydrochloride in the control group are respectively 0.18mg/kg and 1.8mg/kg, the Lurbinectein group is administrated once a week and intravenously, the total dose is 3 times, the topotecan hydrochloride group is administrated twice a week and continuously administrated on days 1 and 2, The administration was carried out 3 times. Body weight and tumor volume were measured three times a week and animal survival was observed during the course of the experiment. The results are shown in FIG. 7 and Table 8, and the 21 st balance average tumor volume of the vehicle blank (01 group) mice at the end of administration was 2773mm ³ FDA027-LE14 (2.5 mg/kg, group 12) treatment group had a 21 st balance average tumor volume of 486mm after termination of dosing ³ FDA027-LE14 (5 mg/kg, group 13) treatment group had a 21 st balance average tumor volume of 305mm after the end of dosing ³ . The 21 st balance average tumor volume of the ch1A5-LE14 treatment group (02 group) with 2.5mg/kg test agent was 607mm after the end of the administration ³ The 21 st balance average tumor volume of the ch1A5-LE14 treatment group (03 group) with 5mg/kg of test drug was 245mm after the end of the administration ³ . Test 2.5mg/kg ch33F11-LE14 treatment group (04 group) with a 21 st balance average tumor volume of 715mm after end of dosing ³ The 21 st balance average tumor volume of the ch33F11-LE14 treatment group (05 group) with 5mg/kg of test drug was 293mm after the end of the administration ³ . Test 2.5mg/kg ch23G2-LE14 treatment group (group 06) at 21 st balance average tumor volume of 1322mm after end of dosing ³ The 21 st balance average tumor volume of the ch23G2-LE14 treatment group (07 group) with 5mg/kg of test drug was 441mm after the end of the administration ³ . 2.5mg/kg of test agent ch32E8-LE14 treatment group (group 08) had a 21 st balance average tumor volume of 945mm after the end of dosing ³ The 21 st balance average tumor volume of the ch32E8-LE14 treatment group (09 group) with 5mg/kg of test drug was 433mm after the end of the administration ³ . FDA031-LE14 (2.5 mg/kg, group 10) treatment group had a balance 21 average tumor volume of 564mm after the end of dosing ³ FDA031-LE14 (5 mg/kg, group 11) treatment group had a 21 st balance average tumor volume of 245mm after the end of dosing ³ . A control Lurbinecedein (from Zhongkeyuan pharmaceutical Co., ltd., lot 000039-87-CE013BA-a 1) at 0.18mg/kg had a balance 21 with a tumor volume of 2268mm after the end of administration ³ 1.8mg/kg control topotecan hydrochloride (available from Saen chemical technology (Shanghai) Co., lot R3URR 4E) had a 21 st balance average tumor volume of 1553mm after the end of dosing ³ . Experimental results show that the treatment groups (especially ch1A5-LE 14) have better in-vivo anti-tumor activity, and meanwhile, the control group hydrochloric acid topotecan is also usedThe experimental mice have no death condition and no weight loss condition, which indicates that each test drug has good safety.

TABLE 8 in vivo anti-tumor Condition of ADC drugs in NCI-H82 model

EXAMPLE 14 in vivo anti-tumor Activity of ADC drugs in NCI-H209 model

Female Balb/c nude mice 6-8 weeks old were selected for right back subcutaneous injection of 1X 10 in 200ul Matrigel ⁷ Human small cell lung cancer cell (NCI-H209) for tumor growth to average volume of 150mm ³ When the mice were left and right, the mice were randomly divided into 7 groups according to tumor size and mouse weight, 6 animals in each group were vehicle blank group, and three dose groups of ch1A5-LE14 and FDA027-LE14 conjugate were 1.5mg/kg, 2.5mg/kg and 5.0mg/kg, respectively, for single administration. Body weight and tumor volume were measured three times a week and animal survival was observed during the course of the experiment. The results are shown in Table 9 below, with the average tumor volume at the end of dosing of 2006mm in the vehicle blank (group 1) mice ³ 1.5mg/kg FDA027-LE14 treatment group (group 5) had a 21 st balance average tumor volume of 0mm after the end of dosing ³ 2.5mg/kg FDA027-LE14 treatment group (group 6) had a 21 st balance average tumor volume of 0mm after the end of dosing ³ 5mg/kg FDA027-LE14 treatment group (group 7) had a balance 21 average tumor volume of 0mm after the end of dosing ³ .1.5mg/kg of ch1A5-LE14 treatment group (group 2) had a 21 st balance average tumor volume of 0mm after the end of the administration ³ 2.5mg/kg of ch1A5-LE14 treatment group (group 3) had a 21 st balance average tumor volume of 0mm after the end of the administration ³ The 5mg/kg ch1A5-LE14 treatment group (group 4) had a 21 st balance average tumor volume of 0mm after the end of the dose ³ . The experimental results show that FDA027-LE14 and ch1A5-LE14 have better in vivo antitumor activity, and all experimental mice have no death condition and no weight loss condition, so that the test medicament has good safety.

TABLE 9 in vivo anti-tumor Activity of ADC drugs in NCI-H209 model

EXAMPLE 15 evaluation of toxicity of ADC drugs in mice

The mice of the KM strain are selected for toxicity evaluation, the mice are randomly divided into 4 groups according to the weight of the mice, 10 mice in each group are half of the mice, 600ul of ch1A5-LE14 conjugate is injected into the tail vein of each group, the doses are respectively 200mg/kg, 400mg/kg, 500mg/kg and 640mg/kg, toxic reaction conditions caused by the ch1A5-LE14 conjugate are observed within 14 days after single administration, the research result shows that only one animal in the 200mg/kg group is recovered and increased after the D3 day, other animals show an increasing trend in the whole experiment process, animals in the other groups are clinically observed to be abnormal except for the 200mg/kg group, the mice in the other groups are recovered normally and are not dying or dying soon after Mao Pengsong days after administration, and the results show that the conjugate has good tolerance in the mice and the maximum tolerance dose of 640mg/kg. In another study evaluating the toxicity of ch1A5-LE14 conjugates in KM mice, the intraperitoneal injection doses were 410mg/kg, 512mg/kg, 640mg/kg, 800mg/kg and 1000mg/kg, respectively, and the animals in each group were seen to be symptomatic of Mao Pengsong, bow-backed, reduced activity, crunched, diarrhea, soft stool and anal Zhou Wuhui, and 5 mice (10 mice per group) died in the 1000mg/kg group, and one mouse in the 800mg/kg group had a weight loss of more than 30% in three consecutive days over the 14 day observation period, and the animals were treated for well-being according to animal welfare, and the other groups did not see animal death, and the results further showed that the conjugates of the present invention had very good tolerability in mice.

EXAMPLE 16 evaluation of toxicity of ADC drugs in rats

The SD rat tail was selected for intravenous injection of the ch1A5-LE14 conjugate of the invention to evaluate the tolerability of ch1A5-LE14 in rats, and the mice were randomly divided into 3 groups of 8 male and female halves based on their body weight, one group was given 100mg/kg once a week, 3 times a week, one group was given 300mg/kg once, while a blank control group was established, the dosing volume was 10ul/g based on their body weight, and 12 days after completion of the last dosing. After the observation period of the last administration for 12 days is over, the animals do not die or die in the whole experiment process; all animals showed no abnormality after administration of the high dose 300mg/kg group by Mao Pengsong and 100mg/kg group; the weight of the high-dose 300mg/kg group is reduced, and the weight of all the surviving animals is recovered after D7 days; all animals were treated for general examination with ease after the end of the observation period, and no abnormalities were found in the viscera. The results of the toxicity studies in rats further indicate that the conjugates of the invention also have very well tolerated doses in rats.

While particular embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely illustrative, and that many changes and modifications may be made to these embodiments without departing from the principles and spirit of the invention. Accordingly, the scope of the invention is defined by the appended claims.

Claims

1. An anti-DLL 3 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein,,

the amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO. 9, 10 and 11, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO. 12, GAS and SEQ ID NO. 14; or alternatively, the first and second heat exchangers may be,

the amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO. 19, 20 and 21, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO. 12, GAS and SEQ ID NO. 24; or alternatively, the first and second heat exchangers may be,

the amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO. 29, 30 and 31, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO. 32, GAT and SEQ ID NO. 34; or alternatively, the first and second heat exchangers may be,

the amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO. 39, 40 and 41, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO. 42, TTS and SEQ ID NO. 44; or alternatively, the first and second heat exchangers may be,

the amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO 59, 60 and 61, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO 62, NAK and SEQ ID NO 64; or alternatively, the first and second heat exchangers may be,

The amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO 69, 70 and 71, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO 72, NAK and SEQ ID NO 74; or alternatively, the first and second heat exchangers may be,

the amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO. 79, 80 and 81, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO. 82, YTS and SEQ ID NO. 84; or alternatively, the first and second heat exchangers may be,

the amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO. 89, 90 and 91, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO. 92, RAN and SEQ ID NO. 94; or alternatively, the first and second heat exchangers may be,

the amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO 99, 100 and 101, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO 102, WAS and SEQ ID NO 104; or alternatively, the first and second heat exchangers may be,

the amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 contained in the VH are respectively shown as SEQ ID NO. 89, 110 and 111, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 contained in the VL are respectively shown as SEQ ID NO. 112, FTS and SEQ ID NO. 114; or alternatively, the first and second heat exchangers may be,

The amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 are shown as SEQ ID NO. 63, 83 and 93 respectively, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 are shown as SEQ ID NO. 103, NAN and 113 respectively.

2. The anti-DLL 3 antibody of claim 1, wherein the heavy chain variable region (VH) further comprises a heavy chain variable region framework region (VH FWR), and/or the light chain variable region (VL) further comprises a light chain variable region framework region (VL FWR); wherein the VH FWR is a heavy chain variable region framework region of a human or murine antibody and the VL FWR is a light chain variable region framework region of a human or murine antibody;

preferably:

the VH comprises an amino acid sequence shown as SEQ ID NO. 15, 25, 35, 45, 65, 75, 85, 95, 105, 13 or 22; and/or the VL comprises an amino acid sequence as set forth in SEQ ID NO 16, 26, 36, 46, 66, 76, 86, 96, 106, 33 or 23;

more preferably:

the VH comprises an amino acid sequence shown as SEQ ID NO. 15, and the VL comprises an amino acid sequence shown as SEQ ID NO. 16; or (b)

The VH comprises an amino acid sequence shown as SEQ ID NO. 25, and the VL comprises an amino acid sequence shown as SEQ ID NO. 26; or (b)

The VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36; or (b)

The VH comprises an amino acid sequence shown as SEQ ID NO. 45, and the VL comprises an amino acid sequence shown as SEQ ID NO. 46; or (b)

The VH comprises an amino acid sequence shown as SEQ ID NO. 65, and the VL comprises an amino acid sequence shown as SEQ ID NO. 66; or (b)

The VH comprises an amino acid sequence shown as SEQ ID NO. 75, and the VL comprises an amino acid sequence shown as SEQ ID NO. 76; or (b)

The VH comprises an amino acid sequence shown as SEQ ID NO. 85, and the VL comprises an amino acid sequence shown as SEQ ID NO. 86; or (b)

The VH comprises an amino acid sequence shown as SEQ ID NO. 95, and the VL comprises an amino acid sequence shown as SEQ ID NO. 96; or (b)

The VH comprises an amino acid sequence shown as SEQ ID NO. 105, and the VL comprises an amino acid sequence shown as SEQ ID NO. 106; or (b)

The VH comprises an amino acid sequence shown as SEQ ID NO. 13, and the VL comprises an amino acid sequence shown as SEQ ID NO. 33; or (b)

The VH comprises the amino acid sequence shown as SEQ ID NO. 22, and the VL comprises the amino acid sequence shown as SEQ ID NO. 23.

3. The anti-DLL 3 antibody of claim 1 or 2, which is a full length antibody, fab ', F (ab') 2, fv preferably scFv, heavy chain antibody or single domain antibody, and/or which is a monoclonal antibody, a bispecific antibody or a multispecific antibody.

4. The anti-DLL 3 antibody of claim 3, which is a full length antibody comprising a heavy chain and a light chain; wherein the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO. 17, 27, 37, 47, 67, 77, 87, 97, 107, 43 or 73 and/or the light chain comprises an amino acid sequence as set forth in SEQ ID NO. 18, 28, 38, 48, 68, 78, 88, 98, 108, 53 or 109;

preferably:

the heavy chain comprises an amino acid sequence shown as SEQ ID NO. 17, and the light chain comprises an amino acid sequence shown as SEQ ID NO. 18; or (b)

The heavy chain comprises the amino acid sequence shown as SEQ ID NO. 27, and the light chain comprises the amino acid sequence shown as SEQ ID NO. 28; or (b)

The heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 37, and the light chain comprises an amino acid sequence as shown in SEQ ID NO. 38; or (b)

The heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 47, and the light chain comprises an amino acid sequence as shown in SEQ ID NO. 48; or (b)

The heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 67, and the light chain comprises an amino acid sequence as shown in SEQ ID NO. 68; or (b)

The heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 77, and the light chain comprises an amino acid sequence as shown in SEQ ID NO. 78; or (b)

The heavy chain comprises the amino acid sequence shown as SEQ ID NO. 87, and the light chain comprises the amino acid sequence shown as SEQ ID NO. 88; or (b)

The heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 97, and the light chain comprises an amino acid sequence as shown in SEQ ID NO. 98; or (b)

The heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 107, and the light chain comprises an amino acid sequence as shown in SEQ ID NO. 108; or (b)

The heavy chain comprises an amino acid sequence shown as SEQ ID NO. 43, and the light chain comprises an amino acid sequence shown as SEQ ID NO. 53; or (b)

The heavy chain comprises the amino acid sequence shown as SEQ ID NO. 73 and the light chain comprises the amino acid sequence shown as SEQ ID NO. 109.

5. An isolated nucleic acid encoding the anti-DLL 3 antibody of any one of claims 1-4.

6. A recombinant expression vector comprising the isolated nucleic acid of claim 5;

Preferably, the expression vector comprises a eukaryotic expression vector and/or a prokaryotic expression vector.

7. A transformant comprising the recombinant expression vector of claim 6;

preferably, the host cells of the transformant are prokaryotic cells, preferably E.coli cells such as TG1, BL21 cells, and/or eukaryotic cells, preferably HEK293 cells or CHO cells.

8. An antibody drug conjugate has a structural general formula Ab- (L) ₃ -L ₂ -L ₁ -D) _m ；

Wherein Ab is an anti-DLL 3 antibody;

d is fineCytotoxic drugs

m is 2-8;

L ₂ is that

L ₃ is that

9. The antibody drug conjugate of claim 8,

the anti-DLL 3 antibody binds to one or more of the following antigen binding epitopes in a DLL3 protein: DSL domain, N-terminal, EGF2 domain and EGF3-EGF6 domain;

and/or, 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 a valine residue and/or an alanine residue, the plurality of L is two or three, and p is 2;

And/or, the R ¹ Is one or more of-NR ^1-1 R ^1-2 Substituted C ₁ ～C ₆ Alkyl, substituted by one or more R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ Alkyl, or C ₁ ～C ₆ Alkyl, said R ^1-1 、R ^1-2 And R is ^1-3 Each independently is C ₁ ～C ₄ An alkyl group;

and/or when L ₁ When the structure of the formula I is shown in the specification, L ₂ Preferably is

And/or when L ₁ When the structure of (C) is shown as formula II, L ₂ Preferably is

And/or when L ₁ When the structure of (C) is shown as formula III, L ₂ Preferably is

And/or when L ₁ When the structure of (C) is shown as formula IV, the L ₂ Preferably is

And/or, the n is independently 8, 9, 10, 11, and 12;

and/or m is an integer or non-integer of 2-8;

and/or, said L ₃ Preferably is

10. The antibody drug conjugate of claim 8 or 9,

the anti-DLL 3 antibody binds to an antigen binding epitope of an EGF2 domain in the DLL3 protein;

and/or, said (L) _p Is that

Wherein the g-terminus is bound to the L via a carbonyl group ₂ C end of (2) is connected;

and/or, said formula III is preferably

11. The antibody drug conjugate of claim 8, which is any one of the compounds shown below:

the Ab is an anti-DLL 3 antibody, wherein the anti-DLL 3 antibody is an anti-DLL 3 antibody according to any one of claims 1 to 4, an anti-DLL 3 antibody having a light chain with an amino acid sequence shown as SEQ ID NO. 1 and a heavy chain with an amino acid sequence shown as SEQ ID NO. 2, or an anti-DLL 3 antibody having a light chain with an amino acid sequence shown as SEQ ID NO. 3 and a heavy chain with an amino acid sequence shown as SEQ ID NO. 4;

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;

preferably, the antibody-conjugated drug is any one of the compounds shown below:

wherein Ab is an anti-DLL 3 antibody comprising a VH having the amino acid sequence shown in SEQ ID NO. 25 and a VL having the amino acid sequence shown in SEQ ID NO. 26,

Wherein Ab is an anti-DLL 3 antibody comprising a VH having the amino acid sequence shown in SEQ ID NO. 25 and a VL having the amino acid sequence shown in SEQ ID NO. 26, < >>

Wherein Ab is an anti-DLL 3 antibody comprising a VH having the amino acid sequence shown in SEQ ID NO. 25 and a VL having the amino acid sequence shown in SEQ ID NO. 26, < > >

Wherein Ab is an anti-DLL 3 antibody comprising an amino acid sequence such asVH shown in SEQ ID NO. 25 and VL shown in SEQ ID NO. 26,

Wherein Ab is an anti-DLL 3 antibody comprising a VH having the amino acid sequence shown in SEQ ID NO. 15 and a VL having the amino acid sequence shown in SEQ ID NO. 16, < >>

Wherein Ab is an anti-DLL 3 antibody comprising a VH having an amino acid sequence shown in SEQ ID NO. 15 and a VL having an amino acid sequence shown in SEQ ID NO. 16,

Wherein Ab is an anti-DLL 3 antibody comprising an amino groupVH with acid sequence shown as SEQ ID NO. 15 and VL with amino acid sequence shown as SEQ ID NO. 16,

Wherein Ab is an anti-DLL 3 antibody comprising a heavy chain having an amino acid sequence shown in SEQ ID NO. 2 and a light chain having an amino acid sequence shown in SEQ ID NO. 1,

Wherein Ab is an anti-DLL 3 antibody comprising a heavy chain having an amino acid sequence shown in SEQ ID NO. 4 and a light chain having an amino acid sequence shown in SEQ ID NO. 3,

Wherein Ab is an anti-DLL 3 antibody comprising a VH having the amino acid sequence shown in SEQ ID NO. 22 and a VL having the amino acid sequence shown in SEQ ID NO. 23

Or (b)

Wherein Ab is an anti-DLL 3 antibody comprising a VH having the amino acid sequence shown in SEQ ID NO. 45 and a VL having the amino acid sequence shown in SEQ ID NO. 46.

12. A chimeric antigen receptor comprising the anti-DLL 3 antibody of any one of claims 1-4.

13. A genetically modified cell comprising an anti-DLL 3 antibody according to any one of claims 1 to 4; preferably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably immune cells such as T cells, or NK cells.

14. A method of preparing an anti-DLL 3 antibody, the method comprising the steps of: culturing the transformant according to claim 7, and obtaining the anti-DLL 3 antibody from the culture.

15. A pharmaceutical composition comprising the anti-DLL 3 antibody of any one of claims 1-4, the antibody drug conjugate of any one of claims 8-11, the chimeric antigen receptor of claim 12, and/or the genetically modified cell of claim 13;

preferably, the pharmaceutical composition is in liquid, gaseous, solid and semi-solid dosage forms, and/or the pharmaceutical composition may be administered orally, by injection, nasally, transdermally or mucosally;

16. Use of an anti-DLL 3 antibody according to any one of claims 1 to 4, an antibody drug conjugate according to any one of claims 8 to 11, a chimeric antigen receptor according to claim 12, a genetically modified cell according to claim 13 and/or a pharmaceutical composition according to claim 15 for the preparation of a medicament, kit and/or administration device for the treatment and/or prevention of diseases associated with abnormal DLL3 expression;

the DLL3 expression abnormality related disease is preferably a tumor, the tumor is preferably a cancer, and the cancer is preferably a neuroendocrine tumor, more preferably a small cell lung cancer.

17. A kit comprising an anti-DLL 3 antibody according to any one of claims 1-4, an antibody drug conjugate according to any one of claims 8-11, a chimeric antigen receptor according to claim 12, a genetically modified cell according to claim 13 and/or a pharmaceutical composition according to claim 15; and optionally, instructions.

18. A drug delivery device, comprising: (1) An infusion module for administering the pharmaceutical composition of claim 15 to a subject in need thereof, and (2) optionally a efficacy monitoring module.

19. A method of detecting DLL3 comprising the step of detecting using an anti-DLL 3 antibody according to any one of claims 1 to 4, said method being for non-diagnostic and/or therapeutic purposes.