Antibody-drug conjugate, linker thereof, and method of use

Abstract

This disclosure provides an antibody-drug conjugate comprising a monoclonal antibody (mAb), a first payload conjugated to the mAb via a first divalent linker, and a second payload conjugated to the mAb via a second divalent linker, wherein the first linker, the second linker, or any combination thereof includes a phosphine-azide linker, an N-succinimidyl S-acetylthioacetate sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SATA sulfo-SMCC) linker, a sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) linker, or a dibromomaleimide (DBM) linker. Furthermore, this disclosure provides compounds, compositions, and methods related to antibody-drug conjugates.

Description

Cross-reference of related applications

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 513,635 filed on 14 July 2023, and the benefit of said Provisional Patent Application, the disclosures of which are expressly incorporated in their entirety by reference.

[0002] Sequence List A sequence listing in accordance with WIPO standard ST.26 is incorporated into this application by reference. The above sequence listing has been submitted to the PatentCenter as an electronic document encoded as XML in UTF-8 text. This electronic document, created on 10 July 2024, is titled "103361-478WO1_ST26.xml" and has a size of 77,922 bytes. [Background technology]

[0003] Conventional antibody-drug conjugates carry only a single payload, making it impossible to target multiple different cell types to improve treatment outcomes. For example, problems with circulatory stability, side effects, and anti-cancer efficacy can arise, particularly in highly aggressive and heterogeneous cancers. The use of only a single payload eliminates the possibility of optimizing or modifying the conjugate to maximize anti-cancer efficacy. Furthermore, there is no method to enhance tumor immunity in addition to cancer-killing effects. [Overview of the project] [Problems that the invention aims to solve]

[0004] The compositions and methods disclosed herein address the above-mentioned needs and other needs. [Means for solving the problem]

[0005] Based on the purpose of the disclosed substances and methods, the subject matter disclosed herein, as embodied and extensively described herein, relates in one embodiment to bioconjugations and related linkers.

[0006] Therefore, in one example, an antibody-drug conjugate is provided comprising a monoclonal antibody (mAb), a first payload conjugated to the mAb via a first divalent linker, and a second payload conjugated to the mAb via a second divalent linker, wherein the first linker, the second linker, or any combination thereof includes a phosphine-azide linker, an N-succinimidyl S-acetylthioacetate sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SATA sulfo-SMCC) linker, an sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) linker, or a dibromomaleimide (DBM) linker.

[0007] Further examples include methods for treating a disease in a subject requiring such treatment, comprising administering a therapeutically effective amount of the antibody-drug conjugate described herein to the subject.

[0008] This specification further provides a method for upregulating tumor immunity in a target in which such upregulation is required, comprising administering a therapeutically effective amount of the antibody-drug conjugate described herein to the target.

[0009] Furthermore, this specification also provides a method for reducing tumor growth in a subject requiring such reduction, which includes administering a therapeutically effective amount of the antibody-drug conjugate described herein to the subject.

[0010] Furthermore, a method is provided for inhibiting a protein in cells that require it, comprising administering a therapeutically effective amount of the antibody-drug conjugate described herein to the cells, wherein the antibody in the antibody-drug conjugate inhibits the protein.

[0011] This specification also provides antibody-drug conjugates comprising a monoclonal antibody (mAb) and a payload conjugated to the mAb via a divalent linker, wherein the divalent linker comprises a phosphine-azide linker or an N-succinimidyl S-acetylthioacetate sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (SATA sulfo-SMCC) linker.

[0012] This specification further states Formula I: [ka] A compound is provided, in which R1 and R2 are selected in a combination such that one of R1 and R2 contains an antibody and the other of R1 and R2 contains a drug; X is selected from -O-, -S-, and -NH-amines, amides, esters, or any combination thereof; and W is selected from -O-, -S-, and -NH-.

[0013] Furthermore, this specification also provides a method for covalently linking a first payload with a second payload, wherein the first payload is given formula III: [ka] [In the formula, R5 is H, C 1~20 Alkyl, C 2~20 Alkenil, C 2~20 Alkynyl, 6-20 member aryl, 7-20 member alkylaryl, 3-20 member cycloalkyl, C 1~20 Ashiru, C 1~20 Alkoxy, 7-20 membered aryloxy, C 1~20 Alkylamino, C 2~20Selected from dialkylamino, halogen, or amino] Reacting with a compound of to form a first precursor; reacting a second payload with a compound of Formula IV:

Chemical formula

Chemical formula

[0014] Also provided herein is a compound of Formula II:

Chemical formula

[0015] Further provided herein is a method of covalently linking a first payload to a second payload, the method comprising reacting the first payload with a compound of Formula VI:

Chemical formula

[0016] Further advantages are some described below, some of which become apparent from that description, or which can be acquired through the practice of the embodiments described below. The advantages described below are realized and achieved by the elements and combinations specifically indicated in the attached claims. Please understand that both the above summary and the following detailed description are for illustrative and illustrative purposes only, and not intended to be limiting.

[0017] The attached drawings are incorporated herein by reference and constitute part of this specification, illustrating some of the embodiments described below. [Brief explanation of the drawing]

[0018] [Figure 1]Figure 1 illustrates the synergistic mechanism of anti-CD276 mAb-MMAF-TLR7 / 8 agonist for the treatment of TNBC. Step 1 is the targeting and binding of DualADC to the surface receptor CD276 on TNBC cells. Step 2 is internalization (internal migration). Step 3 is drug release. Step 4 is inhibition of microtubule polymerization induced by MMAF. Step 5 is cytotoxicity induced by the TLR7 / 8 agonist. Step 6 is activation of immune cells by anti-CD276 mAb and TLR7 / 8 agonist. [Figure 2] Figure 2 shows a synthetic pathway for constructing an exemplary dual-payload antibody-drug conjugate containing a chemotherapy drug, an immunotherapy drug, a DBM linker, and a phosphine-azidrine linker. [Figure 3A] Figures 3A-3E show the construction of a Dual ADC via cysteine ​​and lysine. Figure 3A shows the structure of the CD276 mAb-MMAF / IMQ Dual ADC. [Figure 3B] Figure 3B shows the structure of the DaulADC. [Figure 3C] Figure 3C shows that conjugation between single-payload and dual-payload combinations and the chimeric anti-CD276 mAb was confirmed by HPLC. [Figure 3D] Figure 3D shows MALDI-TOF MS to confirm the exact molecular weight of mAbs, single ADCs (mAb-MMAF, mAb-IMQ), and dual ADCs (mAb-MMAF / IMQ). [Figure 3E] Figure 3E shows the SDS-PAGE of the ADC. M: Marker; 1: CD276 mAb; 2: mAb-MMAF; 3: mAb-IMQ; 4: mAb-MMAF / IMQ. [Figure 4A]Figures 4A-4F show the in vitro evaluation of Dual ADCs using humanized CD276 mAb. TNBC cells MDA-MB-231, MDA-MB-468, and 4T1 cells were used to study cytotoxicity, with free drugs and single-payload ADCs used as controls. Figure 4A shows the anti-TNBC cytotoxicity and IC50 of free DM1 and free MMAF drugs. [Figure 4B] Figure 4B shows the cytotoxicity and IC50 of IMQ. [Figure 4C] Figure 4C shows the EC50 of IMQ in human and mouse TLR8+HEK cells. [Figure 4D] Figure 4D shows the anti-TNBC cytotoxicity and IC50 of a single-payload ADC (mAb-MMAF). [Figure 4E] Figure 4E shows the cytotoxicity and IC50 of a single payload (mAb-IMQ). [Figure 4F] Figure 4F shows the cytotoxicity and IC50 of DualADC mAb-MMAF / IMQ. [Figure 5A] Figures 5A–5D show the antitumor efficacy of DualADC in a TNBC PDX xenograft model. Figure 5A shows the change in tumor volume after treatment with humanized CD276 mAb-derived ADC following a Q7Dx3 schedule, as indicated by the black arrows. Data are shown as mean ± SEM, and n=5–7. Saline solution (○), 16 mg / kg mAb-MMAF (▲), and 16 mg / kg mAb-MMAF / IMQ (●). *P<0.05 (vs. saline, using ANOVA and subsequent Dunnett's t-test). [Figure 5B] Figure 5B shows body weight. [Figure 5C] Figure 5C shows a white light image taken 14 days after the discontinuation of treatment. [Figure 5D] Figure 5D shows IHC staining of TNBC PDX tumor tissue. The scale bar is 20 μm. [Figure 6A]Figures 6A–6E demonstrate the anti-TNBC efficacy of CD276 mAb-MMAF / IMQ in an immunocompetent model. Female BALB / cJ mice xenografted with the mouse TNBC 4T1-FLuc were treated by intravenous injection via the tail vein with DualADC (8, 16, 24 mg / kg mAb-MMAF / IMQ), mAb-MMAF, mAb-IMQ, or saline (control). n=5–8. Figure 6A shows tumor volume after treatment according to a Q7Dx4 schedule, as indicated by the black arrows. Data are shown as mean ± SEM. *P<0.05 (vs. saline, using ANOVA and subsequent Dunnett's t-test). [Figure 6B] Figure 6B shows the final infiltrating tumor weights treated with 16 mg / kg DualADC using chimeric CD276 mAb and humanized CD276 mAb. [Figure 6C] Figure 6C shows H&E staining of major organs. The scale bar is 70 μm. [Figure 6D] Figure 6D shows IHC staining of excised tumors using markers for cell proliferation (Ki67), apoptosis (CCasp3), immune checkpoint inhibition (PD-1), and infiltration and activation of CD8+ T cells, NK cells, and macrophage cells (CD8, CD45, F4 / 80). The scale bar is 20 μm. [Figure 6E] Figure 6E shows HE staining for analyzing TNBC cell death in the treatment group. The scale bar is 40 μm. [Figure 7A] Figures 7A-7B show the analysis of tumor cytokines and systemic toxicity after treatment with dual-payload ADC. The same mice as in Figure 6 were used here as well. (Figure 7A) Luminex assay revealed enhancement of several cytokines and downregulation of PD-1 in TME. [Figure 7B](Figure 7B) Complete blood count. 1: Physiological saline (control); 2: 8 mg / kg mAb-MMAF (control); 3: 8 mg / kg mAb-IMQ (control); 4: 8 mg / kg mAb-MMAF / IMQ; 5: 16 mg / kg mAb-MMAF / IMQ; 6: 24 mg / kg mAb-MMAF / IMQ. [Figure 8A] Figures 8A–8D show the analysis of immune cell infiltration and immune function in TME using single-cell RNA sequencing (scRNA-Seq). Tumor tissue was collected from the same animal experiment as in Figure 6. Figure 8A shows an overview of all cell types in TNBC tumors. [Figure 8B] Figure 8B shows the immune function in TME. [Figure 8C] Figure 8C shows the immune response of macrophages. [Figure 8D] Figure 8D shows the analysis of mitotic activity. [Figure 9] Figure 9 shows a synthetic pathway for constructing an exemplary dual-payload antibody-drug conjugate containing a chemotherapy drug, an immunotherapy drug, a DBM linker, and a SATA sulfo-SMCC linker. [Figure 10A] Figures 10A to 10D illustrate the development and engineering of CD276 mAbs. Figure 10A shows the development of a mouse anti-human CD276 mAb using hybridoma technology. [Figure 10B] Figure 10B shows the structures of the mouse, chimeric, and humanized anti-human CD276 mAb. [Figure 10C] Figure 10C shows the production of humanized anti-CD276 mAb using CHO cells. Dynamis medium supplemented with glucose, L-glutamine, and Feed C. 30 mL of culture was placed in a 125 mL shaking flask and heated at 130 rpm, 5% CO2, and 37°C. [Figure 10D] Figure 10D shows flow cytometry to compare the surface binding of engineered CD276 mAb to normal breast cells and TNBC cells. [Figure 11A]Figures 11A-11C show the evaluation of TNBC targeting and internalization (internal distribution) of chimeric anti-CD276 mAbs in vitro and in vivo. Figure 11A shows confocal imaging to test surface binding 12 hours after incubation of mAb-Cy5.5 (red) with MDA-MB-468 (green) for internalization. [Figure 11B] Figure 11B shows IVIS imaging of live animals 24 hours after tail vein injection of mAb (40 or 50 μg) labeled with the fluorescent dye Cy5.5, to confirm the targeting of mouse and human TNBCs by CD276 mAb. [Figure 11C] Figure 11C shows ex vivo imaging of tumors and organs subsequently obtained after euthanasia. [Figure 12A] Figures 12A–12D show the in vitro cytotoxicity of free TLR agonists and chimeric CD276 mAb-directed ADCs. Ag#2 is the IMQ used in this study. Figure 12A shows the cytotoxicity assays and IC50 values ​​of various free TLR7 / 8 agonists against MDA-MB-468. [Figure 12B] Figure 12B shows the cytotoxic assays and IC50 values ​​of various free TLR7 / 8 agonists against 4T1. [Figure 12C] Figure 12C shows the cytotoxicity and IC50 of a chimeric anti-CD276 mAb conjugate single-payload ADC (ChimAb-MMAF). [Figure 12D]Figure 12D shows the cytotoxicity and IC50 of a chimeric anti-CD276 mAb-based dual-payload ADC (ChimAb-MMAF / IMQ). Data are shown as mean ± STDEV. n=3. MTT assay: Seeding density of 3,000–5,000 cells / well (MDA-MB-468 and MD-MB-231) or 1,000 cells / well (4T1), treatment for 5 days, 37°C, 5% CO2, 96-well plate, relative viability detected with MTT assay kit. [Figure 13A] Figure 13A shows the body weight profile of an immunonormal model treated with DualADC. Female BALB / cJ mice xenografted with 4T1-FLuc were treated with dual-payload ADC (8, 16, or 24 mg / kg of mAb-MMAF / IMQ), mAb-MMAF, mAb-IMQ, or saline (control) via intravenous (iv) injection through the tail vein. n=6–8. [Figure 13B] 13B shows the body weight profile of an immunonormal model treated with DualADC. Female BALB / cJ mice xenografted with 4T1-FLuc were treated with dual-payload ADC (8, 16, 24 mg / kg mAb-MMAF / IMQ), mAb-MMAF, mAb-IMQ, or saline (control) by intravenous injection via the tail vein. n=6-8. [Figure 14] Figure 14 shows further tumor cytokine data collected using a Luminex assay with a custom-designed biomarker standard. Female BALB / cJ mice xenografted with 4T1-FLuc were treated with saline (control) and dual-payload ADC (24 mg / kg mAb-MMAF / IMQ). n=6–8. [Figure 15A]Figures 15A–15C demonstrate the anti-TNBC efficacy of dual-payload ADC (chimeric CD276 mAb-MMAF / IMQ) in an immunocompromised model. Female NSG mice xenografted with MDA-MB-231-FLuc were treated with dual-payload ADC (16 mg / kg mAb-MMAF / IMQ), single-payload ADC (16 mg / kg mAb-MMAF), and saline (control) via intravenous injection through the tail vein. n=5. Figure 15A shows tumor volume after treatment according to a Q5Dx5 schedule as indicated by the black arrows. Tumor volume was measured with calipers and calculated as an ellipsoid. Data are shown as mean ± SEM. *P<0.05 (vs. saline, using ANOVA and subsequent Dunnett's t-test). [Figure 15B] Figure 15B shows the profile of weight change. [Figure 15C] Figure 15C shows bioluminescence and white light images taken with IVIS 14 days after the last treatment injection. [Figure 16A] Figures 16A-16B show pharmacokinetic (PK) studies. Anti-CD276 mAb-MMAF / IMQ in a BALB / cJ mouse model. Six different dosages were used, n=2, totaling 12 mice. Figure 16A shows the serum titer of DualADC. [Figure 16B] Figure 16B shows the PK parameters. [Figure 17A] Figures 17A and 17B show exemplary construction of DualADC via cysteine ​​and lysine. Figure 17A shows exemplary chemical structures of DualADC including DBM linker and sulfo-SMCC linker. [Figure 17B] Figure 17B shows a schematic diagram of the corresponding DualADC. [Figure 18A] Figures 18A–18H show the construction of a single-payload ADC via lysine or cysteine. Figure 18A shows a lysine-mediated mAb-MMAF conjugate using DBM as a crosslinking linker. [Figure 18B]Figure 18B shows the mAb-DM1 conjugate via lysine using sulfo-SMCC as a linker. [Figure 18C] Figure 18C shows a lysine-mediated mAb-TLR agonist conjugate using sulfo-SMCC as a linker and SATA modification. [Figure 18D] Figure 18D shows the mAb-TLR agonist conjugate via the synthetic linker phosphine azide. [Figure 18E] Figure 18E shows the characterization of ADCs (and mAbs) using HPLC, corresponding to Figure 18A. [Figure 18F] Figure 18F shows the characterization of ADCs (and mAbs) using HPLC, corresponding to Figure 18B. [Figure 18G] Figure 18G shows the characterization of ADCs (and mAbs) using HPLC, corresponding to Figure 18C. [Figure 18H] Figure 18H shows the characterization of ADCs (and mAbs) using HPLC, corresponding to Figure 18D. [Modes for carrying out the invention]

[0019] The following descriptions in this disclosure are provided as feasible teachings of the disclosure in the best and currently known embodiments. Those skilled in the art to which the compositions and methods of this disclosure belong, benefiting from the teachings presented in the foregoing description and the accompanying drawings, will come up with numerous modifications and other embodiments disclosed herein. Therefore, it should be understood that this disclosure is not limited to the specific embodiments disclosed, and that modifications and other embodiments are also intended to be included within the scope of the appended claims. Those skilled in the art will recognize numerous variations and adaptations of the embodiments described herein. These variations and adaptations are included in the teachings of this disclosure and are intended to be included within the scope of the claims herein.

[0020] While certain terms are used in this specification, these terms are used only in a general and descriptive sense and are not intended to be limiting.

[0021] As will be apparent to those skilled in the art by reading this disclosure, each of the individual embodiments described and illustrated herein has its own distinct components and features, but they can be readily separated from or combined with any of the features of several other embodiments without departing from the scope or spirit of this disclosure.

[0022] Any method described may be carried out in the order of the events described, or in any other logically possible order. That is, unless otherwise specifically stated, no method or embodiment thereof described herein is intended to be construed as requiring its steps to be carried out in a particular order. Therefore, in a method claim, unless it is specifically stated in the claims or specification that the steps must be limited to a particular order, no order is intended to be implied in any respect. This applies to all implicit grounds for interpretation, such as logical matters concerning the arrangement or flow of operations of the steps, plain meaning derived from grammatical structure or punctuation, or the number or type of embodiments described in the specification.

[0023] All publications referenced herein are incorporated herein by reference to disclose and illustrate methods and / or materials relating to the citation of such publications. Publications referenced herein are provided only for disclosures of publications prior to the filing date of this application. Nothing herein should be construed as admitting that the present invention does not have prior rights to any publication on the grounds of prior invention. Furthermore, publication dates provided herein may differ from actual publication dates and may require separate verification.

[0024] Furthermore, it should be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the compositions and methods of this disclosure belong. In addition, terms as defined in commonly used dictionaries should be interpreted as having the meaning consistent with their meaning in the context of this specification and the related technical field, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0025] The following definitions are provided before describing the various aspects of this disclosure, and will be used as such unless otherwise indicated. Additional terms may be defined elsewhere in this disclosure.

[0026] definition Where used herein, “comprising” should be interpreted as specifying that the features, integers, steps, or components mentioned exist as described, but not as precluding the existence or addition of one or more features, integers, steps, or components, or groups thereof. Furthermore, the terms “by,” “comprising,” “comprises,” “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are each used in their open and non-restrictive sense and may be used interchangeably. Furthermore, the term “comprising” is intended to include examples and aspects that are encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples that are encompassed by the term “consisting of.”

[0027] As used herein and in the appended claims, the singular forms “a, an” and “the” refer to multiple subjects unless explicitly defined by the context. Thus, for example, references to “compound,” “composition,” or “impairment” include, but are not limited to, two or more such compounds, compositions, or impairments.

[0028] Note that in this specification, ratios, concentrations, quantities, and other numerical data may be expressed in the form of ranges. Furthermore, it should be understood that each endpoint of a range is important both in relation to and independently of the other endpoint. It should also be understood that a large number of values ​​are disclosed in this specification, and that each of these values ​​is disclosed not only as the value itself but also as "about" that value. For example, if the value "10" is disclosed, "about 10" is also disclosed. In this specification, ranges may be expressed as "about" a particular value and / or "about" another particular value. Similarly, it should be understood that when a value is expressed as an approximation, the particular value forms a further aspect by using the preceding phrase "about". For example, if the value "about 10" is disclosed, "10" is also disclosed.

[0029] Where a range is expressed, a further aspect may include a range from one specific value to and / or the other specific value. For example, if the range referred to includes one or both of the limit values, the range excluding one or both of these limit values ​​is also included in this disclosure, for example, the phrase "x~y" includes the range from "x" to "y," and the range greater than "x" and less than "y." A range may also be expressed as an upper limit, for example, "about x, y, z, or less than these," which shall be interpreted as including the specific ranges "about x," "about y," and "about z," as well as the ranges "less than x," "less than y," and "less than z." Similarly, the phrase "about x, y, z, or greater than these" shall be interpreted as including the specific ranges "about x," "about y," and "about z," as well as the ranges "greater than x," "greater than y," and "greater than z." Furthermore, where "x" and "y" are numerical values, the phrase "about x~y" includes "about x~about y."

[0030] For convenience and brevity, this range format is used and should be understood flexibly to include not only the numerical limits explicitly stated as range limits, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly stated. For example, the numerical range "approximately 0.1% to 5%" should be interpreted to include not only the explicitly stated values ​​of approximately 0.1% to approximately 5%, but also the individual values ​​within the indicated range (e.g., approximately 1%, approximately 2%, approximately 3%, and approximately 4%) and subranges (e.g., approximately 0.5% to approximately 1.1%; approximately 5% to approximately 2.4%; approximately 0.5% to approximately 3.2%; and approximately 0.5% to approximately 4.4%, as well as any other possible subranges).

[0031] Where used herein, the terms “about,” “approximately,” “just, or about,” and “substantially” mean that the quantity or value in question may be its exact value or a value that provides equivalent results or effects to those described in the claims or taught herein. That is, quantities, sizes, formulations, parameters, and other quantities and characteristics may not be exact, nor do they need to be exact, and may be approximate and / or greater or less, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those skilled in the art, so as necessary, to produce equivalent results or effects. In some circumstances, it may not be possible to reasonably determine a value that provides equivalent results or effects. In such cases, “about” and “just, or about” as used herein are generally understood to mean a variation of ±10% of the nominal value shown, unless otherwise indicated or implied. Generally, quantities, sizes, formulations, parameters, or other quantities or characteristics are “about,” “approximately,” or “just, or about,” whether explicitly stated as such. When “about,” “approximately,” or “just or about” is used before a quantitative value, it is understood that this parameter includes the specific quantitative value itself unless otherwise stated. Where used herein, the term “substantially free” is intended to mean an amount of the substance mentioned that is about 1% by weight or less, based on the total weight of the composition, for example, less than about 0.5% by weight, less than about 0.1% by weight, less than about 0.05% by weight, or less than about 0.01% by weight, when used in reference to a composition or a substantially absent component of a composition.

[0032] The term "subject" preferably refers to a person who requires treatment with anticancer drugs or treatment for any other purpose, more preferably a person who requires the above-mentioned procedures for the treatment of cancer or a precancerous condition or lesion. However, the term "patient" may also refer to an animal that is not a human requiring treatment with anticancer drugs or anticancer treatment, preferably a mammal such as a dog, cat, horse, cattle, pig, sheep, or non-human primate.

[0033] "Reduce," or other forms of this word such as "reducing" or "reduction," means a decrease in an event or characteristic (e.g., tumor growth). This is typically relative to some standard or predicted value, in other words, but it is understood that it is not always necessary to mention the standard or relative value. For example, "reduces tumor growth" means reducing the rate of tumor growth compared to a standard or control (e.g., an untreated tumor).

[0034] The term “treatment” refers to the medical management of a patient with the aim of curing, improving, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed toward the improvement of a disease, pathological condition, or disorder, and causal treatment, i.e., treatment directed toward the removal of the cause of the associated disease, pathological condition, or disorder. Furthermore, this term includes palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, pathological condition, or disorder; preventive treatment, i.e., treatment aimed at minimizing, or partially or completely inhibiting, the progression of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to complement other specific therapies directed toward the improvement of the associated disease, pathological condition, or disorder.

[0035] The term "therapeutically effective amount" refers to the amount of a composition used that is sufficient to improve one or more causes or symptoms of a disease or disorder. Such improvement may require only reduction or modification, not necessarily elimination.

[0036] chemical definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains.

[0037] The organic substructures referred to when defining the variable positions in the general formulas described herein (e.g., the term "halogen") are a collective term for the individual substituents contained within the organic substructure. The prefix C is placed before the group or substructure (moiety). n ~C m In each case, this indicates the number of possible carbon atoms in the group or substructure that follows it.

[0038] As used herein, the term “substituted” is intended to include all acceptable substituents of an organic compound. In a broad sense, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and non-aromatic substituents of an organic compound. Exemplary substituents are, for example, those listed below. There may be one or more acceptable substituents for a given organic compound, and they may be the same or different. For the purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any acceptable substituents of the organic compounds described herein that satisfy the valence of the heteroatom. This disclosure is not intended to be limited in any way by the acceptable substituents of an organic compound. Furthermore, the terms “substitution” or “substituted with” implicitly include the conditions that such substitutions are in accordance with the acceptable valences of the atom being substituted and the substituent, and that the substitution results in a stable compound, such as one that does not spontaneously change through rearrangement, cyclization, elimination, etc.

[0039] "Z 1 "Z 2 "Z 3 " and "Z 4 In this specification, is used as a general symbol to represent various specific substituents. These symbols may represent any substituent not limited to those disclosed herein, and a substituent defined as a specific substituent in one example may be defined as any other substituent in another example.

[0040] As used herein, the term "alkyl" refers to saturated linear or branched saturated hydrocarbon substructures. Unless otherwise specified, C1-C 24 (For example, C1~C 22 , C1~C 20 , C1~C 18 , C1~C 16 , C1~C 14 , C1~C 12 , C1~C10 Alkyl groups (C1-C8, C1-C6, or C1-C4) are intended. Examples of alkyl groups include methyl, ethyl, propyl, 1-methyl-ethyl, butyl, 1-methyl-propyl, 2-methyl-propyl, 1,1-dimethyl-ethyl, pentyl, 1-methyl-butyl, 2-methyl-butyl, 3-methyl-butyl, 2,2-dimethyl-propyl, 1-ethyl-propyl, hexyl, 1,1-dimethyl-propyl, 1,2-dimethyl-propyl, 1-methyl-pentyl, 2-methyl-pentyl, 3-methyl-pentyl, 4-methyl-pentyl, 1,1-dimethyl-propyl Examples include ethyl-butyl, 1,2-dimethyl-butyl, 1,3-dimethyl-butyl, 2,2-dimethyl-butyl, 2,3-dimethyl-butyl, 3,3-dimethyl-butyl, 1-ethyl-butyl, 2-ethyl-butyl, 1,1,2-trimethyl-propyl, 1,2,2-trimethyl-propyl, 1-ethyl-1-methyl-propyl, 1-ethyl-2-methyl-propyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. Alkyl substituents may be unsubstituted or substituted by one or more chemical substructures. The alkyl group may be substituted with one or more groups, including but not limited to hydroxyl, halogen, acetal, acyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate ester, carbamate ester, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, provided that the substituent is conformationally compatible and the rules of chemical bonding and strain energy are satisfied, as described below.

[0041] Throughout this specification, the term "alkyl" is generally used to refer to both unsubstituted and substituted alkyl groups, but this specification also specifically refers to substituted alkyl groups by identifying one or more specific substituents on the alkyl group. For example, the terms "haloalkyl" or "haloalkyl" specifically refer to alkyl groups substituted with one or more halides (halogens; e.g., fluorine, chlorine, bromine, or iodine). The term "alkoxyalkyl" specifically refers to alkyl groups substituted with one or more alkoxy groups, as described below. The term "alkylamino" specifically refers to alkyl groups substituted with one or more amino groups, as described below. The same applies to other terms. Where "alkyl" is used in one example and a specific term such as "alkyl alcohol" is used in another, it is not intended to imply that the term "alkyl" does not also refer to specific terms such as "alkyl alcohol".

[0042] Such conventions also apply to other groups described herein. That is, terms such as “cycloalkyl” refer to both unsubstituted and substituted cycloalkyl substructures, and furthermore, in this specification, substituted substructures may be specifically identified. For example, a particular substituted cycloalkyl can be called, for example, “alkylcycloalkyl.” Similarly, a substituted alkoxy can be called, for example, “halogenated alkoxy,” and a particular substituted alkenyl can be called, for example, “alkenyl alcohol.” Again, the convention of using generic terms such as “cycloalkyl” and specific terms such as “alkylcycloalkyl” is not intended to imply that generic terms do not include specific terms.

[0043] As used herein, the term "alkenyl" refers to an unsaturated linear or branched hydrocarbon substructure containing a double bond. Unless otherwise specified, C2-C 24 (For example, C2~C 22 , C2~C 20 , C2~C18 、C2~C 16 、C2~C 14 、C2~C 12 、C2~C 10The intended group is an alkenyl group (C2-C8, C2-C6, or C2-C4). The alkenyl group may contain two or more unsaturated bonds.Examples include ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 3-methyl-1-pentenyl, 4-methyl-1-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 1-methyl 3-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-1-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl Examples include nyl, 2,3-dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-1-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl, 1-ethyl-2-methyl-1-propenyl, and 1-ethyl-2-methyl-2-propenyl.The term "vinyl" refers to a group with the structure -CH=CH2; 1-propenyl refers to a group with the structure -CH=CH-CH3; and 2-propenyl refers to a group with the structure -CH2-CH=CH2. (Z. 1 Z 2 )C=C(Z 3 Z 4 Asymmetric structures such as ) are intended to include both E and Z isomers. This can be inferred from the structural formulas herein in which asymmetric alkenes exist, or it can be explicitly indicated by the bond symbol C=C. Alkenyl substituents may be unsubstituted or substituted by one or more chemical substructures. Examples of suitable substituents, provided that the substituents are conformationally compatible and the rules of chemical bonding and strain energy are satisfied, include, as described below, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate ester, carbamate ester, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol.

[0044] As used herein, the term "alkynyl" refers to a linear or branched hydrocarbon substructure containing a triple bond. Unless otherwise specified, C2-C 24 (For example, C2~C 24 , C2~C 20 , C2~C 18 , C2~C 16 , C2~C 14 , C2~C 12 , C2~C 10The intended alkynyl groups are C2-C8, C2-C6, or C2-C4. The alkynyl group may contain two or more unsaturated bonds. Examples include C2-C6 alkynyls, e.g., ethynyl, 1-propynyl, 2-propynyl (or propargyl), 1-butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 3-methyl-1-butynyl, 1-methyl-2-butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 3-methyl-1-pentynyl, 4 Examples include methyl-1-pentynyl, 1-methyl-2-pentynyl, 4-methyl-2-pentynyl, 1-methyl-3-pentynyl, 2-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 3,3-dimethyl-1-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl, and 1-ethyl-1-methyl-2-propynyl. The alkynyl substituent may be unsubstituted or substituted by one or more chemical substructures. Examples of suitable substituents, as will be described later, include alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate ester, carbamate ester, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol.

[0045] As used herein, the term "aryl," and derived terms such as "aryloxy," refer to a group comprising a monovalent aromatic carbocyclic group of 3 to 50 carbon atoms. The aryl group may include a single ring or multiple fused rings. In some embodiments, the aryl group is C6-C 10This term includes aryl groups. Examples of aryl groups include, but are not limited to, benzene, phenyl, biphenyl, naphthyl, tetrahydronaphthyl, phenylcyclopropyl, phenoxybenzene, and indanyl. The term "aryl" also includes "heteroaryl," which is defined as an aromatic group containing at least one heteroatom within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term "non-heteroaryl" is also included in the term "aryl," but defines an aromatic group containing no heteroatom. The aryl substituent may be unsubstituted or substituted by one or more chemical substructures. Examples of suitable substituents, as will be discussed later, include alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate ester, carbamate ester, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol. The term "biaryl" refers to a specific type of aryl group and is included in the definition of aryl. A biaryl refers to two aryl groups that are bonded together via a fused ring structure, as in naphthalene, or bonded via one or more carbon-carbon bonds, as in biphenyl.

[0046] As used herein, the term "cycloalkyl" refers to a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term "heterocycloalkyl" refers to a cycloalkyl group as defined above, wherein at least one of the carbon atoms in the ring is substituted with a heteroatom, but is not limited to nitrogen, oxygen, sulfur, or phosphorus. Cycloalkyl groups and heterocycloalkyl groups may be substituted with or without substituted groups, as described below, including but not limited to alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acetal, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, carbonate ester, carbamate ester, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol groups.

[0047] As used herein, the term "acyl" refers to the formula -C(O)Z 1 It is represented as, where Z 1 This can be a hydrogen, hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group. Where used herein, the term "acyl" can be used interchangeably with "carbonyl". Throughout this specification, "C(O)" or "CO" is an abbreviation for C=O.

[0048] As used herein, the term "alkoxy" means an alkyl group linked by a single terminal ether bond, i.e., the "alkoxy" group is of formula Z 1 It can be defined as a base of -O-, where Z 1 is an unsubstituted or substituted alkyl as defined above. Unless otherwise specified, Z 1 C1~C 24(For example, C1~C 22 , C1~C 20 , C1~C 18 , C1~C 16 , C1~C 14 , C1~C 12 , C1~C 10 The intended alkoxy groups are C1-C8, C1-C6, or C1-C4 alkyl groups. Examples include methoxy, ethoxy, propoxy, 1-methyl-ethoxy, butoxy, 1-methyl-propoxy, 2-methyl-propoxy, 1,1-dimethyl-ethoxy, pentoxy, 1-methyl-butyloxy, 2-methyl-butoxy, 3-methyl-butoxy, 2,2-di-methyl-propoxy, 1-ethyl-propoxy, hexoxy, 1,1-dimethyl-propoxy, 1,2-dimethyl-propoxy, 1-methyl-pentoxy, 2-methyl-pentoxy, 3-methyl- Examples include pentoxy, 4-methylpentoxy, 1,1-dimethyl-butoxy, 1,2-dimethyl-butoxy, 1,3-dimethyl-butoxy, 2,2-dimethyl-butoxy, 2,3-dimethyl-butoxy, 3,3-dimethyl-butoxy, 1-ethyl-butoxy, 2-ethyl-butoxy, 1,1,2-trimethyl-propoxy, 1,2,2-trimethyl-propoxy, 1-ethyl-1-methyl-propoxy, and 1-ethyl-2-methyl-propoxy.

[0049] As used herein, the terms "halide," "halogen," or "halo" refer to fluorine, chlorine, bromine, and iodine.

[0050] As used herein, the terms "amine" or "amino" refer to formula NZ 1 Z 2 Z 3 It is represented as, where Z 1 , Z 2 , and Z 3 Each of these substituents may be one of the substituents described herein, such as the hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0051] As used herein, the terms "amide" or "amido" refer to the formula -C(O)NZ 1 Z 2 It is represented as, where Z 1 and Z 2 Each of these substituents may be one of the substituents described in the specification, such as the hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0052] As used herein, the term "ester" refers to the formula -OC(O)Z 1 Or -C(O)OZ 1 It is represented as, where Z 1 The above-mentioned alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups may be present.

[0053] When used herein, "R 1 "R 2 "R 3 "R n (where n is some integer), etc., can independently have one or more of the above-mentioned bases. For example, R 1 If the group is a linear alkyl group, one of the hydrogen atoms of the alkyl group can be optionally substituted with a hydroxyl group, an alkoxy group, an amine group, another alkyl group, a halide, etc. Depending on the selected group, the first group can be incorporated into the second group, or the first group can be a side chain of the second group (i.e., bonded to the second group). For example, in the phrase "alkyl group containing an amino group," the amino group can be incorporated into the backbone of the alkyl group, or the amino group can be bonded to the backbone of the alkyl group. The properties of one or more selected groups determine whether the first group is embedded in or bonded to the second group.

[0054] Unless otherwise specified, formulas containing chemical bonds shown only as solid lines and not as wedge-shaped or dashed lines are intended to represent each possible stereoisomer or mixture of stereoisomers (e.g., each enantiomer, each diastereomer, each meso compound, racemic mixture, or scalemic mixture).

[0055] composition Antibody-drug conjugates Furthermore, this specification provides an antibody-drug conjugate comprising a monoclonal antibody (mAb), a first payload conjugated to the mAb via a first divalent linker, and a second payload conjugated to the mAb via a second divalent linker, wherein the first divalent linker, the second divalent linker, or any combination thereof includes a phosphine-azide linker, an N-succinimidyl S-acetylthioacetate sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SATA sulfo-SMCC) linker, a sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) linker, or a dibromomaleimide (DBM) linker.

[0056] In some examples, DBM linkers are compounds containing dibromomaleimides and carboxylic acids linked by carbon chains having 2 to 13 substituted or unsubstituted carbons.

[0057] In further examples, the SATA sulfo-SMCC linker includes a sulfhydryl substructure, a succinimidyl substructure, an acetate substructure, and a cyclohexane substructure.

[0058] In certain examples, the sulfo-SMCC linker includes a succinimidyl substructure, an acetate substructure, and a cyclohexane substructure.

[0059] In a specific example, a phosphine-azid linker includes an azide substructure and a triphenylphosphine substructure.

[0060] In some examples, the first divalent linker includes a phosphine-azid linker or a SATA sulfo-SMCC linker.

[0061] In further examples, the first divalent linker includes a DBM linker or a sulfo-SMCC linker.

[0062] In certain cases, the second divalent linker includes a phosphine-azid linker or a SATA sulfo-SMCC linker.

[0063] In specific examples, the second divalent linker includes a DBM linker or a sulfo-SMCC linker.

[0064] In some examples, the first divalent linker includes a DBM linker, and the second divalent linker includes one of the following: a sulfo-SMCC linker, a SATA sulfo-SMCC linker, or a phosphine-azid linker.

[0065] In a further example, the first divalent linker is a DBM linker, and the second divalent linker is a phosphine-azid linker.

[0066] In a specific example, the first divalent linker is a DBM linker, and the second divalent linker is a sulfo-SMCC linker.

[0067] In a specific example, the first divalent linker is a DBM linker, and the second divalent linker is a SATA sulfo-SMCC linker.

[0068] In some cases, the first divalent linker covalently links the first payload to a cysteine ​​residue present in the mAb.

[0069] In certain cases, the first divalent linker covalently links the first payload to a lysine residue present in the mAb.

[0070] In some cases, the second divalent linker covalently links the second payload to a cysteine ​​residue present in the mAb.

[0071] In a further example, a second divalent linker covalently links a second payload to a lysine residue present in the mAb.

[0072] Lysine is an amino acid that is a precursor to many proteins. Lysine contains an α-amino group, an α-carboxylic acid group, and a side chain lysine. Lysine is encoded by the codons AAA and AAG. The α-carbon is chiral, and lysine can refer to either an enantiomer or a racemic mixture of both.

[0073] Cysteine ​​is a protein amino acid with the formula HOOC-CH(-NH2)-CH2-SH. The thiol side chain of cysteine ​​can participate in enzymatic reactions as a nucleophile. Cysteine ​​is chiral and is encoded by the codons UGU and UGC.

[0074] In certain cases, cysteine ​​and / or lysine are present on the monoclonal antibody and function as linker binding sites for conjugating the mAb to the first and / or second payload.

[0075] In some cases, mAbs are mouse antibodies, chimeric antibodies, or humanized antibodies.

[0076] In some cases, mAbs inhibit either CD276 or SSTR2.

[0077] In a further example, mAbs inhibit CD276. CD-276 (Cluster of Differentiation 276; B7-H3) is a human protein encoded by the CD276 gene. CD-276 is a type I transmembrane protein with a length of 316 amino acids.

[0078] In some cases, mAbs inhibit SSTR2 (Somatostatin receptor 2). SSTR2 is a protein encoded by SSTR2 in humans. The SSTR2 gene is located at position 25.1 on the long arm of human chromosome 17.

[0079] In certain cases, the first payload includes chemotherapeutic agents. Chemotherapeutic agents exert cytotoxicity by inhibiting cell division (mitosis). Chemotherapeutic agents are means of damaging or stressing target cancer cells. Chemotherapeutic agents include alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors, antineoplastic agents, and cytotoxic antibiotics.

[0080] In specific examples, chemotherapy drugs include anti-cancer agents. Anti-cancer agents are agents that control or kill cancer cells. Anti-cancer agents are cytotoxic and typically cause greater damage to dividing cells than to resting cells.

[0081] Further examples include anti-cancer agents such as monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0082] In some examples, chemotherapy drugs include altretamine, bendamustine, busulfan, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, lomustine, lurubinectin, mechloretamine, melphalan, procarbazine, streptozocin, temozolomide, thiotepa, trabectedin, carboplatin, cisplatin, oxaliplatin, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, Idarubicin, Mitomycin, Mitoxantrone, Plicamycin, Barurubicin, Methotrexate, Pemetrexed, Pralatrexate, Trimethrexate, Azathioprine, Cladribine, Fludarabine, Mercaptopurine, Thioguanine, Azacitidine, Capecitabine, Cytarabine, Decitabine, Furoxuridine, Fluorouracil, Gemcitabine, Trifluridine / Tipiracil (Tipracil), Aldesleukin (IL-2), Denileukin Difutitex, Interferon-γ, Bellinostat, Panobinostat, Romidepsin, Vorinostat, Abiraterone, Apalutamide, Bicalutamide, Cyproterone, Enzalutamide, Flutamide, Nilutamide, Anastrozole, Exemestane, Fulvestrant, Letrozole, Raloxifene, Tamoxifen, Toremifene, Degarelix, Goserelin, Historelli Leuprolide, relugolix, triptorelin, lanreotide, octreotide, pasireotide, alemtuzumab, atezolizumab, avelumab, verantamab, bevacizumab, blinatumomab, brentuximab, semiprimab, cetuximab, daratumumab, dinutuximab, dostallumab, durvalumab, elotuzumab, enfortumab, gemtuzumab, inotuzumab Ozogamicin, Ipilimumab, Mogamulizumab, Moxetumomab Pasdotox, Necitumumab, Nivolumab, Ofatumumab, Oralatumumab, Panitumumab, Pembrolizumab, Pertuzumab, Polatuzumab Vedotin, Ramucirumab, Rituximab, Sacituzumab Govitecan, Tecristamag, Tisotumab Vedotin, Tocitumomab, Trastuzumab, Trastuzumab Deruxtecan, TrastuzumabEmtansine, Tremelimumab, Abemaciclib, Acalabrutinib, Adaglacib, Afatinib, Alectinib, Alpelisib, Aciminib, Axitinib, Binimetinib, Bortezomib, Bosutinib, Brigutinib, Cabozantinib, Carfilzomib, Ceritinib, Cobimetinib, Copanlisib, Crizotinib, Dabrafenib, Dacomitinib, Dasatinib, Dubellisib, Enasidenib, Encorafenib, Entrectinib, Eldafitinib, El Lotinib, Fedratinib, Fucibatinib, Gefitinib, Gilteritinib, Glasdevib, Ibrutinib, Ideralisib, Imatinib, Infiglatinib, Ibosidenib, Ixazomib, Lapatinib, Lalotrectinib, Lenvatinib, Lorlatinib, Midostaurine, Mobosertinib, Momerotinib, Neratinib, Nilotinib, Niraparib, Olaparib, Ortasidenib, Osimertinib, Pacritinib, Palbociclib, Pazopanib, Pemigatinib, Peki Sidartinib, ponatinib, regorafenib, ribocicib, lipretinib, rucaparib, ruxolitinib, selumetinib, sonidegib, sorafenib, sunitinib, thalazoparib, tivozanib, trametinib, trilaciblib, umbralicib, vandetanib, vemurafenib, bismodegib, zanubrutinib, cabazitaxel, docetaxel, paclitaxel, etoposide, irinotecan, teniposide, to Potecan, vinblastine, vincristine, vinorelbine, asparaginase (peguaspargase), verzutifan, bexarotene, sedazulidine, eribulin, everolimus, hydroxyurea, ixabepyrone, lenalidomide, mitotane, omasetaxin, pomalidomide, selinexol, taglaxofusp, tazemetostat, teventafusp, tetrotristat, temsirolimus, thalidomide, venetoclax, or any combination thereof.

[0083] In further examples, the second payload may include immunotherapy drugs. Immunotherapy drugs are a type of cancer treatment that helps the target immune system fight cancer. It is a type of biological therapy that uses substances made from living organisms. Types of immunotherapy include immune checkpoint inhibitors, T-cell transfer therapy, monoclonal antibodies, therapeutic vaccines, and immune system modulators.

[0084] In some cases, immunotherapy drugs include Toll-like receptor agonists. Toll-like receptors are a class of proteins that play important roles in the innate immune system. These are single-span receptors and include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. Humans lack the genes for TLR11, TLR12, and TLR13, and mice lack the functional gene for TLR10. Receptors TLE1, TLR2, TLR4, TLR5, TLR6, and TLR10 are located on the cell membrane, while TLR3, TLR7, TLR8, and TLR9 are located in intracellular vesicles.

[0085] In certain cases, immunotherapeutic drugs include Toll-like receptor 7 or 8 (TLR7 / 8) agonists. TLR7 / 8 agonists stimulate the innate immune system to utilize anti-CTCL effects through cytokine production. TLRs recognize microorganisms by binding to pathogen-associated molecular patterns. This binding activates the immune and inflammatory cascades.

[0086] In specific examples, TLR7 / 8 agonists include imidazoquinolines. Imidazoquinolines are tricyclic organic molecules and include imiquimod, gardiquimod, and rexiquimod.

[0087] In some cases, immunotherapy drugs include brexcabutagen autolucel, trastuzumab, aldesleukin, amivantamab, atezolizumab, avelumab, axicabutagen silolucel, verantamab mahodotin, bevacizumab, blinatumomab, brentuximab vedotin, semiprimab, cetuximab, siltacabutagen, daratumumab, daratumumab, dostallimab, durvalumab, elotuzumab, enfortumab vedotin, epcolitamab, gemtuzumab, grofitamab, ibritumomab tioxetan, idekabutagen bicleucel, inotuzumab ozogamicin, ipilimumab, isatuximab, and lysokabutagen. Maraluucel, Ronkastuximab, Tecilin, Margetuximab, Milbetuximab, Sorabtansine, Mogamulizumab, Mosnetuzumab, Nadofalagen, Philadenobec, Naxitamab, Necitumumab, Nivolumab, Nivolumab and Relatrimab, Obinutuzumab, Ofatumumab, Panitumumab, Pegylated Interferon, Pembrolizumab, Pertuzumab, Polatuzumab, Vedotin, Ramucirumab, Retifanlimab, Rituximab, Sacituzumab, Govitecan, Siltuximab, Cipleucel-T, Tafasitamab, Taguraxofusp, Tarimogen This includes raherpalepbec, toalquetamab, teventafusp, tecristamab, tisagenlecleucel, tisotumab vedotin, trastuzumab, tremelimumab, aflibercept, or any combination thereof.

[0088] This specification also provides antibody-drug conjugates comprising a monoclonal antibody (mAb) and a payload conjugated to the mAb via a divalent linker, wherein the divalent linker comprises a phosphine-azide linker or an N-succinimidyl S-acetylthioacetate sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (SATA sulfo-SMCC) linker.

[0089] In some examples, the divalent linker includes a phosphine-azid linker.

[0090] Further examples include divalent linkers such as SATA sulfo-SMCC linkers.

[0091] In certain cases, the divalent linker covalently links the payload to cysteine ​​residues present in the mAb.

[0092] In a specific example, the divalent linker covalently links the payload to lysine residues present in the mAb.

[0093] In some cases, mAbs are mouse antibodies, chimeric antibodies, or humanized antibodies.

[0094] In some cases, mAbs inhibit either CD276 or SSTR2.

[0095] In another example, mAbs inhibit CD276.

[0096] In a specific example, mAbs inhibit SSTR2.

[0097] In certain cases, the payload includes chemotherapy drugs.

[0098] In specific examples, chemotherapy drugs include anti-cancer agents.

[0099] Further examples include anti-cancer agents such as MMAE or MMAF.

[0100] In some cases, chemotherapy drugs include altretamine, bendamustine, busulfan, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, lomustine, lurubinectin, mechloretamine, melphalan, procarbazine, streptozocin, temozolomide, thiotepa, trabectedin, carboplatin, cisplatin, oxaliplatin, bleomycin, dactinomycin, daunorubicin, doxorubicin, and epidiphen. Rubicin, Idarubicin, Mitomycin, Mitoxantrone, Plicamycin, Barrubicin, Methotrexate, Pemetrexed, Pralatrexate, Trimethrexate, Azathioprine, Cladribine, Fludarabine, Mercaptopurine, Thioguanine, Azacitidine, Capecitabine, Cytarabine, Decitabine, Furoxuridine, Fluorouracil, Gemcitabine, Trifluridine / Tipiracil, Aldesleukin (IL-2), Denileukin Difutitex, Interferon-γ, Bellinostat, Panobinostat, Romidepsin, Vorinostat, Abiraterone, Apalutamide, Bicalutamide, Cyproterone, Enzalutamide, Flutamide, Nilutamide, Anastrozole, Exemestane, Fulvestrant, Letrozole, Raloxifene, Tamoxifen, Toremifene, Degarelix, Goserelin, Historelli Leuprolide, relugolix, triptorelin, lanreotide, octreotide, pasireotide, alemtuzumab, atezolizumab, avelumab, verantamab, bevacizumab, blinatumomab, brentuximab, semiprimab, cetuximab, daratumumab, dinutuximab, dostallumab, durvalumab, elotuzumab, enfortumab, gemtuzumab, inotuzumab Ozogamicin, ipilimumab, mogamulizumab, moxetumomab Pasdotox, necitumumab, nivolumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab Vedotin, ramucirumab, rituximab, sacituzumab Govitecan, tecristamag, tisotumumab Vedotin, tocitumomab, trastuzumab, trastuzumab Deruxtecan, trastuzumabEmtansine, Tremelimumab, Abemaciclib, Acalabrutinib, Adaglacib, Afatinib, Alectinib, Alpelisib, Aciminib, Axitinib, Binimetinib, Bortezomib, Bosutinib, Brigutinib, Cabozantinib, Carfilzomib, Ceritinib, Cobimetinib, Copanlisib, Crizotinib, Dabrafenib, Dacomitinib, Dasatinib, Dubellisib, Enasidenib, Encorafenib, Entrectinib, Eldafi Tinib, erlotinib, fedratinib, futibatinib, gefitinib, gilteritinib, glassegib, ibrutinib, idelalisib, imatinib, infiglatinib, ivosidenib, ixazomib, lapatinib, lalotrectinib, lenbatinib, lorlatinib, midostaurin, mobosertinib, momerotinib, neratinib, nilotinib, niraparib, olaparib, ortasidenib, osimertinib, pacritinib, palbociclib, pazopa Nib, pemigatinib, pexidartinib, ponatinib, regorafenib, ribociclib, lipretinib, rucaparib, ruxolitinib, selumetinib, sonidegib, sorafenib, sunitinib, thalazoparib, tivozanib, trametinib, trilaciclib, umbralicib, vandetanib, vemurafenib, bismodegib, zanubrutinib, cabazitaxel, docetaxel, paclitaxel, etoposide, irinotecan, teniposide, topotecan, bi This includes mblastin, vincristine, vinorelbine, asparaginase (peguaspargase), berzutifan, bexarotene, sedazulidine, eribulin, everolimus, hydroxyurea, ixabepyrone, lenalidomide, mitotane, omasetaxin, pomalidomide, selinexol, taglaxofusp, tazemetostat, teventafusp, tetrotristat, temsirolimus, thalidomide, venetoclax, or any combination thereof.

[0101] In further examples, the payload may include immunotherapy drugs.

[0102] In some cases, immunotherapy drugs include Toll-like receptor agonists, such as Toll-like receptor 7 or 8 (TLR7 / 8) agonists.

[0103] In specific examples, TLR7 / 8 agonists include imidazoquinoline.

[0104] In some cases, immunotherapy drugs include: brexcabutagen (autolucel), trastuzumab, aldezleukin, amivantamab, atezolizumab, avelumab, axicabutagen (silolucel), verantamab (mahodotin), bevacizumab, blinatumomab, brentuximab (vedotin), semiprimab, cetuximab, siltacabutagen, daratumumab, daratumumab, dostallimab, durvalumab, elotuzumab, enfortumab (vedotin), epcolitamab, gemtuzumab, grofitamab, ibritumomab (tiuxetan), idekabutagen (biculucel), inotuzumab (ozogamicin), ipilimumab, isatuximab, lysokabutagen (malulucel), ronkastuximab Tecilin, Margetuximab, Milbetuximab Sorabtansine, Mogamulizumab, Mosnetuzumab, Nadofalagen Filadenobec, Naxitamab, Necitumumab, Nivolumab, Nivolumab and Relatrimab, Obinutuzumab, Ofatumumab, Panitumumab, Pegylated Interferon, Pembrolizumab, Pertuzumab, Polatuzumab Vedotin, Ramucirumab, Retifanlimab, Rituximab, Sacituzumab Govitecan, Siltuximab, Cipleucel-T, Tafacitamab, Taguraxofusp, Tarimogen Rachelpalepbec, Toalquetamab, Teventafusp, Tecristamab, Tisagenlecleucel, Tisotumab This includes vedotin, trastuzumab, tremelimumab, aflibercept, or any combination thereof.

[0105] compound Furthermore, in this specification, Formula I: [ka] A compound is provided, in which R1 and R2 are selected in a combination such that one of R1 and R2 contains an antibody and the other of R1 and R2 contains a drug; X is selected from -O-, -S-, and -NH-; W is selected from -O-, -S-, and -NH-.

[0106] In some examples, X is NH.

[0107] In a further example, W is NH.

[0108] In certain cases, R1 contains an antibody.

[0109] In a specific example, R2 contains drugs.

[0110] In some examples, the antibody is a monoclonal antibody (mAb).

[0111] In further examples, mAbs are mouse antibodies, chimeric antibodies, or humanized antibodies.

[0112] In some cases, mAbs inhibit either CD276 or SSTR2.

[0113] In certain cases, mAbs inhibit CD276.

[0114] In some cases, mAbs inhibit SSTR2.

[0115] A specific example is an immunotherapy drug.

[0116] In some cases, immunotherapy drugs include Toll-like receptor agonists, such as Toll-like receptor 7 or 8 (TLR7 / 8) agonists.

[0117] In specific examples, TLR7 / 8 agonists include imidazoquinoline.

[0118] In certain cases, immunotherapy drugs include: brexcabutagen autolucel, trastuzumab, aldezleukin, amivantamab, atezolizumab, avelumab, axicabutagen silolucel, verantamab mahodotin, bevacizumab, blinatumomab, brentuximab vedotin, semiprimab, cetuximab, siltacabbutagen, daratumumab, daratumumab, dostallimab, durvalumab, elotuzumab, enfortumab vedotin, epcolitamab, gemtuzumab, grofitamab, ibritumomab tiuxetan, idekabutagen bicleucel, inotuzumab ozogamicin, ipilimumab, isatuximab, lysokabutagen maralucel, ronkastuximab Tecilin, Margetuximab, Milbetuximab Sorabtansine, Mogamulizumab, Mosnetuzumab, Nadofalagen Filadenobec, Naxitamab, Necitumumab, Nivolumab, Nivolumab and Relatrimab, Obinutuzumab, Ofatumumab, Panitumumab, Pegylated Interferon, Pembrolizumab, Pertuzumab, Polatuzumab Vedotin, Ramucirumab, Retifanlimab, Rituximab, Sacituzumab Govitecan, Siltuximab, Cipleucel-T, Tafacitamab, Taguraxofusp, Tarimogen Rachelpalepbec, Toalquetamab, Teventafusp, Tecristamab, Tisagenlecleucel, Tisotumab This includes vedotin, trastuzumab, tremelimumab, aflibercept, or any combination thereof.

[0119] This specification further includes Equation II: [ka] A compound is provided, in which R3 and R4 are selected in a combination such that one of R3 and R4 contains an antibody and the other of R3 and R4 contains a drug; Z is selected from -O-, -S-, and -NH-; A is selected from -O-, -S-, and -NH-.

[0120] In some examples, Z is NH.

[0121] In a further example, A is NH.

[0122] In certain cases, R3 contains an antibody.

[0123] In a specific example, R4 contains drugs.

[0124] In some examples, the antibody is a monoclonal antibody (mAb).

[0125] In further examples, mAbs are mouse antibodies, chimeric antibodies, or humanized antibodies.

[0126] In some cases, mAbs inhibit either CD276 or SSTR2.

[0127] In certain cases, mAbs inhibit CD276.

[0128] In some cases, mAbs inhibit SSTR2.

[0129] A specific example is an immunotherapy drug.

[0130] In some cases, immunotherapy drugs include Toll-like receptor agonists, such as Toll-like receptor 7 or 8 (TLR7 / 8) agonists.

[0131] In specific examples, TLR7 / 8 agonists include imidazoquinoline.

[0132] In certain cases, immunotherapy drugs include: brexcabutagen autolucel, trastuzumab, aldezleukin, amivantamab, atezolizumab, avelumab, axicabutagen silolucel, verantamab mahodotin, bevacizumab, blinatumomab, brentuximab vedotin, semiprimab, cetuximab, siltacabbutagen, daratumumab, daratumumab, dostallimab, durvalumab, elotuzumab, enfortumab vedotin, epcolitamab, gemtuzumab, grofitamab, ibritumomab tiuxetan, idekabutagen bicleucel, inotuzumab ozogamicin, ipilimumab, isatuximab, lysokabutagen maralucel, ronkastuximab Tecilin, Margetuximab, Milbetuximab Sorabtansine, Mogamulizumab, Mosnetuzumab, Nadofalagen Filadenobec, Naxitamab, Necitumumab, Nivolumab, Nivolumab and Relatrimab, Obinutuzumab, Ofatumumab, Panitumumab, Pegylated Interferon, Pembrolizumab, Pertuzumab, Polatuzumab Vedotin, Ramucirumab, Retifanlimab, Rituximab, Sacituzumab Govitecan, Siltuximab, Cipleucel-T, Tafacitamab, Taguraxofusp, Tarimogen Rachelpalepbec, Toalquetamab, Teventafusp, Tecristamab, Tisagenlecleucel, Tisotumab This includes vedotin, trastuzumab, tremelimumab, aflibercept, or any combination thereof.

[0133] method Methods of treating diseases In one aspect, this disclosure provides a method for treating a disease in a subject requiring such treatment, comprising administering a therapeutically effective amount of the antibody-drug conjugate described herein to the subject.

[0134] In some cases, the disease is cancer. In further cases, the cancer is triple-negative breast cancer (TNBC), glioblastoma (GBM), or non-small cell lung cancer (NSCLC).

[0135] Triple-negative breast cancer (TNBC) is an aggressive type of invasive breast cancer, accounting for 15% of all breast cancer cases. TNBC is estrogen receptor-negative, progesterone receptor-negative, and HER2-negative. TNBC is often more aggressive than other breast cancers, more difficult to treat than hormone receptor-positive or HER2-positive cancers, and has a higher recurrence rate.

[0136] Glioblastoma (GBM) is a cancer that begins as a proliferation of cells in the brain or spinal cord. Glioblastomas are formed from cells called astrocytes, which support nerve cells. Symptoms of glioblastoma include headache, nausea and vomiting, blurred vision or double vision, difficulty speaking, tactile disturbances, and seizures. Glioblastoma is a grade 4 malignant tumor composed mainly of abnormal astrocyte cells. GBM infiltrates areas of the brain, most commonly the frontal lobe, and can spread to the opposite side of the brain via the corpus callosum or ventricular system.

[0137] Non-small cell lung cancer (NSCLC) is a common type of lung cancer that begins at the cellular level and causes rapid and uncontrolled replication of abnormal cells within the lungs. NSCLC includes carcinomas (e.g., adenocarcinoma, squamous cell carcinoma, large cell carcinoma) that are cancers of the cells lining the surface of the lung airways, such as the bronchi, bronchioles, and alveoli. Symptoms of NSCLC include persistent cough, coughing up blood, chest pain or discomfort, shortness of breath, wheezing, hoarseness, loss of appetite, unexplained weight loss, fatigue, difficulty swallowing, swelling of the facial and / or jugular veins, or any combination thereof.

[0138] Methods to upregulate tumor immunity Furthermore, this specification provides a method for upregulating tumor immunity in a target in which such upregulation is required, comprising administering a therapeutically effective amount of the antibody-drug conjugate described herein to the target.

[0139] Tumor immunity refers to the innate and adaptive immune responses that control tumors.

[0140] Methods to reduce tumor growth This specification further provides a method for reducing tumor growth in a subject requiring such reduction, comprising administering a therapeutically effective amount of the antibody-drug conjugate described herein to the subject.

[0141] In some cases, the tumor may be squamous cell carcinoma, large cell carcinoma, adenocarcinoma, invasive ductal carcinoma, ductal carcinoma in situ, adenoid cystic carcinoma, mucoepidermoid carcinoma, glioblastoma, or any combination thereof.

[0142] Squamous cell carcinoma, also known as cutaneous squamous cell carcinoma, is a malignant tumor and one of the most common types of skin cancer.

[0143] Large cell carcinoma (LCC) is a type of non-steroidal lung cancer (NSCLC), a group of undifferentiated malignant neoplasms. It is the least common type of NSCLC, accounting for approximately 10-15% of all NSCLC diagnoses. Large cell carcinoma may tend to grow and metastasize more rapidly than other forms of lung cancer.

[0144] Adenocarcinomas form in glandular tissue, which covers certain internal organs and produces and releases substances such as mucus, digestive juices, and other bodily fluids. The majority of breast cancers, lung cancers, esophageal cancers, stomach cancers, colon cancers, rectal cancers, pancreatic cancers, prostate cancers, and uterine cancers are adenocarcinomas.

[0145] Invasive ductal carcinoma is the most common form of breast cancer, accounting for 80% of all breast cancer diagnoses. Invasive ductal carcinoma occurs when abnormal cells growing in the inner wall of the milk ducts mutate and invade the breast tissue beyond the duct walls. From there, cancer cells can metastasize to lymph nodes or enter the bloodstream, potentially spreading to other organs and regions of the body, leading to metastatic breast cancer.

[0146] Ductal carcinoma in situ (DSL) is a condition in which abnormal cells are present within the milk ducts of the breast. It is considered the earliest form of breast cancer and is therefore non-invasive. It is usually detected during mammography, which is performed as part of breast cancer screening or to examine a lump in the breast.

[0147] Adenoid cystic carcinoma is a rare malignant tumor that arises from secretory glands, most commonly found in the salivary glands. This tumor typically grows more slowly than other cancers, tends to invade perineural tissues, and metastasizes hematogenously to distant organs. It is most common in older adults.

[0148] Mucoepidermoid carcinoma is a rare salivary gland tumor composed of mucinous cells, squamous epithelial cells, and intermediate cells. It constitutes a distinct group within lung malignancies, accounting for less than 1% of all lung cancers.

[0149] Glioblastoma refers to a tumor that arises from the glioblastoma described above.

[0150] Methods for targeting proteins Furthermore, this specification provides a method for inhibiting a protein in cells that require it, comprising administering a therapeutically effective amount of an antibody-drug conjugate described herein to the cells, wherein the antibody in the antibody-drug conjugate inhibits the protein.

[0151] Proteins contain one or more long folded amino acid chains (each called a polypeptide), and the protein sequence is determined by the DNA sequence of the gene encoding the protein. The structure of a protein has four levels: primary protein structure, secondary protein structure, tertiary protein structure, and quaternary protein structure. Examples of proteins in cells include antibodies, myocardial contractile proteins, enzymes, hormone proteins, structural proteins, storage proteins, and transport proteins.

[0152] Methods of Bioconjugation This specification further provides a method for covalently linking a first payload to a second payload, the method comprising reacting the first payload with a compound of Formula III:

Chemical formula

Chemical formula

Chemical formula

[0153] In some examples, R5 is C1 alkyl.

[0154] In some cases, the first payload contains biomolecules. Biomolecules are molecules related to living organisms, and some examples include large macromolecules such as proteins, carbohydrates, lipids, and nucleic acids, as well as smaller molecules such as vitamins and hormones.

[0155] Further examples include proteins or antibodies.

[0156] In certain cases, the second payload may be a surface, particles, a drug, or a second biomolecule.

[0157] A specific example is an immunotherapy drug.

[0158] In some cases, immunotherapy drugs include Toll-like receptor agonists, such as Toll-like receptor 7 or 8 (TLR7 / 8) agonists.

[0159] In further examples, TLR7 / 8 agonists include imidazoquinoline.

[0160] In certain cases, immunotherapy drugs include: brexcabutagen autolucel, trastuzumab, aldezleukin, amivantamab, atezolizumab, avelumab, axicabutagen silolucel, verantamab mahodotin, bevacizumab, blinatumomab, brentuximab vedotin, semiprimab, cetuximab, siltacabbutagen, daratumumab, daratumumab, dostallimab, durvalumab, elotuzumab, enfortumab vedotin, epcolitamab, gemtuzumab, grofitamab, ibritumomab tiuxetan, idekabutagen bicleucel, inotuzumab ozogamicin, ipilimumab, isatuximab, lysokabutagen maralucel, ronkastuximab Tecilin, Margetuximab, Milbetuximab Sorabtansine, Mogamulizumab, Mosnetuzumab, Nadofalagen Filadenobec, Naxitamab, Necitumumab, Nivolumab, Nivolumab and Relatrimab, Obinutuzumab, Ofatumumab, Panitumumab, Pegylated Interferon, Pembrolizumab, Pertuzumab, Polatuzumab Vedotin, Ramucirumab, Retifanlimab, Rituximab, Sacituzumab Govitecan, Siltuximab, Cipleucel-T, Tafacitamab, Taguraxofusp, Tarimogen Rachelpalepbec, Toalquetamab, Teventafusp, Tecristamab, Tisagenlecleucel, Tisotumab This includes vedotin, trastuzumab, tremelimumab, aflibercept, or any combination thereof.

[0161] A specific example is a chemotherapy drug.

[0162] In some cases, chemotherapy drugs include anti-cancer agents.

[0163] In certain cases, anti-cancer agents include MMAE or MMAF.

[0164] Further examples of chemotherapy drugs include altretamine, bendamustine, busulfan, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, lomustine, lurubinectin, mechloretamine, melphalan, procarbazine, streptozocin, temozolomide, thiotepa, trabectedin, carboplatin, cisplatin, oxaliplatin, bleomycin, dactinomycin, daunorubicin, doxorubicin, and epil. Bicin, Idarubicin, Mitomycin, Mitoxantrone, Plicamycin, Barurubicin, Methotrexate, Pemetrexed, Pralatrexate, Trimethrexate, Azathioprine, Cladribine, Fludarabine, Mercaptopurine, Thioguanine, Azacitidine, Capecitabine, Cytarabine, Decitabine, Furoxuridine, Fluorouracil, Gemcitabine, Trifluridine / Tipiracil, Aldesleukin (IL-2), Denileukin Difutitex, Interferon-γ, Bellinostat, Panobinostat, Romidepsin, Vorinostat, Abiraterone, Apalutamide, Bicalutamide, Cyproterone, Enzalutamide, Flutamide, Nilutamide, Anastrozole, Exemestane, Fulvestrant, Letrozole, Raloxifene, Tamoxifen, Toremifene, Degarelix, Goserelin, Historelli Leuprolide, relugolix, triptorelin, lanreotide, octreotide, pasireotide, alemtuzumab, atezolizumab, avelumab, verantamab, bevacizumab, blinatumomab, brentuximab, semiprimab, cetuximab, daratumumab, dinutuximab, dostallumab, durvalumab, elotuzumab, enfortumab, gemtuzumab, inotuzumab Ozogamicin, ipilimumab, mogamulizumab, moxetumomab Pasdotox, necitumumab, nivolumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab Vedotin, ramucirumab, rituximab, sacituzumab Govitecan, tecristamag, tisotumumab Vedotin, tocitumomab, trastuzumab, trastuzumab Deruxtecan, trastuzumabEmtansine, Tremelimumab, Abemaciclib, Acalabrutinib, Adaglacib, Afatinib, Alectinib, Alpelisib, Aciminib, Axitinib, Binimetinib, Bortezomib, Bosutinib, Brigutinib, Cabozantinib, Carfilzomib, Ceritinib, Cobimetinib, Copanlisib, Crizotinib, Dabrafenib, Dacomitinib, Dasatinib, Dubellisib, Enasidenib, Encorafenib, Entrectinib, Eldafi Tinib, erlotinib, fedratinib, futibatinib, gefitinib, gilteritinib, glassegib, ibrutinib, idelalisib, imatinib, infiglatinib, ivosidenib, ixazomib, lapatinib, lalotrectinib, lenbatinib, lorlatinib, midostaurin, mobosertinib, momerotinib, neratinib, nilotinib, niraparib, olaparib, ortasidenib, osimertinib, pacritinib, palbociclib, pazopa Nib, pemigatinib, pexidartinib, ponatinib, regorafenib, ribociclib, lipretinib, rucaparib, ruxolitinib, selumetinib, sonidegib, sorafenib, sunitinib, thalazoparib, tivozanib, trametinib, trilaciclib, umbralicib, vandetanib, vemurafenib, bismodegib, zanubrutinib, cabazitaxel, docetaxel, paclitaxel, etoposide, irinotecan, teniposide, topotecan, bi This includes mblastin, vincristine, vinorelbine, asparaginase (peguaspargase), berzutifan, bexarotene, sedazulidine, eribulin, everolimus, hydroxyurea, ixabepyrone, lenalidomide, mitotane, omasetaxin, pomalidomide, selinexol, taglaxofusp, tazemetostat, teventafusp, tetrotristat, temsirolimus, thalidomide, venetoclax, or any combination thereof.

[0165] Furthermore, this specification provides a method for covalently linking a first payload with a second payload, wherein the first payload is given formula VI: [ka] [In the formula, R6 is H, C1~20 alkyl, C 2~20 alkenyl, C 2~20 alkynyl, 6- to 20-membered aryl, 7- to 20-membered alkylaryl, 3- to 20-membered cycloalkyl, C 1~20 acyl, C 1~20 alkoxy, 7- to 2o-membered aryloxy, C 1~20 alkylamino, C 2~20 dialkylamino, halogen, or amino selected from] reacting with a compound of to form a first precursor; reacting a second payload with a compound of formula VII:

Chemical formula

Chemical formula

[0166] In some examples, R6 is

Chemical formula

[0167] In a further example, R7 is [[ID=))

Chemical formula

[0168] In some examples, the first payload comprises a biomolecule.

[0169] In further examples, the biomolecule is a protein or an antibody.

[0170] In certain examples, the second payload is a surface, a particle, a drug, or a second biomolecule.

[0171] In a specific example, the drug is an immunotherapy drug.

[0172] In some examples, the immunotherapy drug comprises a Toll-like receptor agonist, such as a Toll-like receptor 7 or 8 (TLR7 / 8) agonist.

[0173] In further examples, the TLR7 / 8 agonist comprises an imidazoquinoline.

[0174] In certain cases, immunotherapy drugs include: brexcabutagen autolucel, trastuzumab, aldezleukin, amivantamab, atezolizumab, avelumab, axicabutagen silolucel, verantamab mahodotin, bevacizumab, blinatumomab, brentuximab vedotin, semiprimab, cetuximab, siltacabbutagen, daratumumab, daratumumab, dostallimab, durvalumab, elotuzumab, enfortumab vedotin, epcolitamab, gemtuzumab, grofitamab, ibritumomab tiuxetan, idekabutagen bicleucel, inotuzumab ozogamicin, ipilimumab, isatuximab, lysokabutagen maralucel, ronkastuximab Tecilin, Margetuximab, Milbetuximab Sorabtansine, Mogamulizumab, Mosnetuzumab, Nadofalagen Filadenobec, Naxitamab, Necitumumab, Nivolumab, Nivolumab and Relatrimab, Obinutuzumab, Ofatumumab, Panitumumab, Pegylated Interferon, Pembrolizumab, Pertuzumab, Polatuzumab Vedotin, Ramucirumab, Retifanlimab, Rituximab, Sacituzumab Govitecan, Siltuximab, Cipleucel-T, Tafacitamab, Taguraxofusp, Tarimogen Rachelpalepbec, Toalquetamab, Teventafusp, Tecristamab, Tisagenlecleucel, Tisotumab This includes vedotin, trastuzumab, tremelimumab, aflibercept, or any combination thereof.

[0175] A specific example is a chemotherapy drug.

[0176] In some cases, chemotherapy drugs include anti-cancer agents such as MMAEs or MMAFs.

[0177] Further examples of chemotherapy drugs include altretamine, bendamustine, busulfan, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, lomustine, lurubinectin, mechloretamine, melphalan, procarbazine, streptozocin, temozolomide, thiotepa, trabectedin, carboplatin, cisplatin, oxaliplatin, bleomycin, dactinomycin, daunorubicin, doxorubicin, and epil. Bicin, Idarubicin, Mitomycin, Mitoxantrone, Plicamycin, Barurubicin, Methotrexate, Pemetrexed, Pralatrexate, Trimethrexate, Azathioprine, Cladribine, Fludarabine, Mercaptopurine, Thioguanine, Azacitidine, Capecitabine, Cytarabine, Decitabine, Furoxuridine, Fluorouracil, Gemcitabine, Trifluridine / Tipiracil, Aldesleukin (IL-2), Denileukin Difutitex, Interferon-γ, Bellinostat, Panobinostat, Romidepsin, Vorinostat, Abiraterone, Apalutamide, Bicalutamide, Cyproterone, Enzalutamide, Flutamide, Nilutamide, Anastrozole, Exemestane, Fulvestrant, Letrozole, Raloxifene, Tamoxifen, Toremifene, Degarelix, Goserelin, Historelli Leuprolide, relugolix, triptorelin, lanreotide, octreotide, pasireotide, alemtuzumab, atezolizumab, avelumab, verantamab, bevacizumab, blinatumomab, brentuximab, semiprimab, cetuximab, daratumumab, dinutuximab, dostallumab, durvalumab, elotuzumab, enfortumab, gemtuzumab, inotuzumab Ozogamicin, ipilimumab, mogamulizumab, moxetumomab Pasdotox, necitumumab, nivolumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab Vedotin, ramucirumab, rituximab, sacituzumab Govitecan, tecristamag, tisotumumab Vedotin, tocitumomab, trastuzumab, trastuzumab Deruxtecan, trastuzumabEmtansine, Tremelimumab, Abemaciclib, Acalabrutinib, Adaglacib, Afatinib, Alectinib, Alpelisib, Aciminib, Axitinib, Binimetinib, Bortezomib, Bosutinib, Brigutinib, Cabozantinib, Carfilzomib, Ceritinib, Cobimetinib, Copanlisib, Crizotinib, Dabrafenib, Dacomitinib, Dasatinib, Dubellisib, Enasidenib, Encorafenib, Entrectinib, Eldafi Tinib, erlotinib, fedratinib, futibatinib, gefitinib, gilteritinib, glassegib, ibrutinib, idelalisib, imatinib, infiglatinib, ivosidenib, ixazomib, lapatinib, lalotrectinib, lenbatinib, lorlatinib, midostaurin, mobosertinib, momerotinib, neratinib, nilotinib, niraparib, olaparib, ortasidenib, osimertinib, pacritinib, palbociclib, pazopa Nib, pemigatinib, pexidartinib, ponatinib, regorafenib, ribociclib, lipretinib, rucaparib, ruxolitinib, selumetinib, sonidegib, sorafenib, sunitinib, thalazoparib, tivozanib, trametinib, trilaciclib, umbralicib, vandetanib, vemurafenib, bismodegib, zanubrutinib, cabazitaxel, docetaxel, paclitaxel, etoposide, irinotecan, teniposide, topotecan, bi This includes mblastin, vincristine, vinorelbine, asparaginase (peguaspargase), berzutifan, bexarotene, sedazulidine, eribulin, everolimus, hydroxyurea, ixabepyrone, lenalidomide, mitotane, omasetaxin, pomalidomide, selinexol, taglaxofusp, tazemetostat, teventafusp, tetrotristat, temsirolimus, thalidomide, venetoclax, or any combination thereof.

[0178] Several embodiments of this disclosure have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are also included in the following claims.

[0179] Examples of specific embodiments of the present disclosure are given below as non-limiting examples. [Examples]

[0180] The following examples are provided to illustrate the methods and results according to the subject matter of this disclosure. These examples are not intended to cover all aspects of the subject matter of this disclosure, but are intended to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the invention that would be apparent to those skilled in the art.

[0181] While efforts are made to ensure accuracy regarding quantities (e.g., volume, temperature), some degree of error and deviation must be considered. Unless otherwise specified, parts refer to parts by weight, temperatures are given in °C or as ambient temperature, and pressure is atmospheric pressure or near atmospheric pressure. Numerous variations and combinations of reaction conditions, such as component concentrations, temperature, pressure, and other reaction ranges and conditions, exist that can be used to optimize the purity and yield of the products obtained from the processes described herein. Only reasonable and conventional experiments are required to optimize such process conditions.

[0182] Example 1: CD276(B7-H3)-targeted dual-payload antibody-drug conjugate for chemoimmunotherapy of triple-negative breast cancer Introduction Triple-negative breast cancer (TNBC) is highly aggressive and heterogeneous, frequently recurring after standard radiotherapy and cytotoxic chemotherapy, and offers little clinical benefit. This specification discusses the development of innovative antibody-dual-payload conjugates (DualADCs) as chemoimmunotherapy for TNBC. Specifically, overexpression of the immune checkpoint transmembrane CD276 (B7-H3), associated with angiogenesis, metastasis, and immune tolerance, has been detected in over 60% of TNBC patients. We have developed and engineered novel monoclonal antibodies (mAbs) that target the extracellular domain of surface CD276, delivering a payload to upregulate tumor immunity.

[0183] Furthermore, we established an innovative platform for the simultaneous conjugation of conventional cytotoxic payloads and immunomodulatory Toll-like receptor 7 / 8 agonists with CD276 mAbs. Evaluation demonstrated that this therapy effectively kills diverse TNBC subtypes, significantly enhances immune function in the tumor microenvironment, and reduces tumor burden (or tumor load) by up to 90–100% in animal studies. Post-treatment analysis using single-cell RNA sequencing, Luminex multiplex cytokine assays, histological examination, and other analyses demonstrated an integrated anti-cancer mechanism. The developed DualADC may offer a promising targeted chemoimmunotherapy for TNBC patients in the future.

[0184] TNBC is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. Numerous surface receptors have been investigated to develop effective targeted therapies. Recently, the transmembrane protein CD276 (B7-H3, Uniprot: Q5ZPR3), composed of two Ig-like V extracellular domains and two Ig-like C2 extracellular domains, was detected in over 80% of breast cancer tissue. In this study, high expression of CD276 was observed in over 60% of TNBC patients (126 cases) and in various cell lines representing different subtypes, while expression was minimal to low in the 33 normal human organs examined. Furthermore, CD276 has been suggested to be associated with angiogenesis, invasion, metastasis, and poor prognosis in cancer patients. Furthermore, CD276 is an immune checkpoint molecule that inhibits the secretion of effector cytokines (IFN-γ, TNF-α, IL-4) and the immune function of natural killer (NK) and T cells. The combination of anti-CD276 enobrituzumab / anti-PD-1 retifanlimab, vobramitamab duocalmazine, and CD276 / CD3-targeted bispecific antibodies were evaluated in a Phase I trial (NCT02475213) for the treatment of head and neck cancer and in Phase I / II trials (MGD009, MGC018) for the treatment of multiple cancers. Herein, it has been demonstrated that targeting CD276 can upregulate tumor immunity across the majority of TNBC patients, indicating that targeting CD276 is a promising therapeutic strategy for aggressive TNBC. Therefore, what is being developed and engineered herein are novel CD276 mAbs for constructing combination chemoimmunotherapy.

[0185] Toll-like receptor (TLR) 7 / 8 agonists play a crucial role in the recruitment and activation of immune cells within the immunologically "cold" tumor microenvironment (TME). These agonists are less toxic than immune checkpoint blockers (ICBs), such as anti-PD-1 / PD-L1 mAbs. Furthermore, TLR7 / 8 agonists have been found to inhibit cancer cell proliferation, induce apoptosis, and stimulate the release of cytokines (e.g., IL-2 / 6 / 8 / 10 / 12 / 18, IFN-α / γ, TNF-α) by immune cells (Table 1). Despite these promising immunotherapeutic effects, administration of free TLR agonists lacking tumor selectivity can induce potentially fatal cytokine storms and other adverse side effects. To overcome this challenge, the present invention promotes the upregulation of tumor immunity in TNBC by precisely delivering a TLR7 / 8 agonist using an anti-CD276 mAb.

[0186] [Table 1] TIFF2026525313000021.tif241169TIFF2026525313000022.tif242169

[0187] The clinical efficacy of single-payload ADCs can be compromised by unpredictable compensatory mechanisms, the development of drug resistance during long-term treatment, and cancer-specific heterogeneity. To overcome these limitations of conventional ADCs, address off-target-induced immunotoxicity caused by free TLR agonists, and thereby improve tumor therapeutic efficacy, we have established an advanced conjugation platform of dual-payload ADCs (named DualADCs) in which a single mAb carries both highly cytotoxic chemotherapy and immunotherapy.

[0188] The objective of the present invention was to develop and evaluate an innovative immune checkpoint CD276-targeted dual-payload ADC for the chemoimmunotherapy of TNBC. A DualADC integrating cancer growth inhibition, enhancement of tumor cytokines, reactivation of immune cells, and regulation of the TME can effectively eradicate TNBC cells in vivo. An original mAb with cross-activity was developed and further engineered to target TNBC patients. This platform was established to conjugate the engineered mAb with synergistic dual therapy via two linkers. This all-in-one ADC reduced the tumor burden by 90 - 100% in TNBC xenograft mouse models, including patient-derived xenograft (PDX) models. The DualADC developed in the present invention is a feasible strategy for the treatment of aggressive TNBC. + This platform was established to conjugate the engineered mAb with synergistic dual therapy via two linkers. This all-in-one ADC reduced the tumor burden by 90 - 100% in TNBC xenograft mouse models, including patient-derived xenograft (PDX) models. The DualADC developed in the present invention is a feasible strategy for the treatment of aggressive TNBC.

[0189] Materials and Methods Cell Lines and Culture Medium Human TNBC cell lines, including MDA-MB-231 (ATCC, Cat# HTB-26, RRID:CVCL_0062, Manassas, Virginia, USA), MDA-MB-468 (ATCC, Cat# HTB-132, RRID:CVCL_0419), MDA-MB-231-FLuc (GenTarget, Cat# SC059-Puro, RRID:CVCL_YZ80, San Diego, California, USA), and MDA-MB-468-FLuc (GeneCopoeia, Cat# SL027, RRID:CVCL_C8XW, Rockville, Maryland, USA), were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS, v / v) and 1% Pen / Strep (v / v). Normal mammary epithelial cell line 184B5 (ATCC, Cat# CRL-8799, RRID: CVCL_4688) was maintained in MEGM BulletKit growth medium (Lonza, Walkersville, Maryland, USA) supplemented with 5% FBS. Mouse TNBC cell lines 4T1 (ATCC, Cat# CRL-2539, RRID: CVCL_0125) and 4T1-FLuc (ATCC, Cat# CRL-2539-LUC2, RRID: CVCL_5I85) were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% P / S. Mice carrying TNBC PDX (Jackson Lab, Cat# J000103917) were purchased from Jackson Lab (Bar Harbor, Maine, USA). The PDX was then collected, subcultured, and maintained in NSG mice or stored frozen in liquid nitrogen tanks. Expi293 cells for chimeric CD276 mAb production were maintained in Expi293 Expression medium supplemented with 4 mM GlutaMAX and 6 g / L glucose. CHO cells producing humanized anti-CD276 mAb were maintained in Dynamis medium supplemented with 4 mM L-glutamine and 6 g / L glucose. All cell lines were incubated in a humidified incubator (Eppendorf, Enfield, Connecticut, USA) at 37°C and under 5% or 8% CO2.All culture media, supplements, and biological reagents used in this study were purchased from Fisher Scientific (Waltham, Massachusetts, USA) unless otherwise specified. All cell lines or PDX cell lines were commercially available, certified by genetic profiling for polymorphic short tandem repeat analysis at University Genomics Core, and confirmed by in-house mycoplasma testing using PCR primers that amplify the 16S rRNA gene sequence. The most recent testing date for all cell banks, containing 30–100 stock vials, was November 21, 2022. The time from thawing of cells in the tested cell banks to their use in our experiments was 2–3 weeks.

[0190] Development, engineering, and production of anti-CD276 mAbs The immune response of BALB / cJ mice (Jackson Lab) was stimulated using a peptide cloned from the extracellular domain (Leu29-Pro245) of human CD276. Blood samples were collected from the tail vein 14–21 days after immunization, and the serum concentration of CD276 mAbs was titrated using ELISA. After mAb detection, splenocytes were collected and fused with multiple myeloma cells Sp2 / O-Ag14 (ATCC) to create hybridomas, which were then limitingly diluted in 96-well plates at a seeding density of 1–4 cells / well. The top hybridoma clones exhibiting high mAb titer and CD276 binding were sequenced. To minimize immunogenicity, improve serum stability, and enhance Fc-mediated antibody effector function, mouse anti-human CD276 mAbs were first engineered by constructing chimeric CD276 mAbs by grafting the complementary-determining region (CDR) with a truncated Fc region of human IgG1. Subsequently, humanized CD276 mAbs were constructed by combining the mouse framework region (FR) with three human CDRs.

[0191] Chimeric CD276 mAbs were produced from Expi293F cells using a transient production system in a 2 L stirred tank bioreactor (temperature 37°C, rotation speed 140 rpm, DO 40%, pH 7.2) or in a shaking flask culture (temperature 37°C, rotation speed 130 rpm, CO 28%). Stable CHO-producing cells were developed, and humanized CD276 mAbs were produced under the same conditions as described above. A liquid chromatography system (Bio-Rad, Hercules, California, USA) equipped with a Bio-Scale Mini UNOsphere SUPrA affinity chromatography cartridge (protein A column, Bio-Rad) was used for mAb purification according to the procedure established by the inventors. A mobile phase A (pH 7.5) of 0.02 M sodium phosphate and 0.02 M sodium citrate and a phase B (elution buffer) (pH 3.0) of 0.1 M sodium chloride and 0.02 M sodium citrate were used.

[0192] CD276 Targeted Single-Payload and Dual-Payload ADC Conjugation Single-payload ADC (CD276 mAb-MMAF) Four MMAF (monomethyl auristatin F) drugs were supported by crosslinking the interchain cysteine ​​of mAb cells using a bifunctional dibromomaleimide (DBM) linker. 5 mM TCEP dissolved in pH 7 DI water and 5 mg / mL of mAb cells in PBS were mixed in a molar ratio of 44:1 and reacted at 37°C for 0.5 hours to completely reduce the disulfide bonds in the mAb cells. A 10 mM commercially available DBM-MMAF payload was prepared in DMSO, and 7 molar equivalents were added to the completely reduced mAb cells and incubated at room temperature for 1 hour. Crude ADCs were purified using a Protein A column in a liquid chromatography system. The LC-purified ADCs were buffered in PBS using a 2 kDa Slide-A-Lyzer dialysis cassette and concentrated to higher concentrations using a 10 kDa MWCO PES concentrator. The purity, drug-antibody ratio (DAR), and homogeneity of ADCs were tested using HPLC (Shimadzu, Columbia, Maryland, USA) equipped with a MAbPac hydrophobic interaction chromatography (HIC)-butyl column (5 μm, 4.6 × 100 mm). Mobile phase A, consisting of 2 M ammonium sulfate and 100 mM sodium phosphate at pH 7.0, and mobile phase B, also consisting of 100 mM sodium phosphate at pH 7.0, were used for HPLC analysis. As an alternative approach, DAR was calculated using UV / Vis spectroscopy. Purified ADCs were filtered through a 0.2 μm PES syringe filter (basix) before intravenous injection into mice and stored at 4°C for short-term storage.

[0193] Single-payload ADC (CD276 mAb-IMQ) In this conjugation platform, mAbs and IMQs were conjugated using NHS-azide and NHS-phosphine reagents (Thermo Scientific). Mixture A was prepared by combining 5 mg / mL of mAb in PBS with 10 mM NHS-phosphine linker in a molar ratio of 1:14. Mixture B was prepared by combining 10 mM NHS-azide linker and 10 mM IMQ in 500 μL of PBS in a molar ratio of 14:22.4. Mixtures A and B were reacted at 37°C for 2 hours. The phosphine-labeled mAb in Mixture A was purified from excess NHS-phosphine using a 10 kDa MWCO PES concentrator. The phosphine-labeled mAb was mixed with 500 μL of Mixture B and incubated at 37°C for 2 hours to synthesize mAb-IMQ ADCs. These purification and concentration steps are the same as those used for mAb-MMAF ADC.

[0194] Dual-payload ADC (CD276 mAb-MMAF / IMQ) Mixture A was prepared by mixing 5 mg / mL of synthetic mAb-MMAF in PBS with 10 mM NHS-phosphine linker in a molar ratio of 1:14. Mixture B was prepared by combining 10 mM NHS-azid linker and 10 mM IMQ in 500 μL of PBS in a molar ratio of 14:22.4. Mixtures A and B were incubated at 37°C for 2 hours. Mixture A was then subjected to a 10 kDa MWCO PES concentrator to remove free linkers. Mixture B was added to phosphine-labeled mAb-MMAF ADC and incubated at 37°C for 2 hours. The crude dual-payload ADC was purified, buffered in PBS, and concentrated as described above.

[0195] Flow cytometry analysis of cell surface binding The surface binding of this anti-CD276 mAb in TNBC cell lines (MDA-MB-231, MDA-MB-468, 4T1) was tested by flow cytometry analysis according to a reported protocol. CD276 mAb was labeled with the fluorescent Alexa Fluor® 647 labeling kit (Life Technologies, Fisher). One million human or mouse TNBC cells were stained with 5 μg of anti-CD276 mAb-AF647 at 37°C for 60 minutes. After washing three times with PBS, the stained cells were analyzed using a BD LSRII flow cytometer (BD Biosciences, San Jose, California, USA), and FlowJo software was used for data processing. In our analysis of the surface binding rate of anti-CD276 mAb, gating was set so that unstained TNBC cells had a fluorescence population of <0.5%. In the analysis of TNBC specificity using anti-CD276 mAbs, standard forward and lateral scattering gating was applied with anti-HER2 mAbs as a negative control.

[0196] Confocal microscopy analysis of living cells Surface binding and internalization of CD276 mAb were evaluated using confocal imaging of living cells according to a protocol established by the inventors. TNBC MDA-MB-468 cells were placed in 35 mm glass-bottom dishes (Cellvis, Mountain View, California, USA) with 1 × 10 cells per dish in 1.5 mL of culture medium. 4 Cells were cultured at a density of 100 cells. To visualize the cytoplasm and nucleus, BacMam GFP Transduction Control (Invitrogen, California, USA) and NucBlue® Live ReadyProbes® Reagent (Invitrogen, California, USA) were used for staining according to the manufacturer's protocol. Cy5.5-labeled CD276 mAb was then added to the cells at a final concentration of 1 μg / mL. Live cell images were taken 2–12 hours after mAb addition using a Nikon A1R-HD25 confocal microscope (Nikon USA, Melville, New York, USA).

[0197] in vitro cytotoxic assay Human and mouse TNBC cells were seeded in 96-well plates at densities of 10,000 cells / well for MDA-MB-468, 1,000 cells / well for MDA-MB-231, and 500 cells / well for 4T1. Free MMAF, mAb-MMAF, and mAb-MMAF / IMQ were added to each well, with final doses ranging from 0 to 300 or 400 nM. Higher concentrations, i.e., 0 to 20 μM of free IMQ and mAb-IMQ, were also tested. Treated cells were incubated at 37°C and 5% CO2 for 5 days, and cell viability was analyzed using the MTT Cell Proliferation Assay Kit.

[0198] Patient-derived xenograft (PDX) models and in vivo treatments CD276 identified from Jackson Lab PDX cell lines by transcript analysis and IHC staining. +++ TNBC PDX was used according to the procedure disclosed by the inventors. Briefly, the PDX tumor was cut into small pieces (1x1x1mm). 3 The sac was cut into pieces, filled into a 1 mL sterile syringe connected to a 13 G needle (BD, Franklin Lakes, New Jersey, USA), and subcutaneously injected into the lateral dorsal region of 5-7 week old female NSG mice at a dose of 40-50 μL per mouse. The anti-TNBC efficacy of single-payload and dual-payload ADCs derived from humanized CD276 mAb was investigated for tumor volumes of 25-50 mm². 3 In the PDX model at the point where it was reached, the tumor volume was >1,500 mm 3 The condition was evaluated by intravenous injection of physiological saline, 16 mg / kg mAb-MMAF, or 16 mg / kg mAb-MMAF / IMQ in a Q7Dx3 schedule until the target was reached (n=5-7).

[0199] Cell line-derived xenotransplantation and in vivo treatment 4T1-FLuc xenotransplant immunonormal model 4T1-FLuc cells (2x10 per mouse) 6 The cells were subcutaneously injected (sc) into 6-week-old female BALB / cJ mice. The average tumor volume was 20-50 mm². 3 When the target was reached, mice were randomly divided into several groups (n=5, or 7 or 8) and intravenously injected with saline (control), 8 mg / kg mAb, mAb-IMQ, mAb-MMAF (control), or 8, 16, or 24 mg / kg mAb-MMAF / IMQ according to the Q7Dx4 schedule. Tumor size was measured by motorized caliper or IVIS imaging, and tumor volume was calculated (length × width). 2 The calculation was performed using the formula ) / 2. Tumors and mouse body weight were monitored twice a week. The control group had a tumor volume >1,000 mm². 3 Mice were sacrificed when the tumor reached a certain size or when the ulcer was >2 mm. All tumors, major organs (brain, heart, lungs, kidneys, liver, and spleen), whole blood, or serum were collected for further analysis.

[0200] MDA-MB-231-FLuc xenotransplant immunodeficiency model 5x10 6 Individual MDA-MB-231-FLuc cells were introduced into 6-week-old female NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl (SzJ) Subcutaneous injection was administered to mice with tumor volume of 50-100 mm. 3 When the tumor volume reached a certain level, the mice were randomly divided into 5 groups (n=5) and intravenously administered either saline (control), 16 mg / kg mAb-MMAF, or 16 mg / kg mAb-MMAF / IMQ according to a Q5Dx5 schedule. Tumor volume was monitored using motorized calipers or IVIS imaging, and mouse body weight was measured twice a week. If the tumor volume was >1,000 mm³, 3 Mice were sacrificed when the ulcer reached >2 mm, and tumors, organs, blood, or serum were collected for post-treatment analysis.

[0201] In vivo imaging system (IVIS) Tumor volume is 50-100 mm 3 When the mice reached a certain stage, 40 μg of engineered anti-CD276 mAb labeled with cyanine (Cy)-5.5 fluorescent dye (Lumiprobe, Hunt Valley, Maryland, USA) was intravenously injected into the mice via the tail vein. Twenty-four hours after injection, FLuc substrate was injected intraperitoneally, and live animals were imaged. The mice were then sacrificed, and major organs, including the brain, heart, lungs, kidneys, spleen, and liver, as well as tumors, were collected for ex vivo imaging. IVIS imaging was performed using excitation / emission wavelengths of 660 / 710 nm and an exposure time of 5 seconds.

[0202] Luminex assay To analyze tumor or serum cytokines, a Luminex-based multiplex assay (Luminex Corporate, Austin, Texas, USA) was used. Ready-made (EPX070-20835-901, EPX260-26088-901) and customized (PPX-03, PPX-13) chemocytokine assay kits covering 3, 7, 13, or 26 plexes were purchased from Thermo Fisher (Waltham, Massachusetts), and all assay reagents were prepared according to the manufacturer's instructions. Luminex assays were performed on tissue or serum samples (n=3) in 96-well plates equipped with the kits, and raw mean fluorescence intensity (MFI) data were read using a Luminex MAGPIX with XPONENT software.

[0203] Immunohistochemistry (IHC) staining TNBC (ER) - / PR - / HER2 -Patient tissue microarrays (TMA, Cat#BR1303, 126 cores) and 33 human normal organ tissue microarrays (Cat#FDA662c) were purchased from US Biomax (Darwood, Maryland, USA), and CD276 expression, or potential nonspecific binding of the inventors' humanized CD276 mAb, was detected by IHC staining (26,27). The collected tumor tissue was frozen fresh or fixed with 4% formalin. The fixed tissue was dehydrated with multiple ethanol solutions in a concentration gradient, embedded in paraffin blocks, sectioned to a thickness of 4-5 μm using a microtome, and placed on glass slides.

[0204] During IHC staining, TMA slides or paraffin-sectioned slides of tumors were first baked overnight at 60°C, deparaffinized with xylene, and hydrated with ethanol and deionized water. Subsequently, the sectioned tissues were subjected to antigen retrieval for 5 minutes in 0.01 M sodium citrate buffer (pH 6.0), gently washed with deionized water, and transferred to a solution (TBST, pH 7.6) consisting of 0.05 M Tris, 0.15 M NaCl, and 0.1% Triton-X-100. Nonspecific background staining was reduced by blocking endogenous peroxidase with 3% H2O2 for 15 minutes and incubating the slides with 5% normal goat serum for 45 minutes. All slides were incubated overnight at 4°C with anti-CD276 antibody (Abcam, rabbit monoclonal, Cat#ab226256, RRID:AB_3069232, 1 / 500 dilution) or our humanized CD276 mAb. After washing with TBST, the slides were incubated with HRP-conjugated goat anti-rabbit secondary antibody (Abcam, Cat#ab6721, RRID:AB_856214, 1:1000). Finally, the stained TMA slides were scanned with a Lionheart FX Automated Microscope (BioTek, Winooski, Vermont, USA), and the images were processed with Gen5 software. CD276 expression in the stained TMA was scored using ImageJ according to our previously established method (26). Briefly, the CD276 expression score was (red). 強度 -blue 強度 ) / blue 強度 The values ​​were calculated using x100, and scores greater than 10 were defined as high expression, scores between 6 and 10 as moderate expression, scores between 3 and 6 as low expression, and scores between 0 and 3 as no expression or minimal expression.

[0205] Characterization of ADCs by HPLC The purity and drug-antibody ratio (DAR) of ADCs were analyzed using a high-performance liquid chromatography (HPLC) system (Shimadzu, Columbia, Maryland, USA) with a 5 μm, 4.6 × 100 mm MAbPac HIC-butyl column. Two mobile phases were used in this analysis: mobile phase A, pH 7.0, containing 1.5 M ammonium sulfate and 50 mM sodium phosphate, and mobile phase B, pH 7.0, containing 50 mM sodium phosphate. The procedure was performed at a constant flow rate of 1.0 mL / min at a temperature of 25°C.

[0206] ADC characterization using MOLDI-TOF MS Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry was used to characterize and confirm the structure of ADCs by the inventors. Both ADC and mAb samples were diluted to 2 mg / mL with HPLC-grade water. 10 mg of sinapic acid was dissolved in 1 mL of HPLC-grade water containing 50% ACN (acetonitrile) and 0.1% TFA (trifluoroacetic acid). 10 μL of the sample was mixed with 10 μL of the sinapic acid matrix solution, and 4 μL of the mixture was spotted onto an MSP96 target and air-dried. The samples were analyzed using a linear positive-mode MALDI-TOF mass spectrometer (microflex LRF, BRUKER). Data were processed using flexAnalysis software.

[0207] Western blotting TNBC cells were washed three times with cold PBS and lysed in RIPA buffer to extract cellular proteins. Protein concentration was quantified using the bicinchoninic acid (BCA) method with the Pierce protein assay kit (Pierce, Cambridge, New Jersey). Next, 30 μg of protein per lane, along with a protein size marker (Bio-Rad Precision Plus), was loaded onto gradient SDS-PAGE and NuPAGE 4-12% gradient gels (Invitrogen, California, USA), and proteins were separated by molecular weight. Then, the separated proteins were transferred to a PVDF membrane using a 100V constant voltage Bio-Rad power supply (Bio-Rad Laboratories, California, USA) for 90 minutes. After transfer, the PVDF membrane was blocked with 5% skim milk in TBST buffer and stirred at room temperature (RT) for 1 hour. For the primary antibodies, CD276 (dilution 1:1000, AB134161, Abcam, Cambridge, UK) and β-actin (1:2000, sc-47778, Santa Cruz, California, USA) were used. The membrane was then incubated overnight at 4°C with continuous agitation. The following day, the primary antibodies were discarded, and the membrane was washed three times with TBST buffer on a shaker for 5 minutes, 5 minutes, and 10 minutes, respectively. After the washing step, a horseradish peroxidase (HRP) conjugate secondary antibody (dilution 1:2000) specific to mice or rabbits, manufactured by Cell Signaling Technology (CST Inc, Danvers, Massachusetts, USA), in 3% skim milk was applied to the membrane at room temperature (RT) for 1 hour. After discarding the secondary antibody, the membrane was washed three times with TBST for 5 minutes, 5 minutes, and 10 minutes. Finally, protein bands were visualized and quantified using the Odyssey Fc imaging system (LI-COR Biosciences, Nebraska, USA). CD276 expression in TNBC cells was compared to that of the internal control, β-actin.

[0208] whole blood analysis To evaluate the potential peripheral toxicity of the anti-CD276 mAb, free TLR7 / 8 agonist, and ADC developed by the inventors, whole blood analysis was performed using BALB / cJ mice. Specifically, 8 mg / kg of the mAb, 8 mg / kg of the IMQ, and 8–24 mg / kg of single-payload or dual-payload ADC were intravenously injected into each mouse via the tail vein using physiological saline as a control. Ten or 21 days after injection, blood samples were collected by cardiac puncture for whole blood analysis using HemaVet 950FS (Drew Scientific, Miami Lakes, Florida, USA). Blood levels of leukocytes (white blood cells, neutrophils, lymphocytes, monocytes, eosinophils), erythrocytes (red blood cells, hemoglobin, mean corpuscular hemoglobin), and thrombocytes (platelets) were measured and analyzed.

[0209] Hematoxylin and Eosin (H&E) staining Paraffin-sectioned organ slides were dewaxed with xylene, hydrated with multiple concentrations of ETOH (100% to 50%) and dH2O, and then stained with hematoxylin. The slides were immersed in 1% HCl and 70% ETOH, then immersed in 1% NH4OH to develop a blue color, and finally stained with eosin for 30 seconds. Lastly, the stained slides were dehydrated with 95% and 100% ethanol and cleared with xylene. The H&E-stained slides were imaged using a Lionheart FX Automated Microscope (BioTek, Winooski, Vermont, USA).

[0210] Single-cell RNA sequencing and data analysis Preparation of single-cell samples A rapidly frozen tissue sample (25 mg) was collected and stored at -80°C until processing. Using the Chromium Next GEM RNA Profiling Sample Fixation Kit (PN-1000414, 10x Genomics, Pleasanton, California, USA), the tissue was fixed by mixing 25 mg of tissue with 1 mL of fixation buffer. The tissue was then finely chopped and incubated at 4°C for 16–24 hours without agitation. After fixation, the tissue sample was centrifuged, washed with cooled PBS, and resuspended in 1 mL of tissue resuspension buffer. The fixed tissue, to which pre-warmed dissociation buffer had been added, was dissociated using an Octo Dissociator according to the manufacturer's instructions. The dissociated tissue sample was filtered through a 30 μm filter, centrifuged, and resuspended in 1 mL of cooled quenching buffer. Cell concentration was determined using an automated cell counter along with fluorescent nucleic acid staining. The dissociated tumor samples were stored at -80°C in 50% glycerol containing an enhancer until single-cell libraries were prepared.

[0211] Building a single-cell library A 10X single-cell library was prepared using the Chromium Fixed RNA Kit (Cat#1000497, 10X Genomics). Briefly, approximately 16,500 cells were hybridized by probe hybridization targeting polyadenylated RNA, and loaded onto Chromium X (PN-1000326, 10X Genomics) to create barcode-labeled single-cell gel beads-in-emulsion (GEM) containing approximately 10,000 target cells. The GEM was then lysed using a recovery agent to release the hybridized RNA. cDNA was synthesized and amplified, and then indexed by sample index PCR. The quality control of the cDNA was evaluated using an Agilent Bioanalyzer, targeting the final library molecule containing P5 and P7 primer binding sites used in Illumina sequencers. For each cDNA sample, single-cell library construction was initiated using a 10x barcoded and ligated probe product. Sequencing was performed using a NovaSeq 6000 flow cell 100-cycle kit (Illumina, San Diego, California, USA) in 10X Genomics with a Read1:i7:i5:Read2 format of 28:10:10:90 bp. Subsequently, the demultiplexed fastq files were used for analysis.

[0212] Data preprocessing and cell annotation Three FASTQ files, containing raw read information for index reads, forward reads from paired-end sequences, and reverse reads from paired-end sequences, were processed using 10X Genomics Cloud Analysis, integrated into Cell Ranger Multi version 7.1.0. The mouse reference genome mm10 2020-A was used for alignment. Sequence quality control (QC) was performed using Windows and Linux®-based FastQC (version 0.12.1). Count-by-gene matrices were analyzed using the "Seurat" package (version 4.3.0.1, PMID:34062119) in R (version 4.3.0). Data normalization and scaling were performed using the SCTransform function developed by Christoph Hafemeister and Rahul Satija. Annotation was performed on different cell types using CellMarker 2.0, PanglaoDB, and the Tabula Muris database.

[0213] Differential gene expression and GO pathway enrichment Differential expression genes were identified across all cell types and integrated cell populations using Seurat's built-in functions, PrepSCTFindMarkers and FindMarkers. All tests were performed based on the Wilcoxon rank-sum test. In particular, the min.pct and logfc.threshold parameters were set to 0 for further GSEA pathway enrichment. GSEA pathway enrichment was performed using the gseGO function, which is included in the clusterProfiler package (version 4.8.2), with an adjusted p-value of 0.05 sourced from the gene ontology database (https: / / geneontology.org / ).

[0214] Pharmacokinetics (PK) Five doses of CD276 mAb-MMAF / IMQ, including 4, 8, 16, 24, and 32 mg / kg, were intravenously injected into 4T1 xenografted BALB / cJ mice. Approximately 10–15 μL of blood samples were collected via the tail vein, submandibular, or submental vein at 0.5, 2, 8, 24, 48, 72, and 120 hours after injection. The supernatant was used to titrate DualADC using ELISA with human CD276 receptor and anti-MMAF antibody (Fisher, Cat#PIMA542537, RRID:AB_2687990). The following key parameters were calculated using reported PK models:

number

number

number

number

number

[0215] result Concept Overview of Dual Payload ADC Figure 1 illustrates the concept of this CD276-targeted Dual ADC for chemoimmunotherapy. In this study, an engineered CD276 mAb, named CD276 mAb-MMAF / IMQ, carrying either the potent molecule MMAF, the immunoenhancing reagent TLR7 / 8 agonist IMQ, or both payloads, targets the overexpressed surface receptor CD276 on TNBCs. Following internalization (internal translocation) via receptor-mediated endocytosis and lysosomal degradation, the payloads MMAF and IMQ are released into the cytoplasm. Intracellular MMAF induces direct cancer cell death by inhibiting microtubule polymerization, while IMQ, as previously reported, targets TLR7 / 8 in endosomes and then inhibits cell proliferation via the MyD88 / IRF7 signaling pathway. IMQ (the free drug released by TNBC cell death, or the drug conjugated in the ADC) upregulates cytokine secretion by immune cells in the TME. Furthermore, inhibition of the CD276 immune checkpoint by mAbs reactivates NK cells and T cells in tumors.

[0216] Development of a dual-payload ADC A novel sequential conjugation procedure for constructing DualADCs was developed by linking mAbs to MMAF using a re-bridging dibromomaleimide (DBM) linker, followed by conjugation of IMQ using the phosphine-azide linker designed in this study (Figures 3A-B). HPLC characterization demonstrated the success of both single-payload (IMQ or MMAF) and double-payload (IMQ and MMAF) conjugations (Figure 3C). MS evaluation confirmed the precise structures of mAbs, mAb-MMAF, mAb-IMQ, and mAb-MMAF / IMQ with the expected MZ values ​​(Figure 3D). The integrity of the mAbs and ADCs was confirmed by SDS-PAGE (Figure 3E). Site-directed conjugations of MMAF exhibited homogeneous structure, 95–100% purity, and a DAR of 3–4, while random conjugations of IMQ exhibited heterogeneous structure and a DAR of 7–14. Using random conjugations, the characteristics and DAR (8–12) of IMQ were optimized to achieve high solubility, stability, and tumor immunity.

[0217] In vitro anti-TNBC cytotoxicity of ADC The cytotoxicity of free MMAF and TLR7 / 8 agonists (control), CD276 mAb-MMAF and mAb-IMQ (control), and dual-payload mAb-MMAF / IMQ was tested using MDA-MB-231, MDA-MB-468, and 4T1 cells. MMAF showed IC50 levels of 151.0, 143.0, and 103.7 nM for these three cell lines, respectively. 50 This was shown (Figure 4A). From the TLR7 / 8 agonists tested, two IMQs with high TNBC cytotoxicity were identified, including AXC715 and R848 (Figure 12). IC50 was 10.4, 6.2, and 11.4 μM in three TNBC cell lines. 50 The IMQ1 values ​​shown (Figure 4B) were used in single and dual-payload ADCs. IMQ was found to have EC values ​​of 24.5 and 6.7 μM in human and mouse HEK293 cells overexpressing TLR8. 50It had a value (Figure 4C). Humanized CD276 mAb (Hu276 mAb)-MMAF IC 50 The values ​​were 175.0 nM (MDA-MB-231), 50.7 nM (MDA-MB-468), and 125.4 nM (4T1), as shown in Figure 4D. Similar to the free IMQ, the Hu276 mAb-IMQs were 7.7 μM (MDA-MB-231), 6.9 μM (MDA-MB-468), and 13.1 μM (4T1) for IC. 50 The values ​​were shown for ICs with 18.8nM (MDA-MB-231), 16.9nM (MDA-MB-468), and 40.8nM (4T1). 50 DualADC Hu276 mAb-MMAF / IMQ (Figure 4F) exhibited the highest cytotoxicity or efficacy against TNBC cells compared to free-release drugs and single-payload ADCs.

[0218] In vivo anti-TNBC efficacy of DualADC in a TNBC PDX model PDX models that replicate tumor heterogeneity and the tumor microenvironment are essential for fully evaluating newly developed targeted therapies. As shown in Figure 5A, the PDX tumor volume of the untreated (saline group) was approximately 1,700 mm³ at 29 days post-treatment. 3 The results showed that both Hu276 mAb-MMAF and Hu276 mAb-MMAF / IMQ completely inhibited tumor growth, with a final volume of approximately 0 mm³ on day 7. 3 Furthermore, no recurrence was observed 15 to 29 days after discontinuation of treatment (Figures 5A and 5C). There was no difference in body weight profile between the ADC and saline groups (Figure 5B). Positive expression of CD276 was confirmed by IHC staining of untreated TNBC PDX (Figure 5D). These data demonstrate that our humanized CD276 mAb-directed ADC can effectively target and treat TNBC PDX.

[0219] In vivo anti-TNBC efficacy of DualADC in a TNBC immunonormal model. The synergistic chemoimmunotherapy of dual-payload ADCs was further evaluated in a female BALB / cJ mouse model (n=5-8) xenografted with 4T1-FLuc. The final tumor volume was 642 mm². 3 Compared to the saline group, CD276 mAb-MMAF / IMQ at 16 and 24 mg / kg reduced tumor load by 142 mm 25 days after the first injection. 3 It can be seen that the tumor volume was reduced to this extent (Figure 6A). mAb-MMAF / IMQ at 8 mg / kg, mAb-MMAF, and mAb-MMAF / IMQ showed moderate therapeutic effects, with final tumor volumes ranging from 241 to 376 mm². 3 There were no significant differences in body weight between the DualADC, singleADC, and saline groups (Figure 13). The two engineered mAb (chimeric and humanized) directional DualADCs (16 mg / kg) had comparable anti-TNBC efficacy (Figure 6B). Further pathological evaluation by H&E staining of major organs (brain, heart, liver, kidney, lung, and spleen) revealed that in the 24 mg / kg DualADC group, splenomegaly was observed at the endpoint, but there was no inflammation, apoptosis, necrosis, injury, or toxicity (Figure 6C).

[0220] Anti-cancer mechanism of dual-payload ADCs Underlying anti-cancer mechanisms were investigated using diverse research approaches, including H&E staining for tumor cell death, IHC staining with various antibodies to analyze tumor immunity, Luminex assays to titrate tumor cytokine secretion and test cytokine storms, whole blood analysis, and single-cell RNA sequencing (scRNA-Seq) to analyze immune cell infiltration, immune response, and mitotic activity in TMEs. All data suggested combined chemo-immunotherapy with dual ADCs for the treatment of TNBC.

[0221] Firstly, IHC staining of tumor tissue showed clear infiltration of activated cytotoxic CD8 T cells (CD8, CD45), activated NK cells (CD45), and macrophages (F4 / 80) in the 24 mg / kg DualADC group (Figure 6D). DualADC also reduced tumor intensity and slightly downregulated PD-1 expression. In TNBC tumors treated with DualADC, the expression of the proliferation marker (Ki67) was downregulated, and the apoptosis marker (CCasp3) was significantly upregulated. These data demonstrate that our CD276-targeted DualADC mAb-MMAF / IMQ effectively modulates TNBC tumor immunity. H&E staining of tumor tissue showed healthy TNBC cells in the saline group, a moderate level of cell death in the single-payload ADC (mAb-MMAF, mAb-IMQ) group, and severe cell death in the Dual ADC (mAb-MMAF / IMQ) group (Figure 6E).

[0222] Secondly, Luminex assays of tumor tissue collected at the end of treatment showed significant enhancement of several oncokinetic cytokines, including TFN-γ, TNF-α, and IL-6, confirming targeted delivery of IMQ by mAbs and its immunotherapy in TNBC tumors (Figure 7A). Further oncokinetic cytokine (TFN-γ, TNF-α, IL-6, IL-10, IL-2, IL-4, MCP-1) profiles from TNBC obtained from the DualADC group are summarized in Figure 14. These results confirm that TLR7 / 8 agonists enhance cytokine secretion in TME. At the end of the animal experiment, approximately 4.2–6.0 μg of IMQ was detected in TME (not cell lysate) from 1 mg of tumor sample, but further advanced analysis of the dynamic distribution of the drug is needed in future studies.

[0223] Thirdly, CBC analysis of whole blood samples, as summarized in Figure 7B, showed that neither mAb-MMAF, mAb-IMQ, nor mAb-MMAF / IMQ significantly altered erythrocytes (red blood cells, hemoglobin, hematocrit) nor thrombocytes (platelets, platelet crit). The leukocyte count (white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils) in mice treated with 24 mg / kg dual-payload ADC was higher than in mice treated with 16 mg / kg dual-payload ADC. No anemia or thrombosis was observed during ADC treatment. Chemocytokine analysis of serum samples demonstrated that, with the exception of increased CCL4 in mice treated with 16 and 24 mg / kg DualADCs, and increased CXCL1 in the 24 mg / kg DualADC group, most of the 25 titrated chemokines showed no significant differences across the groups (Table 1). All of these data demonstrate the safety and minimal systemic toxicity of CD276-targeted mAbs, single-payload ADCs, and DualADCs at doses of 8–24 mg / kg.

[0224] Finally, scRNA-Seq of TNBC identified tumor cells, myoepithelial cells, endothelial cells, fibroblasts, stromal cells, and CD8 +All cell types in tumor tissue, including T cells, dendritic cells, neutrophils, macrophages, and leukocytes, were identified and quantified (Figure 8A). The percentage of immune cells infiltrating the TME in the 16 mg / kg DualADC group was 43.1%, significantly higher than the 15.3% in the saline group. These data were consistent with IHC staining shown in Figure 6D, but provided accurate numbers of immune cells. Figure 8B summarizes the count and gene ratio of tumor cells involved in various immune functions, including spindle midzone assembly, mast cell activation, adaptive immune response, immune response regulatory cell surface receptor signaling pathways, immune response activation, and immunity mediated by B cells, immunoglobulins, leukocytes, myeloid leukocytes, and lymphocytes. Macrophages (Figure 8C), neutrophils, CD8 + Immunomodulatory functions of T cells, dendritic cells, leukocytes, and other cells were observed in the tumor. Analysis of tumor cell distribution at different mitotic stages confirmed that MMAF inhibits cell proliferation (Figure 8D).

[0225] Verification of DualADC's anti-TNBC efficacy in immunodeficiency models. The anti-TNBC efficacy of 276 mAb-MMAF / IMQ was investigated in an immunodeficiency model using female NSG mice xenografted with MDA-MB-231-FLuc. Figure 15A shows that the TNBC tumor volume was 15-17 mm on day 22 in the group treated with 16 mg / kg ADC. 3 It was reduced to 660 mm in the physiological saline group. 3 This indicates that the following was the case. Fourteen days after discontinuation of treatment, no recurrence of TNBC was observed. There was no significant difference in weight change between the treatment group and the saline group (Figure 15B). IVIS imaging (Figure 15C, left) and white light imaging (Figure 15C, right) at the endpoint verified the high anti-TNBC efficacy of CD276-targeted ADCs, such as the reduction in tumor volume and tumor burden.

[0226] Pharmacokinetics (PK) PK parameters were evaluated using a BALB / cJ model with xenotransplanted 4T1 cells. The serum concentration profiles of CD276 mAb-MMAF / IMQ are shown in Figure 16A. As summarized in Figure 16B, t 1 / 2 =1.21~2.99 days, C max The values ​​were 21.39–93.37 μg / mL, D=6.48–16.66 mg / kg, and τ=5.13–7.10 days. None of the tested doses of DualADC had any significant effect on the mice's body weight, survival rate, or overall health, including water intake, respiration, and exercise, and none reached the maximum toxic dose.

[0227] Consideration While targeted therapies such as mAbs, ADCs, and small molecule inhibitors have been developed for the treatment of solid tumors, none have been proposed for the treatment of primary and metastatic TNBC due to the lack of promising targets. In this example, CD276 receptor overexpression was confirmed in the majority (over 60%) of TNBC patient tissue and in various TNBC subtypes, which is consistent with TCGA transcript analysis and literature reports that CD276 is found in 80% of breast cancers. Furthermore, CD276 inhibits the immune function of NK cells and T cells and reduces the secretion of effector cytokines as an immune checkpoint. In this example, an innovative CD276 targeted therapy was developed by establishing a novel platform that conjugates a dual payload with an mAb, aiming to eliminate TNBC cells in vivo through a synergistic anti-cancer mechanism. Cross-activation of CD276 mAbs allowed the inventors to evaluate the anti-TNBC efficacy and mechanism of DualADC in a mouse model. This therapy utilizes CD276 + It demonstrated high specificity against tumors, effective tumor cell death, clear immune cell infiltration, and cytokine secretion in TEM. Tumor burden (tumor load) was significantly reduced in all animal studies, highlighting its great potential as a targeted therapy for the treatment of TNBC.

[0228] Unlike conventional ADCs that use mAbs carrying a single payload, our DualADC consists of a CD276 mAb, a highly potent drug (MMAF), and an immune booster TLR agonist (IMQ). The concept of targeting and treating TNBC (and other cancers) with DualADC is innovative for several advantages. First, DualADC combines multiple different drugs with synergistic cancer treatment mechanisms to upregulate tumor immunity while simultaneously inducing direct cancer cell death. Integrating chemotherapy and immunotherapy into a single molecule improves circulatory stability, reduces side effects, and enhances anticancer efficacy, particularly against highly aggressive and heterogeneous cancers. Second, the DualADC platform established here allows for the conjugation of two different drugs at two sites (e.g., cysteine ​​and lysine). Unlike site-selective conjugation or the fusion of two payloads, this conjugation strategy allows for the optimization of the ratio of the two payloads. Because cancer cells and immune cells may have different responses to dual payloads, the flexibility to adjust the ratio of the two payloads allows for the achievement of optimal anticancer efficacy. In the future, it will be necessary to evaluate the impact of conjugation strategies, linkers, different payload combinations, and the ratio of the two payloads on circulatory stability, potential toxicity, and cancer treatment efficacy. Thirdly, by targeting CD276, 60% of TNBC patients (and other CD276 + It can cover cancer, improve therapeutic efficacy through targeted drug delivery to the tumor microenvironment, and reduce dose and dosage. Fourthly, engineered anti-CD276 mAbs can improve their plasma stability, biological function, and translational potential, which require further evaluation in advanced animal models.

[0229] Importantly, investigations into the anti-cancer mechanisms of DualADC using scRNA-Seq, Luminex assays, histological analysis, whole blood analysis, and others identified multiple anti-TNBC mechanisms: 1) direct cancer cell killing and proliferation inhibition by a potent payload; 2) combined tumor immunity by CD276 mAb and TLR7 / 8 agonists; and 3) TLR agonist-mediated enhancement of oncokinetic cytokines.

[0230] First, anti-CD276 mAbs can effectively and specifically target TNBC xenografts with minimal off-target effects, delivering the payload in vivo. Furthermore, clinical data and literature reports have indicated that blocking CD276 can neutralize inhibitory signaling and reactivate immune cells to restore effector immune function. This study demonstrated that anti-CD276 mAbs reactivate immune function in TMEs by increasing the infiltration of activated NK cells and T cells. In addition to treating cancer as monotherapy, combinations of CD276 mAbs with other therapies such as enobrituzumab / retifanlimab and vobramitamab duocalmazine show great potential in preclinical or clinical research. In the future, to benefit most (75-90%) of TNBC cancer patients who do not respond to ICB due to primary resistance, acquired resistance, and relapse during treatment, we plan to conduct further investigations in our future research using our proprietary ADC to dually target CD276 and PD-1.

[0231] Pattern recognition receptor (PRR) agonists, such as TLR agonists, have been developed to treat cancer and other diseases. They target the innate immune system and stimulate immune responses via MYD88 and other pathways. PRR agonists, including TLR7 / 8 / 9 agonists, RIG-I, MDA-5, and STING, have been investigated in preclinical and clinical studies. Administration of free TLR agonists lacking tumor selectivity can cause lethal cytokine storms and other adverse effects. The conjugation linker and strategy developed in this study enable direct delivery of TLR agonists to tumors, overcoming the challenge of systemic toxicity. Consistent with the literature, this embodiment demonstrates that a TLR7 / 8 agonist targeted and delivered using an anti-CD276 mAb inhibits TNBC cancer growth, induces apoptosis in tumor cells, and stimulates the production of multiple cytokines by activated immune cells in the TME. Furthermore, tumor-associated antigens released by dead tumor cells can trigger a cascade of adaptive immune responses through antibody-dependent cell phagocytosis.

[0232] In addition to their immunotherapeutic properties, both the potent payload (MMAF) and the immunoenhancing reagent (IMQ) demonstrated cytotoxicity against TNBC cells. MMAF effectively blocks microtubule polymerization, inhibiting the mitotic process and proliferation of TNBC cells. This study confirmed the cytotoxicity and direct cell death of both free MMAF and single-payload ADCs (mAb-MMAF). In addition to upregulating immune function, TLR7 / 8 agonists and single-payload ADCs (mAb-IMQ) also demonstrated cytotoxicity against TNBC cells by inhibiting proliferation, reducing cell viability, and increasing apoptosis. Combining MMAF and IMQ in a single Dual ADC is more efficient in killing TNBC cells by integrating multiple different anti-cancer mechanisms, which has great potential to circumvent drug resistance, overcome cancer recurrence, and improve survival rates. Alternatively, heterogeneous TNBCs with low CD276 expression may be targeted by other mAbs via alternative receptors such as EGFR, Trop-2, LSR, or GRP56, which could be investigated in future studies.

[0233] This example demonstrated the high plasma stability of DualADC in PK studies and its high TNBC specificity in in vivo distribution studies. These characteristics could benefit the therapeutic efficacy of TBNC in future preclinical or clinical studies. Although the maximum tolerated dose (MTD) has not been reached, all doses of DualADC tested did not show toxicity in major organs, blood cell counts, serum cytokines, body weight, survival rate, or overall health. To define a safe dose for cancer treatment, future studies on the maximum tolerated dose (MTD) are necessary. Toxicological studies are also strongly desired to fully evaluate potential toxicity, including its effects on antigen-presenting cells with low CD276 expression.

[0234] In summary, this embodiment developed an innovative therapy—a CD276-targeted dual-payload antibody-drug conjugate—combining chemotherapy and immunotherapy into a single molecule, with the aim of eliminating aggressive and heterogeneous TNBCs. Promising anti-TNBC efficacy and minimal side effects were validated in multiple mouse models and post-treatment analyses. The concept of targeted delivery of a synergistic dual payload is novel and readily translateable to clinical practice.

[0235] Example 2: Purification of mAbs and ADCs using liquid chromatography (LC) mAb: Monoclonal antibody ADC: Antibody-drug conjugate LC: Liquid Chromatography Column selection for one-step or two-step purification: Primary purification: UNOsphere SUPrA Cartridge (Affinity) and other Protein A columns Intermediate and final purification: Nuvia S (cation exchange)

[0236] [Table 2]

[0237] [Table 3] Note: Confirm the isoelectric point (pI) of the humanized antibody.

[0238] Steps: 1. Purification procedure for mAb / ADC - Protein A column: Note: If two-step purification is required, follow the equilibration, loading, washing, and elution steps of Step 2, which are detailed in Table 2.

[0239] 1.1 Column equilibration: Use 10 CV of buffer A1. 1.2 Injection of mAb sample: Add the mAb sample as is, or diluted 1:10 or 1:5, to buffer A1. 1.3 Column washing: Wash with buffer A1 until absorbance returns to baseline, or 5-6 times the column volume. 1.4 Elution of mAb: Linear gradient to 100% B or desired % of buffer B at 10 or 5 CV. Elute mAb with neutralizing buffer. 1.5 strips: Use buffer B with 5 CV. 1.6 Desalting and Formulation of Purified mAbs: Remove salt using a PD SpinTrap® G-25 Desalting Column, or dialyze for buffer exchange immediately after LC / protein A purification. Adjust pH to match the formulation buffer. Add 10% 10X formulation buffer for long-term storage at -20°C or 4°C. Note: Formulation buffer and pH may need to be optimized.

[0240] The mAb was titrated using NanoDrop, and its purity / quality was confirmed using SDS-PAGE.

[0241] Note: Buffer A1 was used for binding human and guinea pig IgG. Buffer A2 was used for all other samples.

[0242] The recommended column equilibration interval was excessive under most conditions, but it was used as the default until specific equilibration requirements were established.

[0243] Furthermore, attention was paid to the solubility limits (A2) of antibodies that require high salt concentrations for binding. If the antibody could not remain completely dissolved for the longest possible period from equilibration to the completion of sample loading after adjusting for the loading conditions, online dilution techniques were used for loading.

[0244] Initial selective screening was performed using a linear gradient.

[0245] Citric acid is chosen as the low-pH buffer because it allows for a wide pH range to be achieved with a phosphate / citric acid system. If a higher pH range is required, boric acid is added to the binding buffer.

[0246] Cleaning of the LC system (every 6 months, or as needed) To ensure that all bound substances are released and washed away from the column, the following cleaning-in-place (CIP) protocol was recommended. The following protocol is proposed for removing precipitated or denatured substances from the bed.

[0247] 2.1 Wash the bed with a reverse flow of 2 to 5 times the column volume using one of the following solutions: 6M guanidine hydrochloride 10 mM hydrochloric acid 0.1M sodium hydroxide 1M acetic acid / 20% ethanol 2.2 Next, wash with a reverse flow of a binding buffer (e.g., buffer A1) with a neutral pH (7-8) at least 5 times the column volume. 2.3 To remove any hydrophobicly bound substances from the bed, the column is washed with a reverse flow of nonionic surfactant / detergent in a volume 2 to 5 times the column volume, followed by a reverse flow of a neutral pH binding buffer in a volume at least 5 times the column volume. 2.4 A contact time of 15 minutes per cycle is proposed at room temperature.

[0248] Note: -Disinfection (if necessary)- To reduce the possibility of microbial contamination of the cartridge, the column can be periodically washed with a solution consisting of 0.1 M sodium hydroxide. After standing for 1 hour, wash with buffer until the pH becomes neutral. -Column Storage- To store UNOsphere SUPrA for extended periods, equilibrate the medium with 20% ethanol / water and store at 4°C. - Preparing the Column for Use - All columns are ready for use once they have been equilibrated with the selected buffer solution. To perform a buffer change, connect the column to the liquid chromatography system or peristaltic pump and condition it as follows: - Set the pump flow rate to 0.5-1 mL / min (59.5-119 cm / hour) for a 1 mL column, or 2.5-5 mL / min (297.5-595 cm / hour) for a 5 mL column. - Wash the column with degassed low-salt buffer solution in an amount equal to twice the column volume. Then wash it again with degassed low-salt buffer solution in an amount equal to five times the column volume. - Equilibrate with a low-salt buffer solution five times the volume of the column. - Reduce the flow rate to the flow rate that will be used in the purification protocol.

[0249] Sample preparation For consistent and reproducible results, the correct pH and ionic strength were required. Samples were either replaced with the initial buffer or diluted to the concentration of the initial buffer. This was achieved by diluting the sample to the ionic strength of the initial buffer, dialyzing against the initial buffer, or replacing the sample with the initial buffer. All samples were filtered through a 0.45 μm filter before being applied to the column.

[0250] Stationary cleaning and disinfection If reproducible results can no longer be obtained from the column, it may be necessary to subject the medium to thorough CIP and disinfection to remove strongly bound contaminants. Acceptable CIP agents include 25% acetic acid, 8M urea, 1% Trition X-100, 6M potassium thiocyanate, 70% ethanol, 30% isopropyl alcohol, 1N NaOH, and 6M guanidine hydrochloride.

[0251] 1. Disinfect the support in the column with 1.0N NaOH in an amount 2 to 4 times the bed volume, and maintain contact time for at least 40 minutes. 2. To re-equilibrium the column, wash it with a 0.5-2M NaCl solution (containing 50-100 mM buffer) in an amount 2-4 times the bed volume. 3. If lipid removal is necessary, wash the column with a 20-70% ethanol solution.

[0252] Example 3: Dual Payload ADC DualADC anti-cancer mechanism Figure 1 shows the synergistic mechanism of anti-CD276 mAb-MMAF(DM1) / TLR7 / 8 agonist for the treatment of TNBC, where 1. shows the targeting and binding of DualADC to the surface receptor CD276 on TNBC cells, 2. shows internalization, 3. shows drug release, 4. shows inhibition of microtubule polymerization induced by MMAF, 5. shows cytotoxicity induced by the TLR7 / 8 agonist, and 6. shows activation of immune cells by anti-CD276 mAb and TLR7 / 8 agonist.

[0253] DualADC Construction 1 Figures 3A-3E show the construction of Dual ADCs via cysteine ​​and lysine. Figure 3A shows the structure of the Dual ADC, CD276 mAb-MMAF / IMQ. Figure 3B shows the structure of the Dual ADC. Figure 3C shows that conjugation of single-payload and dual-payload with chimeric anti-CD276 mAb was confirmed by HPLC. Figure 3D shows MALDI-TOF MS to confirm the exact molecular weight of the mAb, single ADC (mAb-MMAF, mAb-IMQ), and Dual ADC (mAb-MMAF / IMQ). Figure 3E shows SDS-PAGE of the ADCs. M: Marker; 1: CD276 mAb; 2: mAb-MMAF; 3: mAb-IMQ; 4: mAb-MMAF / IMQ.

[0254] Figure 2 shows the synthesis pathway of DualADC construct 1. In this figure, the step resulting in the conjugation of the DBM linker is performed first, followed by the conjugation of phosphine and azide to the agonist. In some examples, the resulting phosphine-azide linker is conjugated first, and the DBM linker is obtained from the second conjugation step. The conjugation may be performed in any order. Furthermore, phosphine and azide may be conjugated to the antibody and drug in any order (e.g., phosphine first, followed by azide second, or azide first, followed by phosphine second). The concept of conjugating a potent payload, such as a chemotherapeutic agent like MMAF, and an immunoenhancing reagent into a single monoclonal antibody is noteworthy and enables the superior anticancer efficacy of this DualADC compared to conventional antibody-drug conjugations.

[0255] Dual ADC Construction 2 Figures 17A and 17B show exemplary construction of DualADC via cysteine ​​and lysine. Figure 17A shows the corresponding schematic diagram of DualADC including DBM linker and phosphine-azide linker. Figure 17B shows exemplary chemical structure of DualADC including DBM linker and sulfo-SMCC linker.

[0256] Figure 9 shows the synthesis pathway for DualADC construct 2. In this figure, the step resulting in DBM linker conjugation is performed first, followed by the conjugation of SATA and sulfo-SMCC to the agonist. In some examples, the resulting SATA sulfo-SMCC linker is conjugated first, and the DBM linker is obtained from the second conjugation step. The conjugation may be performed in any order. Furthermore, SATA and sulfo-SMCC may be conjugated to the antibody and drug in any order (e.g., SATA first, followed by sulfo-SMCC second, or sulfo-SMCC first, followed by SATA second). The concept of conjugating a potent payload, such as a chemotherapeutic agent like MMAF, and an immunoenhancing reagent into a single monoclonal antibody is noteworthy and enables the superior anticancer efficacy of this DualADC compared to conventional antibody-drug conjugations.

[0257] Single-payload ADC construct Figures 18A-18H show the construction of single-payload ADCs via lysine or cysteine. Figure 18A shows a lysine-mediated mAb-MMAF conjugate using DBM as a crosslinking linker. Figure 18B shows a lysine-mediated mAb-DM1 conjugate using sulfo-SMCC as a linker. Figure 18C shows a lysine-mediated mAb-TLR agonist conjugate using sulfo-SMCC as a linker and SATA modification. Figure 18D shows an mAb-TLR agonist conjugate via phosphine azide as a synthetic linker. Figures 18E-18H show the HPLC-based characterization of the ADCs (and mAbs) corresponding to Figures 18A-18D, respectively.

[0258] in vitro cytotoxicity Figures 4A-4F show the in vitro evaluation of Dual ADCs using humanized CD276 mAb. MDA-MB-231, MDA-MB-468, and 4T1 cells, which are TNBCs, were used for cytotoxicity studies, with free drugs and single-payload ADCs used as controls. Figure 4A shows the anti-TNBC cytotoxicity and IC of free DM1 and free MMAF drugs. 50 Figure 4B shows the cytotoxicity and IC of IMQ. 50 Figure 4C shows human and mouse TLR8. + EC of IMQ in HEK cells 50 Figure 4D shows the anti-TNBC cytotoxicity and IC of a single-payload ADC (mAb-MMAF). 50 Figure 4E shows the cytotoxicity and IC of a single payload (mAb-IMQ). 50 Figure 4F shows the cytotoxicity and IC of DualADC mAb-MMAF / IMQ. 50 This indicates.

[0259] DualADC anti-TNBC in the TNBC PD model Figures 5A–5D show the antitumor efficacy of DualADC in a TNBC PDX xenograft model. Figure 5A shows the change in tumor volume after treatment with humanized CD276 mAb-derived ADC following a Q7Dx3 schedule, as indicated by the black arrows. Data are shown as mean ± SEM, and n=5–7. Saline solution (○), 16 mg / kg mAb-MMAF (▲), and 16 mg / kg mAb-MMAF / IMQ (●). *P<0.05 (vs. saline, using ANOVA and subsequent Dunnett's t-test). Figure 5B shows body weight. Figure 5C shows a white light image 14 days after discontinuation of treatment. Figure 5D shows IHC staining of TNBC PDX tumor tissue. Scale bar is 20 μm.

[0260] DualADC anti-TNBC model in a normal TNBC immunological model Figures 6A–6E demonstrate the anti-TNBC efficacy of 276 mAb-MMAF / IMQ in an immunocompetent model. Female BALB / cJ mice xenografted with the mouse TNBC 4T1-FLuc were treated with DualADC (8, 16, 24 mg / kg mAb-MMAF / IMQ), mAb-MMAF, mAb-IMQ, or saline (control) via intravenous injection through the tail vein. n=5–8. Figure 6A shows tumor volume after treatment according to the Q7Dx4 schedule as indicated by the black arrows. Data are shown as mean ± SEM. *P<0.05 (vs. saline, using ANOVA and subsequent Dunnett's t-test). Figure 6B shows the final infiltrating tumor weight after treatment with 16 mg / kg DualADC using chimeric CD276 mAb and humanized 276 mAb. Figure 6C shows H&E staining of major organs. The scale bar is 70 μm. Figure 6D shows the markers for cell proliferation (Ki67), apoptosis (CCasp3), immune checkpoint inhibition (PD-1), and CD8 + Figure 6E shows IHC staining of excised tumors using markers for T cell, NK cell, and macrophage cell filtration and activation (CD8, CD45, F4 / 80). The scale bar is 20 μm. Figure 6E shows HE staining for analysis of TNBC cell death in the treatment group. The scale bar is 40 μm.

[0261] Comparison of single-payload ADCs and dual-payload ADCs Figure 13 shows the body weight profile of an immunonormal model treated with DualADC. Female BALB / cJ mice xenografted with 4T1-FLuc were treated with dual-payload ADC (8, 16, or 24 mg / kg of mAb-MMAF / IMQ), mAb-MMAF, mAb-IMQ, or saline (control) via intravenous injection through the tail vein. n=6–8.

[0262] Tumor Immunology Figures 7A-7B show the analysis of tumor cytokines and systemic toxicity after treatment with dual-payload ADC. The same mice as in Figure 6 were used here as well. (A) Luminex assay revealed enhancement of several cytokines and downregulation of PD-1 in TME. (B) Complete blood count. 1: Physiological saline (control); 2: 8 mg / kg mAb-MMAF (control); 3: 8 mg / kg mAb-IMQ (control); 4: 8 mg / kg mAb-MMAF / IMQ; 5: 16 mg / kg mAb-MMAF / IMQ; 6: 24 mg / kg mAb-MMAF / IMQ.

[0263] scRNA-seq of TNBC treated with DualADC Figures 8A-8D show the analysis of immune cell infiltration and immune function in TME using single-cell RNA sequencing (scRNA-Seq). Tumor tissue was collected from the same animal experiment as in Figure 6. Figure 8A shows an overview of all cell types in TNBC tumors. Figure 8B shows immune function in TME. Figure 8C shows the immune response of macrophages. Figure 8D shows the analysis of mitotic activity.

[0264] DualADC in immunodeficiency models Figures 15A–15C demonstrate the anti-TNBC efficacy of dual-payload ADC (chimeric CD276 mAb-MMAF / IMQ) in an immunocompromised model. Female NSG mice xenografted with MDA-MB-231-FLuc were treated with dual-payload ADC (16 mg / kg mAb-MMAF / IMQ), single-payload ADC (16 mg / kg mAb-MMAF), and saline (control) via intravenous injection through the tail vein. n=5. Figure 15A shows tumor volume after treatment according to a Q5Dx5 schedule as indicated by the black arrows. Tumor volume was measured with calipers and calculated as an ellipsoid. Data are shown as mean ± SEM. *P<0.05 (vs. saline, using ANOVA and subsequent Dunnett's t-test). Figure 15B shows the profile of weight change. Figure 15C shows bioluminescence and white light images obtained using IVIS 14 days after the final injection.

[0265] Pharmacokinetics - CD276 mAb - MMAF / IMQ Figures 16A and 16B show pharmacokinetic (PK) studies of anti-CD276 mAb-MMAF / IMQ in a BALB / cJ mouse model. Six different dosages were used, with n=2 in total, involving 12 mice. Figure 16A shows the serum titer of DualADC. Figure 16B shows the PK parameters.

[0266] Other obvious advantages inherent in the present invention will be self-evident to those skilled in the art. It will be understood that certain features and partial combinations are useful and can be adopted without reference to other features and partial combinations. This is intended and included in the claims. Since numerous possible embodiments of the present invention can be made without departing from the scope of the present invention, it should be understood that all matters described herein or shown in the accompanying drawings should be construed as illustrative and not as restrictive.

[0267] TIFF2026525313000030.tif236169 TIFF2026525313000031.tif254169 TIFF2026525313000032.tif253169 TIFF2026525313000033.tif216170 TIFF2026525313000034.tif241169 References

[0268] TIFF2026525313000035.tif240170TIFF2026525313000036.tif254170TIFF2026525313000037.tif252170TIFF2026525313000038.tif219170

Claims

1. An antibody-drug conjugate comprising a monoclonal antibody (mAb), a first payload conjugated to the mAb via a first divalent linker, and a second payload conjugated to the mAb via a second divalent linker, wherein the first linker, the second linker, or any combination thereof comprises a phosphine-azide linker, an N-succinimidyl S-acetylthioacetate sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SATA-SMCC) linker, a sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) linker, or a dibromomaleimide (DBM) linker.

2. The antibody-drug conjugate according to claim 1, wherein the first divalent linker comprises a phosphine-azid linker or a SATA sulfo-SMCC linker.

3. The antibody-drug conjugate according to claim 1, wherein the first divalent linker comprises a DBM linker or a sulfo-SMCC linker.

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the second divalent linker comprises a phosphine-azid linker or a SATA sulfo-SMCC linker.

5. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the second divalent linker comprises a DBM linker or a sulfo-SMCC linker.

6. The antibody-drug conjugate according to claim 1, wherein the first divalent linker comprises a DBM linker, and the second divalent linker comprises one of a sulfo-SMCC linker, a SATA sulfo-SMCC linker, or a phosphine-azid linker.

7. The antibody-drug conjugate according to claim 1, wherein the first divalent linker is a DBM linker and the second divalent linker is a phosphine-azid linker.

8. The antibody-drug conjugate according to claim 1, wherein the first divalent linker is a DBM linker and the second divalent linker is a sulfo-SMCC linker.

9. The antibody-drug conjugate according to claim 1, wherein the first divalent linker is a DBM linker and the second divalent linker is a SATA sulfo-SMCC linker.

10. The antibody-drug conjugate according to any one of claims 1 to 9, wherein the first divalent linker covalently links the first payload to a cysteine ​​residue present in the mAb.

11. The antibody-drug conjugate according to any one of claims 1 to 9, wherein the first divalent linker covalently links the first payload to a lysine residue present in the mAb.

12. The antibody-drug conjugate according to any one of claims 1 to 11, wherein the second divalent linker covalently links the second payload to a cysteine ​​residue present in the mAb.

13. The antibody-drug conjugate according to any one of claims 1 to 11, wherein the second divalent linker covalently links the second payload to a lysine residue present in the mAb.

14. The antibody-drug conjugate according to any one of claims 1 to 13, wherein the mAb is a mouse antibody, a chimeric antibody, or a humanized antibody.

15. The mAb inhibits CD276 or SSTR2, according to any one of claims 1 to 14.

16. The antibody-drug conjugate according to any one of claims 1 to 15, wherein the first payload comprises a chemotherapeutic agent.

17. The antibody-drug conjugate according to claim 16, wherein the chemotherapeutic agent comprises an anti-cancer agent such as monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

18. The aforementioned chemotherapy drugs include altretamine, bendamustine, busulfan, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, lomustine, lurubinectin, mechloretamine, melphalan, procarbazine, streptozocin, temozolomide, thiotepa, trabectedin, carboplatin, cisplatin, oxaliplatin, bleomycin, dactinomycin, daunorubicin, doxorubicin, and epirubicin. Idarubicin, Mitomycin, Mitoxantrone, Plicamycin, Barurubicin, Methotrexate, Pemetrexed, Pralatrexate, Trimethrexate, Azathioprine, Cladribine, Fludarabine, Mercaptopurine, Thioguanine, Azacitidine, Capecitabine, Cytarabine, Decitabine, Furoxuridine, Fluorouracil, Gemcitabine, Trifluridine / Tipiracil, Aldesleukin (IL-2), Denileukin Difutitex, Interferon-γ, Bellinostat, Panobinostat, Romidepsin, Vorinostat, Abiraterone, Apalutamide, Bicalutamide, Cyproterone, Enzalutamide, Flutamide, Nilutamide, Anastrozole, Exemestane, Fulvestrant, Letrozole, Raloxifene, Tamoxifen, Toremifene, Degarelix, Goserelin, Historelli Leuprolide, relugolix, triptorelin, lanreotide, octreotide, pasireotide, alemtuzumab, atezolizumab, avelumab, verantamab, bevacizumab, blinatumomab, brentuximab, semiprimab, cetuximab, daratumumab, dinutuximab, dostallumab, durvalumab, elotuzumab, enfortumab, gemtuzumab, inotuzumab Ozogamicin, ipilimumab, mogamulizumab, moxetumomab Pasdotox, necitumumab, nivolumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab Vedotin, ramucirumab, rituximab, sacituzumab Govitecan, tecristamag, tisotumumab Vedotin, tocitumomab, trastuzumab, trastuzumab Deruxtecan, trastuzumabEmtansine, Tremelimumab, Abemaciclib, Acalabrutinib, Adaglacib, Afatinib, Alectinib, Alpelisib, Aciminib, Axitinib, Binimetinib, Bortezomib, Bosutinib, Brigutinib, Cabozantinib, Carfilzomib, Ceritinib, Cobimetinib, Copanlisib, Crizotinib, Dabrafenib, Dacomitinib, Dasatinib, Dubellisib, Enasidenib, Encorafenib, Entrectinib, Erdafitinib, E Lulotinib, Fedratinib, Futivatinib, Gefitinib, Gilteritinib, Glasdevib, Ibrutinib, Idelalisib, Imatinib, Infiglatinib, Ibosidenib, Ixazomib, Lapatinib, Lalotrectinib, Lenvatinib, Lorlatinib, Midostaurine, Mobosertinib, Momerotinib, Neratinib, Nilotinib, Niraparib, Olaparib, Ortasidenib, Osimertinib, Pacritinib, Palbociclib, Pazopanib, Pemigatinib, Pe Xidaritinib, ponatinib, regorafenib, ribociclib, lipretinib, rucaparib, ruxolitinib, selumetinib, sonidegib, sorafenib, sunitinib, thalazoparib, tivozanib, trametinib, trilaciclib, umbralicib, vandetanib, vemurafenib, bismodegib, zanubrutinib, cabazitaxel, docetaxel, paclitaxel, etoposide, irinotecan, teniposide, topotecan, vinblastine, vincristine, vino The antibody-drug conjugate according to claim 16, comprising relbin, asparaginase (peguaspargase), berzutifan, bexarotene, sedazulidine, eribulin, everolimus, hydroxyurea, ixabepyrone, lenalidomide, mitotane, omasetaxin, pomalidomide, selinexol, taglaxofusp, tazemetostat, teventafusp, tetrotristat, temsirolimus, thalidomide, venetoclax, or any combination thereof.

19. The antibody-drug conjugate according to any one of claims 1 to 18, wherein the second payload comprises an immunotherapy agent.

20. The antibody-drug conjugate according to claim 19, wherein the immunotherapy agent comprises a Toll-like receptor agonist, such as a Toll-like receptor 7 or 8 (TLR7 / 8) agonist.

21. The antibody-drug conjugate according to claim 20, wherein the TLR7 / 8 agonist comprises imidazoquinoline.

22. The aforementioned immunotherapy drugs include brexcabutagen autolucel, trastuzumab, aldesleukin, amivantamab, atezolizumab, avelumab, axicabutagen silolucel, verantamab mahodotin, bevacizumab, blinatumomab, brentuximab vedotin, semiprimab, cetuximab, siltacabbutagen, daratumumab, daratumumab, dostallimab, durvalumab, elotuzumab, enfortumab vedotin, epcolitamab, gemtuzumab, grofitamab, ibritumomab tiuxetan, idekabutagen bicleucel, inotuzumab ozogamicin, ipilimumab, isatuximab, lysokabutagen maralucel, and ronkastuximab. Tecilin, Margetuximab, Milbetuximab, Sorabtansine, Mogamulizumab, Mosnetuzumab, Nadofalagen, Filadenobec, Naxitamab, Necitumumab, Nivolumab, Nivolumab and Relatrimab, Obinutuzumab, Ofatumumab, Panitumumab, Pegylated Interferon, Pembrolizumab, Pertuzumab, Polatuzumab, Vedotin, Ramucirumab, Retifanlimab, Rituximab, Sacituzumab, Govitecan, Siltuximab, Cipleucel-T, Tafacitamab, Taguraxofusp, Tarimogen, Rachelpalepbec, Toalquetamab, Teventafusp, Tecristamab, Tisagenlecleucel, Tisotumab An antibody-drug conjugate according to any one of claims 19 to 21, comprising vedotin, trastuzumab, tremelimumab, aflibercept, or any combination thereof.

23. A method for treating a disease in a target area, comprising administering a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1 to 22 to the target area.

24. The method according to claim 23, wherein the disease is cancer.

25. The method according to claim 24, wherein the cancer is triple-negative breast cancer, glioblastoma, or non-small cell lung cancer.

26. A method for upregulating tumor immunity in a target area, comprising administering a therapeutically effective amount of an antibody-drug conjugate described in any one of claims 1 to 22 to the target area.

27. A method for reducing tumor growth in a target area, comprising administering a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1 to 22 to the target area.

28. The method according to claim 27, wherein the tumor is squamous cell carcinoma, large cell carcinoma, adenocarcinoma, invasive ductal carcinoma, non-invasive ductal carcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, glioblastoma, or any combination thereof.

29. A method for inhibiting a protein in cells in which it is needed, comprising administering a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1 to 22 to the cells, wherein the antibody in the antibody-drug conjugate inhibits the protein.

30. An antibody-drug conjugate comprising a monoclonal antibody (mAb) and a payload conjugated to the mAb via a divalent linker, wherein the divalent linker comprises a phosphine-azid linker or an N-succinimidyl S-acetylthioacetate sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SATA sulfo-SMCC) linker.

31. The antibody-drug conjugate according to claim 30, wherein the divalent linker comprises a phosphine-azid linker.

32. The antibody-drug conjugate according to claim 30, wherein the divalent linker comprises a SATA sulfo-SMCC linker.

33. The antibody-drug conjugate according to any one of claims 30 to 32, wherein the divalent linker covalently links the payload to a cysteine ​​residue present in the mAb.

34. The antibody-drug conjugate according to any one of claims 30 to 32, wherein the divalent linker covalently links the payload to a lysine residue present in the mAb.

35. The antibody-drug conjugate according to any one of claims 30 to 34, wherein the mAb is a mouse antibody, a chimeric antibody, or a humanized antibody.

36. The mAb inhibits CD276 or SSTR2, according to any one of claims 30 to 35.

37. The antibody-drug conjugate according to any one of claims 30 to 36, wherein the payload comprises a chemotherapeutic agent.

38. The antibody-drug conjugate according to claim 37, wherein the chemotherapy agent comprises an anti-cancer agent such as MMAE or MMAF.

39. The aforementioned chemotherapy drugs include altretamine, bendamustine, busulfan, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, lomustine, lurubinectin, mechloretamine, melphalan, procarbazine, streptozocin, temozolomide, thiotepa, trabectedin, carboplatin, cisplatin, oxaliplatin, bleomycin, dactinomycin, daunorubicin, doxorubicin, and epirubicin. Idarubicin, Mitomycin, Mitoxantrone, Plicamycin, Barurubicin, Methotrexate, Pemetrexed, Pralatrexate, Trimethrexate, Azathioprine, Cladribine, Fludarabine, Mercaptopurine, Thioguanine, Azacitidine, Capecitabine, Cytarabine, Decitabine, Furoxuridine, Fluorouracil, Gemcitabine, Trifluridine / Tipiracil, Aldesleukin (IL-2), Denileukin Difutitex, Interferon-γ, Bellinostat, Panobinostat, Romidepsin, Vorinostat, Abiraterone, Apalutamide, Bicalutamide, Cyproterone, Enzalutamide, Flutamide, Nilutamide, Anastrozole, Exemestane, Fulvestrant, Letrozole, Raloxifene, Tamoxifen, Toremifene, Degarelix, Goserelin, Historelli Leuprolide, relugolix, triptorelin, lanreotide, octreotide, pasireotide, alemtuzumab, atezolizumab, avelumab, verantamab, bevacizumab, blinatumomab, brentuximab, semiprimab, cetuximab, daratumumab, dinutuximab, dostallumab, durvalumab, elotuzumab, enfortumab, gemtuzumab, inotuzumab Ozogamicin, ipilimumab, mogamulizumab, moxetumomab Pasdotox, necitumumab, nivolumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab Vedotin, ramucirumab, rituximab, sacituzumab Govitecan, tecristamag, tisotumumab Vedotin, tocitumomab, trastuzumab, trastuzumab Deruxtecan, trastuzumabEmtansine, Tremelimumab, Abemaciclib, Acalabrutinib, Adaglacib, Afatinib, Alectinib, Alpelisib, Aciminib, Axitinib, Binimetinib, Bortezomib, Bosutinib, Brigutinib, Cabozantinib, Carfilzomib, Ceritinib, Cobimetinib, Copanlisib, Crizotinib, Dabrafenib, Dacomitinib, Dasatinib, Dubellisib, Enasidenib, Encorafenib, Entrectinib, Erdafitinib, E Lulotinib, Fedratinib, Futivatinib, Gefitinib, Gilteritinib, Glasdevib, Ibrutinib, Idelalisib, Imatinib, Infiglatinib, Ibosidenib, Ixazomib, Lapatinib, Lalotrectinib, Lenvatinib, Lorlatinib, Midostaurine, Mobosertinib, Momerotinib, Neratinib, Nilotinib, Niraparib, Olaparib, Ortasidenib, Osimertinib, Pacritinib, Palbociclib, Pazopanib, Pemigatinib, Pe Xidaritinib, ponatinib, regorafenib, ribociclib, lipretinib, rucaparib, ruxolitinib, selumetinib, sonidegib, sorafenib, sunitinib, thalazoparib, tivozanib, trametinib, trilaciclib, umbralicib, vandetanib, vemurafenib, bismodegib, zanubrutinib, cabazitaxel, docetaxel, paclitaxel, etoposide, irinotecan, teniposide, topotecan, vinblastine, vincristine, vino The antibody-drug conjugate according to claim 37, comprising relbin, asparaginase (peguaspargase), berzutifan, bexarotene, sedazulidine, eribulin, everolimus, hydroxyurea, ixabepyrone, lenalidomide, mitotane, omasetaxin, pomalidomide, selinexol, taglaxofusp, tazemetostat, teventafusp, tetrotristat, temsirolimus, thalidomide, venetoclax, or any combination thereof.

40. The antibody-drug conjugate according to any one of claims 30 to 37, wherein the payload comprises an immunotherapy agent.

41. The antibody-drug conjugate according to claim 40, wherein the immunotherapy agent comprises a Toll-like receptor agonist, such as a Toll-like receptor 7 or 8 (TLR7 / 8) agonist.

42. The antibody-drug conjugate according to claim 41, wherein the TLR7 / 8 agonist comprises imidazoquinoline.

43. The aforementioned immunotherapy drugs include brexcabutagen autolucel, trastuzumab, aldesleukin, amivantamab, atezolizumab, avelumab, axicabutagen silolucel, verantamab mahodotin, bevacizumab, blinatumomab, brentuximab vedotin, semiprimab, cetuximab, siltacabbutagen, daratumumab, daratumumab, dostallimab, durvalumab, elotuzumab, enfortumab vedotin, epcolitamab, gemtuzumab, grofitamab, ibritumomab tiuxetan, idekabutagen bicleucel, inotuzumab ozogamicin, ipilimumab, isatuximab, lysokabutagen maralucel, and ronkastuximab. Tecilin, Margetuximab, Milbetuximab, Sorabtansine, Mogamulizumab, Mosnetuzumab, Nadofalagen, Filadenobec, Naxitamab, Necitumumab, Nivolumab, Nivolumab and Relatrimab, Obinutuzumab, Ofatumumab, Panitumumab, Pegylated Interferon, Pembrolizumab, Pertuzumab, Polatuzumab, Vedotin, Ramucirumab, Retifanlimab, Rituximab, Sacituzumab, Govitecan, Siltuximab, Cipleucel-T, Tafacitamab, Taguraxofusp, Tarimogen, Rachelpalepbec, Toalquetamab, Teventafusp, Tecristamab, Tisagenlecleucel, Tisotumab An antibody-drug conjugate according to any one of claims 40 to 42, comprising vedotin, trastuzumab, tremelimumab, aflibercept, or any combination thereof.

44. Compound of formula I: 【Chemistry 1】 [In the formula, R １ and R ２ R １ and R ２ One of them contains an antibody, R １ and R ２ One of the two is selected in such a combination that it contains a drug; X is selected from -O-, -S-, and -NH-; W is selected from -O-, -S-, and -NH-.

45. The compound according to claim 44, wherein X is NH.

46. The compound according to claim 44 or 45, wherein W is NH.

47. R １ The compound according to any one of claims 44 to 46, wherein the compound comprises an antibody.

48. R ２ The compound according to any one of claims 44 to 47, wherein the compound includes a drug.

49. The compound according to any one of claims 44 to 48, wherein the antibody is a monoclonal antibody (mAb).

50. The compound according to claim 49, wherein the mAb is a mouse antibody, a chimeric antibody, or a humanized antibody.

51. The mAb is the compound according to claim 49 or 50, which inhibits CD276 or SSTR2.

52. The compound according to any one of claims 44 to 51, wherein the drug is an immunotherapy agent.

53. The immunotherapy agent comprises a Toll-like receptor agonist, such as a Toll-like receptor 7 or 8 (TLR7 / 8) agonist, according to claim 52.

54. The compound according to claim 53, wherein the TLR7 / 8 agonist comprises imidazoquinoline.

55. The aforementioned immunotherapy drugs include brexcabutagen autolucel, trastuzumab, aldesleukin, amivantamab, atezolizumab, avelumab, axicabutagen silolucel, verantamab mahodotin, bevacizumab, blinatumomab, brentuximab vedotin, semiprimab, cetuximab, siltacabbutagen, daratumumab, daratumumab, dostallimab, durvalumab, elotuzumab, enfortumab vedotin, epcolitamab, gemtuzumab, grofitamab, ibritumomab tiuxetan, idekabutagen bicleucel, inotuzumab ozogamicin, ipilimumab, isatuximab, lysokabutagen maralucel, and ronkastuximab. Tecilin, Margetuximab, Milbetuximab, Sorabtansine, Mogamulizumab, Mosnetuzumab, Nadofalagen, Filadenobec, Naxitamab, Necitumumab, Nivolumab, Nivolumab and Relatrimab, Obinutuzumab, Ofatumumab, Panitumumab, Pegylated Interferon, Pembrolizumab, Pertuzumab, Polatuzumab, Vedotin, Ramucirumab, Retifanlimab, Rituximab, Sacituzumab, Govitecan, Siltuximab, Cipleucel-T, Tafacitamab, Taguraxofusp, Tarimogen, Rachelpalepbec, Toalquetamab, Teventafusp, Tecristamab, Tisagenlecleucel, Tisotumab A compound according to any one of claims 52 to 54, comprising vedotin, trastuzumab, tremelimumab, aflibercept, or any combination thereof.

56. A method for covalently linking a first payload with a second payload, the method being: The first payload is given by equation III: 【Chemistry 2】 [wherein, R ５ is H, C １～２０ alkyl, C ２～２０ alkenyl, C ２～２０ alkynyl, 6-20 member aryl, 7-20 member alkylaryl, 3-20 member cycloalkyl, C １～２０ acyl, C １～２０ alkoxy, 7-20 member aryloxy, C １～２０ alkylamino, C ２～２０ dialkylamino, halogen, or amino]. A step of reacting with a compound to form a first precursor; Formula IV for the second payload: 【Transformation 3】 The step of reacting with a compound to form a second precursor; and The first precursor and the second precursor are coupled to form formula V: 【Chemistry 4】 Steps to form the compound The method, including the method described above.

57. The method according to claim 56, wherein the first payload comprises a biomolecule.

58. The method according to claim 57, wherein the biomolecule is a protein or an antibody.

59. The method according to any one of claims 56 to 58, wherein the second payload is a surface, particles, a drug, or a second biomolecule.

60. The method according to claim 59, wherein the drug is an immunotherapy agent.

61. The method according to claim 60, wherein the immunotherapy agent comprises a Toll-like receptor agonist, such as a Toll-like receptor 7 or 8 (TLR7 / 8) agonist.

62. The method according to claim 61, wherein the TLR7 / 8 agonist comprises imidazoquinoline.

63. The aforementioned immunotherapy drugs include brexcabutagen autolucel, trastuzumab, aldesleukin, amivantamab, atezolizumab, avelumab, axicabutagen silolucel, verantamab mahodotin, bevacizumab, blinatumomab, brentuximab vedotin, semiprimab, cetuximab, siltacabbutagen, daratumumab, daratumumab, dostallimab, durvalumab, elotuzumab, enfortumab vedotin, epcolitamab, gemtuzumab, grofitamab, ibritumomab tiuxetan, idekabutagen bicleucel, inotuzumab ozogamicin, ipilimumab, isatuximab, lysokabutagen maralucel, and ronkastuximab. Tecilin, Margetuximab, Milbetuximab, Sorabtansine, Mogamulizumab, Mosnetuzumab, Nadofalagen, Filadenobec, Naxitamab, Necitumumab, Nivolumab, Nivolumab and Relatrimab, Obinutuzumab, Ofatumumab, Panitumumab, Pegylated Interferon, Pembrolizumab, Pertuzumab, Polatuzumab, Vedotin, Ramucirumab, Retifanlimab, Rituximab, Sacituzumab, Govitecan, Siltuximab, Cipleucel-T, Tafacitamab, Taguraxofusp, Tarimogen, Rachelpalepbec, Toalquetamab, Teventafusp, Tecristamab, Tisagenlecleucel, Tisotumab The method according to any one of claims 60 to 62, comprising vedotin, trastuzumab, tremelimumab, aflibercept, or any combination thereof.

64. The method according to claim 59, wherein the drug is a chemotherapy drug.

65. The method according to claim 64, wherein the chemotherapy agent includes an anti-cancer agent such as MMAE or MMAF.

66. The aforementioned chemotherapy drugs include altretamine, bendamustine, busulfan, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, lomustine, lurubinectin, mechloretamine, melphalan, procarbazine, streptozocin, temozolomide, thiotepa, trabectedin, carboplatin, cisplatin, oxaliplatin, bleomycin, dactinomycin, daunorubicin, doxorubicin, and epirubicin. Idarubicin, Mitomycin, Mitoxantrone, Plicamycin, Barurubicin, Methotrexate, Pemetrexed, Pralatrexate, Trimethrexate, Azathioprine, Cladribine, Fludarabine, Mercaptopurine, Thioguanine, Azacitidine, Capecitabine, Cytarabine, Decitabine, Furoxuridine, Fluorouracil, Gemcitabine, Trifluridine / Tipiracil, Aldesleukin (IL-2), Denileukin Difutitex, Interferon-γ, Bellinostat, Panobinostat, Romidepsin, Vorinostat, Abiraterone, Apalutamide, Bicalutamide, Cyproterone, Enzalutamide, Flutamide, Nilutamide, Anastrozole, Exemestane, Fulvestrant, Letrozole, Raloxifene, Tamoxifen, Toremifene, Degarelix, Goserelin, Historelli Leuprolide, relugolix, triptorelin, lanreotide, octreotide, pasireotide, alemtuzumab, atezolizumab, avelumab, verantamab, bevacizumab, blinatumomab, brentuximab, semiprimab, cetuximab, daratumumab, dinutuximab, dostallumab, durvalumab, elotuzumab, enfortumab, gemtuzumab, inotuzumab Ozogamicin, ipilimumab, mogamulizumab, moxetumomab Pasdotox, necitumumab, nivolumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab Vedotin, ramucirumab, rituximab, sacituzumab Govitecan, tecristamag, tisotumumab Vedotin, tocitumomab, trastuzumab, trastuzumab Deruxtecan, trastuzumabEmtansine, Tremelimumab, Abemaciclib, Acalabrutinib, Adaglacib, Afatinib, Alectinib, Alpelisib, Aciminib, Axitinib, Binimetinib, Bortezomib, Bosutinib, Brigutinib, Cabozantinib, Carfilzomib, Ceritinib, Cobimetinib, Copanlisib, Crizotinib, Dabrafenib, Dacomitinib, Dasatinib, Dubellisib, Enasidenib, Encorafenib, Entrectinib, Erdafitinib Erlotinib, Fedratinib, Futivatinib, Gefitinib, Gilteritinib, Glasdevib, Ibrutinib, Idelalisib, Imatinib, Infiglatinib, Ibosidenib, Ixazomib, Lapatinib, Lalotrectinib, Lenvatinib, Lorlatinib, Midostaurin, Mobosertinib, Momerotinib, Neratinib, Nilotinib, Niraparib, Olaparib, Ortasidenib, Osimertinib, Pacritinib, Palbociclib, Pazopanib, Pemiga Tinib, pexidartinib, ponatinib, regorafenib, ribociclib, lipretinib, rucaparib, ruxolitinib, selumetinib, sonidegib, sorafenib, sunitinib, thalazoparib, tivozanib, trametinib, trilaciclib, umbralicib, vandetanib, vemurafenib, bismodegib, zanubrutinib, cabazitaxel, docetaxel, paclitaxel, etoposide, irinotecan, teniposide, topotecan, vinblastine, vin The method according to claim 64, comprising cristine, vinorelbine, asparaginase (peguaspargase), berzutifan, bexarotene, sedazulidine, eribulin, everolimus, hydroxyurea, ixabepyrone, lenalidomide, mitotane, omasetaxin, pomalidomide, selinexol, taglaxofusp, tazemetostat, teventafusp, tetrotristat, temsirolimus, thalidomide, venetoclax, or any combination thereof.

67. R ５ is C １ The method according to any one of claims 56 to 66, wherein the alkyl group is alkyl.

68. Compounds of formula II: 【Transformation 5】 [In the formula, R ３ and R ４ R ３ and R ４ One of them contains an antibody, R ３ and R ４ One of the two is selected in such a combination that it contains a drug; Z is selected from -O-, -S-, and -NH-; A is selected from -O-, -S-, and -NH-.

69. The compound according to claim 68, wherein Z is NH.

70. The compound according to claim 68 or 69, wherein A is NH.

71. R ３ The compound according to any one of claims 68 to 70, wherein the compound comprises an antibody.

72. R ４ The compound according to any one of claims 68 to 71, wherein the compound includes a drug.

73. The compound according to any one of claims 68 to 72, wherein the antibody is a monoclonal antibody (mAb).

74. The compound according to claim 73, wherein the mAb is a mouse antibody, a chimeric antibody, or a humanized antibody.

75. The compound according to claim 73 or 74, wherein the mAb inhibits CD276 or SSTR2.

76. The compound according to any one of claims 68 to 75, wherein the drug is an immunotherapy agent.

77. The immunotherapy agent comprises a Toll-like receptor agonist, such as a Toll-like receptor 7 or 8 (TLR7 / 8) agonist, according to claim 76.

78. The compound according to claim 77, wherein the TLR7 / 8 agonist comprises imidazoquinoline.

79. The aforementioned immunotherapy drugs include brexcabutagen autolucel, trastuzumab, aldesleukin, amivantamab, atezolizumab, avelumab, axicabutagen silolucel, verantamab mahodotin, bevacizumab, blinatumomab, brentuximab vedotin, semiprimab, cetuximab, siltacabbutagen, daratumumab, daratumumab, dostallimab, durvalumab, elotuzumab, enfortumab vedotin, epcolitamab, gemtuzumab, grofitamab, ibritumomab tiuxetan, idekabutagen bicleucel, inotuzumab ozogamicin, ipilimumab, isatuximab, lysokabutagen maralucel, and ronkastuximab. Tecilin, Margetuximab, Milbetuximab, Sorabtansine, Mogamulizumab, Mosnetuzumab, Nadofalagen, Filadenobec, Naxitamab, Necitumumab, Nivolumab, Nivolumab and Relatrimab, Obinutuzumab, Ofatumumab, Panitumumab, Pegylated Interferon, Pembrolizumab, Pertuzumab, Polatuzumab, Vedotin, Ramucirumab, Retifanlimab, Rituximab, Sacituzumab, Govitecan, Siltuximab, Cipleucel-T, Tafacitamab, Taguraxofusp, Tarimogen, Rachelpalepbec, Toalquetamab, Teventafusp, Tecristamab, Tisagenlecleucel, Tisotumab A compound according to any one of claims 76 to 78, comprising vedotin, trastuzumab, tremelimumab, aflibercept, or any combination thereof.

80. A method for covalently linking a first payload with a second payload, the method being: The first payload is given by equation VI: 【Transformation 6】 [In the formula, R ６ H, C １～２０ Alkyl, C ２～２０ Alkenil, C ２～２０ Alkynyl, 6-20 membered aryl, 7-20 membered alkylaryl, 3-20 membered cycloalkyl, C １～２０ Ashiru, C １～２０ Alkoxy, 7-20 membered aryl oxy, C １～２０ Alkylamino, C ２～２０ [Selected from dialkylamino, halogen, or amino] A step of reacting with a compound to form a first precursor; The second payload is given by equation VII: 【Transformation 7】 [In the formula, R ７ H, C １～２０ Alkyl, C ２～２０ Alkenil, C ２～２０ Alkynyl, 6-20 membered aryl, 7-20 membered alkylaryl, 3-20 membered cycloalkyl, C １～２０ Ashiru, C １～２０ Alkoxy, 7-20 membered aryl oxy, C １～２０ Alkylamino, C ２～２０ [Selected from dialkylamino, halogen, or amino] The step of reacting with a compound to form a second precursor; and The first precursor and the second precursor are coupled to form formula VIII: 【Transformation 8】 Steps to form the compound The method, including the method described above.

81. The method according to claim 80, wherein the first payload comprises a biomolecule.

82. The method according to claim 81, wherein the biomolecule is a protein or an antibody.

83. The method according to any one of claims 80 to 82, wherein the second payload is a surface, particles, a drug, or a second biomolecule.

84. The method according to claim 83, wherein the drug is an immunotherapy agent.

85. The method according to claim 84, wherein the immunotherapy agent comprises a Toll-like receptor agonist, such as a Toll-like receptor 7 or 8 (TLR7 / 8) agonist.

86. The method according to claim 85, wherein the TLR7 / 8 agonist comprises imidazoquinoline.

87. The aforementioned immunotherapy drugs include brexcabutagen autolucel, trastuzumab, aldesleukin, amivantamab, atezolizumab, avelumab, axicabutagen silolucel, verantamab mahodotin, bevacizumab, blinatumomab, brentuximab vedotin, semiprimab, cetuximab, siltacabbutagen, daratumumab, daratumumab, dostallimab, durvalumab, elotuzumab, enfortumab vedotin, epcolitamab, gemtuzumab, grofitamab, ibritumomab tiuxetan, idekabutagen bicleucel, inotuzumab ozogamicin, ipilimumab, isatuximab, lysokabutagen maralucel, and ronkastuximab. Tecilin, Margetuximab, Milbetuximab, Sorabtansine, Mogamulizumab, Mosnetuzumab, Nadofalagen, Filadenobec, Naxitamab, Necitumumab, Nivolumab, Nivolumab and Relatrimab, Obinutuzumab, Ofatumumab, Panitumumab, Pegylated Interferon, Pembrolizumab, Pertuzumab, Polatuzumab, Vedotin, Ramucirumab, Retifanlimab, Rituximab, Sacituzumab, Govitecan, Siltuximab, Cipleucel-T, Tafacitamab, Taguraxofusp, Tarimogen, Rachelpalepbec, Toalquetamab, Teventafusp, Tecristamab, Tisagenlecleucel, Tisotumab The method according to any one of claims 84 to 86, comprising vedotin, trastuzumab, tremelimumab, aflibercept, or any combination thereof.

88. The method according to claim 83, wherein the drug is a chemotherapy agent.

89. The method according to claim 88, wherein the chemotherapy agent includes an anti-cancer agent such as MMAE or MMAF.

90. The aforementioned chemotherapy drugs include altretamine, bendamustine, busulfan, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, lomustine, lurubinectin, mechloretamine, melphalan, procarbazine, streptozocin, temozolomide, thiotepa, trabectedin, carboplatin, cisplatin, oxaliplatin, bleomycin, dactinomycin, daunorubicin, doxorubicin, and epirubicin. Idarubicin, Mitomycin, Mitoxantrone, Plicamycin, Barurubicin, Methotrexate, Pemetrexed, Pralatrexate, Trimethrexate, Azathioprine, Cladribine, Fludarabine, Mercaptopurine, Thioguanine, Azacitidine, Capecitabine, Cytarabine, Decitabine, Furoxuridine, Fluorouracil, Gemcitabine, Trifluridine / Tipiracil, Aldesleukin (IL-2), Denileukin Difutitex, Interferon-γ, Bellinostat, Panobinostat, Romidepsin, Vorinostat, Abiraterone, Apalutamide, Bicalutamide, Cyproterone, Enzalutamide, Flutamide, Nilutamide, Anastrozole, Exemestane, Fulvestrant, Letrozole, Raloxifene, Tamoxifen, Toremifene, Degarelix, Goserelin, Historelli Leuprolide, relugolix, triptorelin, lanreotide, octreotide, pasireotide, alemtuzumab, atezolizumab, avelumab, verantamab, bevacizumab, blinatumomab, brentuximab, semiprimab, cetuximab, daratumumab, dinutuximab, dostallumab, durvalumab, elotuzumab, enfortumab, gemtuzumab, inotuzumab Ozogamicin, ipilimumab, mogamulizumab, moxetumomab Pasdotox, necitumumab, nivolumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab Vedotin, ramucirumab, rituximab, sacituzumab Govitecan, tecristamag, tisotumumab Vedotin, tocitumomab, trastuzumab, trastuzumab Deruxtecan, trastuzumabEmtansine, Tremelimumab, Abemaciclib, Acalabrutinib, Adaglacib, Afatinib, Alectinib, Alpelisib, Aciminib, Axitinib, Binimetinib, Bortezomib, Bosutinib, Brigutinib, Cabozantinib, Carfilzomib, Ceritinib, Cobimetinib, Copanlisib, Crizotinib, Dabrafenib, Dacomitinib, Dasatinib, Dubellisib, Enasidenib, Encorafenib, Entrectinib, Erdafitinib Erlotinib, Fedratinib, Futivatinib, Gefitinib, Gilteritinib, Glasdevib, Ibrutinib, Idelalisib, Imatinib, Infiglatinib, Ibosidenib, Ixazomib, Lapatinib, Lalotrectinib, Lenvatinib, Lorlatinib, Midostaurin, Mobosertinib, Momerotinib, Neratinib, Nilotinib, Niraparib, Olaparib, Ortasidenib, Osimertinib, Pacritinib, Palbociclib, Pazopanib, Pemiga Tinib, pexidartinib, ponatinib, regorafenib, ribociclib, lipretinib, rucaparib, ruxolitinib, selumetinib, sonidegib, sorafenib, sunitinib, thalazoparib, tivozanib, trametinib, trilaciclib, umbralicib, vandetanib, vemurafenib, bismodegib, zanubrutinib, cabazitaxel, docetaxel, paclitaxel, etoposide, irinotecan, teniposide, topotecan, vinblastine, vin The method according to claim 88, comprising cristine, vinorelbine, asparaginase (peguaspargase), berzutifan, bexarotene, sedazulidine, eribulin, everolimus, hydroxyurea, ixabepyrone, lenalidomide, mitotane, omasetaxin, pomalidomide, selinexol, taglaxofusp, tazemetostat, teventafusp, tetrotristat, temsirolimus, thalidomide, venetoclax, or any combination thereof.

91. R ６ teeth, 【Chemistry 9】 The method according to any one of claims 80 to 90.

92. R ７ teeth, 【Chemistry 10】 The method according to any one of claims 80 to 91.

Images