Anti-DLL3 and b7-h3 bispecific antibody-drug conjugate

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

Dual antibody drug conjugate against DLL3 and B7-H3

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. CN202410743383.5, filed on June 7, 2024, and entitled “Dual antibody drug conjugate against DLL3 and B7-H3”, and Chinese Patent Application No. CN202411832563.7, filed on December 12, 2024, and entitled “Dual antibody drug conjugate against DLL3 and B7-H3”, the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present disclosure relates to the field of biomedicine, in particular to a dual antibody drug conjugate. BACKGROUND

[0004] Antibody-drug conjugate (ADC) is a small molecule drug with biological activity coupled to an antibody through a linker. Currently, most ADCs are formed by coupling tumor antigen-targeting antibodies to highly cytotoxic small molecule chemical drugs through a linker, which takes advantage of the specific binding of antibodies to target antigens to target small molecule drugs to tumor cells and then kill tumor cells. There are also specific binding of antibodies, antigens and polypeptides, and other proteins and polypeptides with similar specific binding properties as antibodies.

[0005] DLLs(Delta-like ligands) are ligands of Notch signaling pathway, which are often in an uncontrolled state in tumors and affect tumor growth, tumor blood vessels and tumor immunity. Notch signaling pathway is a relatively conservative signaling pathway in human evolution, which mainly controls the progress of cells through cell-cell interaction. Notch receptor is transported to the cell surface and forms a transmembrane heterodimer after being enzymatically cut in the Golgi body. After interacting with the Notch ligand on the adjacent cell, the intracellular part of the Notch ligand is enzymatically cut, and the Notch intracellular domain(NICD) is released from the cell, and then enters the nucleus and binds to the DNA binding protein CSL(CBF-1), which then recruits MAML(Mastermind-like protein) and activates the transcription of Notch targeted genes, including Hes and Hey families. DLL3 is a highly tumor-selective cell surface target, mainly expressed in neural or neuroendocrine tumors, including small cell lung cancer(SCLC), large cell neuroendocrine carcinoma(LCNEC), gastrointestinal neuroendocrine tumor(GI-NEC), small cell bladder cancer(SCBC), glioblastoma multiforme, metastatic castration-resistant prostate cancer, lung neuroendocrine tumor, etc., especially SCLC, more than 80% of SCLC has positive expression of DLL3, while normal lung cancer tissue and paracancerous tissue do not express.(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 Target for the Treatment of Neuroendocrine Neoplasms. Oncologist. 2022 Nov 3;27(11):940-951. doi: 10.1093 / oncolo / oyac161. PMID: 35983951; PMCID: PMC9632312. Leonetti A, Facchinetti F, Minari R, Cortellini A, Rolfo CD, Giovannetti E, Tiseo M. Notch pathway in small-cell lung cancer: from preclinical evidence to therapeutic challenges. Cell Oncol (Dordr). 2019 Jun;42(3):261-273. doi: 10.1007 / s13402-019-00441-3. Epub 2019 Apr 9. PMID: 30968324.).

[0006] Target point DLL3 is highly expressed on the surface of various neuroendocrine tumor cells and is lowly expressed in normal tissues and cells. Drugs targeting DLL3 are already in the clinical stage. Typical representative drugs include: Rova-T, an ADC drug targeting DLL3; AMG757, a dual antibody targeting DLL3 and CD3, which has been approved for marketing; HPN328, a dual antibody targeting DLL3 and CD3; and BI764532, a dual antibody targeting DLL3 and CD3. In addition, there are multiple ADCs and CAR-Ts under development.

[0007] B7-H3 is expressed on the surface of immune cells or tumor cells, and its corresponding receptor ligand can be multiple, and none of the major ligands or receptors have been found, and the major biological function is unknown, although immunosuppressive or immunostimulatory effects have been found. At the same time, in a variety of tumors, high expression of B7-H3 in tumor tissues is negatively correlated with the prognosis of survival period to a certain extent. B7-H3 is widely expressed in tissues such as heart, liver, pancreas, prostate, small intestine and colon, but the expression amount is very low. It is expressed in immune cells, but the expression amount is very low, and it is not constitutive expression but induced expression. B7-H3 is expressed in a variety of malignant tumors, including melanoma, glioma, lung cancer, pancreatic cancer, renal cancer, colon cancer, ovarian cancer, breast cancer, gastric cancer, endometrial cancer and some hematological tumors. (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 Role of CD276 in Cancers. Front Oncol. 2021 Mar 26;11:654684. doi: 10.3389 / fonc.2021.654684. PMID: 33842369; PMCID: PMC8032984. Wang L, Kang FB, Shan BE. B7-H3-mediated tumor immunology: Friend or foe? Int J Cancer. 2014 Jun 15;134(12):2764-71. doi: 10.1002 / ijc.28474. Epub 2013 Sep 30. PMID: 24013874.)

[0008] The target B7-H3 is highly expressed on the surface of a variety of solid tumor cells and is lowly expressed in normal tissues and cells. Drugs targeting B7-H3 are already in the clinical stage. Typical representative drugs include: early large molecule drugs targeting B7-H3 (not including cell therapy) such as radionuclide conjugated antibody iodine 131-Omburtamab, followed by MGD009, MGA271, MGC018 and DS-7300, etc. Camptothecin ADC; ABBV-155 (Mirzotamab Clezutoclax) is an antibody conjugate targeting B7-H3, conjugated with a small molecule BCL inhibitor; MGA271 (Enoblituzumab) is a monoclonal antibody drug targeting B7-H3; MGC018 is an antibody conjugate targeting B7-H3, conjugated with a small molecule duocarmycin. SUMMARY

[0009] The present disclosure first provides a dual-antibody drug conjugate against DLL3 and B7-H3 and its application.

[0010] ADC resistance has target-related resistance and load-related resistance. If there is heterogeneity in the expression of the target on the surface of tumor cells, target resistance will be a problem, and the combination of dual targets can increase the expression heterogeneity of the target to homogeneity, to a certain extent, to solve the target-related resistance. If the dual target combination achieves homogeneity, load-related resistance can be further solved by conjugating multiple loads of ADC or sequential treatment or combination therapy of single load ADC. Ultimately, dual-antibody ADC is expected to solve the resistance to a certain extent.

[0011] The PSMA / STEAP1 dual-antibody ADC (ABBV-969) of AbbVie is a representative of this design. PSMA and STEAP1 have high expression in prostate cancer and have a certain complementarity, so that the dual-antibody ADC can more comprehensively eliminate tumor cells and is not prone to drug resistance.

[0012] The present disclosure provides specific antibodies with high affinity to DLL3 and significant cellular endocytosis. Compared with the positive control of AbbVie, the antibodies in the present disclosure have higher affinity and more significant cellular endocytosis, and can be used for further toxin conjugation for tumor treatment. Therefore, the antibodies provided by the present disclosure have important value for the treatment of tumors expressing DLL3.

[0013] The target B7-H3 and DLL3 are tumor-associated antigens / tumor-specific antigens in function, and have some biological functions, but the functions are not clear. It is very unexpected to find that the two have a synergistic effect in the dual-antibody ADC, 1+1>2. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present disclosure, and other drawings can also be obtained by those skilled in the art without creative labor.

[0015] Figure 1: ELISA assay of DLL3 hybridoma antibody binding to DLL3-hFc protein EC 50 .

[0016] Figures 2A-2C: FACS assay of DLL3 hybridoma antibody binding to 293T-DLL3 cells EC 50 .

[0017] Figure 3A-3B: EC of DLL3 hybridoma antibody binding to SHP-77 cells by FACS assay 50 .

[0018] Figure 4A-4C: EC of DLL3 hybridoma antibody internalization in 293T-DLL3 cells by FACS assay 50 .

[0019] Figure 5A-5B: EC of DLL3 hybridoma antibody internalization in SHP-77 cells by FACS assay 50 .

[0020] Figure 6: EC of DLL3 humanized antibody binding to DLL3-mFc protein by ELISA assay 50 .

[0021] Figure 7A-7B: EC of DLL3 humanized antibody binding to 293T-DLL3 cells by FACS assay 50 .

[0022] Figure 8A-8B: EC of DLL3 humanized antibody binding to SHP-77 cells by FACS assay 50 .

[0023] Figure 9A-9B: EC of DLL3 humanized antibody internalization in 293T-DLL3 cells by FACS assay 50 .

[0024] Figure 10A-10B: EC of DLL3 humanized antibody internalization in SHP-77 cells by FACS assay 50 .

[0025] Figure 11A-11C: ELISA assay demonstrating DLL3 humanized antibody species cross- reactivity.

[0026] Figure 12A-12C: ELISA assay demonstrating DLL3 humanized antibody lack of cross- reactivity with cognate proteins.

[0027] Figure 13: Architecture of DLL3 and B7-H3 bispecific molecule targeting.

[0028] Figure 14: SHP-77 model saline control tumor cell growth curve.

[0029] Figure 15: Tumor cell growth curves for the 3103-59H4-LD-38 molecule at 1.5 mg / kg, the 3103-15A2-LD-38 molecule at 1.5 mg / kg, the 3103-59H4-LD-38 and 3103-15A2-LD-38 molecules at 0.75 mg / kg each, the 3103-59H4-LD-38 and 3103-15A2-LD-38 molecules at 1.5 mg / kg each, in the SHP-77 model.

[0030] Figure 16: Tumor cell growth curves for the 3103-0259H415A2-LD-38 molecule at 4.0 mg / kg, the 3103-0259H415A2-LD-38 molecule at 2.0 mg / kg, the 3103-037B759H4-LD-38 molecule at 4.0 mg / kg, the 3103-037B759H4-LD-38 molecule at 2.0 mg / kg, in the SHP-77 model.

[0031] Figure 17: Tumor cell growth curves for the 3103-05-56G10-15A2-LD-38 molecule at 2.0 mg / kg, the 3103-06-71B11-15A2-LD-38 molecule at 2.0 mg / kg, the 3103-07-59H4-15A2-LD-38 molecule at 2.0 mg / kg, the 3103-08-7B7-59H4-LD-38 molecule at 2.0 mg / kg, the 3103-09-59H47B7-LD-38 molecule at 2.0 mg / kg, in the SHP-77 model.

[0032] Figure 18: Tumor cell growth curve for the saline control in the NCI-H82 model.

[0033] Figure 19: Tumor cell growth curves for the 3103-59H4-LD-38 molecule at 1.5 mg / kg, the 3103-15A2-LD-38 molecule at 1.5 mg / kg, the 3103-59H4-LD-38 and 3103-15A2-LD-38 molecules at 0.75 mg / kg each, the 3103-59H4-LD-38 and 3103-15A2-LD-38 molecules at 1.5 mg / kg each, in the NCI-H82 model.

[0034] Figure 20: NCI-H82 tumor model of 3103-0259H415A2-LD-38 molecule at 4.0 mg / kg, 3103-0259H415A2-LD-38 molecule at 2.0 mg / kg, 3103-037B759H4-LD-38 molecule at 4.0 mg / kg, 3103-037B759H4-LD-38 molecule at 2.0 mg / kg, tumor cell growth curves.

[0035] Figure 21: NCI-H82 tumor model of 3103-05-56G10-15A2-LD-38 molecule at 2.0 mg / kg, 3103-06-71B11-15A2-LD-38 molecule at 2.0 mg / kg, 3103-07-59H4-15A2-LD-38 molecule at 2.0 mg / kg, 3103-08-7B7-59H4-LD-38 molecule at 2.0 mg / kg, 3103-09-59H47B7-LD-38 molecule at 2.0 mg / kg, tumor cell growth curves.

[0036] Figures 22A-22D: RKO, JIMT-1, SHP-77, and Calu-6 tumor models of B7H3 antibody ADC molecules, 7B7-LD38 molecule, tumor cell growth curves. DETAILED DESCRIPTION

[0037] Reference will now be made in detail to embodiments of the application, one or more examples of which are illustrated in the drawings and described below. Each example is provided by way of explanation of the application and not as a limitation. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present application without departing from the scope or spirit of the application. For instance, features illustrated or described as part of one embodiment, can be used with another embodiment to yield a still further embodiment.

[0038] Unless otherwise defined, all terms (including technical and scientific terms) used in the disclosure of the application are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. By further guidance, the following definitions are used for better understanding of the teachings of the present application. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0039] The selection scope of the terms "and / or", "or / and", "and / or" used in the present disclosure includes any one of the two or more relevant listed items, and also includes any and all combinations of the relevant listed items, including any two relevant listed items, any more relevant listed items, or all relevant 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 the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B, and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (i.e., the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C, and D, i.e., includes the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (i.e., the technical solution connected by "logical and").

[0040] The terms "containing", "including", and "comprising" used in the present disclosure are synonymous terms, which are inclusive or open, and do not exclude additional, unmentioned members, elements, or method steps.

[0041] The numerical ranges represented by endpoints in the present disclosure include all numerical values and fractions within the range, and the mentioned endpoints.

[0042] In the present disclosure, the concentration numerical values include fluctuations within a certain range. For example, it can fluctuate within a corresponding accuracy range. For example, 2%, it can be allowed to fluctuate within ±0.1%. For numerical values that are larger or do not need to be controlled too finely, it is also allowed that the meaning includes larger fluctuations. For example, 100mM, it can be allowed to fluctuate within ±1%, ±2%, ±5%, etc. For molecular weight, it is allowed that the meaning includes ±10% fluctuations.

[0043] In the present disclosure, descriptions such as "multiple", "various", etc. refer to greater than or equal to 2 in quantity, unless otherwise specified.

[0044] In the present disclosure, among the technical features described in an open manner, both the closed technical solution consisting of the listed features and the open technical solution containing the listed features are included.

[0045] In the present disclosure, "preferably", "more preferably", "most preferably", "suitably" are only used to describe better embodiments or examples, and it should be understood that they do not constitute a limitation on the protection scope of the present disclosure. In the present disclosure, "optionally", "optional", "optionally" means that it can or can not exist, that is, it means to select any one from the two parallel schemes of "yes" or "no". If there are multiple "options" in a technical solution, unless otherwise specified, and there is no contradictory or mutual restrictive relationship, each "option" is independent of each other.

[0046] All the documents mentioned in the present disclosure are cited in the present application as references, just as each document is cited as a reference. Unless and the invention purpose and / or technical scheme of the present application conflict, the reference documents involved in the present disclosure are cited in their entirety, all purposes. When the present disclosure refers to the reference documents, the definition of the relevant technical features, terms, nouns, phrases, etc. in the reference documents is also cited. When the present disclosure refers to the reference documents, the examples, preferred modes of the relevant technical features cited can also be incorporated into the present application as a reference, but limited to the implementation of the present disclosure. It should be understood that when the cited content conflicts with the description in the present application, the present application is used as the reference or is modified according to the description in the present application.

[0047] The present disclosure relates to the application of a DLL3 and B7-H3 dual-targeting antibody drug conjugate.

[0048] The term "targeting antibody" is a macromolecular compound that can target and bind to the antigen or receptor associated with the target cell. The role of the antibody is to deliver drugs to the target cell population bound to the antibody, which includes but is not limited to protein hormones, lectins, growth factors, antibodies, polypeptides with binding ability or other molecules capable of binding to cells. In the embodiments of the present disclosure, the targeting antibody is represented as Ab, and the targeting antibody can form a linkage with the linking unit through the heteroatom on the antibody.

[0049] The term "pharmaceutically acceptable salt" refers to a salt of the antibody-drug conjugate of the present disclosure, meaning a salt that is acceptable for administration to a patient (e.g., a mammal) for a given dosage regimen (which is a salt that comprises a counterion having acceptable mammalian safety). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. The antibody-drug conjugate of the present disclosure contains at least one amino group, and thus can form a salt with an acid, non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, malate, hydrogenphosphate, dihydrogenphosphate, salicylate, bicarbonate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate. Especially when the conjugated drug is LD-38, the preferred pharmaceutically acceptable salt thereof is LD-38 salt.

[0050] The term "solvate" as used throughout this disclosure refers to a combination of a compound of the present disclosure with solvent molecules, either by solvation. In certain instances, solvate refers to hydrate, i.e., the solvent molecule is water, the combination of a compound of the present disclosure with water forms a hydrate.

[0051] The DLL3 antagonistic antibody of the present application comprises a group of DLL3 monoclonal antibodies or antigen binding fragments thereof, including heavy chain and 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 the amino acid sequences spanning CDR1 to CDR3 of the heavy chain and light chain of the antibody, respectively.

[0052] Further, the present application discloses the above-mentioned DLL3 monoclonal antibody or antigen binding fragment thereof, characterized in that the amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NO: 1, 3, 5; the amino acid sequence of the light chain variable region is selected from one of SEQ ID NO: 2, 4, 6.

[0053] Further, the application discloses the DLL3 monoclonal antibody or antigen binding fragment thereof, characterized in that the heavy chain and the light chain are humanized; the amino acid sequence of the humanized heavy chain variable region is selected from one of SEQ ID NO: 25, 27 and 29; and the amino acid sequence of the humanized light chain variable region is selected from one of SEQ ID NO: 26, 28 and 30.

[0054] Further, the application discloses the use of the DLL3 monoclonal antibody or antigen binding fragment thereof in preparation of a drug with high affinity to DLL3 and significant intracellular endocytosis.

[0055] Further, the application discloses the DLL3 monoclonal antibody or antigen binding fragment thereof, characterized in that the heavy chain and the light chain are humanized; the amino acid sequence of the humanized heavy chain variable region is selected from one of SEQ ID NO: 25, 27 and 29; and the amino acid sequence of the humanized light chain variable region is selected from one of SEQ ID NO: 26, 28 and 30.

[0056] A group of DLL3 monoclonal antibodies or antigen binding fragments thereof, comprising a heavy chain and a light chain, are characterized in that the amino acid sequence of CDR1 of the heavy chain is selected from one of SEQ ID NO: 7, 13 and 19; the amino acid sequence of CDR2 of the heavy chain is selected from one of SEQ ID NO: 8, 14 and 20; the amino acid sequence of CDR3 of the heavy chain is selected from one of SEQ ID NO: 9, 15 and 21; the amino acid sequence of CDR1 of the light chain is selected from one of SEQ ID NO: 10, 16 and 22; the amino acid sequence of CDR2 of the light chain is selected from one of SEQ ID NO: 11, 17 and 23; and the amino acid sequence of CDR3 of the light chain is selected from one of SEQ ID NO: 12, 18 and 24; wherein the heavy chain and the light chain of the antigen binding fragment comprise the amino acid sequences of CDR1 to CDR3 of the heavy chain and the light chain of the antibody, respectively.

[0057] A group of DLL3 monoclonal antibodies or antigen binding fragments thereof are characterized in that the amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NO: 1, 3 and 5; and the amino acid sequence of the light chain variable region is selected from one of SEQ ID NO: 2, 4 and 6.

[0058] A group of DLL3 monoclonal antibodies or antigen binding fragments thereof are 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 and 29; and the amino acid sequence of the humanized light chain variable region is selected from one of SEQ ID NO: 26, 28 and 30.

[0059] The conjugated moiety is one or more selected from the group consisting of a radionuclide, a drug, a toxin, a cytokine, a cytokine receptor fragment, an enzyme, a fluorescein, and a biotin, preferably the linking unit and the conjugated drug moiety comprise a linker payload, the linker payload is LD-38.

[0060] Further, the application discloses a monoclonal antibody conjugate comprising a monoclonal antibody and a conjugated moiety, wherein the monoclonal antibody is the group of B7-H3 monoclonal antibodies or antigen binding fragments thereof in claim 11, the linking unit and the conjugated drug moiety comprise a linker payload, the linker payload is LD-38.

[0061] Further, the application discloses the use of the monoclonal antibody conjugate in the preparation of a drug for preventing and / or treating and / or adjuvant treating tumors.

[0062] The DLL3 and B7-H3 dual-targeting antibody of the application comprises: 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; and 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 the light chain of the antigen binding fragment comprise the amino acid sequences of CDR1 to CDR3 of the heavy chain and the light chain 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.

[0063] Further, the DLL3 and B7-H3 dual targeting antibody of the present application 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.

[0064] Further, the present application discloses a dual targeting antibody comprising a heavy chain constant region, characterized in that the heavy chain constant region amino acid sequence is selected from SEQ ID NO: 47 or 49.

[0065] Further, the present application discloses a dual targeting antibody comprising a heavy chain and a light chain, characterized in that the heavy chain amino acid sequence 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 light chain amino acid sequence 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.

[0066] Further, the present application discloses a dual targeting antibody or an antigen binding fragment thereof, which is selected from a rabbit-derived antibody, a mouse-derived antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

[0067] The antibody or an antigen binding fragment thereof of the dual drug conjugate of the present application comprises a first domain and a second domain, characterized in that the first domain and the second domain are selected from an anti-DLL3 monoclonal antibody or an anti-DLL3 and anti-B7-H3 dual targeting antibody or an antigen binding fragment thereof.

[0068] The present application uses a mammalian cell expression system to prepare recombinant DLL3 as an antigen to immunize mice, and fuses the mouse spleen cells with myeloma cells to obtain hybridoma cells. After multiple cloning and screening of a large number of hybridoma cells, some monoclonal hybridoma cell strains are obtained. These hybridoma cells can produce monoclonal antibodies with high specificity and affinity to DLL3, and show significant endocytosis. The genes encoding the light chain and heavy chain variable regions of the antibodies are cloned by RT-PCR, and the humanized antibodies are constructed by the complementarity determining region grafting method. In vitro functional tests show that these humanized DLL3 antibodies can specifically and highly bind to DLL3 protein and show significant endocytosis. The above experimental results show that the monoclonal antibody or an antigen binding fragment thereof of the present application, or the conjugate comprising the monoclonal antibody or an antigen binding fragment thereof of the present application has a good application prospect in the preparation of a tumor treatment drug for tumors expressing DLL3, and in the prevention and treatment or adjuvant therapy of tumors.

[0069] In some embodiments, the drug can be selected from the group consisting of:

[0070] alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, inhibitors of chromatin function, antiangiogenic agents, antiestrogens, antiandrogens, immunomodulators;

[0071] Alkylating agents include, without limitation, mechlorethamine, chlorambucil, melphalan, bromopiperazine, prednimustine, ifosfamide, cyclophosphamide, hexamethylmelamine, chloroethycyclophosphamide, thioepa, triethylenephosphoramide, carmustine, streptozocin, fotemustine, lomustine, busulfan, thiotepa, improsulfan, dacarbazine, cisplatin, oxaliplatin, carboplatin;

[0072] Antimetabolites include, without limitation, methotrexate, 5-uracil, fluorouracil, 5-deoxyuracil, capecitabine, cytarabine, cladribine, 6-mercaptopurine (6-MP), 6-mercaptopurine (6-TG), 2-chlorodeoxyadenosine, 5-azacytidine, 2,2-fluorodeoxycytidine, cladrabine, deoxycoformycin, pentostatin;

[0073] Antitumor antibiotics include, without limitation, doxorubicin, daunorubicin, idarubicin, valrubicin, mitoxantrone, dactinomycin, plicamycin, mithramycin, mitomycin C, bleomycin, procarbazine;

[0074] Mitotic inhibitors include, without limitation, paclitaxel, vinblastine, vincristine, vindesine, vinorelbine, halichondrin B, eribulin;

[0075] Inhibitors of chromatin function include, without limitation, camptothecin, topotecan, irinotecan, other camptothecin derivatives, etoposide, etoposide phosphate, epipodophyllotoxin; used in the present application is irinotecan (CAS 171335-80-1).

[0076] Antiangiogenic agents include, without limitation, prolyl hydroxylase inhibitors, marimastat, batimastat, prinomastat, tanomastat, ilomastat, CGS-27023A, brivanib, COL-3, neovastat, BMS-275291, thalidomide;

[0077] Antiestrogens include, without limitation, tamoxifen, toremifene, raloxifene, droloxifene, ormeloxifene, anastrozole, letrozole, exemestane;

[0078] Antiandrogens include, without limitation, flutamide, nilutamide, bicalutamide, spironolactone, cyproterone acetate, finasteride, cimetidine;

[0079] Immunomodulators include, without limitation, interferons, interleukins, tumor necrosis factors, lentinan, sizofiran, roquinimex, pidoxil, methoxypolyethylene glycol succinimidyl oleate adenosine deaminase, thymic peptide preparations.

[0080] The term "antibody" as used herein is used in the broadest sense, and includes immunoglobulin or other types of molecules that comprise one or more antigen binding domains that specifically bind an antigen, a protein or polypeptide that exhibits binding specificity to a particular antigen. Specific examples of antibodies can include intact antibodies (e.g., classical four-chain antibody molecules), single chain antibodies, single domain antibodies, bispecific antibodies, multispecific antibodies, and the like. A classical antibody molecule typically is a tetramer composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds. The heavy and light chains are divided into regions of hypervariability at the amino terminal portion, the variable region (V), and regions that are more conserved at the carboxy terminal portion, the constant region (C), based on differences in the conservation of the amino acid sequences. The variable region is responsible for antigen recognition and binding, and the constant region (e.g., the Fc fragment) is responsible for mediating down-stream effects such as antibody-dependent cell-mediated cytotoxicity (ADCC). Within the variable regions of the heavy and light chains, there are three local regions of hypervariability in the arrangement and sequence of the amino acids, which are referred to as complementarity determining regions (CDRs) because they are the key positions for antibody-antigen binding. The amino acid sequences of the CDRs can be readily determined using art-recognized numbering schemes, such as Kabat, Chothia, IMGT, AbM, or Contact. The antibodies can be IgG, IgM, IgD, IgE, or IgA antibodies.

[0081] An "antigen binding fragment" of an antibody refers to a fragment of an antibody molecule that is involved in antigen-specific binding, such as one of F(ab')2, Fab, and scFv.

[0082] The term "F(ab')2" is derived from the digestion of whole full-length antibody by pepsin to remove most of the Fc region while leaving some of the hinge region intact. The F(ab')2 fragment has two antigen-binding Fab portions that are linked together by disulfide bonds, and thus the F(ab')2 fragment is a bivalent antibody. For example, F(ab')2 prepared from IgG antibodies has a molecular weight of about 110 kDa.

[0083] The term "Fab" is an antibody structure that can still bind to an antigen, which is monovalent and does not contain the Fc portion. Papain digestion of full-length antibodies results in two Fab fragments, each of about 50 kDa, and an Fc fragment.

[0084] The term "scFv" is a single chain antibody composed of the variable region of the heavy chain and the variable region of the light chain connected by a short peptide. Through proper folding, the variable regions from the heavy chain and the light chain interact with each other through non-covalent bonds to form a Fv fragment, so that the scFv can well retain its affinity activity to the antigen.

[0085] The term "diabody" refers to an antibody that binds to two different antigens or two epitopes of the same antigen.

[0086] In some embodiments, the antibody or antigen-binding fragment thereof is selected from a rabbit-derived antibody, a murine-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0087] "Rabbit-derived / murine-derived antibody" refers to an antibody whose variable region and constant region (if present) are derived from rabbit / murine immunoglobulin sequences. Rabbit-derived / murine-derived antibodies can be conveniently obtained by immunizing a rabbit / mouse (including a mouse or a rat) with a corresponding antigen and isolating the antibody of interest from the rabbit / mouse. Alternatively, after immunizing a rabbit / mouse with a corresponding antigen, cells (such as B cells) expressing the antibody of interest are isolated and cultured to obtain the antibody. Alternatively, after immunizing a rabbit / mouse with a corresponding antigen, cells expressing the antibody of interest are isolated and cultured, and the cells are fused with immortalized cells such as myeloma cells to obtain hybridoma cells, and the antibody of interest (such as a monoclonal antibody) can be obtained in large quantities and for a long time by culturing the hybridoma cells.

[0088] The term "chimeric antibody" is an antibody in which the variable region of the antibody of the first animal species is fused with the constant region of the antibody of the second animal species. To establish a chimeric antibody, a hybridoma secreting a specific monoclonal antibody of the first animal species is first established, and then the variable region gene is cloned from the hybridoma cells, and the constant region gene of the antibody of the second animal species 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, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present disclosure, the first animal species is rabbit or mouse, and the second animal species is preferably human, which can reduce the immune response induced by the first animal-derived antibody. The antibody light chain of the chimeric antibody further comprises the light chain constant region of the human κ, λ chain or a variant thereof. The antibody heavy chain of the chimeric antibody further comprises the heavy chain constant region of the human IgG1, IgG2, IgG3, IgG4 or a variant thereof. The subtypes of antibody constant regions, different human isomers, and mutations based on changes in the effector function of the constant region do not affect the preparation of antibody conjugates. Linker toxin conjugation sites such as cysteine, lysine, glutamine, carboxyl terminal of the peptide chain, and glycosylation sites, including natural sites and engineered sites, are generally used for conjugation.

[0089] The term "humanized antibody" also known as CDR-grafted antibody refers to an antibody produced by grafting the CDR sequences of a first animal origin into a human antibody variable region framework, i.e. a different type of human germline antibody framework sequence. This can overcome the heterogeneity reactions induced by chimeric antibodies due to carrying a large amount of first animal-derived protein components. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. The 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 the decrease in immunogenicity while causing a decrease in activity, the human antibody variable region framework sequence can be subjected to minimal back-mutation or back-mutation to maintain activity. The humanized antibody of the present disclosure also includes a humanized antibody further subjected to affinity maturation of CDR by phage display. In a preferred embodiment of the present disclosure, the first animal origin is rabbit or mouse origin. The human antibody variable region framework is designed and selected. To avoid the decrease in immunogenicity while causing a decrease in activity, the human antibody variable region can be subjected to minimal back-mutation to maintain activity.

[0090] A fully humanized antibody refers to a human antibody gene by transgenic or transchromosomal technology, transferring human antibody-encoding genes to genetically engineered antibody gene-deficient animals to express human antibodies in animals, achieving the purpose of fully humanizing antibodies.

[0091] In some embodiments, the antibody is selected from the group consisting of an anti-CD3 antibody, an anti-FOLRl antibody, an anti-RORl antibody, an anti-TNFa antibody, an anti-tissue factor (TF) antibody, an anti-EpCAM antibody, an anti-EGFRvIII antibody, an anti-DLL-3 antibody, an anti-PSMA antibody, an anti-MUC16 antibody, an anti-ENPP3 antibody, an anti-TDGF1 antibody, an anti-ETBR antibody, an anti-MSLN antibody, an anti-TIM-1 antibody, an anti-LRRC15 antibody, an anti-LIV-1 antibody, an anti-CanAg / AFP antibody, an anti-Claudin 6 antibody, an anti-Claudin 9 antibody, an anti-Claudin 18.2 antibody, an anti-Mesothelin antibody, an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-c-MET antibody, an anti-SLITRK6 antibody, an anti-KIT / CD117 antibody, an anti-STEAP1 antibody, an anti-SLAMF7 / CS1 antibody, an anti-NaPi2B / SLC34A2 antibody, an anti-GPNMB antibody, an anti-HER3 (ErbB3) antibody, an anti-MUC1 / CD227 antibody, an anti-AXL antibody, an anti-CD166 antibody, an anti-B7-H3 (CD276) antibody, an anti-PTK7 / CCK4 antibody, an anti-PRLR antibody, an anti-EFNA4 antibody, an anti-5T4 antibody, an anti-NOTCH3 antibody, an anti-Nectin 4 antibody, an anti-TROP-2 antibody, an anti-CD142 antibody, an anti-CA6 antibody, an anti-GPR20 antibody, an anti-CD174 antibody, an anti-CD70 antibody, an anti-CD71 antibody, an anti-EphA2 antibody, an anti-LYPD3 antibody, an anti-FGFR2 antibody, an anti-FGFR3 antibody, an anti-FR alpha antibody, an anti-CEACAMs antibody, an anti-GCC antibody, an anti-Integrin Av antibody, an anti-CAIX antibody, an anti-P-cadherin antibody, an anti-GD3 antibody, an anti-Cadherin 6 antibody, an anti-LAMP1 antibody, an anti-FLT3 antibody, an anti-BCMA antibody, an anti-CD79b antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD33 antibody, an anti-CD56 antibody, an anti-CD74 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD37 antibody, an anti-CD47 antibody, an anti-CD138 antibody, an anti-CD352 antibody, an anti-CD25 antibody, and an anti-CD123 antibody.

[0092] In some specific embodiments, the antibody is selected from the group consisting of:

[0093] Anti-GD2 antibody 3F8, Abagovomab, Abciximab, ACZ885 (canakinumab), Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Anrukinzumab (IMA-638), Apolizumab, Arcitumomab, Aselizumab, Atezolizumab, Atorlimumab, Avelumab, Bapineuzumab, Basiliximab, Bavituximab, Beectumomab, Belimumab, Bertilimumab, Besilesomab, Bevacizumab, Biciromab, Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin, Briakinumab, Canakinumab, Cantuzumab mertansine, Capromab pendetide, Catuxomab, Cedelizumab, Certolizumabpegol, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clenoliximab, Clivatuzumab tetraxetan, CNTO148 (golimumab), CNTO1275(ustekinumab), Conatumumab, Dacetuzumab, Daclizumab, Denosumab, Detumomab, Dorlimomab aritox, Dorlixizumab, Durvalumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elsilimomab, Enlimomab pegol, Epitumomabcituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Exbivirumab, Fanolesomab, Faralimomab, Felvizumab, Fezakinumab, Figitumumab, Fontolizumab, Foravirumab, Fresolimumab, Galiximab, Gavilimomab, Gemtuzumab ozogamicin, Golimumab, Gomiliximab, Ibalizumab, Ibritumomab tiuxetan, Igovomab, Imciromab, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Isatuximab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icratuzumab, Icrozogamicin), Ipilimumab, Iratumumab, Keliximab, Labetuzumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Lintuzumab, Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Morolimumab, Motavizumab, Muromonab-CD3, MY0-029 (stamulumab), Nacolomab tafenatox, Naptumomab estafenatox, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Ocrelizumab, Odulimomab, Ofatumumab, Omalizumab, Oportuzumab, Oregovomab, Orteronel, Osidem, Oxelumab, Ozelimomab, Pablizumab, Padsecalikimab, Pantiumomab, Pembrolizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pidtriximab, Pidriximab, Pinatuzumab, Pintumomab, Pitronath, Pritumumab, Prostascint, Prostascint Planitia, Prostascint TM, Prostascint VHH, Prostascint VHH2, Prostascint VHH3, Prostascint VHH4, Prostascint VHH5, Prostascint VHH6, Prostascint VHH7, Prostascint VHH8, Prostascint VHH9, Prostascint VHH10, Prostascint VHH11, Prostascint VHH12, Prostascint VHH13, Prostascint VHH14, Prostascint VHH15, Prostascint VHH16, Prostascint VHH17, Prostascint VHH18, Prostascint VHH19, Prostascint VHH20, Prostascint VHH21, Prostascint VHH22, Prostascint VHH23, Prostascint VHH24, Prostascint VHH25, Prostascint VHH26, Prostascint VHH27, Prostascint VHH28, Prostascint VHH29, Prostascint VHH30, Prostascint VHH31, Prostascint VHH32, Prostascint VHH33, Prostascint VHH34, Prostascint VHH35, Prostascint VHH36, Prostascint VHH37, Prostascint VHH38, Prostascint VHH39, Prostascint VHH40, Prostascint VHH41, Prostascint VHH42, Prostascint VHH43, Prostascint VHH44, Prostascint VHH45, Prostascint VHH46, Prostascint VHH47, Prostascint VHH48, Prostascint VHH49, Prostascint VHH50, Prostascint VHH51, Prostascint VHH52, Prostascint VHH53, Prostascint VHH54, Prostascint VHH55, Prostascint VHH56, Prostascint VHH57, Prostascint VHH58, Prostascint VHH59, Prostascint VHH60, Prostascint VHH61, Prostascint VHH62, Prostascint VHH63, Prostascint VHH64, Prostascint VHH65, Prostascint VHH66, Prostascint VHH67, Prostascint VHH68, Prostascint VHH69, Prostascint VHHMonatox, Oregovomab, Otelixizumab, Pagibaximab, JQ, Palivizumab, Panitumumab, Panobacumab, Pascolizumab, Pembrolizumab, Pemtumomab, Pertuzumab, Pexelizumab, Pintumomab, Priliximab, Pritumumab, PRO 140. Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Rontalizumab, Rovelizumab, Rupliz Satumomab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Siplizumab, Solanezumab, Sonepizumab, Sontuzumab, Stamulumab, Sulesomab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Tapitutumomab paptox, Tefibazumab, Telimomabaritox, tenatumomab, teneliximab, teplizumab, TGN1412, ticilimumab, tremelilimumab, tigatuzumab, ibalizumab (TNX-355), talizumab (TNX-650), talizumab (TNX-901), tocilizumab, toralizumab, tositumomab, trastuzumab, tremelimumab, tucotuzumab The following are listed: celmoleukin, Tuvirumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vedolizumab, Veltuzumab, Vepalimomab, Visilizumab, Volociximab, Votumumab, Zalutumumab, Zanomimumab, Ziralimumab, and Zolimomab aritox.

[0094] In some embodiments, the antibody is selected from:

[0095] i) Antibody; anti-DLL3 antibody or;

[0096] ii) Antibody; anti-B7-H3 antibody.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] In some embodiments, the anti-DLL3 antibody has a heavy chain variable region HCVR as set forth in SEQ ID NO: 25 and a light chain variable region LCVR as set forth in SEQ ID NO: 26.

[0101] In some embodiments, the anti-DLL3 antibody has a heavy chain variable region HCVR as set forth in SEQ ID NO: 27 and a light chain variable region LCVR as set forth in SEQ ID NO: 28.

[0102] In some embodiments, the anti-DLL3 antibody has a heavy chain variable region HCVR as set forth in SEQ ID NO: 29 and a light chain variable region LCVR as set forth in SEQ ID NO: 30.

[0103] In some embodiments, the anti-B7-H3 antibody has a heavy chain variable region HCVR as set forth in SEQ ID NO: 31 and a light chain variable region LCVR as set forth in SEQ ID NO: 32.

[0104] In some embodiments, the anti-B7-H3 antibody has a heavy chain variable region HCVR as set forth in SEQ ID NO: 33 and a light chain variable region LCVR as set forth in SEQ ID NO: 34.

[0105] In some embodiments, the anti-B7-H3 antibody has a heavy chain constant region as set forth in SEQ ID NO: 47 or 49 and a light chain constant region as set forth in SEQ ID NO: 48.

[0106] Variants of the above amino acid sequences are also within the scope of the application, and corresponding variants comprise up to 3 amino acid mutations in at least one CDR region compared to any of the polypeptides of SEQ ID NOs: 7-24 and 35-46, respectively; or, variants can comprise fewer than 3 or more mutations relative to the overall sequence of SEQ ID NOs: 1-6, SEQ ID NOs: 25-34, and SEQ ID NOs: 47-87, for example, a sequence that is at least 80%, 85%, 90%, 93%, 95%, 97%, or 99% identical to any of the polypeptides of SEQ ID NOs: 54-87. The mutations can be substitutions, deletions, or additions of amino acids, or any combination thereof; preferably, the mutations are conservative substitutions.

[0107] A "conservative substitution" is one in which the amino acid is replaced with a residue that has similar features (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) to the original residue, such that the change can frequently be made without altering the biological activity of the protein.

[0108] Substitutions that are generally considered conservative are replacements within groups of aliphatic amino acids Ala, Val, Leu and He, exchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, substitution between amide residues Asn and Gin, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe and Tyr. As is known to those skilled in the art, in general, a single amino acid substitution in non-essential regions of a polypeptide will not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). Additionally, substitution of amino acids with similar properties is less likely to disrupt biological activity.

[0109] The present disclosure also relates to a method of preparing an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described above, comprising:

[0110] After reduction of the targeting antibody, a coupling reaction is carried out with the pre-synthesized -L-D to obtain a compound represented by the general formula Ab-L-D.

[0111] The reducing agent is preferably TCEP, in particular, it is preferred to reduce the disulfide bond on the targeting antibody.

[0112] The present disclosure also relates to a pharmaceutical composition comprising an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described above, and a pharmaceutically acceptable excipient, diluent or carrier.

[0113] As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes any material that allows the active ingredient to retain its biological activity and does not interact with the immune system of the subject when combined with the active ingredient.

[0114] The present disclosure also relates to the use of an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described above in the manufacture of a medicament for treating a tumor.

[0115] The term "cancer" refers to a physiological condition or disease characterized by unregulated cell growth. "Tumor" includes cancerous cells. In some embodiments, the tumor is a solid tumor or a hematological tumor, such as a 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, neural tumor, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, squamous carcinoma, myeloma, lymphoma, and leukemia.

[0116] The present disclosure also relates to a method of treating a medical condition in a subject comprising administering a safe and effective amount of an antibody-drug conjugate as described above.

[0117] Preferably wherein the medical condition is cancer.

[0118] The phrase "safe and effective amount". As used herein, means an amount of a compound or composition large enough to significantly effect a treatment of the condition or disorder for which it is administered, but small enough to avoid serious side effects (at a reasonable benefit / risk ratio). The safe and effective amount of active ingredient in the pharmaceutical compositions used in the methods of the present disclosure will vary with the particular condition being treated, the age and physical condition of the patient being treated, the severity of the disease, the duration of the treatment, the use or non-use of concurrent treatment, the particular active ingredient being used, the particular pharmaceutical formulation being used, and like factors including the knowledge and expertise of the treating physician.

[0119] It should be understood that contemplated methods of treatment will also include administration of other therapeutic entities, particularly preferred immunotherapeutic entities, including viral cancer vaccines (e.g., adenoviral vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., non- pyrogenic E. coli expressing one or more cancer-specific antigens), yeast cancer vaccines, N-803 (also known as ALT-803, ALTOR Biosciences), chemotherapeutic drugs, antibodies (e.g., binding to tumor-associated antigens or patient-specific tumor neoantigens), stem cell grafts (e.g., allogeneic or autologous), and tumor-targeted cytokines (e.g., NHS-IL12, IL-12 conjugated to a tumor-targeting antibody or fragment thereof). In some embodiments, contemplated methods of treatment further include radiation treatment of the patient. In some embodiments, contemplated methods of treatment further include surgery of the patient, e.g., tumor resection surgery.

[0120] The antibody-drug conjugate can also be administered and / or co-formulated in combination with an antiviral agent, an antibiotic, an analgesic, a corticosteroid, a steroid, oxygen, an antioxidant, a COX inhibitor, a cardioprotectant, a metal chelator, IFN-gamma, and / or an NSAID. The aforementioned therapeutic entities can be included in the pharmaceutical composition.

[0121] The terms "subject" and "patient" are used interchangeably herein and refer to any animal, such as any mammal, including (but not limited to) humans, non-human primates, rodents, dogs, cats, chimpanzees, orangutans, gorillas, macaques, marmosets, pigs, horses, pandas, elephants, and the like.

[0122] As used herein, "treatment" refers to any improvement in the disease outcome, such as increased survival, less morbidity, and / or lessened side effects as a byproduct of an alternative treatment modality. As is readily appreciated in the art, complete eradication of the disease is preferred, but is not a necessary condition of the act of treatment. As used herein, "treatment" refers to administration of the antibody-drug conjugate to a subject, e.g., a patient. Treatment can be curative, therapeutic, palliative, alleviative, altering, ameliorative, diminishing, lessening, improving, or affecting a condition, a symptom of a condition, or a predisposition to, e.g., cancer.

[0123] The pharmaceutical compositions of the present disclosure can be administered by any route, as will be appreciated by those in the art. In some embodiments, the pharmaceutical compositions of the present disclosure are administered intravenously (IV).

[0124] Embodiments of the present disclosure will be described in detail with reference to the following examples. It is to be understood that the examples are only intended to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods in the following examples, for which specific conditions are not indicated, are preferably referred to the guidelines given in the present disclosure, but can also be performed according to the protocols in the art or according to the conditions suggested by the manufacturers or according to other experimental methods known in the art.

[0125] In the following specific examples, the measurement parameters of the raw material components, such as the amount, can have slight deviations within the range of the weighing accuracy, if not otherwise specified. The temperature and time parameters are allowed to have acceptable deviations caused by the instrument testing accuracy or the operation accuracy.

[0126] Example 1 Antigen molecule amino acid sequence

[0127] The antigen human B7-H3 is the amino acid of Uniprot database Q5ZPR3-1 gene, the sequence is as follows:

[0128] The human B7-H3 protein is purchased from Acrobiosystem, item number B7B-H52E7, and the extracellular region of 27-461 amino acids can also be expressed by fusion with GGGGSHHHHHH. The human antibody IgG1 or mouse antibody IgG2a Fc constant region can also be fused for expression.

[0129] The antigen human DLL3 is the amino acid of Uniprot database Q9NYJ7-1, the sequence is as follows:

[0130] Human DLL3 protein was purchased from Acrobiosystem, Cat No: DL3-H52H4, and the extracellular domain of 27-492 amino acids was also expressed with GGGGSHHHHHH. Human IgG1 or mouse IgG2a Fc constant region was also fused to express.

[0131] Preparation, screening and subcloning of mouse hybridoma antibodies

[0132] (1) Mouse immunization and hybridoma cell fusion. Human DLL3-ECD-mFc fusion protein was used as antigen, and was emulsified with an equal volume of complete Freund's adjuvant (Sigma, Cat No: F5581) to immunize 6-8 week old Balb / c mice (purchased from Zhaoyan (Suzhou) New Drug Research Center Co., Ltd.) subcutaneously, and the amount of antigen was 20 μg per mouse. Then the mice were immunized subcutaneously with the same dose of antigen emulsified with incomplete Freund's adjuvant (Sigma, Cat No: F5506) every two weeks for three times. The serum titer of the mice was determined after three times of immunization, and the mice were boosted intraperitoneally three days before fusion. PEG Hybri-Max (Sigma, Cat No: 7181) was used as the fusion agent, and the mouse spleen cells were mixed with SP2 / 0 cells at a ratio of 4:1. The fused cells were added to 96-well plates (1 x 10 5 cells / well), and each well contained 0.1 mL of 1X HAT medium (Invitrogen, Cat No: 21060-017). On the third day, 0.1 mL of HT (Invitrogen, Cat No: 11067-030) medium was added, and on the seventh day, the medium in the 96-well plate was aspirated and 0.2 mL of fresh HT medium was added. On the ninth day, the supernatant was collected for ELISA and FACS detection.

[0133] (2) ELISA screening of DLL3-ECD binding antibodies. 50 μL of DLL3-hFc (final concentration: 2 μg / mL) coated 96-well ELISA plates (Corning, Cat. No.: 9018) were added and incubated at room temperature overnight; after washing 3 times with washing buffer (PBS + 0.05% Tween 20), blocking buffer (PBS + 2% BSA) was added and incubated at room temperature for 1 hour, and the ELISA plate was washed 3 times with washing buffer; hybridoma supernatant was added and incubated at room temperature for 1 hour, and washed 3 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, incubated at room temperature for 1 hour in the dark, and washed 3 times; 100 μL of TMB (Beijing Baosai Bio, Cat. No.: ES-002) was added to each well, and color development was performed at room temperature for 1-3 minutes; 100 μL / well of stop solution (2N H2SO4) was added to terminate the color development reaction, and the OD450 value of each well was read using an enzyme-labeled instrument (Tecan Spark).

[0134] (3) FACS screening of DLL3-ECD binding antibodies. FACS screening of hybridoma antibodies binding to 293T-DLL3: 50 μL of hybridoma supernatant positive in the above detection was mixed with 50 μL of 293T-DLL3 cells (2 x 105cells / well) and added to a 96-well U-bottom cell plate, and incubated at 4°C for 1 hour; washed twice with FACS buffer (PBS + 3% FBS) and centrifuged; 100 μL of 400-fold diluted PE-labeled goat anti-mouse secondary antibody (Biolegend, Cat. No.: 405307) was added, and incubated at 4°C for 40 minutes in the dark; washed twice with FACS buffer and centrifuged; and the particle signal value in the PE channel was detected using a flow cytometer (SinoCyte). 5

[0135] (4) Subcloning of hybridomas. Hybridomas with strong binding activity were subcloned using limited dilution, and then ELISA and FACS were used to analyze the binding of hybridoma antibodies to DLL3, and hybridoma monoclonal antibodies with strong binding were screened.

[0136] Example 3 Testing of DLL3 hybridoma monoclonal antibodies

[0137] ​1) ELISA method for determining the binding activity of purified monoclonal antibodies to DLL3 protein: in the experiment, 100 μL of DLL3-hFc (final concentration: 2 μg / mL) was added to a 96-well ELISA plate (Corning, Cat. No.: 9018) coated with DLL3-hFc, and incubated overnight at room temperature; after washing 3 times with washing buffer (PBS + 0.05% Tween 20), blocking buffer (PBS + 2% BSA) was added and incubated at room temperature for 1 hour, and the ELISA plate was washed 3 times with washing buffer; gradient-diluted antibody diluent was added and incubated at room temperature for 1 hour, and washed 3 times; 100 μL of 10000-fold diluted HRP-conjugated goat anti-mouse IgG secondary antibody (Thermo, Cat. No.: 31439) was added to each well, and incubated at room temperature for 1 hour in the dark, and washed 3 times; 100 μL of TMB (Beijing Baosai Bio, Cat. No.: ES-002) was added to each well, and color developed at room temperature for 1-3 minutes, and 100 μL / well of stop solution (2N H2SO4) was added to terminate the color development reaction, and the OD450 values of each well were read with an enzyme-labeled instrument (Tecan Spark), and the EC 50 values were calculated by Prism software.

[0138] The results are shown in Table 1 and Figure 1, and the three candidate DLL3 hybridoma monoclonal antibodies, 59H4, 71B11, and 56G10, showed strong DLL3 antigen binding activity (based on EC 50 values, stronger than the positive control of Abbvie).

[0139] 2) FACS method for determining the binding activity of purified monoclonal antibodies to DLL3-expressing cells: in the experiment, the monoclonal antibodies were gradient-diluted with FACS diluent, 50 μL of the gradient-diluted antibody was mixed with 50 μL of 293T-DLL3 or SHP-77 cells (2 x 10 5 cells / well) and gently blown to mix, and added to a 96-well U-bottom cell plate and incubated at 4°C for 1 hour, washed twice with FACS buffer (PBS + 3% FBS) and centrifuged, and 400-fold diluted PE-labeled goat anti-mouse secondary antibody (Biolegend, Cat. No.: 405307) was added, and incubated at 4°C in the dark for 40 minutes, washed twice with FACS buffer and centrifuged, and the cell particles were detected in the PE channel signal value with a flow cytometer (Zhongsheng SinoCyte). The EC 50 values were calculated by Prism software.

[0140] The results are shown in Tables 2, 3 and Figures 2A-3B, and the three candidate DLL3 hybridoma monoclonal antibodies, 59H4, 71B11, and 56G10, showed strong binding activity to DLL3-expressing cells (based on EC 50 values, stronger than or close to the positive control of Abbvie).

[0141] 3) FACS assay of endocytosis activity of purified monoclonal antibodies in cells: in the experiment, the monoclonal antibodies were diluted with FACS diluent gradient, 50 μL of antibody gradient diluent was mixed with 50 μL of 293T-DLL3 or SHP-77 cells (2 x 10 5 cells / well) and blown gently to mix, added to a 96-well U-bottom cell plate, incubated at 4°C for 1 hour, washed twice with FACS buffer (PBS + 3% FBS) and centrifuged, added with Goat anti-mFc (Jackson, Cat. No.: 115-006-071) secondary antibody labeled with pH-dependent fluorescent dye CypHer5E (Cytiva, Cat. No.: PA15401), incubated at 4°C in the dark for 40 minutes, washed with FACS buffer and centrifuged twice, added with the corresponding cell complete medium (DMEM + 10% FBS or RPMI1640 + 10% FBS), incubated in the incubator for about 3 hours, centrifuged to remove the culture medium, resuspended the cells with PBS (pH 9.0), and detected the signal value of cell particles in the APC channel (CypHer5E) with a flow cytometer (SinoCyte). The Prism software was used to fit and calculate the EC 50 value.

[0142] The results are shown in Figures 4A-4C and Figures 5A-5B. The three candidate DLL3 hybridoma monoclonal antibodies, 59H4, 71B11, and 56G10, showed strong endocytosis activity (based on the EC 50 value, stronger or close to the positive control of Abbvie).

[0143] Table 1. EC 50

[0144] Table 2. EC 50

[0145] Table 3. EC 50

[0146] Example 4 Gene cloning of DLL3 antibody variable region

[0147] The present application carries out gene cloning and sequence analysis of the variable region of three monoclonal hybridoma antibodies 59H4, 71B11, 56G10; the specific method is as follows: the TRIzon (Cwbiotech, Cat No: CW0580) is used to lyse the DLL3 monoclonal hybridoma cell strain, and the total RNA of the hybridoma cell is extracted. The hybridoma cell RNA is reversely transcribed into cDNA by using the HiFi Script cDNA synthesis kit (Cwbiotech, Cat No: CW2569). The cDNA is used as a template, and the variable region genes of the heavy chain and light chain of the antibody are amplified by PCR method (Kettleborough et al. (1993) Eur J Immunology 23: 206-211; Strebe et al. (2010) Antibody Engineering 1: 3-14). After the PCR amplification product is connected to the T / A vector, the DH5a competent cells are transformed, plated and cultured at 37°C overnight. The single colony is picked from the culture plate, and the plasmid is extracted after the culture is expanded, and the gene sequence of the antibody is determined. According to the gene sequence of the antibody, the complementarity determining region (CDR) and the framework region are analyzed. The variable region gene sequence and amino acid sequence number of the heavy chain and light chain of some antibodies are shown in Table 4 below.

[0148] Table 4. Sequence list of DLL3 hybridoma antibody and CDR region

[0149] Example 5 - Humanization of murine DLL3 antibodies 59H4, 71B11, 56G10

[0150] Humanization of mouse-derived DLL3 antibodies was performed by grafting the complementarity determining regions. First, the human germline antibody sequences with the highest sequence homology to the light and heavy chain variable regions of mouse-derived 59H4, 56G10 and 71B11 antibodies were searched in IMGT database. For 59H4, the germline for light chain variable region humanization was IGKV4-1*01 and for heavy chain variable region humanization was IGHV7-4-1*02. For 56G10, the germline for light chain variable region humanization was IGKV4-1*01 and for heavy chain variable region humanization was IGHV7-4-1*02. For 71B11, the germline for light chain variable region humanization was IGHV2-29*02 and for heavy chain variable region humanization was IGHV7-4-1*02. The CDR regions of the mouse-derived antibodies were kept unchanged, and the framework regions of the mouse-derived antibodies were replaced by the framework regions of the human germline antibodies. The structure model of the mouse-derived antibodies was established, and the amino acid at each site in the framework region of the humanized antibody was compared with that of the corresponding mouse-derived antibody. If the human amino acid sequence at a certain site in the framework region did not result in the destruction or change of the spatial structure of the CDR region, the human amino acid sequence was used at that site, otherwise the corresponding mouse-derived sequence was used at that site (i.e., back mutation to the mouse-derived sequence).

[0151] The sequences of the humanized antibodies are shown in Table 5.

[0152] Table 5. Amino acid sequence numbers of humanized DLL3 antibodies

[0153] The nucleic acid sequences encoding the light and heavy chains of the humanized 59H4, 71B11 and 56G10 antibodies were synthesized and inserted into the expression vector pcDNA3.1. 200 milliliters of 293 cells (cell density 1 x 10 6 ) were co-transfected with 0.1 mg of each of the light and heavy chain expression plasmids, cultured at 37°C with shaking for 6 days, and the supernatant was collected by centrifugation. The humanized antibodies were purified using Protein A. The purified humanized antibodies were used for activity detection.

[0154] Example 6 Activity detection of humanized DLL3 antibodies and detection of species cross-reactivity and binding reaction with homologous proteins

[0155] 1) Activity detection of humanized DLL3 antibodies: The binding of the purified humanized antibody sample to DLL3 was detected by ELISA and FACS, and the endocytosis activity of the humanized antibody was detected by FACS. The specific methods are described in Example 2.

[0156] Results are shown in Figures 6, 7A-7B, 8A-8B, 9A-9B, 10A-10B. Overall, the three candidate antibodies after humanization maintained strong antigen activity and cellular endocytosis. This laid a foundation for further conjugation and drug development.

[0157] 2) Detection of species cross-reactivity and binding to homologous proteins

[0158] A) Species cross-reactivity of humanized antibodies

[0159] ELISA method was used to determine the binding activity of purified monoclonal antibodies to monkey, rat, and mouse DLL3 proteins, respectively: 100 μL of monkey, rat, and mouse DLL3 proteins (final concentration: 2 μg / mL) were used to coat 96-well ELISA plates (Corning, Cat. No.: 9018) overnight at room temperature; after washing 3 times with washing buffer (PBS + 0.05% Tween 20), blocking buffer (PBS + 2% BSA) was added and incubated at room temperature for 1 hour, and the ELISA plate was washed 3 times; gradient-diluted antibody diluent was added and incubated at room temperature for 1 hour, and washed 3 times; 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 incubated at room temperature in the dark for 1 hour, and washed 3 times; 100 μL of TMB (Beijing Baosai Bio, Cat. No.: ES-002) was added to each well, and color developed at room temperature for 1-3 minutes, and 100 μL / well of stop solution (2N H2SO4) was added to stop the color development reaction, and the OD450 values of each well were read using an enzyme-labeled instrument (Tecan Spark), and the Prism software was used to fit and calculate the EC 50 values.

[0160] Results are shown in Figures 11A-11C, which show that among the three humanized antibodies, humanized 59H4 and 71B11 have strong binding activity to monkey, rat, and mouse DLL3 proteins; while humanized 56G10 can still bind to monkey DLL3, but the binding to rat DLL3 protein is weakened to a certain level, and no binding occurs with mouse DLL3 protein.

[0161] B) Detection of binding of humanized antibodies to homologous proteins

[0162] The purified monoclonal antibodies were respectively determined for binding activity with human DLL1, DLL3 and DLL4 proteins by ELISA method: 100 μL of human DLL1, DLL3 and DLL4 proteins (final concentration: 2 μg / mL) were respectively coated on 96-well ELISA plates (Corning, Cat. No.: 9018) at room temperature overnight; after washing 3 times with washing buffer (PBS + 0.05% Tween 20), blocking buffer (PBS + 2% BSA) was added for incubation at room temperature for 1 hour, and the ELISA plate was washed 3 times with washing buffer; gradient-diluted antibody diluent was added for incubation at room temperature for 1 hour, and washing was performed 3 times; 100 μL of 3000-fold diluted HRP-conjugated mouse anti-human IgG secondary antibody (BD Biosciences, Cat. No.: 555788) was added to each well for incubation at room temperature in the dark for 1 hour, and washing was performed 3 times; 100 μL of TMB (Beijing Baosai Bio, Cat. No.: ES-002) was added to each well for color development at room temperature for 1-3 minutes, 100 μL / well of stop solution (2N H2SO4) was added to terminate the color development reaction, and the OD450 value of each well was read by an enzyme-labeled instrument (Tecan Spark).

[0163] The results, as shown in FIGS. 12A-12C, indicate that the three humanized antibodies 59H4, 71B11 and 56G10 do not bind to the homologous proteins. It is shown that the three antibodies only bind to the DLL3 protein and have good specificity.

[0164] Example 7 Targeting B7-H3 antibody candidate clones and targeting DLL3 candidate clones

[0165] The humanized antibodies of candidate clones 7B7 and 15A2 have been disclosed in the patent CN113402610A and its homologous patents, and the amino acids of individual sites are appropriately optimized, and the SEQ ID NO. sequence is rewritten in the present application, and the sequence is excerpted as shown in Table 6.

[0166] Table 6. Amino acid sequence number of humanized B7-H3 antibody

[0167] Example 8 Targeting DLL3 and B7-H3 double antibody

[0168] The above 5 clones, one candidate clone for each target point in the form of a conventional complete human IgG1 antibody, the heavy and light chain variable regions of the other target point in a (G4S)*3 or (G4S)*4 or (G4S)*5 tandem to form a single-chain antibody ScFv, fused with the carbon terminal of IgG1, to form a double-target antibody as shown in Figure 13. The terminal amino acids are adjusted accordingly as needed. The glycosylation site N297 can be selected to be adjusted to N297A (A can also be other amino acids other than N). The length of G4S, the adjustment of terminal amino acids, the adjustment of glycosylation sites, these optimization adjustments, belong to the prior art. For the ScFv structure, the amino acids in the heavy chain variable region VH44 and the light chain variable region VL100 can be mutated to cysteine to form disulfide bonds, or mutated to positively and negatively charged aspartate and lysine to form salt bonds, to stabilize the ScFv structure.

[0169] The architecture of the double antibody is shown in Figure 13.

[0170] The sequence of the double antibody is shown in Table 7:

[0171] Table 7

[0172] According to the designed architecture and sequence, the antibody light and heavy chains were constructed in pCDNA vectors respectively, and plasmids were extracted. The suspension-adapted CHO-K1 cells were resuscitated in OPM-CD TransCHO medium, and cultured to a density of 2 million cells / ml, with a viability of more than 95%, and a volume of 1000 ml. The light chain plasmid 0.5 mg and the heavy chain plasmid 0.5 mg were combined and dissolved in 10 ml of medium, 3 mg of PEI dissolved in medium was diluted in 10 ml of medium, the plasmid and PEI solution were mixed and placed at room temperature for 10 minutes, then added dropwise to 1000 ml of cell culture solution, and placed at 37°C for 5 days, then centrifuged at 12000g for 15 minutes to harvest the supernatant. The supernatant was purified with HiTrap Mabselect SuRe, and eluted with 50 mM acetic acid, and the neutralized collection peak was replaced in PBS pH 7.4 with a 30KD ultrafiltration tube. The antibody was determined by absorbance at 280 nm, and the absorbance value was divided by the theoretical extinction coefficient to obtain the concentration value. The antibody expression and purity are as follows in Table 8:

[0173] Table 8. DLL3 and B7-H3 double antibody expression and purity

[0174] Binding activity of antibodies to antigen and endocytosis activity of antibodies

[0175] Binding activity of antibodies to antigen (ELISA): 100 μL of DLL3-mFc or B7H3-mFc (final concentration: 2 μg / mL) was added to a 96-well ELISA plate (Corning, Cat. No.: 9018) respectively, and incubated overnight at room temperature; after washing 3 times with washing buffer (PBS + 0.05% Tween 20), blocking buffer (PBS + 2% BSA) was added and incubated at room temperature for 1 hour, and the ELISA plate was washed 3 times with washing buffer; gradient-diluted antibody diluent was added and incubated at room temperature for 1 hour, and washed 3 times; 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 incubated at room temperature for 1 hour in the dark, and washed 3 times; 100 μL of TMB (Beijing Baosai Bio, Cat. No.: ES-002) was added to each well, and incubated at room temperature for 1-3 minutes to develop color, and 100 μL / well of stop solution (2N H2SO4) was added to stop the color development reaction, and the OD450 value of each well was read with an enzyme-labeled instrument (Tecan Spark), and the EC 50 value was calculated by fitting.

[0176] Binding activity of antibodies to cells (FACS): the test antibody was gradient-diluted with a flow cytometer, 50 μL of antibody diluent was mixed with 50 μL of SHP-77 tumor cells (200,000 cells / well) and gently blown to mix, added to a 96-well U-bottom cell plate and incubated for 1 hour, washed twice with FACS buffer (PBS + 3% FBS) and centrifuged, 100 μL of 400-fold diluted PE-labeled goat anti-human secondary antibody (eBioscience, Cat. No.: 12-4998-82) was added, and incubated in the dark for 40 minutes, washed twice with FACS buffer and centrifuged, and the cell particles in the PE channel signal value were detected with a flow cytometer (Zhongsheng SinoCyte). The EC 50 value was calculated by fitting.

[0177] Endocytosis activity of antibody (FACS): dilute the test bispecific antibody with flow cytometry dilution gradient, take 50 μL antibody gradient dilution and mix with 50 μL SHP-77 or DMS53 cells (20,000 cells / well) and gently blow to mix, add to 96-well U-bottom cell plate, incubate at 4°C for 1 hour, wash twice with FACS buffer (PBS+3% FBS) and centrifuge, add 100 μL pH-dependent fluorescent dye CypHer5E (Cytiva, Cat.No.: PA15401) labeled goat anti-hFc (Jackson, Cat.No.: 109-006-098) secondary antibody, incubate at 4°C in the dark for 40 minutes, wash twice with FACS buffer and centrifuge, add complete medium (RPMI1640+10% FBS), place in incubator at 37°C for about 3 hours, centrifuge to remove culture medium, resuspend cells with PBS (pH 9.0), and detect the signal value of cell particles in the APC channel (CypHer5E) with a flow cytometer (SinoCyte). Fit and calculate EC 50 values.

[0178] The results of the binding activity and endocytosis activity detection of the above bispecific antibody are shown in Table 9 below:

[0179] Table 9. Binding activity and endocytosis activity of bispecific antibody

[0180] Example 10 linker payload synthesis

[0181] The patent application number of the linker payload technical solution is PCT / CN2023 / 106385 (WO2025011419A1, Chinese counterpart application CN119264213A). Applicant: Shanghai SiJian Biotechnology Co., Ltd.

[0182] LD-38 structure:

[0183] For more detailed synthesis information or other linker payload information, and coupling detection, please refer to the patent PCT / CN2023 / 106385.

[0184] Example 11 antibody conjugation

[0185] Take the antibody, dilute to 5 mg / ml with 10 mM sodium phosphate buffer (pH 7.4), add a TCEP hydrochloride stock solution, ensure that the molar final concentration of TCEP is 6:1 of the molar final concentration of the antibody, and reduce the antibody at 25°C for 2 hours. Add a linker toxin dissolved in DMSO stock solution to a molar final concentration of linker toxin that is 20:1 of the molar final concentration of the antibody, and couple the antibody at 25°C for 2 hours. Then, replace the ADC with a 30KD ultrafiltration tube with sodium phosphate buffer, sterile filter, determine the concentration, and freeze and store in aliquots. To obtain products with different coupling ratios, adjust the molar ratio of antibody: reducing agent: linker toxin to be between 1:1 and 10:2-50, and keep the rest of the conditions unchanged. The DAR value of the final coupling product is determined according to the test results.

[0186] The antibody and conjugate are detected by SEC-HPLC molecular sieve method, and the analysis column is TSKgel G3000SWXL, 7.8mm x 30cm, 5 microns, item number 08541. Take about 0.3 mg of the sample to be tested, centrifuge at 10000g for 5 minutes, and take the supernatant. The mobile phase is 50 mM sodium phosphate + 0.1 M sodium chloride pH 6.8. The analysis column is connected to an Agilent 1260 high performance liquid chromatograph, the flow rate is 0.8 ml / min, the mobile phase is flushed for more than 30 minutes until the 280 nm ultraviolet baseline is stable, 100 micrograms of sample is injected, and the mobile phase is flushed for 20 minutes. According to the peak area, calculate the percentage of each peak of high molecule, monomer, and low molecule. Take the monomer as the sample purity value.

[0187] The average number of linkers per antibody molecule coupled, the DAR, was determined by hydrophobic interaction chromatography (HIC-HPLC) using a TSKgel Butyl-NPR, 4.6 mm x 10 cm, 2.5 microns, Cat# 042168. About 0.3 mg of sample was taken and 150 microliters of mobile phase A was added. The sample was centrifuged at 10,000 g for 5 minutes and the supernatant was taken. Mobile phase A was 20 mM sodium phosphate + 1.5 M ammonium sulfate pH 7.0 and mobile phase B was 20 mM sodium phosphate + 20% (v / v) acetonitrile pH 7.0. The column was attached to an Agilent 1260 high performance liquid chromatograph and the flow rate was 0.6 ml / min. The column was flushed with mobile phase A for 30 minutes or more until the UV baseline at 280 nm was stable. A 50 microliter sample was injected and the column was flushed with mobile phase A for 2 minutes before a gradient elution was performed (0% B - 100% B) for 18 minutes. The column was then flushed with 100% mobile phase B for 5 minutes before the run was ended. The percentage of each DAR value was calculated from the peak area of each DAR value. The percentage of each peak was multiplied by the DAR value and then summed and divided by the total percentage to give the average DAR value. It was clear that although the DAR was set to 8 for this experiment, the HIC-HPLC method gave a single peak for the conjugate and the DAR was considered to be 8. Other methods could be used as alternatives. The average DAR value could be adjusted between 2 and 8 to balance efficacy and safety. The conjugate of the application is not limited to a DAR of 8 and can be extended to a DAR of 2 to 8.

[0188] Table 10. Purity of ADC molecules

[0189] Example 12. In vitro activity assay

[0190] Human tumor cell lines SHP-77 and NCI-H82 were separately recovered and cultured to the logarithmic growth phase. When the viability was greater than 90%, 500-5000 cells were inoculated into each well of a 96-well culture plate, and the volume was 150 microliters. The plate was incubated in a 37°C, 5% CO2 incubator. After about 20 hours, 50 microliters of culture medium containing different antibody-drug conjugates was added to each well, so that the final concentration of the drug was 12 dilution concentrations between 0 and 1000 nM (the first well had a concentration of 1000 nM, and the subsequent 10 gradients were diluted by 5 times, and the last gradient had a drug concentration of 0, with duplicate wells for each sample). After co-culturing for 4-7 days, the culture plate was removed, 50 microliters of CTG detection reagent (CellTiterGlo, manufacturer Promega, Cat# G7575) was added to each well, and the reaction was allowed to proceed for 2 minutes according to the recommended operation of the kit. Then the fluorescence value was determined using a Tecan Spark. The average value of the duplicate wells was taken as the Y axis, and the log10 value of the dilution gradient was taken as the X axis. A smooth curve was drawn, and the EC / IC values were calculated according to the four-parameter fitting. 50 ​

[0191] Table 11. ADC inhibits SHP-77 growth activity

[0192] Example 13 in vivo activity

[0193] The antibody and conjugate correspondence is as follows Table 12:

[0194] Table 12

[0195] SHP-77 or NCI-H82 tumor cell lines were resuscitated to the logarithmic growth phase, with a viability of greater than 90%, and were inoculated into immunodeficient mice (mouse species) in varying amounts from 10,000 to 1 million cells. When the tumor size grew to 90-150 square millimeters, the mice were grouped, with 5 mice in each group, and dosing was performed, with tumor size being measured twice per week. All groups were dosed once, intravenously. The activity of each drug group in inhibiting tumor cells was determined by comparing the tumor size between each drug group or with the normal saline group. Groups G1-G14 were completed in one experiment, and the number of groups was large, so that the results could be clearly displayed by separating several groups. The horizontal axis is the treatment time of the grouped dosing, and the vertical axis is the tumor size. At the same time, the tumor size and tumor inhibition rate TGI at a certain midpoint day were statistically analyzed in the table.

[0196] Both target points B7-H3 and DLL3 are tumor-related antigens or tumor-specific antigens, and the target points have biological functions and insufficient biological function research. The dual antibody ADC combined with the two has unexpectedly observed a certain degree of synergistic inhibition of tumor growth in a tumor transplantation model, i.e., 1+1>2, as shown in the table.

[0197] The antibody conjugate inhibits SHP-77 tumor cell growth as follows:

[0198] Table 13

[0199] The SHP-77 tumor cell growth inhibition curve is shown in Figures 14-17.

[0200] The antibody conjugate inhibits NCI-H82 tumor cell growth as follows:

[0201] Table 14

[0202] The NCI-H82 tumor cell growth inhibition curve is shown in Figures 18-21.

[0203] Example 14 preparation and comparison of other antibody conjugates

[0204] Methods of double antibody conjugation and in vivo activity evaluation model. The 7B7 clone of B7H3 antibody was conjugated with LD38 to prepare 7B7-LD38 conjugate, and the DAR was detected 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 RKO, JIMT-1, SHP-77 and Calu-6 models. Therefore, it is preliminarily determined that LD38 is superior to Deruxtecan, so the control sample DS-7300 is not always placed in some tumor transplantation models (CDX models).

[0205] The above-described embodiments only express several implementation manners of the present disclosure, which are described in a more specific and detailed manner, but cannot be understood as limiting the scope of the patent application. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present disclosure, which are all within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure patent should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A dual drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the antibody-drug conjugate having a general formula of Ab-(L-D)n; Ab is a dual targeting antibody, L is a linker unit, D is a drug, and n is a positive integer or a decimal number from 1 to 20, the dual targeting antibody comprising a first domain and a second domain having binding specificity, the first domain and the second domain are capable of binding to target DLL3 or B7-H3, respectively. characterized in that the drug is selected from one or more of the following effectors or derivatives: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, antiangiogenic agents, antiestrogens, antiandrogens, immunomodulators.

2. The dual-action drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 1, characterized in that, the chromatin function inhibitor and the linker unit form a linker payload, the linker payload is LD-38.

3. The dual-action drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 2, wherein, n is a positive integer or a decimal number from 2 to 8.

4. The dual-action drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein 5. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to DLL3, comprising CDRs of a heavy chain and CDRs of a light chain, wherein the amino acid sequence of CDR1 of the heavy chain comprises SEQ ID NO: 7, 13, or 19; the amino acid sequence of CDR2 of the heavy chain comprises SEQ ID NO: 8, 14, or 20; the amino acid sequence of CDR3 of the heavy chain comprises SEQ ID NO: 9, 15, or 21; the amino acid sequence of CDR1 of the light chain comprises SEQ ID NO: 10, 16, or 22; the amino acid sequence of CDR2 of the light chain comprises SEQ ID NO: 11, 17, or 23; and the amino acid sequence of CDR3 of the light chain comprises SEQ ID NO: 12, 18, or 24.

6. The antibody or antigen-binding fragment thereof of claim 5, comprising 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 NOs: 1, 3, and 5; and the amino acid sequence of the light chain variable region is selected from any one of SEQ ID NOs: 2, 4, and 6.

7. The antibody or antigen-binding fragment thereof of claim 6, comprising 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.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the amino acid sequence of the humanized heavy chain variable region is selected from any one of SEQ ID NOs: 25, 27, and 29; and the amino acid sequence of the humanized light chain variable region is selected from any one of SEQ ID NOs: 26, 28, and 30. the antibody is the monoclonal antibody or antigen-binding fragment thereof that specifically binds to DLL3 of any one of claims 5-8.

9. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, characterized in that, the linker unit and the drug moiety form a linker payload, the linker payload is LD-38.

10. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 9, wherein the targeting antibody or antigen-binding fragment thereof that specifically binds to DLL3 comprises the heavy chain CDRs and the light chain CDRs recited in claim 5; 11. 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 B7-H3 comprises a heavy chain and a light chain, the targeting antibody or antigen-binding fragment thereof that specifically binds to B7-H3 comprises a heavy chain and a light chain, 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.

12. The dual targeting antibody or antigen binding fragment thereof of claim 11, comprising 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.

13. The dual targeting antibody or antigen binding fragment thereof of claim 12, comprising a heavy chain constant region, characterized in that, the heavy chain constant region amino acid sequence is selected from SEQ ID NO: 47 or 49.

14. The dual targeting antibody or antigen binding fragment thereof of claim 13, 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.

15. The dual targeting antibody or antigen binding fragment thereof according to claim 11, comprising 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 be mutated to cysteine to form a disulfide bond, or to aspartate and lysine of opposite charges to form a salt bond, to stabilize the ScFv structure.

16. The antibody or antigen-binding fragment thereof of any one of claims 5-6, 11-15, which is selected from a rabbit-derived antibody, a murine-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

17. The dual-action drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the first domain and the second domain are selected from the antibody or antigen-binding fragment thereof of any one of claims 5-8, 11-16.

18. An antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, according to claim 17, wherein the monoclonal antibody or antigen-binding fragment thereof part thereof is a targeting antibody or antigen-binding fragment thereof that specifically binds to B7-H3 as described in claim 11, which is connected to a linker unit and a drug moiety to form a linker payload, the linker payload being LD-38.

19. A method of preparing the dual antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof of any one of claims 1-4, comprising: after reduction of the dual targeting antibody, coupling with the pre-synthesized -L-D to obtain a compound represented by the general formula Ab-(L-D)n.

20. A pharmaceutical composition comprising the dual antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof of any one of claims 1-4, and a pharmaceutically acceptable excipient, diluent or carrier.

21. Use of the dual antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof of any one of claims 1-4 in the manufacture of a medicament for treating a tumor.

22. The use according to claim 21, wherein the tumor is a solid or hematological tumor of 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, neural tumor, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, squamous carcinoma, myeloma, lymphoma and leukemia.

23. The use according to claim 22, wherein the cancer is selected from the group consisting of 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, anaplastic lung cancer, ovarian teratocarcinoma, ovarian epithelial tumor, ovarian sex cord-stromal tumor, ovarian germ cell tumor, ovarian metastatic tumor, neuroblastoma, glioma, glioblastoma multiforme, head and neck squamous cell carcinoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, multiple myeloma; preferably, the cancer is selected from the group consisting of triple negative breast cancer, HER-2 positive breast cancer, rhabdomyosarcoma, fibrosarcoma, non-small cell lung cancer, anaplastic lung cancer, acute leukemia.

Images