WO2025252164A1 - Anti-dll3 and b7-h3 bispecific antibody-drug conjugate - Google Patents

Abstract

The present invention relates to the technical field of antibody-drug conjugates, and specifically relates to an anti-DLL3 and B7-H3 bispecific ADC molecule. Two candidate antibody clones targeting B7-H3 and three candidate antibody clones targeting DLL3 are paired across different targets so as to form multiple bispecific antibody molecules, and the bispecific ADC molecule is screened therefrom on the basis of the activity.

Description

Bispecific antibody-drug conjugates against DLL3 and B7-H3

Cross-references to related applications

This application claims priority to Chinese patent applications filed on June 7, 2024, with application number CN202410743383.5 entitled "Bispecific Antidrug Conjugate Against DLL3 and B7-H3", and on December 12, 2024, with application number CN202411832563.7 entitled "Bispecific Antidrug Conjugate Against DLL3 and B7-H3", the contents of which are incorporated herein by reference in their entirety.

Technical Field

This disclosure relates to the field of biomedical technology, and more specifically, to a bispecific antibody drug conjugate.

Background Technology

Antibody-drug conjugates (ADCs) are produced by linking biologically active small-molecule drugs to antibodies via linkers. Currently, the vast majority of ADCs are formed by linking antibodies targeting tumor antigens to highly cytotoxic small-molecule chemical drugs via linkers. Utilizing the specific binding characteristic of antibodies to target antigens, the small-molecule drug is delivered directly to tumor cells to exert its tumor-killing effect. Other ADCs combine specifically binding antibodies, antigens, and peptides—proteins and peptides with similar specific binding properties to antibodies—with linker toxins.

Delta-like ligands (DLLs) are ligands of the Notch signaling pathway. They are often uncontrolled in tumors and influence tumor growth, angiogenesis, and immunity. The Notch signaling pathway is a relatively conserved pathway in human evolution, primarily controlling cellular processes through cell-cell interactions. The Notch receptor, after enzymatic cleavage in the Golgi apparatus, is transported to the cell surface and forms a transmembrane heterodimer. Upon interaction with Notch ligands on neighboring cells, the intracellular portion of the Notch ligand is cleaved, leading to the release of the Notch intracellular domain (NICD) from the cell surface. This NICD then enters the nucleus and binds to the DNA-binding protein CSL (CBF-1). CBF-1 subsequently recruits Mastermind-like proteins (MAMLs) and activates transcription of Notch-targeted genes, including the Hes and Hey families. DLL3 is a highly tumor-selective cell surface target, mainly expressed in neuro or neuroendocrine tumors, including small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), gastrointestinal neuroendocrine tumor (GI-NEC), small cell bladder cancer (SCBC), glioma multiforme, metastatic castration prostate cancer, and pulmonary neuroendocrine tumors, especially SCLC, where more than 80% of SCLCs show positive expression of DLL3, while it is not expressed in normal lung cancer tissue or adjacent tissues. (Yao J, Bergsland E, Aggarwal R, Aparicio A, Beltran H, Crabtree JS, Hann CL, Ibrahim T, Byers LA, Sasano H, Umejiego J, Pavel M. DLL3 as an Emerging T target for the Treatment of Neuroendocrine Neoplasms.Oncologist.2022 Nov 3;27(11):940-951.doi:10.1093/oncolo/oyac161.PMID:35983951;PMCID:PMC9 632312.Leonetti A,Facchinetti F,Minari R,Cortellini A,Rolfo CD,Giovannetti E,Tiseo M.Notch pathway in small-cell lung cancer:from preclinic al evidence to therapeutic challenges.Cell Oncol(Dordr).2019Jun;42(3):261-273.doi:10.1007/s13402-019-00441-3.Epub 2019 Apr 9.PMID:30968324.)

DLL3, a target of neuroendocrine tumors, is highly expressed on the surface of various cells but lowly expressed in normal tissues and cells. Drugs targeting DLL3 are already in clinical trials. Typical representative drugs include: Rova-T, an ADC targeting DLL3; AMG757, a bispecific antibody targeting DLL3 and CD3, which is currently approved for marketing; HPN328, a bispecific antibody targeting DLL3 and CD3; and BI764532, a bispecific antibody targeting DLL3 and CD3. In addition, several other ADCs and CAR-T therapies are under development.

B7-H3 is expressed on the surface of immune cells or tumor cells. It may have multiple corresponding receptor ligands, but no single dominant ligand or receptor has been identified. Although immunosuppressive or immunoactivating effects have been observed, its primary biological function remains unknown. Furthermore, in various tumors, high expression of B7-H3 in tumor tissue is negatively correlated with prognosis and survival to some extent. B7-H3 is widely expressed in tissues such as the heart, liver, pancreas, prostate, small intestine, and colon, but at very low levels. It is expressed in immune cells, but at very low levels, and is induced rather than constitutively expressed. B7-H3 is expressed in various malignant tumors, including melanoma, glioma, lung cancer, pancreatic cancer, kidney cancer, colon cancer, ovarian cancer, breast cancer, gastric cancer, endometrial cancer, and some hematologic malignancies. (Liu S, Liang J, Liu Z, Zhang C, Wang Y, Watson AH, Zhou C, Zhang F, Wu K, Zhang F, Lu Y, Wang X.The Rol e of CD276 in Cancers.Front Oncol.2021 Mar 26;11:654684.doi:10.3389/fonc.2021.654684.PMID:338 42369; PMCID: PMC8032984.Wang L, Kang FB, Shan BE.B7-H3-mediated tumor immunology: Friend or foe? Int J Cancer.2014Jun 15;134(12):2764-71.doi:10.1002/ijc.28474.Epub 2013 Sep 30.PMID:24013874.)

The target B7-H3 is highly expressed on the surface of various solid tumor cells and lowly expressed in normal tissues and cells. Drugs targeting B7-H3 are already in clinical trials. Typical representative drugs include: early large molecule drugs targeting B7-H3 (excluding cell therapy) such as the radionuclide-conjugated antibody iodine-131-Omburtamab, followed by several camptothecin ADCs such as MGD009, MGA271, MGC018, and DS-7300; ABBV-155 (Mirzotamab Clezutoclax) is an antibody-drug conjugate targeting B7-H3, with the conjugated small molecule being a BCL inhibitor; MGA271 (Enoblituzumab) is a monoclonal antibody drug targeting B7-H3; and MGC018 is an antibody-drug conjugate targeting B7-H3, with the conjugated small molecule being ducardycin.

Summary of the Invention

This disclosure provides, for the first time, a bispecific antidote conjugate against DLL3 and B7-H3 and its application.

ADC resistance can be categorized into target-related resistance and load-related resistance. If the expression of a target on the surface of tumor cells exhibits heterogeneity, target resistance becomes a problem. Combining two targets may improve the homogeneity of target expression, thus mitigating target-related resistance to some extent. If homogeneity is achieved through the dual-target combination, load-related resistance can be further addressed through sequential or combination therapy with ADCs conjugated to multiple loads or single-load ADCs. Ultimately, bispecific antibody ADCs hold promise for overcoming a certain degree of resistance.

AbbVie's PSMA/STEAP1 bispecific antibody ADC (ABBV-969) is a representative of this design. PSMA and STEAP1 are both highly expressed in prostate cancer and have a certain degree of complementarity, so the bispecific antibody ADC can more comprehensively eliminate tumor cells and is less likely to develop drug resistance.

This invention provides a specific antibody with high affinity for DLL3 and significant endocytosis activity. Compared with AbbVie's positive control, the antibody of this invention exhibits higher affinity and more significant endocytosis activity, and can be used for further toxin conjugation for tumor treatment. Therefore, the antibody provided by this invention has significant value for the treatment of DLL3-expressing tumors.

Targets B7-H3 and DLL3 are tumor-associated antigens/tumor-specific antigens with some biological functions, but these functions are not clear. Surprisingly, it was found that the two have a synergistic effect in bispecific antibody ADCs, with 1+1>2.

Attached Figure Description

To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

Figure 1: ELISA measurement of _EC50 of DLL3 hybridoma antibody binding to DLL3-hFc protein.

Figures 2A-2C: FACS assay of _EC50 of DLL3 hybridoma antibody binding to 293T-DLL3 cells.

Figures 3A-3B: FACS determination of _EC50 of DLL3 hybridoma antibody binding to SHP-77 cells.

Figures 4A-4C: FACS assay of _EC50 of DLL3 hybridoma antibody endocytosis in 293T-DLL3 cells.

Figures 5A-5B: FACS assay of _EC50 of DLL3 hybridoma antibody endocytosis in SHP-77 cells.

Figure 6: ELISA assay of _EC50 of humanized DLL3 antibody binding to DLL3-mFc protein.

Figures 7A-7B: FACS assay of _EC50 of humanized DLL3 antibody binding to 293T-DLL3 cells.

Figures 8A-8B: FACS assay of _EC50 of DLL3 humanized antibody binding to SHP-77 cells.

Figures 9A-9B: FACS assay of _EC50 of humanized DLL3 antibody in 293T-DLL3 cells.

Figures 10A-10B: FACS assay of _EC50 of DLL3 humanized antibody endocytosis in SHP-77 cells.

Figures 11A-11C: ELISA assay for species cross-reactivity of humanized DLL3 antibodies.

Figures 12A-12C: ELISA assays show that the humanized DLL3 antibody does not cross-react with its homologous proteins.

Figure 13: Structure of the dual antibody molecule targeting DLL3 and B7-H3.

Figure 14: Tumor cell growth curves in the saline control group of the SHP-77 model.

Figure 15: Tumor cell growth curves of the SHP-77 model at the following dosage groups: 1.5 mg/kg of 3103-59H4-LD-38 molecule, 1.5 mg/kg of 3103-15A2-LD-38 molecule, 0.75 mg/kg of each of 3103-59H4-LD-38 and 3103-15A2-LD-38 molecules, and 1.5 mg/kg of each of 3103-59H4-LD-38 and 3103-15A2-LD-38 molecules.

Figure 16: Tumor cell growth curves of the above SHP-77 model groups at doses of 4.0 mg/kg, 2.0 mg/kg, 4.0 mg/kg, and 2.0 mg/kg for 3103-0259H415A2-LD-38 molecules.

Figure 17: Tumor cell growth curves of the above SHP-77 model groups at doses of 2.0 mg/kg for the following groups: 3103-05-56G10-15A2-LD-38 molecule, 3103-06-71B11-15A2-LD-38 molecule, 3103-07-59H4-15A2-LD-38 molecule, 3103-08-7B7-59H4-LD-38 molecule, and 3103-09-59H47B7-LD-38 molecule.

Figure 18: Tumor cell growth curves in the saline control group of the NCI-H82 model.

Figure 19: Tumor cell growth curves of each group in the NCI-H82 model, including the following dosage groups: 1.5 mg/kg of 3103-59H4-LD-38, 1.5 mg/kg of 3103-15A2-LD-38, 0.75 mg/kg of each of 3103-59H4-LD-38 and 3103-15A2-LD-38, and 1.5 mg/kg of each of 3103-59H4-LD-38 and 3103-15A2-LD-38.

Figure 20: Tumor cell growth curves of the above NCI-H82 model groups at doses of 4.0 mg/kg, 2.0 mg/kg, 4.0 mg/kg, and 2.0 mg/kg for 3103-0259H415A2-LD-38 molecules.

Figure 21: Tumor cell growth curves of the above NCI-H82 model groups at doses of 2.0 mg/kg for the following groups: 3103-05-56G10-15A2-LD-38 molecule, 3103-06-71B11-15A2-LD-38 molecule, 3103-07-59H4-15A2-LD-38 molecule, 3103-08-7B7-59H4-LD-38 molecule, and 3103-09-59H47B7-LD-38 molecule.

Figures 22A-22D: ADC molecules of B7H3 antibody and 7B7-LD38 molecule after administration to RKO, JIMT-1, SHP-77 and Calu-6 tumor models, and tumor cell growth curves in each group.

Detailed Implementation

Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "and/or," "or/and," and "and/or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and/or," "or/and," and "and/or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and/or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and/or, B, and/or, C, and/or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

The terms “containing,” “including,” and “comprise” as used in this disclosure are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

The range of values represented by endpoints in this disclosure includes all values and fractions contained within that range, as well as the endpoints referenced.

The concentration values mentioned in this disclosure include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may allow fluctuations within ±0.1%. For larger values or values that do not require overly precise control, even greater fluctuations are allowed. For example, 100mM may allow fluctuations within the ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations within ±10% are allowed.

In this disclosure, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

In this disclosure, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

In this disclosure, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments and should be understood not to limit the scope of protection of this disclosure. In this disclosure, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" options appear in a technical solution, unless otherwise specified and without contradictions or mutual constraints, each "optional" option is independent.

All documents mentioned in this disclosure are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and/or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this disclosure, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this disclosure, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this disclosure. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

This disclosure relates to the use of a dual-targeting antibody-drug conjugate of DLL3 and B7-H3.

The term "targeting antibody" refers to a large molecular compound that can specifically recognize and bind to antigens or receptors associated with target cells. The role of antibodies is to present drugs to a target cell population that has bound to the antibody. These antibodies include, but are not limited to, protein hormones, lectins, growth factors, antibodies, binding peptides, or other molecules that can bind to cells. In embodiments of this disclosure, a targeting antibody is referred to as Ab, and the targeting antibody can form a linker bond between heteroatoms on the antibody and a linker unit.

The term "pharmaceutically acceptable salt" refers to a salt of the antibody-drug conjugate of this disclosure, meaning a salt that is acceptable for administration to a patient (e.g., a mammal) (for a given dosage regimen, it is a salt containing an anti-counterionic ion with acceptable mammalian safety). Such a salt can be derived from pharmaceutically acceptable inorganic or organic bases, as well as from pharmaceutically acceptable inorganic or organic acids. The antibody-drug conjugates of this disclosure contain at least one amino group, and therefore can form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate. Especially when the conjugated drug is LD-38, its preferred pharmaceutically acceptable salt is an LD-38 salt.

As used in this disclosure, the term "solvent" refers to a combination of the disclosed compound and solvent molecules formed through solvation. In some cases, a solvate refers to a hydrate, i.e., the solvent molecule is a water molecule, and the combination of the disclosed compound and water forms a hydrate.

The DLL3 antagonistic antibody of the present invention comprises: a group of DLL3 monoclonal antibodies or antigen-binding fragments thereof, including a heavy chain and a light chain, characterized in that: the amino acid sequence of CDR1 of the heavy chain is selected from one of SEQ ID NO: 7, 13, 19; the amino acid sequence of CDR2 of the heavy chain is selected from one of SEQ ID NO: 8, 14, 20; the amino acid sequence of CDR3 of the heavy chain is selected from one of SEQ ID NO: 9, 15, 21; the amino acid sequence of CDR1 of the light chain is selected from one of SEQ ID NO: 10, 16, 22; the amino acid sequence of CDR2 of the light chain is selected from one of SEQ ID NO: 11, 17, 23; the amino acid sequence of CDR3 of the light chain is selected from one of SEQ ID NO: 12, 18, 24; wherein the heavy chain and light chain of the antigen-binding fragment comprise amino acid sequences spanning CDR1 to CDR3 of the heavy chain and light chain of the antibody, respectively.

Furthermore, the present invention discloses the above-mentioned DLL3 monoclonal antibody or its antigen-binding fragment, characterized in that the amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NO: 1, 3, 5; and the amino acid sequence of the light chain variable region is selected from one of SEQ ID NO: 2, 4, 6.

Furthermore, the present invention discloses the above-mentioned DLL3 monoclonal antibody or its antigen-binding fragment, characterized in that the heavy chain and light chain are humanized; the amino acid sequence of the variable region of the humanized heavy chain is selected from one of SEQ ID NO: 25, 27, 29; and the amino acid sequence of the variable region of the humanized light chain is selected from one of SEQ ID NO: 26, 28, 30.

Furthermore, this invention discloses the use of the above-mentioned DLL3 monoclonal antibody or its antigen-binding fragment in the preparation of a drug with high affinity for DLL3 and significant endocytosis activity.

Furthermore, the present invention discloses the above-mentioned monoclonal antibody conjugate, comprising a monoclonal antibody and a conjugation portion, wherein the monoclonal antibody is selected from the following:

A group of DLL3 monoclonal antibodies or antigen-binding fragments thereof, comprising a heavy chain and a light chain, characterized in that the amino acid sequence of CDR1 of the heavy chain is selected from one of SEQ ID NO: 7, 13, 19; the amino acid sequence of CDR2 of the heavy chain is selected from one of SEQ ID NO: 8, 14, 20; the amino acid sequence of CDR3 of the heavy chain is selected from one of SEQ ID NO: 9, 15, 21; the amino acid sequence of CDR1 of the light chain is selected from one of SEQ ID NO: 10, 16, 22; the amino acid sequence of CDR2 of the light chain is selected from one of SEQ ID NO: 11, 17, 23; the amino acid sequence of CDR3 of the light chain is selected from one of SEQ ID NO: 12, 18, 24; wherein the heavy chain and light chain of the antigen-binding fragment comprise amino acid sequences spanning CDR1 to CDR3 of the heavy chain and light chain of the antibody, respectively;

Or a group of DLL3 monoclonal antibodies or their antigen-binding fragments, characterized in that the amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NO: 1, 3, 5; and the amino acid sequence of the light chain variable region is selected from one of SEQ ID NO: 2, 4, 6;

Or a group of DLL3 monoclonal antibodies or their antigen-binding fragments, characterized in that the amino acid sequence is humanized; the amino acid sequence of the humanized heavy chain variable region is selected from one of SEQ ID NO: 25, 27, 29; the amino acid sequence of the humanized light chain variable region is selected from one of SEQ ID NO: 26, 28, 30;

The coupling portion is selected from one or more of radionuclides, drugs, toxins, cytokines, cytokine receptor fragments, enzymes, fluorescein, and biotin. Preferably, the linker unit and the coupled drug portion constitute the linker payload, and the linker payload is LD-38.

Furthermore, the present invention discloses a monoclonal antibody conjugate, comprising a monoclonal antibody and a conjugation portion, wherein the monoclonal antibody is one of the B7-H3 monoclonal antibodies or its antigen-binding fragment as described in claim 11, and its linker unit and the conjugated drug portion constitute a linker payload, wherein the linker payload is LD-38.

Furthermore, the present invention discloses the use of the monoclonal antibody conjugate in the preparation of drugs for the prevention and/or treatment and/or adjuvant treatment of tumors.

The DLL3 and B7-H3 dual-targeting antibody of the present invention comprises: a group of DLL3 monoclonal antibodies or antigen-binding fragments thereof, including a heavy chain and a light chain, characterized in that: the amino acid sequence of CDR1 of the heavy chain is selected from one of SEQ ID NO: 7, 13, 19; the amino acid sequence of CDR2 of the heavy chain is selected from one of SEQ ID NO: 8, 14, 20; the amino acid sequence of CDR3 of the heavy chain is selected from one of SEQ ID NO: 9, 15, 21; the amino acid sequence of CDR1 of the light chain is selected from one of SEQ ID NO: 10, 16, 22; the amino acid sequence of CDR2 of the light chain is selected from one of SEQ ID NO: 11, 17, 23; and the amino acid sequence of CDR3 of the light chain is selected from SEQ ID NO: 12, 13, 23. One of 18 and 24; wherein the heavy and light chains of the antigen-binding fragment comprise amino acid sequences CDR1 to CDR3 spanning the heavy and light chains of the antibody, respectively; and a group of B7-H3 monoclonal antibodies or antigen-binding fragments thereof, comprising a heavy chain and a light chain, characterized in that the amino acid sequence of CDR1 of the heavy chain comprises SEQ ID NO: 35 or 41; the amino acid sequence of CDR2 of the heavy chain comprises SEQ ID NO: 36 or 42; the amino acid sequence of CDR3 of the heavy chain comprises SEQ ID NO: 37 or 43; the amino acid sequence of CDR1 of the light chain comprises SEQ ID NO: 38 or 44; the amino acid sequence of CDR2 of the light chain comprises SEQ ID NO: 39 or 45; and the amino acid sequence of CDR3 of the light chain comprises SEQ ID NO: 40 or 46.

Furthermore, the DLL3 and B7-H3 dual-targeting antibody of the present invention comprises a heavy chain variable region and a light chain variable region, characterized in that the heavy chain variable region sequence is selected from SEQ ID NO:31 or 33; and the light chain variable region sequence is selected from SEQ ID NO:32 or 34.

Furthermore, the present invention discloses a dual-targeting antibody comprising a heavy chain constant region, characterized in that the amino acid sequence of the heavy chain constant region is selected from SEQ ID NO:47 or 49.

Furthermore, the present invention discloses a dual-targeting antibody comprising a heavy chain and a light chain, characterized in that the amino acid sequence of the heavy chain is selected from any one of SEQ ID NO: 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 86; and the amino acid sequence of the light chain is selected from any one of SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 and 87.

Furthermore, the present invention discloses a dual-targeting antibody or its antigen-binding fragment, selected from rabbit-derived antibodies, mouse-derived antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

The antibody or antigen-binding fragment thereof of the bispecific antibody-drug conjugate of the present invention comprises a first domain and a second domain, characterized in that the first domain and the second domain are selected from anti-DLL3 monoclonal antibodies, or antibodies or antigen-binding fragments thereof that target both anti-DLL3 and anti-B7-H3.

This invention utilizes a mammalian cell expression system to prepare recombinant DLL3 as an antigen for immunizing mice. Hybridoma cells are obtained by fusing mouse spleen cells with myeloma cells. Through multiple cloning and screening of a large number of hybridoma cells, several monoclonal hybridoma cell lines were obtained. These hybridoma cells can produce monoclonal antibodies with specific high affinity for DLL3 and exhibit significant endocytosis. Genes encoding the variable regions of the antibody light and heavy chains were cloned using RT-PCR, and humanized antibodies were constructed using a complementarity determinant grafting method. In vitro functional experiments showed that these humanized DLL3 antibodies can specifically and with high affinity bind to DLL3 protein and exhibit significant endocytosis. These experimental results indicate that the monoclonal antibody or its antigen-binding fragment described in this invention, or conjugates containing the monoclonal antibody or its antigen-binding fragment described in this invention, have promising applications in the preparation of tumor therapeutic drugs expressing DLL3, as well as in the prevention, treatment, or adjuvant therapy of tumors.

In some implementations, the drug may be selected from:

Alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, antiangiogenic agents, antiestrogens, antiandrogens, and immunomodulators;

Alkylating agents include, but are not limited to, dichloroethyl methylamine, chlorambucil, phenylalanine mustard, bromoperazine, pine oxychloride, phosphatidylcholine mustard, cyclophosphamide, hexamethyl melamine, chlorocyclophosphamide, isophosphamide, thiamethoxam, carmustine, streptozomycin, fotiam, cyclohexanenitrosourea, busulfan, sulfan, indomethacin, dacarbazine, cisplatin, oxaliplatin, and carboplatin;

Antimetabolites include, but are not limited to, methotrexate, 5-uridine, fluoroglycoside, 5-deoxyuridine, capecitabine, cytarabine, clodarabine, 6-mercaptopurine (6-MP), 6-mercaptoguanine (6-TG), 2-chlorodeoxyadenosine, 5-azacytidine, 2,2-fluorodeoxycytidine nucleoside, cladribine, deoxycofromycin, and pentostatin;

Antitumor antibiotics include, but are not limited to, doxorubicin, daunorubicin, methoxybenzoic acid, pentorubicin, mitoxin hydrochloride, styromycin, styromycin, mitomycin C, bleomycin, and procarbazine.

Mitosis inhibitors include, but are not limited to, paclitaxel, vincristine, vinblastine, vinorelbine, leukorelbine B, and eribulin;

Chromatin function inhibitors include, but are not limited to, camptothecin, topotecan, irinotecan, other camptothecin derivatives, etopoxetine, etopoxetine phosphate, and podophyllotoxin; the one used in this invention is irinotecan (CAS171335-80-1).

Anti-angiogenic agents include, but are not limited to, propylthiouracil, mamastastat, balmastastat, prrinstat, tannostat, ilomastastat, CGS-27023A, bromochloropiperazine, COL-3, neovastat, BMS-275291, and thalidomide;

Anti-estrogens include, but are not limited to, tamoxifen, toremifene, raloxifene, droloxifene, oldoxifene, anatozol, letrozol, and exemestane;

Antiandrogens include, but are not limited to, flutamethasone, nilumethasone, bicalutamide, spironolactone, cyproterone acetate, phenacetin, and cimetidine;

Immunomodulators include, but are not limited to, interferon, interleukin, tumor necrosis factor, mushroom polysaccharide, cizole, roquinomeprazole, pidotimod, methoxypolyethylene glycol succinamide adenosine deaminase, and thymopeptide preparations.

The term "antibody" as used herein is used in its broadest sense to include immunoglobulins or other types of molecules containing one or more antigen-binding domains that specifically bind to antigens, and proteins or peptides that exhibit binding specificity to a particular antigen. Specific examples of antibodies can include intact antibodies (e.g., classic tetrachain antibody molecules), single-chain antibodies, single-domain antibodies, bispecific antibodies, multispecific antibodies, etc. Classic antibody molecules are typically tetramers composed of two identical heavy chains and two identical light chains linked together by disulfide bonds. Based on the conservation of amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxyl terminus. The variable region is used to recognize and bind antigens, while the constant region (such as the Fc fragment) is used to initiate downstream effects, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Within the variable regions of the heavy and light chains, there are three local regions with a higher degree of variability in amino acid composition and sequence, which are key sites for antibody-antigen binding and are therefore also called complementarity-determining regions (CDRs). The amino acid sequence of the CDR can be readily determined using numbering schemes recognized in the art, such as Kabat, Chothia, IMGT, AbM, or Contact. The antibody can be an IgG, IgM, IgD, IgE, or IgA antibody.

The "antigen-binding fragment" of an antibody refers to an amino acid fragment in the antibody molecule that participates in the specific binding of the antigen, such as one of F(ab') ₂ , Fab, and scFv.

The term "F(ab') ₂ " refers to the fragment obtained by digesting the entire full-length antibody with pepsin to remove most of the Fc region while retaining some hinge regions intact. The F(ab') ₂ fragment has two antigen-binding Fab moieties linked together by disulfide bonds, thus the F(ab') ₂ fragment is a bivalent antibody. Taking F(ab') ₂ prepared from IgG antibody as an example, its molecular weight is approximately 110 kDa.

The term "Fab" refers to an antibody structure that can still bind to the antigen; it is monovalent and does not contain an Fc portion. Papain digestion of a full-length antibody yields two Fab fragments and one Fc fragment, each Fab fragment being approximately 50 kDa.

The term "scFv" is formed by linking the variable regions of the antibody heavy chain and light chain into a single peptide chain via short peptides. Through proper folding, the variable regions from the heavy and light chains interact non-covalently to form the Fv segment, thus allowing scFv to better retain its affinity activity for the antigen.

The term "dual antibody" refers to an antibody that binds to two different antigens or two epitopes of the same antigen simultaneously.

In some embodiments, the antibody or its antigen-binding fragment is selected from rabbit antibodies, mouse antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

"Rabbit/mouse antibodies" refer to antibodies whose variable and constant regions (if present) are derived from rabbit/mouse immunoglobulin sequences. Rabbit/mouse antibodies can be conveniently obtained by immunizing rabbits/mice (including mice or rats) with the corresponding antigen and then isolating the target antibody from them. Alternatively, after immunizing rabbits/mouse with the corresponding antigen, cells expressing the target antibody (such as B cells) can be isolated and cultured. Alternatively, after immunizing rabbits/mouse with the corresponding antigen, cells expressing the target antibody can be isolated and cultured, and then fused with immortalized cells such as myeloma cells to obtain hybridoma cells. Cultured hybridoma cells can then yield the target antibody (such as monoclonal antibodies) in a long-term and large-scale manner.

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a first animal-derived antibody with the constant region of a second animal-derived antibody. To establish a chimeric antibody, a hybridoma secreting a specific monoclonal antibody from the first animal-derived antibody is first established. Then, the variable region gene is cloned from the hybridoma cells, and the constant region gene of the second animal-derived antibody is cloned as needed. The first animal-derived variable region gene and the second animal-derived constant region gene are linked to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system. In a preferred embodiment of this disclosure, the first animal-derived antibody is rabbit or mouse-derived, and the second animal-derived antibody is preferably human, which can mitigate the immune response induced by the first animal-derived antibody. The antibody light chain of the chimeric antibody further includes the constant region of the human κ, λ chain, or variants thereof. The antibody heavy chain of the chimeric antibody further includes the constant region of the human IgG1, IgG2, IgG3, IgG4, or variants thereof. Subtypes of the antibody constant region, isoforms in different individuals, and mutations resulting from changes in the effector function of the constant region do not affect the preparation of antibody-drug conjugates. Linker toxin conjugation sites, such as cysteine, lysine, glutamine, the carboxyl terminus of the peptide chain, and glycosylation sites, include both natural and engineered sites; these sites are generally used for conjugation.

The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting a first-animal-derived CDR sequence into the variable region framework of a human antibody, i.e., a human germline antibody framework sequence of different types. This can overcome the heterologous response induced by chimeric antibodies carrying a large number of first-animal-derived protein components. Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be obtained from the VBase human germline sequence database (www.mrccpe.com.ac.uk/vbase) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the aforementioned human antibody variable region framework sequence to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display to enhance affinity for CDR. In a preferred embodiment of this disclosure, the first animal origin is rabbit or mouse. The human antibody variable region framework is designed and selected. To avoid a decrease in activity along with a decrease in immunogenicity, the human antibody variable region may be subjected to minimal reverse mutations to maintain activity.

A fully humanized antibody is an antibody created by transferring the human antibody gene into a genetically engineered animal that lacks the antibody gene, through transgenic or transchromosomal techniques. This allows the animal to express human antibodies, thus achieving the goal of fully humanizing the antibody.

In some embodiments, the antibody is selected from: anti-CD3 antibody, anti-FOLR1 antibody, anti-ROR1 antibody, anti-TNFα antibody, anti-tissue factor (TF) antibody, anti-EpCAM antibody, anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg/AFP antibody, anti-Cl Anti-Audin 6 antibody, anti-Claudin 9 antibody, anti-Claudin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT/CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7/CS1 antibody, anti-NaPi2B/SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1/CD227 antibody, anti-AXL antibody, anti-CD1 Anti-66 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7/CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibody, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody Anti-Integrin Av antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, and anti-CD123 antibody.

In some specific embodiments, the antibody is selected from the group consisting of the following antibodies:

Anti-GD2 antibody 3F8, Abagovomab, Abciximab, ACZ885 (canakinumab), Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, and other anti-GD2 antibodies. Anrukinzumab (IMA-638), Apolizumab, Arcitumomab, Aselizumab, Atezolizumab, Atorlimumab, Avelumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab, Bertilimu mab), Besilesomab, Bevacizumab, Biciromab, Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin, Briakinumab, Canakinumab, Cantuzumab mertansine, Capromab pendetide, Catuxomab, Cilizumab edelizumab), pesezumab (Certolizumabpegol), cetuximab, citatuzumab bogatox, cixutumumab, cleliximab, clivatuzumab tetraxetan, CNTO148 (golimumab), CNTO 1275 (ustekinumab), conatumumab, dacetuzumab, daxetuzumab (D aclizumab, denosumab, detumomab, atorumumab (aritox), dorlixizumab, durvalumab, ecromeximab, eculizumab, edrecolomab, efalizumab, efungumab, elsilimomab, enlimomab (pegol) Epitumomabcituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Exbivirumab, Fanolesomab, Faralimomab, Felvizumab, Fezakinumab, Figitumumab, Fontolizumab, Foravirumab virumab, fusomumab, galiximab, gavilimomab, gemtuzumab ozogamicin, golimumab, gomiliximab, ilalizumab, ilbritumomab tiuxetan, ilgovomab, imciromab, infliximab, intetumumab, enoxamer Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Keliximab, Labetuzumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Lintuzumab, Lucatumuma b) Lumiliximab, Mapatumumab, Maslimomab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Morolimumab, Motavizumab, Muromonab_CD3, MY0-029 (saturamumab) Tamulumab, Nacolomab tafenatox, Naptumomab estafenatox, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Ocrelizumab, Odulimomab, Ofa Ofatumumab, Omalizumab, Oportuzumab monatox, Oregovomab, Otelixizumab, Pagibaximab, Palivizumab, Panitumumab, Panobacumab, Pascolizumab, Pembrolizumab, Pemtumomab, Pertuzumab Pexelizumab, Pintumomab, Priliximab, Pritumumab, PRO 140, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab ), Rontalizumab, Rovelizumab, Ruplizumab, Satumomab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Siplizumab, Solanezumab, Sonepizumab, Sontuzumab, Stamulumab, Thioxalumab (Sulesomab), Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Tapitumomab paptox, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, TGN1412, Ticilimumab Tremelimumab, Tigatuzumab, TNX-355 (ibalizumab), TNX-650, TNX-901 (talizumab), Tocilizumab, Toralizumab, Tositumomab, Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, and others. Urtoxazumab, Ustekinumab, Vapaliximab, Vedolizumab, Veltuzumab, Vepalimomab, Visilizumab, Volociximab, Votumumab, Zalutumumab, Zanomimumab, Ziralimumab, and Zolimomab aritox.

In some embodiments, the antibody is selected from:

i) Antibody; anti-DLL3 antibody or;

ii) Antibody; anti-B7-H3 antibody.

In some embodiments, the heavy chain variable region HCVR of the anti-DLL3 antibody is as shown in SEQ ID NO:1, and the light chain variable region LCVR is as shown in SEQ ID NO:2.

In some embodiments, the heavy chain variable region HCVR of the anti-DLL3 antibody is as shown in SEQ ID NO:3, and the light chain variable region LCVR is as shown in SEQ ID NO:4.

In some embodiments, the heavy chain variable region HCVR of the anti-DLL3 antibody is shown in SEQ ID NO:5, and the light chain variable region LCVR is shown in SEQ ID NO:6.

In some embodiments, the heavy chain variable region HCVR of the anti-DLL3 antibody is shown in SEQ ID NO:25, and the light chain variable region LCVR is shown in SEQ ID NO:26.

In some embodiments, the heavy chain variable region HCVR of the anti-DLL3 antibody is shown in SEQ ID NO:27, and the light chain variable region LCVR is shown in SEQ ID NO:28.

In some embodiments, the heavy chain variable region HCVR of the anti-DLL3 antibody is shown in SEQ ID NO:29, and the light chain variable region LCVR is shown in SEQ ID NO:30.

In some embodiments, the heavy chain variable region HCVR of the anti-B7-H3 antibody is as shown in SEQ ID NO:31, and the light chain variable region LCVR is as shown in SEQ ID NO:32.

In some embodiments, the heavy chain variable region HCVR of the anti-B7-H3 antibody is as shown in SEQ ID NO:33, and the light chain variable region LCVR is as shown in SEQ ID NO:34.

In some embodiments, the heavy chain constant region of the anti-B7-H3 antibody is as shown in SEQ ID NO:47 or 49, and the light chain constant region is as shown in SEQ ID NO:48.

Variants of the above-mentioned amino acid sequences are also within the scope of this invention. The corresponding variants, compared to any one of the polypeptides SEQ ID NO:7-24 and 35-46, each contain a mutation of up to three amino acids occurring in at least one CDR region; or, the variants, relative to the overall sequences of SEQ ID NO:1-6, SEQ ID NO:25-34, and SEQ ID NO:47-87, may contain three or more mutations, for example, sequences having at least 80%, 85%, 90%, 93%, 95%, 97%, or 99% identity with any one of the polypeptides SEQ ID NO:54-87. The mutation can be an amino acid substitution, deletion, or addition, or any combination thereof; preferably, the mutation is a conserved substitution.

"Conservative substitution" refers to the substitution of amino acids in a protein with other amino acids that have similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity), so that the protein can be frequently modified without changing its biological activity.

Substitutions generally considered conserved are substitutions between aliphatic amino acids Ala, Val, Leu, and Ile; interchange of hydroxyl residues Ser and Thr; exchange of acidic residues Asp and Glu; substitution between amide residues Asn and Gln; exchange of basic residues Lys and Arg; and substitution between aromatic residues Phe and Tyr. Those skilled in the art will recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. (1987), *Molecular Biology of the Gene*, The Benjamin/Cummings Pub. Co., p. 224, 4th edition). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

This disclosure also relates to methods for preparing antibody-drug conjugates as described above or pharmaceutically acceptable salts or solvates thereof, including:

After the target antibody is reduced, it is coupled with a pre-synthesized -L-D to obtain a compound with the general formula Ab-L-D.

The reducing agent is preferably TCEP, and in particular, it is preferred to reduce the disulfide bonds on the target antibody.

This disclosure also relates to pharmaceutical compositions comprising the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described above, and pharmaceutically acceptable excipients, diluents or carriers.

As used in this disclosure, "pharmaceuticalally acceptable carrier, diluent or excipient" includes any material that, when combined with an active ingredient, allows the ingredient to remain biologically active and does not react with the immune system of a subject.

This disclosure also relates to the use of antibody-drug conjugates as described above or pharmaceutically acceptable salts or solvates thereof in the preparation of medicaments for treating tumors.

The term "cancer" refers to a physiological condition or disease characterized by disordered cell growth. "Tumor" includes cancer cells. In some embodiments, the tumor is a solid tumor or hematologic malignancy such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, head and neck cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, neurotumor, glioma, neuroblastoma, glioma multiforme, sarcoma, squamous cell carcinoma, myeloma, lymphoma, and leukemia.

This disclosure also relates to a method for treating a subject’s medical condition, comprising administering a safe and effective amount of the antibody-drug conjugate as described above.

Preferably, the medical condition described is cancer.

The phrase "safe and effective amount" means, as used herein, a compound or composition in an amount sufficient to significantly and effectively relieve the symptoms or condition being treated, but small enough to avoid serious side effects (with a reasonable benefit/risk ratio), within reasonable pharmaceutical modulation. The safe and effective amount of the active ingredient in the pharmaceutical composition used in the methods of this disclosure varies depending on the specific symptoms being treated, the patient's age and physical condition, the severity of the disease, the duration of treatment, concurrent treatments, the specific active ingredient used, the specific pharmaceutically acceptable excipients used, and factors including the knowledge and skills of the physicians involved in the treatment.

It should be understood that the contemplated treatment methods will also include the administration of other therapeutic entities, particularly preferably immunotherapeutic entities, including viral cancer vaccines (e.g., adenovirus vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., nonpyrogenic Escherichia coli expressing one or more cancer-specific antigens), yeast cancer vaccines, N-803 (also known as ALT-803, ALTOR Biosciences), chemotherapeutic agents, antibodies (e.g., those binding to tumor-associated antigens or patient-specific tumor neoantigens), stem cell grafts (e.g., allogeneic or autologous), and tumor-targeting cytokines (e.g., NHS-IL12, IL-12 conjugated to a tumor-targeting antibody or a fragment thereof). In some embodiments, the contemplated treatment methods also include radiation therapy to the patient. In some embodiments, the contemplated treatment methods also include surgery on the patient, such as tumor resection surgery.

Antibody-drug conjugates can also be administered in combination with and/or co-formulated with antiviral agents, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotective agents, metal chelators, IFN-γ, and/or NSAIDs. The pharmaceutical composition may contain the aforementioned therapeutic entities.

The terms “subject” and “patient” are used interchangeably in this document and refer to any animal, such as any mammal, including (but not limited to) humans, non-human primates, rodents, dogs, cats, chimpanzees, orangutans, gibbons, macaques, marmosets, pigs, horses, pandas, and elephants, etc.

As used herein, “treatment” means any improvement in the outcome of a disease, such as prolonged survival, lower morbidity, and/or reduced side effects as a byproduct of alternative treatment. As is readily understood in the art, complete eradication of the disease is preferred, but not a necessary condition for treatment. As used herein, “treatment” refers to the administration of the antibody-drug conjugate to a subject (e.g., a patient). Treatment can be curative, therapeutic, alleviating, reducing, altering, improving, weakening, improving, or affecting a condition, its symptoms, or, for example, a predisposition to cancer.

The pharmaceutical compositions disclosed herein can be administered by any route, as those skilled in the art will understand. In some embodiments, the pharmaceutical compositions disclosed herein are administered intravenously (IV).

The embodiments of this disclosure will now be described in detail with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this disclosure. For experimental methods in the following embodiments where specific conditions are not specified, reference should be made to the guidelines given in this disclosure, or to experimental manuals or conventional conditions in the art, or to other experimental methods known in the art, or to the conditions recommended by the manufacturer.

In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

Example 1: Antigen molecule amino acid sequence

The antigen human B7-H3 is an amino acid sequence of the Q5ZPR3-1 gene from the Uniprot database, as follows:

The Human B7-H3 protein was purchased from Acrobiosystems, catalog number B7B-H52E7. It can also be expressed by fusing the extracellular region of amino acids 27-461 with GGGGSHHHHHH. It can also be expressed by fusing the Fc constant region of human antibody IgG1 or mouse antibody IgG2a.

The antigen human DLL3 is amino acid Q9NYJ7-1 from the Uniprot database, and its sequence is as follows:

The human DLL3 protein was purchased from Acrobiosystems, catalog number DL3-H52H4. It can also be expressed by fusing the extracellular region of amino acids 27-492 with GGGGSHHHHHH. It can also be expressed by fusing the Fc constant region of human antibody IgG1 or mouse antibody IgG2a.

Example 2: Preparation, screening, and subcloning of murine hybridoma antibodies

(1) Mouse immunization and hybridoma cell fusion. Human DLL3-ECD-mFc fusion protein was used as the antigen, and after thorough emulsification with an equal volume of complete Freund's adjuvant (Sigma, Cat No: F5581), 6-8 week old Balb/c mice (purchased from Joinn Laboratories (Suzhou) New Drug Research Center Co., Ltd.) were subcutaneously immunized with an antigen dose of 20 μg/mouse. Subsequently, every 2 weeks, the same dose of antigen was used to thoroughly emulsify with incomplete Freund's adjuvant (Sigma, Cat No: F5506) and the mice were subcutaneously immunized three times. Serum titers were measured after three immunizations, and a booster immunization was performed intraperitoneally 3 days before fusion. Mouse spleen cells and SP2/0 cells were mixed at a ratio of 4:1 using PEG Hybri-Max (Sigma, Cat No: 7181) as the fusion agent. The fused cells were added to 96-well plates (1 × ^10⁵ cells/well), with each well containing 0.1 mL of 1X HAT medium (Invitrogen, Cat No: 21060-017). On day 3, 0.1 mL of HT medium (Invitrogen, Cat No: 11067-030) was added. On day 7, the medium in the 96-well plates was aspirated, and 0.2 mL of fresh HT medium was added. On day 9, the supernatant was collected for ELISA and FACS analysis.

(2) ELISA screening of antibodies that bind to DLL3-ECD. Add 50 μL of DLL3-hFc (final concentration: 2 μg/mL) to coat a 96-well ELISA plate (Corning, Cat. No.: 9018) and incubate overnight at room temperature. Wash three times with washing buffer (PBS + 0.05% Tween 20), then add blocking buffer (PBS + 2% BSA) and incubate at room temperature for 1 hour. Wash the ELISA plate three times with washing buffer. Add hybridoma supernatant and incubate at room temperature for 1 hour, then wash three times. Add 100 μL of 10,000-fold diluted HRP-conjugated goat anti-mouse IgG secondary antibody (Thermo, Cat. No.: 31439) to each well and incubate at room temperature in the dark for 1 hour, then wash three times. Add 100 μL of TMB (Beijing Bio-Science, Cat. No.: ES-002) to each well and incubate at room temperature for 1-3 minutes. Add 100 μL/well of stop solution (2N _{H2SO4 ).} Terminate the colorimetric reaction and read the OD450 value of each well using a microplate reader (Tecan Spark).

(3) FACS screening of antibodies binding to DLL3-ECD. FACS screening of hybridoma antibodies binding to 293T-DLL3: Take 50 μL of hybridoma supernatant that is positive in the above test and mix it with 50 μL of 293T-DLL3 cells (2× ^10⁵ cells/well), add it to a 96-well U-bottom cell plate and incubate at 4°C for 1 hour. Wash with FACS buffer (PBS+3% FBS) and centrifuge twice. Add 100 μL of PE-labeled goat anti-mouse secondary antibody (Biolegend, Cat. No.: 405307) diluted 400 times, incubate at 4°C in the dark for 40 minutes, wash with FACS buffer and centrifuge twice. Detect the signal value of particles in the PE channel using a flow cytometer (SinoCyte).

(4) Subcloning of hybridomas. Hybridomas with strong binding activity were subcloned using the limiting dilution method. Then, the binding affinity between the hybridoma antibody and DLL3 was analyzed by ELISA and FACS to screen for hybridoma monoclonal antibodies with strong binding affinity.

Example 3: Testing of DLL3 hybridoma monoclonal antibodies

1) The binding activity of the purified monoclonal antibody to DLL3 protein was determined by ELISA: In the experiment, 100 μL of DLL3-hFc (final concentration: 2 μg/mL) was added to coat a 96-well ELISA plate (Corning, Cat. No.: 9018) and incubated overnight at room temperature; after washing three times with washing buffer (PBS + 0.05% Tween 20), blocking buffer (PBS + 2% BSA) was added and incubated at room temperature for 1 hour, followed by washing three times with washing buffer; serially diluted antibody buffer was added and incubated at room temperature for 1 hour, followed by washing three times; 100 μL of 10,000-fold diluted HRP-conjugated goat anti-mouse IgG secondary antibody (Thermo, Cat. No.: 31439) was added to each well and incubated at room temperature in the dark for 1 hour, followed by washing three times; 100 μL of [unclear text - possibly a continuation of the previous sentence] was added to each well. TMB (Beijing Bio-Science, Cat.No.: ES-002), develop color at room temperature for 1-3 minutes, add 100 μL/well of stop solution (2N H ₂ SO ₄ ) to terminate the color development reaction, read the OD450 value of each well using a microplate reader (Tecan Spark), and fit and calculate the EC ₅₀ value using Prism software.

The results are shown in Table 1 and Figure 1. The three candidate DLL3 hybridoma monoclonal antibodies, 59H4, 71B11, and 56G10, showed strong DLL3 antigen-binding activity (stronger than the Abbvie positive control based on _EC50 values).

2) The binding activity of purified monoclonal antibody to DLL3-expressing cells was determined using the FACS method: In the experiment, the monoclonal antibody was serially diluted with FACS dilution buffer. 50 μL of the antibody dilution buffer was mixed with 50 μL of 293T-DLL3 or SHP-77 cells (2 × ^10⁵ cells/well) and gently pipetted. The mixture was then added to 96-well U-bottom cell plates and incubated at 4°C for 1 hour. The cells were washed with FACS buffer (PBS + 3% FBS) and centrifuged twice. 400-fold diluted PE-labeled goat anti-mouse secondary antibody (Biolegend, Cat. No.: 405307) was added, and the cells were incubated at 4°C in the dark for 40 minutes. The cells were washed with FACS buffer and centrifuged twice. The signal value of cell particles in the PE channel was detected using a flow cytometer (SinoCyte). The _EC50 value was calculated using Prism software.

The results are shown in Tables 2 and 3 and Figures 2A-3B. The three candidate DLL3 hybridoma monoclonal antibodies, 59H4, 71B11, and 56G10, showed strong binding activity to DLL3-expressing cells (stronger than or close to the positive control Abbvie based on _EC50 values).

3) Assess the endocytic activity of purified monoclonal antibodies in cells using FACS: In the experiment, monoclonal antibodies were serially diluted with FACS dilution buffer. 50 μL of the antibody dilution buffer was mixed with 50 μL of 293T-DLL3 or SHP-77 cells (2 × ^10⁵ cells/well) and gently pipetted to mix. The mixture was then added to 96-well U-bottom cell plates and incubated at 4°C for 1 hour. The cells were washed with FACS buffer (PBS + 3% FBS) and centrifuged twice. Goat's antibody labeled with the pH-dependent fluorescent dye CypHer5E (Cytiva, Cat. No.: PA15401) was added. Anti-mFc (Jackson, Cat. No.: 115-006-071) secondary antibody was incubated at 4°C in the dark for 40 minutes. Cells were washed with FACS buffer and centrifuged twice. The appropriate complete cell culture medium (DMEM + 10% FBS or RPMI 1640 + 10% FBS) was added, and the cells were incubated for approximately 3 hours. The culture medium was removed by centrifugation, and the cells were resuspended in PBS (pH 9.0). The signal value of cell granules in the APC channel (CypHer5E) was detected using a flow cytometer (SinoCyte). _EC50 values were calculated and fitted using Prism software.

The results are shown in Figures 4A-4C and 5A-5B. The three candidate DLL3 hybridoma monoclonal antibodies, 59H4, 71B11, and 56G10, showed strong endocytic activity (stronger than or close to the positive control of Abbvie based on _EC50 values).

Table 1. _EC50 of DLL3 hybridoma antibody binding to DLL3 detected by ELSI method

Table 2. Detection of _EC50 in 293T-DLL3 cells by DLL3 hybridoma antibody binding using FACS method

Table 3. Detection of _EC50 of DLL3 hybridoma antibody binding to SHP-77 tumor cells using FACS method

Example 4: Gene Cloning of the DLL3 Antibody Variable Region

This invention performed gene cloning and sequence analysis on the variable regions of three monoclonal hybridoma antibodies, 59H4, 71B11, and 56G10. The specific methods are as follows: DLL3 monoclonal hybridoma cell lines were lysed using TRIzon (Cwbiotech, Cat No: CW0580) to extract total RNA. The RNA from the hybridoma cells was reverse transcribed into cDNA using a HiFi Script cDNA synthesis kit (Cwbiotech, Cat No: CW2569). Using the cDNA as a template, the variable regions of the heavy and light chains of the antibodies were amplified by PCR using degenerate primers (Kettleborough et al. (1993) Eur J Immunology 23:206-211; Strebe et al. (2010) Antibody Engineering 1:3-14). The PCR amplification products were ligated into a T/A vector, transformed into DH5α competent cells, plated, and incubated overnight at 37°C. Single clones were picked from culture plates, expanded, and plasmids were extracted to determine the antibody gene sequence. Based on the antibody gene sequence, its complementarity determinant (CDR) and backbone region were analyzed. The variable region gene sequences and amino acid sequence numbers of some antibody heavy and light chains are shown in Table 4 below.

Table 4. Sequence listing of DLL3 hybridoma antibody and CDR region

Example 5 – Humanization of mouse DLL3 antibodies 59H4, 71B11, and 56G10

Humanization of the murine DLL3 antibody was performed using a complementarity determinant grafting method. First, the IMGT database was searched for human germline antibody sequences with the highest homology to the light and heavy chain variable regions of the murine 59H4, 56G10, and 71B11 antibodies. For the 59H4 antibody, the germline selected for light chain variable region humanization was IGKV4-1*01, and for the heavy chain variable region humanization, it was IGHV7-4-1*02. For the 56G10 antibody, the germline selected for light chain variable region humanization was IGKV4-1*01, and for the heavy chain variable region humanization, it was IGHV7-4-1*02. For the 71B11 antibody, the germline selected for light chain variable region humanization was IGHV2-29*02, and for the heavy chain variable region humanization, it was IGHV7-4-1*02. The CDR region of the murine antibody is retained, and the frame region sequence of the murine antibody is replaced with the frame region sequence of the human germline antibody. A structural model of the murine antibody is established, and the amino acids at each site in the frame region of the human antibody and the corresponding murine antibody are compared one by one. If using a human amino acid sequence at a certain site in the frame region does not cause damage or change to the spatial structure of the CDR region, then the human amino acid sequence is used at that site; otherwise, the corresponding murine sequence is used at that site (i.e., a reversion mutation to the murine sequence).

The humanized sequences are shown in Table 5.

Table 5. Amino acid sequence numbers of humanized DLL3 antibodies

Nucleic acid sequences encoding the light and heavy chains of humanized antibodies 59H4, 71B11, and 56G10 were synthesized and inserted into the expression vector pcDNA3.1. 200 mL of 293 cells (cell density 1 × ^10⁶ ) were co-transfected with 0.1 mg each of the antibody light and heavy chain expression plasmids. The cells were cultured at 37°C with shaking for 6 days. The supernatant was collected by centrifugation, and the humanized antibodies were purified using Protein A. The purified humanized antibodies were used for activity assays.

Example 6: Detection of the activity of humanized DLL3 antibody and detection of species cross-linking and binding reactions with homologous proteins.

1) Activity detection of humanized DLL3 antibody: The binding of purified humanized antibody sample to DLL3 was detected by ELISA and FACS, and the endocytic activity of humanized antibody was detected by FACS. For specific methods, please refer to Example 2.

The results are shown in Figures 6, 7A-7B, 8A-8B, 9A-9B, and 10A-10B. Overall, the three humanized candidate antibodies maintained strong antigenic activity and endocytosis. This lays the foundation for further conjugation and drug development.

2) Detection of species cross-reaction and binding reaction with homologous proteins

A) Species cross-reactivity of humanized antibodies

The binding activity of purified monoclonal antibodies to monkey, rat, and mouse DLL3 protein was determined by ELISA: 100 μL of monkey, rat, and mouse DLL3 protein (final concentration: 2 μg/mL) was coated onto each well of a 96-well ELISA plate (Corning, Cat. No.: 9018) and incubated overnight at room temperature. The plates were washed three times with washing buffer (PBS + 0.05% Tween 20), then incubated with blocking buffer (PBS + 2% BSA) at room temperature for 1 hour, followed by three washes with washing buffer. Serially diluted antibody buffer was added, and the plates were incubated at room temperature for 1 hour, followed by three washes. 100 μL of 3000-fold diluted HRP-conjugated mouse anti-human IgG secondary antibody (BD Biosciences, Cat. No.: 555788) was added to each well, and the plates were incubated at room temperature in the dark for 1 hour, followed by three washes. 100 μL of [unspecified ingredient] was added to each well. TMB (Beijing Bio-Science, Cat.No.: ES-002), develop color at room temperature for 1-3 minutes, add 100 μL/well of stop solution (2N H ₂ SO ₄ ) to terminate the color development reaction, read the OD450 value of each well using a microplate reader (Tecan Spark), and fit and calculate the EC ₅₀ value using Prism software.

The results, shown in Figures 11A-11C, indicate that among the three humanized antibodies, humanized 59H4 and 71B11 exhibited strong binding activity to DLL3 proteins in monkeys, rats, and mice; while humanized 56G10 showed acceptable binding to DLL3 in monkeys, but its binding to DLL3 protein in rats was weakened to a certain level, and it did not bind to DLL3 protein in mice.

B) Detection of the binding reaction between humanized antibodies and homologous proteins

The binding activity of purified monoclonal antibodies to human DLL1, DLL3, and DLL4 proteins was determined by ELISA: 100 μL of human DLL1, DLL3, and DLL4 protein (final concentration: 2 μg/mL) was coated onto each well of a 96-well ELISA plate (Corning, Cat. No.: 9018) and incubated overnight at room temperature. The plates were washed three times with washing buffer (PBS + 0.05% Tween 20), then incubated with blocking buffer (PBS + 2% BSA) at room temperature for 1 hour, followed by three washes with washing buffer. Serially diluted antibody buffer was added, and the plates were incubated at room temperature for 1 hour, followed by three washes. 100 μL of 3000-fold diluted HRP-conjugated mouse anti-human IgG secondary antibody (BD Biosciences, Cat. No.: 555788) was added to each well, and the plates were incubated at room temperature in the dark for 1 hour, followed by three washes. 100 μL of [unspecified ingredient] was added to each well. TMB (Beijing Bio-Science, Cat.No.:ES-002), develop color at room temperature for 1-3 minutes, add 100μL/well of stop solution (2N H ₂ SO ₄ ) to terminate the color development reaction, and read the OD450 value of each well using a microplate reader (Tecan Spark).

The results, shown in Figures 12A-12C, indicate that the three humanized antibodies, 59H4, 71B11, and 56G10, did not bind to their homologous proteins. This suggests that these three antibodies bind only to the DLL3 protein and exhibit good specificity.

Example 7: Candidate clones of antibodies targeting B7-H3 and candidate clones targeting DLL3

Humanized antibodies of candidate clones 7B7 and 15A2 were selected, which have been disclosed in patent CN113402610A and its family of patents. The amino acids at certain sites were appropriately optimized. The SEQ ID NO. was rewritten in this invention, and its sequence is excerpted in Table 6.

Table 6. Amino acid sequence numbers of humanized B7-H3 antibodies

Example 8: Targeting DLL3 and B7-H3 bispecific antibodies

From the five clones mentioned above, one candidate clone targeting one target was used as a traditional, intact human IgG1 antibody. The heavy and light chain variable regions of the other target were tandemly linked with (G4S)*3, (G4S)*4, or (G4S)*5 to form a single-chain antibody ScFv, which was then fused to the C-terminus of IgG1 to form a dual-target antibody as shown in Figure 13. The terminal amino acids were adjusted as needed. The glycosylation site N297 could be adjusted to N297A (A could also be any amino acid other than N). The G4S length, terminal amino acid adjustments, and glycosylation site adjustments are existing technologies. For the ScFv structure, the amino acids in the heavy chain variable region VH44 and the light chain variable region VL100 could be mutated to cysteine to form a disulfide bond, or mutated to positively and negatively charged aspartic acid and lysine to form a salt bond, thus stabilizing the ScFv structure.

The architecture of the dual-antibody system is shown in Figure 13.

The sequences of the bispecific antibodies are shown in Table 7:

Table 7

Following the designed architecture and sequence, the antibody light and heavy chains were constructed separately into pCDNA vectors, and plasmids were extracted. Suspension-acclimated CHO-K1 cells were revived in OPM-CD TransCHO medium and cultured to a density of 2 million cells/ml with a viability of over 95% in a volume of 1000 ml. 0.5 mg of the light chain plasmid and 0.5 mg of the heavy chain plasmid were combined and dissolved in 10 ml of medium. 3 mg of PEI dissolved in the medium was diluted in 10 ml of medium. The plasmid and PEI solutions were mixed and incubated at room temperature for 10 minutes, then added dropwise to 1000 ml of cell culture medium. After incubation at 37°C for 5 days, the supernatant was collected by centrifugation at 12000g for 15 minutes. The supernatant was purified using HiTrap Mabselect SuRe and eluted with 50 mM acetic acid. The collected peak after neutralization was replaced with PBS (pH 7.4) using a 30 kDa ultrafiltration tube. The absorbance of the antibody at 280 nm was measured, and the absorbance value divided by the theoretical extinction coefficient was used to determine the concentration. The antibody expression levels and purity are shown in Table 8 below:

Table 8. Expression levels and purity of DLL3 and B7-H3 bispecific antibodies

Example 9: Antibody-antigen binding activity and antibody endocytosis activity

Antibody-antigen binding activity (ELISA): Add 100 μL of DLL3-mFc or B7H3-mFc (final concentration: 2 μg/mL) to a 96-well ELISA plate (Corning, Cat. No.: 9018) and incubate overnight at room temperature; wash three times with washing buffer (PBS + 0.05% Tween 20), add blocking buffer (PBS + 2% BSA) and incubate at room temperature for 1 hour, then wash the ELISA plate three times with washing buffer; add serially diluted antibody buffer, incubate at room temperature for 1 hour, and wash three times; add 100 μL of 3000-fold diluted HRP-conjugated mouse anti-human IgG secondary antibody (BD Biosciences, Cat. No.: 555788) to each well, incubate at room temperature in the dark for 1 hour, and wash three times; add 100 μL of... TMB (Beijing Bio-Science, Cat.No.: ES-002), incubate at room temperature for 1-3 minutes, add 100 μL/well of stop solution (2N H ₂ SO ₄ ) to terminate the color development reaction, read the OD450 value of each well using a microplate reader (Tecan Spark), and calculate the EC ₅₀ value by fitting.

Antibody-cell binding activity (FACS): The tested bispecific antibody was serially diluted with flow cytometry dilution buffer. 50 μL of antibody dilution buffer was mixed with 50 μL of SHP-77 tumor cells (200,000 cells/well) and gently pipetted to form a 96-well U-bottom cell plate. The plate was incubated for 1 hour, washed with FACS buffer (PBS + 3% FBS), and centrifuged twice. 100 μL of 400-fold diluted PE-labeled goat anti-human secondary antibody (eBioscience, Cat. No.: 12-4998-82) was added, and the plate was incubated in the dark for 40 minutes. The plate was washed with FACS buffer and centrifuged twice. The signal value of cell particles in the PE channel was detected using a flow cytometer (SinoCyte). The _EC50 value was then fitted and calculated.

Antibody endocytosis activity (FACS): The target bispecific antibody was serially diluted with flow cytometry dilution buffer. 50 μL of the antibody dilution buffer was mixed with 50 μL of SHP-77 or DMS53 cells (20,000 cells/well) and gently pipetted to form a 96-well U-bottom cell plate. The plate was incubated at 4°C for 1 hour. The cells were washed with FACS buffer (PBS + 3% FBS) and centrifuged twice. 100 μL of pH-dependent fluorescent dye CypHer5E (Cytiva, Cat. No.: PA15401) labeled with Goat anti-hFc (Jackson, Cat. No.: 109-006-098) secondary antibody was added. The plate was incubated at 4°C in the dark for 40 minutes. The cells were washed with FACS buffer and centrifuged twice. Complete culture medium (RPMI 1640 + 10% FBS) was added, and the plate was incubated at 37°C for approximately 3 hours. The culture medium was removed by centrifugation, and PBS (pH...) was added... 9.0) Cells were resuspended, and the signal values of cell granules in the APC channel (CypHer5E) were detected using a flow cytometer (SinoCyte). The _EC50 values were then fitted and calculated.

The results of the binding and endocytic activities of the above-mentioned bispecific antibodies are shown in Table 9 below:

Table 9. Binding and endocytic activities of bispecific antibodies

Example 10: Connection Sub-load Synthesis

Patent application number for the connecting sub-payload technology: PCT/CN2023/106385 (WO2025011419A1, Chinese application CN119264213A). Applicant: Shanghai Shijian Biotechnology Co., Ltd.

LD-38 structure:

More detailed information on synthesis or other connector payloads, as well as coupling detection, can be found in patent PCT/CN2023/106385.

Example 11 Antibody Conjugation

Antibody was diluted to 5 mg/ml with 10 mM sodium phosphate buffer (pH 7.4), and tris(2-hydroxyethyl)phosphonic acid hydrochloride (TCEP) stock solution was added to ensure a final TCEP molar ratio of 6:1 to antibody. The antibody was then incubated at 25°C for 2 hours for reduction. Next, dimethyl sulfoxide (DMSO) was added to dissolve the linker toxin stock solution until the final linker toxin molar ratio of antibody was 20:1. The antibody was then incubated at 25°C for 2 hours for conjugation. The ADC was then replaced with sodium phosphate buffer in a 30 kDa ultrafiltration tube, aseptically filtered, and the concentration was determined. The resulting aliquots were then frozen. To obtain products with different conjugation ratios, the molar ratio of antibody:reducing agent:linker toxin was adjusted between 1:1, 10:2, and 50, while keeping other conditions constant. The final DAR value of the conjugated product was determined based on the detection results.

Antibodies and conjugates were detected using the SEC-HPLC molecular sieve method. The analytical column was a TSKgel G3000SWXL (7.8 mm × 30 cm, 5 μm, catalog number 08541). Approximately 0.3 mg of the sample was centrifuged at 10000g for 5 minutes in a volume of 300 μL, and the supernatant was collected. The mobile phase was 50 mM sodium phosphate + 0.1 M sodium chloride, pH 6.8. The analytical column was connected to an Agilent 1260 HPLC system at a flow rate of 0.8 mL/min. The mobile phase was eluted for at least 30 minutes until the 280 nm UV baseline stabilized. 100 μg of sample was injected, and the mobile phase was eluted for 20 minutes. The percentage of each peak (polymer, monomer, low molecular weight) was calculated based on the peak area. The purity value was determined using the monomer peak.

The average number of linker loads conjugated to each antibody molecule is the coupling ratio (DAR), which is detected by hydrophobic chromatography-high-performance liquid chromatography (HIC-HPLC). The analytical column is a TSKgel Butyl-NPR, 4.6 mm × 10 cm, 2.5 μm, catalog number 042168. Approximately 0.3 mg of the sample to be tested was taken in a volume of 150 μL and added to 150 μL of mobile phase A. The mixture was centrifuged at 10000 g for 5 minutes, and the supernatant was collected. Mobile phase A consisted of 20 mM sodium phosphate + 1.5 M ammonium sulfate at pH 7.0, and mobile phase B consisted of mobile phase A consisting of 20 mM sodium phosphate + 20% (v/v) acetonitrile at pH 7.0. The analytical column was connected to an Agilent 1260 high-performance liquid chromatograph. The flow rate was 0.6 ml/min. Mobile phase A was used for rinsing for at least 30 minutes until the 280 nm UV baseline stabilized. 50 μg of sample was injected, followed by rinsing with mobile phase A for 2 minutes, then gradient elution (0% B–100% B) for 18 minutes, and finally rinsing with 100% mobile phase B for 5 minutes. The percentage of each DAR value was calculated based on the peak area of each DAR value. The percentage of each peak was multiplied by the DAR value, summed, and then divided by the total percentage to obtain the average DAR value. Clearly, although the coupling ratio DAR of 8 was achieved in this experiment, this is only a result obtained by the HIC-HPLC method. Since the coupled products all had only one distinct peak, a DAR of 8 was considered appropriate. Other methods can be considered alternatives. Furthermore, adjusting the average DAR between 2 and 8 can achieve a balance between effectiveness and safety. The couplings of this invention do not limit the DAR to 8; they can be extended to 2–8.

Table 10. ADC Molecular Purity

Example 12 In vitro activity assay

Human tumor cell lines SHP-77 and NCI-H82 were resuscitated and, when they reached the logarithmic growth phase and viability was greater than 90%, were seeded into 96-well culture plates at a density of 500–5000 cells per well (150 μL). The plates were incubated at 37°C in a 5% CO2 incubator. After approximately 20 hours, 50 μL of culture medium containing different antibody-drug conjugates were added to achieve 12 dilutions between 0 and 1000 nM (1000 nM for the first well, followed by 10 five-fold dilutions, with the last dilution containing 0 nM; each sample was replicated). The plates were co-cultured for 4–7 days. Then, 50 μL of CTG detection reagent (CellTiterGlo, Promega, catalog number G7575) was added to each well. The reaction was allowed to proceed for 2 minutes, following the kit's recommended procedure. Fluorescence was then measured using Tecan Spark. The average value of the multiple wells is used as the Y-axis, and the dilution gradient log10 value is used as the X-axis to create a smooth curve. The EC/IC ₅₀ value is then calculated using a four-parameter fitting method.

Table 11. Inhibition of SHP-77 growth activity by ADC

Example 13 In vivo activity

The correspondence between antibodies and conjugates is shown in Table 12 below:

Table 12

SHP-77 or NCI-H82 tumor cell lines were resuscitated to the logarithmic growth phase with a viability greater than 90%. Immunodeficient mice (various species) were inoculated with varying numbers of 10,000 to 1,000,000 cells. When tumors reached 90-150 square millimeters, mice were divided into groups of five. All mice were administered the drug once daily via intravenous administration. The tumor size of each drug group was compared with that of the saline group to determine the inhibitory activity on tumor cells. Groups G1-G14 were completed in a single experiment; due to the large number of groups, several groups were plotted separately for clearer visualization. The horizontal axis represents the treatment time for each group, and the vertical axis represents tumor size. The table also includes statistics on tumor size and tumor inhibition rate (TGI) at a specific midpoint day.

Both targets B7-H3 and DLL3 are tumor-associated antigens or tumor-specific antigens, and while these targets possess biological functions, their biological functions are not fully understood. Surprisingly, the combination of these two bispecific antibody ADCs showed a certain degree of synergistic inhibition of tumor growth in a tumor transplantation model, i.e., 1+1>2, as detailed in the table.

The antibody-drug conjugates inhibit the growth of SHP-77 tumor cells as shown in the table below:

Table 13

The growth curve of SHP-77 tumor cells was inhibited, as shown in Figure 14-17.

The antibody-drug conjugates inhibit the growth of NCI-H82 tumor cells as shown in the table below:

Table 14

The growth curve of NCI-H82 tumor cells was inhibited, as shown in Figure 18-21.

Example 14: Preparation and Comparison of Other Antibody Conjugates

Methods such as bispecific antibody conjugation and in vivo activity assessment models were used. A 7B7-LD38 conjugate was prepared by conjugating LD38 with the 7B7 clone monoclonal antibody of B7H3, and the DAR was measured to be 8. The conjugate 7B7-LD38 (DAR8) and DS-7300 (DAR4) (Daiichi Sankyo, self-made) were compared in different tumor transplantation models (CDX models). The tumor cell growth inhibition curves are shown in Figures 22A-22D. It was found that 7B7-LD38 was more effective than DS-7300 in the RKO, JIMT-1, SHP-77, and Calu-6 models. Therefore, it was preliminarily determined that LD38 was superior to trastuzumab (Deruxtecan), so the control sample DS-7300 was not consistently placed in some tumor transplantation models (CDX models).

The embodiments described above are merely illustrative of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the protection scope of this disclosure. Therefore, the protection scope of this patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims (23)

A bispecific antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the general formula of the antibody-drug conjugate is Ab-(L-D)n; The feature is that Ab is a dual-targeting antibody, L is a linker unit, D is a drug, and n is a positive integer or decimal from 1 to 20. The dual-targeting antibody includes a first structural domain and a second structural domain with binding specificity, and the first structural domain and the second structural domain can bind to the target DLL3 or B7-H3, respectively. According to claim 1, the bispecific antidote conjugate or its pharmaceutically acceptable salt or solvate is characterized in that the drug is selected from one or more of the following effector molecules or derivatives: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, antiangiogenic agents, antiestrogens, antiandrogens, and immunomodulators. According to claim 2, the bispecific antibiotic conjugate or its pharmaceutically acceptable salt or solvate is characterized in that the chromatin function inhibitor and the linker unit constitute a linker payload, wherein the linker payload is LD-38. According to claim 1, the bispecific antidote conjugate or its pharmaceutically acceptable salt or solvate is characterized in that n is a positive integer or decimal of 2-8. A monoclonal antibody that specifically binds to DLL3, or an antigen-binding fragment thereof, comprising a heavy chain CDR and a light chain CDR, wherein... The amino acid sequence of CDR1 of the heavy chain includes SEQ ID NO: 7, 13 or 19; The amino acid sequence of CDR2 of the heavy chain includes SEQ ID NO: 8, 14 or 20; The amino acid sequence of CDR3 of the heavy chain includes SEQ ID NO: 9, 15 or 21; The amino acid sequence of CDR1 of the light chain includes SEQ ID NO: 10, 16 or 22; The amino acid sequence of CDR2 in the light chain includes SEQ ID NO: 11, 17, or 23; and The amino acid sequence of CDR3 in the light chain includes SEQ ID NO: 12, 18, or 24. The antibody or antigen-binding fragment thereof according to claim 5 comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is selected from any one of SEQ ID NO: 1, 3 and 5; and the amino acid sequence of the light chain variable region is selected from any one of SEQ ID NO: 2, 4 and 6. The antibody or antigen-binding fragment thereof according to claim 6 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are humanized. The antibody or antigen-binding fragment thereof according to claim 7, wherein the amino acid sequence of the humanized heavy chain variable region is selected from any one of SEQ ID NO: 25, 27 and 29; and the amino acid sequence of the humanized light chain variable region is selected from any one of SEQ ID NO: 26, 28 and 30. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, characterized in that the antibody is a monoclonal antibody or antigen-binding fragment thereof that specifically binds DLL3 as described in any one of claims 5-8. The antibody-drug conjugate according to claim 9, or a pharmaceutically acceptable salt or solvate thereof, is characterized in that its linker unit and drug portion constitute a linker payload, said linker payload being LD-38. A dual-targeting antibody or antigen-binding fragment thereof that specifically binds to DLL3 and B7-H3, characterized in that the targeting antibody or antigen-binding fragment thereof that specifically binds to DLL3 comprises the heavy chain CDR and light chain CDR as described in claim 5; Targeting antibodies that specifically bind to B7-H3, or their antigen-binding fragments, contain both heavy and light chains. The amino acid sequence of CDR1 in the heavy chain contains SEQ ID NO: 35 or 41; The amino acid sequence of CDR2 in the heavy chain contains SEQ ID NO: 36 or 42; The amino acid sequence of CDR3 in the heavy chain contains SEQ ID NO: 37 or 43; The amino acid sequence of the light chain CDR1 contains SEQ ID NO: 38 or 44; The amino acid sequence of the light chain CDR2 contains SEQ ID NO: 39 or 45; and The amino acid sequence of the light chain CDR3 contains SEQ ID NO:40 or 46. The dual-targeting antibody or its antigen-binding fragment according to claim 11 comprises a heavy chain variable region and a light chain variable region, characterized in that the heavy chain variable region sequence is selected from SEQ ID NO: 31 or 33; and the light chain variable region sequence is selected from SEQ ID NO: 32 or 34. The dual-targeting antibody or its antigen-binding fragment according to claim 12, comprising a heavy chain constant region, characterized in that the amino acid sequence of the heavy chain constant region is selected from SEQ ID NO:47 or 49. The dual-targeting antibody or its antigen-binding fragment according to claim 13 comprises a heavy chain and a light chain, characterized in that the amino acid sequence of the heavy chain is selected from any one of SEQ ID NO: 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 86; and the amino acid sequence of the light chain is selected from any one of SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 and 87. The dual-targeting antibody or its antigen-binding fragment according to claim 11 includes an ScFv structure, a heavy chain variable region, and a light chain variable region, characterized in that the amino acid site VH44 of the heavy chain variable region and the amino acid site VL100 of the light chain variable region can mutate the amino acid to cysteine to form a disulfide bond, or mutate it to positively and negatively charged aspartic acid and lysine to form a salt bond, so as to stabilize the ScFv structure. The antibody or antigen-binding fragment thereof according to any one of claims 5-6 and 11-15 is selected from rabbit antibodies, mouse antibodies, chimeric antibodies, humanized antibodies or fully human antibodies. According to claim 1, the bispecific antibody conjugate or its pharmaceutically acceptable salt or solvate is characterized in that the first and second structural domains are selected from the antibodies or their antigen-binding fragments according to any one of claims 5-8 and 11-16. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, characterized in that its monoclonal antibody or antigen-binding fragment portion is the B7-H3-specific targeting antibody or antigen-binding fragment thereof as described in claim 11, and its linker unit and drug portion constitute a linker payload, said linker payload being LD-38. A method for preparing the bispecific antibody drug conjugate of any one of claims 1-4 or a pharmaceutically acceptable salt or solvate thereof, comprising: After the dual-targeting antibody is reduced, it is coupled with a pre-synthesized -L-D to obtain a compound with the general formula Ab-(L-D)n. A pharmaceutical composition comprising the bispecific antidote conjugate of any one of claims 1-4 or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient, diluent or carrier. Use of the bispecific antibody conjugate or its pharmaceutically acceptable salt or solvate as described in any one of claims 1-4 in the preparation of a medicament for treating tumors. According to the use described in claim 21, the tumor is a solid tumor or hematologic malignancy such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, head and neck cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, neurotumor, glioma, neuroblastoma, glioma multiforme, sarcoma, squamous cell carcinoma, myeloma, lymphoma, and leukemia. According to the use described in claim 22, the cancer is selected from triple-negative breast cancer, HER-2 positive breast cancer, Luminal A breast cancer, Luminal B breast cancer, rhabdomyosarcoma, fibrosarcoma, chronic leukemia, acute leukemia, metastatic castration-resistant prostate cancer, non-small cell lung cancer, small cell lung cancer, degenerative lung cancer, ovarian teratoma, ovarian epithelial tumor, ovarian sex cord-stromal tumor, ovarian germ cell tumor, ovarian metastatic tumor, neuroblastoma, glioma, glioma multiforme, head and neck squamous cell carcinoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, and multiple myeloma; preferably, the cancer is selected from triple-negative breast cancer, HER-2 positive breast cancer, rhabdomyosarcoma, fibrosarcoma, non-small cell lung cancer, degenerative lung cancer, and acute leukemia.