Antibody drug conjugates containing STING agonists

Abstract

We provide antibody-drug conjugates (ADCs). [Solution] We provide a scaffold and ADC containing interferon gene stimulating factor (STING). We also provide the use of ADC in treatment, for example, in cancer treatment.

Description

[Technical Field]

[0001] Related applications This application claims priority and benefits relating to U.S. Provisional Application No. 63 / 004,108, filed on 2 April 2020, U.S. Provisional Application No. 63 / 040,755, filed on 18 June 2020, and U.S. Provisional Application No. 63 / 111,820, filed on 10 November 2020. The contents of each of these applications are incorporated herein by reference in their entirety.

[0002] Sequence listing by reference The contents of the text file named "MRSN-033_001WO_SeqList.txt", created on March 31, 2021, with a size of 49KB, are incorporated in their entirety by reference herein. [Background technology]

[0003] background Interferon gene stimulators (STINGs) are intraendoplasmic reticulum receptors that transmit innate immune detection of cytosolic pathogen-derived DNA and autologous DNA. STINGs are 378-amino acid proteins primarily containing three structural domains: (i) an N-terminal transmembrane domain (aa1-154); (ii) a central globular domain (aa155-341); and (iii) a C-terminal tail (aa342-379). STINGs can form symmetric dimers in a V-conformation when bound to their ligands, but they do not completely encapsulate the bound ligand. STING agonists can enter and bind to the pocket region of STINGs. However, in some severe disease conditions, the STING activation process is readily inhibited, resulting in inactivation of the STING pathway. Therefore, screening and design of potent STING agonists are crucial for cancer immunotherapy and other infectious disease treatments, including but not limited to obesity, liver injury, glucose-lipid metabolism, and viral infections. Specific targeting of immune pathways offers opportunities for cancer therapy, potentially providing greater specificity than cell population-based therapeutic approaches.

[0004] Antibody-drug conjugates (ADCs) consist of a drug, such as a small molecule, covalently bound to an antibody. The antibody is a targeting mechanism aligned to a specific site of action. Upon reaching this site, the ADC releases the small molecule drug, allowing it to perform its intended function precisely, in contrast to systemic diffusion throughout the target body. This targeted approach makes it possible to treat patients with drugs that would require doses high enough to be toxic if administered systemically without a targeted approach.

[0005] An important feature of the innate immune system is to recognize and eliminate foreign substances. The identification of these pathogenic invaders is done by the host recognizing evolutionarily conserved microbial structures known as pathogen-associated molecular patterns (PAMPs). Host recognition can ultimately lead to downstream signaling events and may be carried out by multiple pathways such as activation of pattern recognition receptors (PRRs) that initiate an immune response.

[0006] The antibody-drug conjugates of the present disclosure modulate STING activity and thus may be beneficial in the treatment of diseases, disorders, and / or conditions including, but not limited to, inflammation, allergic diseases and autoimmune diseases, infectious diseases, cancer, and pre-cancerous syndromes, where modulation of STING (stimulator of interferon genes) is beneficial, and may provide a therapeutically beneficial effect as a vaccine adjuvant. New immunotherapies for treating diseases, particularly cancer, are still needed.

Summary of the Invention

[0007] Summary In some aspects, the present disclosure provides Formula (I): PBRM-[A 1 -(L C ) 0または1 -D]d 15 (I) a conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein PBRM represents a protein-based recognition molecule, L C when present, is a linker unit, A 1 when L C is present, is a bivalent linker moiety that connects PBRM to L C , or when L C is absent, is a bivalent linker moiety that connects PBRM to D, D is a STING agonist drug moiety, d 15 is an integer from about 1 to about 20, The present invention provides a conjugate, or a pharmaceutically acceptable salt or solvation compound thereof.

[0008] In some contexts, this disclosure is, Equation (II) is useful for conjugating with PBRM: A 1’ -(L C ) 0または1 -D (II) A scaffold of, or a pharmaceutically acceptable salt or solvated compound thereof, wherein, PBRM represents a protein-based recognition molecule. L C When present, it is a linker unit. A 1’ This is a monovalent linker moiety containing a functional group that can form a covalent bond with the functional group of PBRM, D is the STING agonist drug portion, d 15 These are integers ranging from approximately 1 to approximately 20. The present invention provides a scaffold, or a pharmaceutically acceptable salt or solvation compound thereof.

[0009] In some aspects, this disclosure provides pharmaceutical compositions comprising a conjugate described herein and one or more pharmaceutically acceptable carriers or excipients.

[0010] In certain aspects, this disclosure provides a method for activating or enhancing the activity of interferon gene stimulating factor (STING) in a subject, comprising the step of administering the subject to a conjugate described herein or a pharmaceutically acceptable salt thereof.

[0011] In certain aspects, this disclosure provides a method for preventing or treating a disease or disorder in a subject, comprising the step of administering a therapeutically effective amount of the conjugate described herein or a pharmaceutically acceptable salt thereof to the subject.

[0012] In certain aspects, this disclosure provides conjugates or pharmaceutically acceptable salts thereof described herein for activating or enhancing STING activity in a subject.

[0013] In certain aspects, this disclosure provides conjugates or pharmaceutically acceptable salts thereof described herein for preventing or treating diseases or disorders in a subject.

[0014] In certain aspects, this disclosure provides the use of the conjugates or pharmaceutically acceptable salts thereof described herein in the manufacture of a pharmacopoeia for activating or enhancing STING activity in a subject.

[0015] In certain aspects, this disclosure provides the use of the conjugates or pharmaceutically acceptable salts thereof described herein in the manufacture of a medicament for the prevention or treatment of a disease or disorder in a subject.

[0016] 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 this disclosure belongs. In this specification, singular nouns also include plural nouns unless explicitly defined by context. Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. References cited herein are not considered prior art to the present invention. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to limit. In case of any conflict between a chemical structure and the name of a compound disclosed herein, the chemical structure shall prevail.

[0017] Other features and advantages of this disclosure are evident from the following detailed description and claims. [Brief explanation of the drawing]

[0018] [Figure 1A] Figure 1A plots the red object confluence as a function of time for conjugate 8b-1 and conjugate 8f (100 nM, 10 nM, and 1 nM, respectively) and conjugate 8c-1 and compound 1 (100 nM and 10 nM, respectively) (conjugate concentrations are based on payload). [Figure 1B] Figure 1B plots the red object confluence as a function of time for conjugate 8l and compound 1 (100 nM, 10 nM, and 1 nM, respectively) and conjugate 8m (100 nM) (conjugate concentration based on payload). [Figure 2] Figure 2 shows CD14- / CD3+ cells in PBMCs, enriched monocytes, and CD16-depleted monocyte populations. [Figure 3] Figures 3A and 3B plot the red object confluence as a function of time and show the death of STING wild-type (sgNT-2) and knockout (sg#3-2) SKBR3 NucRed cells by PBMCs against conjugates 8a-3, 8j, 8c-2, and compound 1 at 100 nM, 25 nM, 5 nM, and 1 nM, respectively (conjugate concentrations based on payload). [Figure 4] Figure 4A plots red object confluence as a function of dose-response for STING wild-type SKBR3 cancer cell killing activity in SKBR3 cancer cell / PBMC co-cultures for conjugate 8a-3, conjugate 8-j, wild-type Fc trastuzumab, and AAG Fc mutant trastuzumab. Figure 4B plots red object confluence as a function of dose-response for STING knockout SKBR3 cancer cell killing activity in SKBR3 cancer cell / PBMC co-cultures for conjugate 8a-3, conjugate 8-j, wild-type Fc trastuzumab, and AAG Fc mutant trastuzumab. [Figure 5A]Figure 5A plots the change in red object confluence of OVCAR3-NucRed cancer cells as a function of time by PBMCs, targeting conjugate 8b-1 and compound 1, both at 20 nM and 4 nM (conjugate concentrations based on payload). [Figure 5B] Figure 5B plots the change in red object confluence of OVCAR3-NucRed cancer cells as a function of time, by enriched monocytes, targeting conjugate 8b-1 and compound 1, both at 20 nM and 4 nM (conjugate concentrations based on payload). [Figure 5C] Figure 5C plots the change in red object confluence of OVCAR3-NucRed cancer cells as a function of time, targeting CD16-depleted monocytes, with conjugates 8b-1 and compound 1, both at 20 nM and 4 nM (conjugate concentrations based on payload). [Figure 6] Figure 6 is a graph showing the antitumor efficacy of trastuzumab (3 / 0 mg / kg), diABZI STING agonist (0 / 5 mg / kg), conjugate 8c-1 (1 / 0.04 mg / kg or 3 / 0.12 mg / kg), and conjugate 8a-1 (1 / 0.03 mg / kg or 3 / 0.09 mg / kg) (all doses are indicated by antibody / payload) in a SKOV3 xenograft mouse model. [Figure 7-1]Figures 7A–7H show cytokine levels for mouse CXCL-10 (IP-10; Figure 7A), IL-6 (Figure 7B), TNFα (Figure 7C), IFNγ (Figure 7D), CXCL1 (KC) (Figure 7E), MIG (Figure 7F), MIP-1a (Figure 7G), and RANTES (Figure 7H) as a function of time after administration of diABZI STING agonist (0 / 5 mg / kg), conjugate 8c-1 (3 / 0.12 mg / kg), or conjugate 8a-1 (3 / 0.09 mg / kg) (all doses are written as antibody / payload) to the SKOV3 mouse model. The insets in each plot show cytokine levels induced by conjugate 8a-1 and conjugate 8c-1 compared to the vehicle. [Figure 7-2] See the explanation in Figure 7-1. [Figure 7-3] See the explanation in Figure 7-1. [Figure 7-4] See the explanation in Figure 7-1. [Figure 8A] Figures 8A–8C show the levels of mouse CXCL10 (Figure 8A), interferon-β (Figure 8B), and IL-6 (Figure 8C) mRNA in SKOV3 xenograft mouse models 12 and 72 hours after administration of conjugate 8c-1 (3 / 0.12 mg / kg), conjugate 8a-1 (3 / 0.09 mg / kg), conjugate 8c-1 (3 / 0.12 mg / kg), or conjugate 8a-1 (3 / 0.09 mg / kg) (all doses are listed as antibody / payload). [Figure 8B] See the explanation in Figure 8A. [Figure 8C] See the explanation in Figure 8A. [Figure 9] Figure 9 shows CD45 immunohistochemistry (IHC) staining with rabbit anti-CD45 monoclonal antibody at 12 and 72 hours for conjugate 8a-1 (3 / 0.09 mg / kg), conjugate 8c-1 (3 / 0.12 mg / kg), or vehicle. [Figure 10]Figure 10 is a graph showing the circulating plasma concentrations of the total antibody and conjugated drug after administration of conjugate 8a-1 (3 / 0.1 mg / kg) (dose indicated by antibody / payload) to CB.17 SCID mice. [Figure 11] Figure 11 shows the effects of IFNλ1 (IL29) or IFNλ2 (IL28A) neutralizing antibodies (10 μg / mL, 2 μg / mL, 0.4 μg / mL, 0.08 μg / mL) on the cell death activity of conjugate 8a-3 (1 nM or 0.1 nM, payload-based) in co-culture of cancer cells and PBMCs. [Figure 12] Figure 12 is a graph showing the antitumor efficacy of diABZI STING agonist (0 / 5 mg / kg), conjugate 8c-1 (3 / 0.12 mg / kg), conjugate 8b-1 (3 / 0.09 mg / kg), or conjugate 8f (3 / 0.12 mg / kg) (all doses are indicated by antibody / payload) in OVCAR3 xenografts in mice. [Figure 13] Figure 13 is a graph showing the antitumor efficacy of conjugate 8d-2 (3 / 0.1 mg / kg) or conjugate 8e (3 / 0.1 mg / kg) (all doses are shown as antibody / payload) in OVCAR3 xenografts in mice. [Figure 14] Figure 14 is a graph showing the antitumor efficacy of diABZI STING agonist (0 / 5 mg / kg), conjugate 8c-1 (1 / 0.04 mg / kg), or conjugate 8h (1 / 0.04 mg / kg) (all doses are shown as antibody / payload) in triple-negative breast cancer xenografts in mice. [Figure 15A] Figure 15A is a graph showing the antitumor efficacy of conjugate 8d-3 (1 / 0.04 mg / kg) or conjugate 8g (0.88 / 0.04 mg / kg) (all doses are shown as antibody / payload) in a syngenic mouse model of colon cancer. [Figure 15B]Figure 15B shows Kaplan-Meier survival curves for conjugate 8d-3 (1 / 0.04 mg / kg) or conjugate 8g (0.88 / 0.04 mg / kg) (all doses are shown as antibody / payload) in a syngenic mouse model of colon cancer. [Figure 16-1] Figure 16A is a graph showing the antitumor efficacy of 8 g (0.9 / 0.04 mg / kg) of the conjugate (all doses are shown as antibody / payload) in individual mice of a colon cancer syngenic mouse model. [Figure 16-2] Figure 16B is a graph showing the antitumor efficacy of 8 g (0.9 / 0.04 mg / kg) of the conjugate (all doses are shown as antibody / payload) when reloaded with syngenic mouse colon cancer cells. Figure 16C is a graph showing the antitumor efficacy of 8 g (0.9 / 0.04 mg / kg) of the conjugate (all doses are shown as antibody / payload) when reloaded with syngenic mouse lung cancer cells. [Figure 17] Figure 17 is a graph showing the antitumor efficacy of diABZI STING agonist (0 / 5 mg / kg) conjugate 8d-3 (5.5 / 0.18 mg / kg) or conjugate 8i (3.2 / 0.18 mg / kg) (all doses are shown as antibody / payload) in a syngenic mouse embryonic cancer model. [Figure 18] Figure 18 is a graph showing the antitumor efficacy of conjugate 20a (3 / 0.09 mg / kg), conjugate 34a (3 / 0.11 mg / kg), conjugate 34 (3 / 0.11 mg / kg), or conjugate 20-1 (3 / 0.10 mg / kg) (all doses are shown as antibody / payload) in SKOV3 xenografts in mice. [Figure 19]Figure 19 is a graph showing the antitumor efficacy of conjugate 28 (0.3 / 0.01 mg / kg or 1 / 0.03 mg / kg), conjugate 29 (0.2 / 0.01 mg / kg or 0.8 / 0.02 mg / kg), conjugate 8-2 (0.3 / 0.01 mg / kg or 1 / 0.04 mg / kg), conjugate 25 (0.3 / 0.01 mg / kg or 1 / 0.04 mg / kg), or conjugate 45 (0.3 / 0.01 mg / kg or 1 / 0.04 mg / kg) (all doses are shown as antibody / payload) in SKOV3 xenografts in mice. [Figure 20] Figure 20 shows the results in SKOV3 xenografts in mice for diABZI STING agonist (1.5 mg / kg q3dx3 or 0.128 mg / kg qdx1), compound 30 (1.5 mg / kg q3dx3 or 0.128 mg / kg qdx1), conjugate 32b-2 (3.42 / 0.128 mg / kg qdx1), XMT-1519 (3.00 mg / kg qdx1), conjugate 32-5 (0.100 / 0.004, 0.300 / 0.013, 1.00 / 0.042, or 3.00 / 0.128 mg / kg qdx1), and conjugate 32e (1.00 / 0.039 or 3.00 / 0.117 mg / kg). This graph shows the antitumor efficacy of qdx1)) (all doses are shown as antibody / payload). [Figure 21] Figure 21A is a graph showing the antitumor efficacy of conjugate 8d-3 (1 / 0.04 mg / kg) or conjugate 8k (0.9 / 0.04 mg / kg) in syngenic mouse models. Figure 21B shows the antitumor efficacy of conjugate 8d-3 (1 / 0.04 mg / kg) in individual mice in syngenic mouse models. Figure 21C shows the antitumor efficacy of conjugate 8k (0.9 / 0.04 mg / kg) in individual mice in syngenic mouse models. [Figure 22]Figure 22 is a graph showing the antitumor efficacy of conjugate 8c-2 (3.17 / 0.10 mg / kg), conjugate 8a-2 (2.7 / 0.10 mg / kg or 0.81 / 0.03 mg / kg), conjugate 8j (2.71 / 0.10 mg / kg or 0.81 / 0.03 mg / kg), or diABZI IV STING agonist (0 / 5 mg / kg) (all doses are shown as antibody / payload) in SKOV3 xenografts in mice. [Figure 23] Figure 23 is a graph showing the antitumor efficacy of conjugate 32b-1 (3.39 / 0.10 mg / kg), conjugate 32a (0.93 / 0.03 or 3.12 / 0.10 mg / kg), conjugate 32c (2.12 / 0.10 mg / kg), conjugate 32d (2.20 / 0.10 mg / kg), or diABZI IV STING agonist (0 / 5 mg / kg) (all doses are shown as antibody / payload) in OVCAR3 xenografts in mice. [Figure 24]Figure 24 shows the combinations of rituximab AF-HPA ADC (0.75 / 0.023 mg / kg) and conjugate 8c-2 (4.0 / 0.126 mg / kg) in OVCAR3 xenografts in mice; XMT-1535 AF-HPA ADC (0.75 / 0.024 mg / kg); conjugate 8b-2 (2.0 / 0.071 or 4.0 / 0.142 mg / kg); XMT-1535 AF-HPA ADC (0.75 / 0.024 mg / kg) and conjugate 8c-2 (4.0 / 0.126 mg / kg); rituximab AF-HPA ADC (0.75 / 0.023 mg / kg) and conjugate 8b-2 (4.0 / 0.142 mg / kg); rituximab AF-HPA This graph shows the antitumor efficacy of the following combinations: ADC (0.75 / 0.023 mg / kg) and conjugate 8b-2 (2.0 / 0.071 mg / kg); XMT-1535 AF-HPA ADC (0.75 / 0.024 mg / kg) and conjugate 8b-2 (4.0 / 0.142 mg / kg); XMT-1535 AF-HPA ADC (0.75 / 0.024 mg / kg) and conjugate 8b-2 (2.0 / 0.071 mg / kg); XMT-1535 AF-HPA ADC (0.75 / 0.024 mg / kg) and XMT-1535 (4.0 / 0 mg / kg); or XMT-1535 (4.75 / 0 mg / kg). [Figure 25] Figure 25 is a graph showing the antitumor efficacy of conjugate 32b (0.85 / 0.03 mg / kg), conjugate 32-2 (0.90 / 0.03 mg / kg), conjugate 88 (0.87 / 0.03 mg / kg), conjugate 85 (2.87 / 0.10 mg / kg), conjugate 92 (2.36 / 0.10 mg / kg), conjugate 100 (2.23 / 0.10 mg / kg), conjugate 89 (0.99 / 0.030 mg / kg), conjugate 85a (2.59 / 0.10 mg / kg), conjugate 93 (2.85 / 0.10 mg / kg), or conjugate 101 (2.70 / 0.10 mg / kg) in the SKOV3 xenograft mouse model. [Figure 26]Figure 26 is a graph showing the antitumor efficacy of vehicle, conjugate 28 (0.99 / 0.0325 mg / kg), or conjugate 62 (0.92 / 0.0325 mg / kg) in the SKOV3 xenograft mouse model. [Figure 27] Figure 27 is a graph showing the circulating plasma concentrations of conjugate drugs after administration of conjugate 28 (3.0 / 0.10 mg / kg) or conjugate 62 (2.84 / 0.10 mg / kg) (doses are indicated by antibody / payload) to CB.17SCID mice. [Modes for carrying out the invention]

[0019] Detailed explanation This disclosure provides novel antibody-drug conjugates, methods for synthesizing conjugates or scaffolds, pharmaceutical compositions containing them, and various uses of the conjugates.

[0020] definition The chemical names given to the intermediates and / or compounds of this disclosure described herein may refer to any one of the tautomeristic notations of such compounds (in some cases, such aliases are given to experimental compounds). Any reference to named compounds (intermediates or compounds of this disclosure) or compounds whose structures are illustrated (intermediates or compounds of this disclosure) should be understood to be intended to encompass all tautomeristic forms, including the zwitterionic form of such compounds and any mixture thereof.

[0021] The terms “in certain embodiments,” “in certain embodiments of the present disclosure,” and “in certain embodiments of the compounds of the present disclosure” should be understood to be synonymous as appropriate.

[0022] When the terms “about,” “approximately,” or “approximate” are used in relation to a number, they mean that a set or range of numbers is included. In some embodiments, “about X” includes a range of values ​​of ±25%, ±20%, ±15%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the number X. In some embodiments, the term “about” refers to a range of values ​​that are 5% more or 5% less than a given value. In some embodiments, the term “about” refers to a range of values ​​that are 2% more or 2% less than a given value. In some embodiments, the term “about” refers to a range of values ​​that are 1% more or 1% less than a given value.

[0023] The enumeration of value ranges is intended to serve as a shorthand way of referring to each distinct value within that range, unless otherwise specified herein. Each distinct value is incorporated herein as being enumerated one by one. As used herein, ranges include two limits unless otherwise specified. In some embodiments, the expressions “x being an integer between 1 and 6” and “x being an integer of 1 to 6” both mean “x is 1, 2, 3, 4, 5, or 6.” That is, the terms “between X and Y” and “range from X to Y” include X and Y and the integers in between.

[0024] As used herein, the term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. The numbering of antibody amino acids follows the Kabat EU Index (see Kabat, EA, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)).

[0025] The term "antibody fragment" refers to a molecule other than the intact antibody that contains a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0026] As used herein, the terms "reference antibody" and "antibody that binds to the same epitope" refer to an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competitive assay. Conversely, the reference antibody blocks 50% or more of the binding of the "antibody that binds to the same epitope" as the reference antibody to its antigen in a competitive assay. An exemplary competitive assay is provided herein.

[0027] The term "class" of an antibody refers to the type of constant domain or constant region present in the antibody heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0028] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, for example, the individual antibodies constituting the population are identical and / or bind to the same epitope, except for possible variants, such as those containing native mutations or arising during the preparation of the monoclonal antibody preparation. Such variants are generally present in trace amounts. Typically, in contrast to polyclonal antibody preparations, which contain different antibodies directed against different determinants (e.g., epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Therefore, the modifier “monoclonal” should not be interpreted as indicating that the antibody is obtained from a substantially homogeneous population of antibodies and that it requires the production of the antibody by a specific method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus. Such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0029] The term "epitope" refers to a specific site on an antigen molecule to which an antibody binds.

[0030] The term “protein-based recognition molecule” or “PBRM” refers to a molecule that recognizes and binds to cell surface markers or receptors, such as transmembrane proteins, surface-immobilized proteins, or proteoglycans. In some embodiments, PBRMs include engineered cysteine. Examples of PBRMs include, but are not limited to, antibodies, peptides, lipocalins, proteins, peptides, or peptide mimics. In addition to targeting conjugates to specific cells, tissues, or locations, protein-based recognition molecules may also have certain therapeutic effects, such as antiproliferative (cell division arrest and / or cytotoxic) activity against target cells or target pathways. Protein-based recognition molecules may include, or be engineered to include, at least one chemically reactive group, such as -COOH, primary amines, secondary amines -NHR, -SH, or a chemically reactive amino acid moiety or side chain, such as tyrosine, histidine, cysteine, or lysine. In some embodiments, the PBRM may be a ligand (LG) or targeting moiety that specifically binds to or complexes with a cell surface molecule of a given target cell population, such as a cell surface receptor or antigen. After the ligand specifically binds to or complexes with its receptor, the cell allows uptake of the ligand or ligand-drug conjugate, which is then internally transferred into the cell. Ligands used herein that "specifically bind to or complexe" or "target" cell surface molecules preferentially associate with cell surface molecules via intermolecular forces. In some embodiments, ligands can preferentially associate with cell surface molecules at Kd values ​​less than about 50 nM, less than about 5 nM, or less than 500 pM. Techniques for measuring the binding affinity of a ligand to a cell surface molecule are well known. For example, one suitable technique is called surface plasmon resonance (SPR). In some embodiments, the ligand is used for targeting and does not have a detectable therapeutic effect when separated from the drug that the ligand delivers.In some embodiments, the ligand functions as a targeting portion, either as a therapeutic agent or an immunomodulatory agent (for example, to enhance the activity of an active drug or prodrug). The term “PEG unit” as used herein refers to a formula. This refers to a polyethylene glycol subunit having TIFF2026086770000002.tif10128. In some embodiments, the PEG unit comprises multiple PEG subunits.

[0031] As used herein, the term "alkyl" refers to a saturated, linear or branched hydrocarbon group having a specified number of carbon atoms. "C1-C6 alkyl" or "C 1~6 The term "alkyl" refers to a linear or branched alkyl moiety containing a methyl moiety or 2 to 6 carbon atoms. Exemplary alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, pentyl, and hexyl.

[0032] As used herein, the term "halo(alkyl)" refers to a saturated linear or branched hydrocarbon group having a specified number (n) of carbon atoms and one or more (up to 2n+l) halogen atoms. 1~4 Examples of alkyl groups include, but are not limited to, -CF3 (trifluoromethyl), -CCl3 (trichloromethyl), 1,1-difluoroethyl, 2,2,2-trifluoroethyl, and hexafluoroisopropyl.

[0033] As used herein, the term “alkenyl” refers to a linear or branched hydrocarbon group having a specified number of carbon atoms and at least one to three carbon-carbon double bonds. Examples include ethenyl and propenyl.

[0034] As used herein, the term "alkynyl" refers to a linear or branched hydrocarbon group having a specified number of carbon atoms and at least one to three carbon-carbon triple bonds. Examples include ethynyl and propynyl.

[0035] As used herein, the terms “alkoxy-” or “(alkyl)oxy-” refer to an “alkyl-oxy-” group that contains an alkyl moiety having a specified number of carbon atoms, attached via an oxygen-linked atom. 1~4 Alkoxy-" or "(C 1~4 The alkyl)oxy- group includes, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, s-butoxy, and t-butoxy.

[0036] As used herein, the term "halo(alkoxy)-" refers to a saturated linear or branched hydrocarbon group having a specified number (n) of carbon atoms and one or more (up to 2n+l) halogen atoms attached via an oxygen-linked atom. An example of "halo(C)" is... 1~4 The alkoxy-" group includes, but is not limited to, -OCHF2 (difluoromethoxy), -OCF3 (trifluoromethoxy), -OCH2CF3 (trifluoroethoxy), and -OCH(CF3)2 (hexafluoroisopropoxy).

[0037] As used herein, the term "amino" refers to substituents containing at least one nitrogen atom. Specifically, -NH2, -NH(C 1~4 Alkyl), alkylamino, or (C 1~4 Alkyl)amino- or (C 1~4 Alkyl)(C 1~4 The term "amino" includes alkyl)amino- or dialkylamino, amide-, carbamide-, urea, and sulfamide substituents.

[0038] As used herein, the term "carbocyclic group or carbocyclic moiety" refers to a cyclic group or cyclic moiety whose ring members are carbon atoms, which may be saturated, partially unsaturated (non-aromatic), or fully unsaturated (aromatic).

[0039] As used herein, the term “cycloalkyl” refers to a non-aromatic saturated hydrocarbon ring group containing a specified number of carbon atoms in the ring. For example, “C 3~6 The term "cycloalkyl" refers to a cyclic group having 3 to 6 ring carbon atoms. An example is "C 3~6 The "cycloalkyl" group includes cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0040] As used herein, the term “aryl” refers to an aromatic group having one or more aromatic rings and containing no heteroatoms in the ring structure, including conjugated or polycyclic structures. The term aryl includes both monovalent and divalent chemical species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, and naphthyl. In some embodiments, the aryl is phenyl.

[0041] As used herein, the term “heterocyclic group or heterocyclic moiety” means a cyclic group or cyclic moiety having atoms of at least two different elements as ring members, and the cyclic group or cyclic moiety may be saturated, partially unsaturated (non-aromatic), or fully unsaturated (aromatic).

[0042] As used herein, the term “heteroatom” refers to a nitrogen, sulfur, or oxygen atom, for example, a nitrogen atom or an oxygen atom.

[0043] As used herein, the term “heterocycloalkyl” refers to a non-aromatic monocyclic or bicyclic group containing 3 to 10 ring atoms and one or more (generally one or two) heteroatom ring members independently selected from oxygen, sulfur, and nitrogen. The heterocycloalkyl group may be attached to any suitable carbon or nitrogen atom.

[0044] As used herein, the term “heteroaryl” refers to an aromatic monocyclic or bicyclic group containing 5 to 10 ring atoms, each containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, where at least a portion of the group is aromatic. For example, the term encompasses bicyclic heterocyclic-aryl groups containing a heteroaryl ring fused with a heterocyclic moiety or a heteroaryl ring fused with a carbocyclic moiety. The heteroaryl group may be attached to any suitable carbon or nitrogen atom.

[0045] As used herein, the terms "halogen" and "halo" refer to halogen radicals, such as fluoro, chloro, bromo, or iodo substituents.

[0046] As used herein, the term "oxo" refers to a double-bonded oxygen moiety. For example, if it is directly bonded to a carbon atom, it forms a carbonyl moiety (C=O).

[0047] As used herein, the terms "hydroxy" or "hydroxyl" are intended to mean the radical -OH.

[0048] As used herein, the term "cyano" refers to a nitrile group -C≡N.

[0049] As used herein, the term “may be substituted” means that a group (e.g., an alkyl, cycloalkyl, alkoxy, heterocycloalkyl, aryl, or heteroaryl group), ring, or moiety may be unsubstituted, or may be substituted with one or more substituents. If a group can be selected from a number of alternative groups, the selected groups may be the same or different. Suitable substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphonato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfamide, nitro, trifluoromethyl, cyano, azide, heterocyclyl, alkylaryl, or aromatic or heterocyclic aromatic moieties.

[0050] As used herein, the term "independently" means that multiple substituents are selected from a number of possible substituents, which may be the same or different.

[0051] As used herein, the term “pharmaceutically acceptable” means a compound, conjugate, material, composition, and dosage form that, within the bounds of appropriate medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, other problems, or complications, corresponding to a reasonable benefit-risk ratio.

[0052] As used herein, the terms “treating” or “treat” mean managing and caring for a patient in order to combat a disease, condition, or disorder, and include administering the compounds of the Disclosure or any pharmaceutically acceptable salts, polymorphs, or solvated compounds thereof to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term “treat” may also include, in vitro, the treatment of cells or animal models.

[0053] As used herein, the terms “preventing,” “prevent,” or “protect from” refer to reducing or eliminating the onset of symptoms or complications of such disease, condition, or disorder.

[0054] The term "subject" refers to an animal, preferably a mammal, most preferably a human, that is the subject of treatment, observation, or experimentation.

[0055] The term "therapeutic effective dose" refers to the amount of an active compound or pharmaceutical agent containing the conjugate of this disclosure that elicits a biological or pharmacokinetic response in a tissue system, animal, or human, which is sought after by researchers, veterinarians, physicians, or other clinicians, including relief or partial relief of symptoms of the disease, syndrome, condition, or disorder being treated.

[0056] The therapeutic “effective dose” is intended to mean an amount of conjugate sufficient to effectively treat or prevent, as defined herein, when administered to a patient requiring such treatment. A given amount of conjugate corresponding to such a dose is a specific conjugate (e.g., efficacy (pIC)). so ), effectiveness (EC 50The duration of treatment and the duration of administration of the conjugate (the biological half-life of a particular conjugate), the disease state and its severity, and the personal information of the patient requiring treatment (e.g., age, size, and weight) vary depending on these factors, but can nevertheless be routinely determined by those skilled in the art. Similarly, the duration of treatment and the duration of administration of the conjugate (the timing of administration, e.g., the period before / during / after meals) vary depending on the personal information of the mammal requiring treatment (e.g., weight), the particular conjugate and its properties (e.g., pharmacokinetic properties), the disease or disorder and its severity, and the particular composition and method used, but can nevertheless be determined by those skilled in the art.

[0057] The term "composition" refers to any product containing a specified component in a therapeutically effective amount, as well as any product directly or indirectly resulting from a combination of the specified components in specified amounts.

[0058] As used herein, the term “pharmaceutically acceptable excipient” generally means an excipient that is safe, non-toxic, not biologically undesirable, and not otherwise undesirable, and is useful in preparing a pharmaceutical composition, and includes excipients that are acceptable for veterinary and human pharmaceutical use. As used herein and in the claims, “pharmaceutically acceptable excipient” includes both one and more such excipients.

[0059] As used herein, the term “STING agonist” refers to a compound or moiety that can interact with STING, for example, by binding to STING, and / or by inducing downstream signaling (for example, characterized by activation of molecules related to STING function). This includes direct phosphorylation of STING, IRF3, and / or NF-κB, and may also include STAT6. In some embodiments, activation of the STING pathway leads to increased production of type 1 interferons (primarily IFN-α and IFN-β) and / or increased expression of interferon-stimulated genes.

[0060] As used herein, the term “STING agonist drug portion” refers to a portion derived from a STING agonist that is capable of interacting with STING. In some embodiments, the STING agonist drug portion is a portion derived from a STING agonist that enables the portion derived from the STING agonist to be coupled with the rest of the conjugate of this disclosure.

[0061] The conjugates of the present disclosure are useful in methods for treating or relieving viral infections, diseases, syndromes, conditions, or disorders affected by STING agonism. Such methods essentially consist of a step of administering a therapeutically effective amount of the conjugate of the present disclosure, or its enantiomers, diastereomers, solvates, or pharmaceutically acceptable salts, to a subject, including animals, mammals, and humans, that requires such treatment, remission, and / or prevention.

[0062] In some embodiments, the conjugates of the present disclosure, or their enantiomers, diastereomers, solvated compounds, or pharmaceutically acceptable salt forms thereof, may be useful in treating or relieving diseases, syndromes, conditions, or disorders, such as melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B.

[0063] As used herein, the terms “conjugate(s) of the disclosure” or “conjugate(s) of the present disclosure” mean conjugates as defined herein, taking any form, i.e., any tautomer form, any isomer form, any salt or non-salt form (e.g., in the form of a free acid or free base, or a salt, in particular a pharmaceutically acceptable salt thereof) and any physical form thereof (e.g., a non-solid form (e.g., a liquid or semi-solid form), and a solid form (e.g., a amorphous or crystalline form, certain polymorphic forms, a solvated compound form including a hydrate form (e.g., monohydrate, dihydrate, and hemihydrate)), as well as mixtures of various forms.

[0064] Accordingly, the disclosure herein includes conjugates in any salt or non-salt form and any physical form thereof, as well as mixtures in various forms. While such conjugates in any salt or non-salt form and any physical form thereof are included in the disclosure, it is understood that the conjugates of the disclosure in any salt or non-salt form and any physical form thereof may have varying levels of activity, varying bioavailability, and varying handling properties for formulation purposes.

[0065] As used herein, expressions such as “one or more A, B, or C,” “one or more A, B, or C,” “one or more A, B, and C,” “one or more A, B, and C,” “selected from the group consisting of A, B, and C,” and “selected from A, B, and C” are used synonymously and, unless otherwise specified, all refer to a selection from the group consisting of A, B, and / or C, i.e., one or more A, one or more B, one or more C, or any combination thereof.

[0066] Throughout this specification, where a composition is described as having, including, or comprising certain components, it is understood that it is intended to consist essentially of or comprise the listed components. Similarly, where a method or process is described as having, including, or comprising certain process steps, the process is similarly intended to consist essentially of or comprise the listed process steps. Furthermore, it should be understood that the order of the steps, or the order in which certain actions are performed, is not important as long as the invention is implementable. Moreover, two or more steps or actions may be performed simultaneously.

[0067] All percentages and ratios used herein are weight percentages and weight ratios unless otherwise specified. Other features and advantages of this disclosure are evident from different examples. The examples provided illustrate different components and methodologies useful in carrying out this disclosure. The examples do not limit the claimed disclosure. Based on this disclosure, a person skilled in the art can identify and use other components and methodologies useful in carrying out this disclosure.

[0068] All publications and patent documents cited herein are incorporated herein by reference in the same way that each such publication or document is specifically and individually indicated so as to be incorporated herein by reference. References to publications and patent documents are not intended to constitute an endorsement of the prior art relating thereto, nor do they constitute an endorsement of the content or date of the publications and patent documents. Now that the present invention has been described in writing, those skilled in the art will recognize that the invention can be carried out in various embodiments, and that the foregoing description and the following examples are illustrative and not intended to limit the scope of the following claims.

[0069] Conjugates and scaffolds of this disclosure In some contexts, this disclosure is, Equation (I): PBRM-[A 1 -(L C ) 0または1 -D]d 15 (I) A conjugate of, or a pharmaceutically acceptable salt or solvated compound thereof, wherein, PBRM represents a protein-based recognition molecule. L C When present, it is a linker unit. A 1 L C When PBRM exists, C This is the divalent linker portion that connects to L C When it does not exist, it is the divalent linker part that connects PBRM to D. D is the STING agonist drug portion, d 15 These are integers ranging from approximately 1 to approximately 20. The present invention provides a conjugate, or a pharmaceutically acceptable salt or solvation compound thereof.

[0070] In some embodiments, the conjugate is Equation (IA): PBRM-[A 1 -D]d 15(IA) It is a conjugate of, or a pharmaceutically acceptable salt or solvated compound thereof.

[0071] In some embodiments, the conjugate is Formula (IB): PBRM-[A 1 -L C -D]d 15 (IB) It is a conjugate of, or a pharmaceutically acceptable salt or solvated compound thereof.

[0072] In some embodiments, the conjugate is given by formula (I-B'): It is a conjugate of TIFF2026086770000003.tif19128, or a pharmaceutically acceptable salt or solvated compound thereof.

[0073] In some contexts, this disclosure is useful for conjugating with PBRM, formula (II): A 1’ -(L C ) 0または1 -D (II) A scaffold of, or a pharmaceutically acceptable salt or solvated compound thereof, wherein, PBRM represents a protein-based recognition molecule. L C When present, it is a linker unit. A 1’ This is a monovalent linker moiety containing a functional group that can form a covalent bond with the functional group of PBRM, D is the STING agonist drug portion. The present invention provides a scaffold, or a pharmaceutically acceptable salt or solvation compound thereof.

[0074] In some embodiments, the scaffold is given by formula (II-A): A 1’ -D (II-A) It is a scaffold of, or a pharmaceutically acceptable salt or solvated compound thereof.

[0075] In some embodiments, the scaffold is given by formula (II-B): A 1’ -L C -D (II-B) It is a scaffold of, or a pharmaceutically acceptable salt or solvated compound thereof.

[0076] In some embodiments, the scaffold is given by formula (II-B'): It is a scaffold of TIFF2026086770000004.tif17128, or a pharmaceutically acceptable salt or solvated compound thereof.

[0077] For any one conjugate of formula (I), (IA), (IB), (I-B'), (II), (II-A), (II-B), or (II-B') or any pharmaceutically acceptable salt or solvated compound thereof, the variables PBRM, L C , A 1 , T 1 M A , L D , D, and d 15 Each of these may be selected from the groups described herein, where applicable, and the variables PBRM, L C , A 1 , T 1 M A , L D , D, and d 15 For any of the groups described herein, if applicable, the variables PBRM, L C , A 1 , T 1 M A , L D , D, and d 15 It is understood that this can be combined with one or more of the remaining groups described herein.

[0078] variable d 15 In some embodiments, d15 is an integer of about 2 to about 14, about 2 to about 12, about 2 to about 10, about 2 to about 8, about 2 to about 6, about 2 to about 4, about 4 to about 10, about 4 to about 8, about 4 to about 6, about 6 to about 14, about 6 to about 12, about 6 to about 10, about 6 to about 8, about 8 to about 14, about 8 to about 12, or about 8 to about 10.

[0079] In some embodiments, d 15 is an integer of about 2 to about 8.

[0080] In some embodiments, d 15 is 2, 4, 6, or 8. In some embodiments, d 15 is 6 or 8.

[0081] In some embodiments, d 15 is 8. In some embodiments, d 15 is 6.

[0082] Variable A 1 and A 1’ In some embodiments, each A 1 is, independently, a divalent linker moiety that connects PBRM to L when L is present, or a divalent linker moiety that connects PBRM to D when L is absent. C When L is present, it is a divalent linker moiety that connects PBRM to L. C When L is absent, it is a divalent linker moiety that connects PBRM to D. C When L is present, it is a divalent linker moiety that connects PBRM to L, or when L is absent, it is a divalent linker moiety that connects PBRM to D.

[0083] In some embodiments, each A 1 is, independently, TIFF2026086770000005.tif49128, where R 7 is -O-, -NR 8 -, -(C1-C 10 alkyl)-, -(C1-C 10 alkenyl)-, -(C1-C 10 alkynyl)-, -(C3-C8 cycloalkyl)-, -aryl-, -O-(C1-C8 alkyl)-, -O-(C1-C 10 alkenyl)-, -O-(C1-C 10-alkynyl)-, -(C1-C 10 -alkyl)-(C3-C8 cycloalkyl)-, -(C1-C 10 -alkyl)-aryl-, -(C2-C 10 -alkenyl)-(C3-C8 cycloalkyl)-, -(C2-C 10 -alkenyl)-aryl-, -(C2-C 10 -alkynyl)-(C3-C8 cycloalkyl)-, -(C2-C 10 -alkynyl)-aryl-, -(C3-C8 cycloalkyl)-(C1-C 10 -alky(alky))-, -aryl-(C1-C 10 -alky)-, -(C3-C8 cycloalkyl)-(C2-C 10 -alkenyl)-, -aryl-(C2-C 10 -alkenyl)-, -(C3-C8 cycloalkyl)-(C2-C 10 -alkynyl)-, -aryl-(C2-C 10 -alkynyl)-, -(3-8 membered heterocycloalkyl)-, -(5-8 membered heteroaryl)-, -(C1-C 10 -alkyl)-(3-8 membered heterocycloalkyl)-, -(C1-C 10 -alkyl)-(5-8 membered heteroaryl)-, -(C2-C 10 -alkenyl)-(3-8 membered heterocycloalkyl)-, -(C2-C 10 -alkenyl)-(5-8 membered heteroaryl)-, -(C2-C 10 -alkynyl)-(3-8 membered heterocycloalkyl)-, -(C2-C 10 -alkynyl)-(5-8 membered heteroaryl)-, -(3-8 membered heterocycloalkyl)-(C1-C 10 -alkyl)-, -(5-8 membered heteroaryl)-(C1-C 10 -alkyl)-, -(3-8 membered heterocycloalkyl)-(C2-C 10 -alkenyl)-, -(5-8 membered heteroaryl)-(C2-C 10 -alkenyl)-, -(5-8 membered heteroaryl)-(C2-C 10 -alkynyl)-, -(5-8 membered heteroaryl)-(C2-C 10Alkinyl)-,-OC(O)-(CH2CH2O) r -(CH2)2-, -(CH2CH2O) r -, or -(CH2CH2O) r The compound is -(CH2)2-, and alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl may be substituted. R 8 is H, hydroxy, or C 1~4 It is alkyl, r is an integer between approximately 1 and approximately 12. * This indicates that it is attached to the PBRM. ** L C When L exists, C It indicates that it is attached to L C If it does not exist, it indicates that it is attached to D.

[0084] In some embodiments, R 7 -O-, -NR 8 ,-(C1~C 10 Alkyl)-, -(C3~C8 cycloalkyl)-, -aryl-, -O-(C1~C8 alkyl)-, -(C1~C 10 Alkyl)-aryl-, -aryl-(C1~C 10 Alkyl)-,-(C1~C 10 Alkyl)-(C3~C8 cycloalkyl)-,-(C3~C8 cycloalkyl)-(C1~C 10 Alkyl)-, -(3-8 membered heterocycloalkyl)-, -(5-8 membered heteroaryl)-, -(C1-C 10 Alkyl)-(3-8 membered ring heterocycloalkyl)-,-(C1-C 10 Alkyl)-(5-8 membered heteroaryl ring)-,-(3-8 membered heterocycloalkyl)-(C1-C 10 Alkyl)-,-(5-8 membered ring heteroaryl)-(C1-C 10 Alkyl)-,-OC(O)-(CH2CH2O) r -(CH2)2-, -(CH2CH2O) r -, or -(CH2CH2O)r It is -(CH2)2-.

[0085] In some embodiments, R 7 is, -(C1~C 10 Alkyl)-, -O-(C1~C8 alkyl)-, -(CH2CH2O) r -, -OC(O)-(CH2CH2O) r -(CH2)2-, or -(CH2CH2O) r It is -(CH2)2-.

[0086] In some embodiments, R 7 These are -O-, -NH, -N(CH3), -CH2-, -(CH2)2-, -(CH2)5-, -OC(O)-(CH2CH2O)6-(CH2)2-, -(CH2CH2O)-(CH2)2-, -(CH2CH2O)2-(CH2)2-, -(CH2CH2O)4-(CH2)2-, or -(CH2CH2O)6-(CH2)2-.

[0087] In some embodiments, each A 1 Independently, The file is TIFF2026086770000006.tif48128, and in the formula, R 8 is H, hydroxy, or C 1~4 It is an alkyl group, and r is an integer between approximately 4 and approximately 6. * This indicates that it is attached to the PBRM. ** L C When L exists, C It indicates that it is attached to L C If it does not exist, it indicates that it is attached to D.

[0088] Each A 1 Before being connected to PBRM, independently, one valence part A 1’ This is understood to correspond to the above.

[0089] In some embodiments, each A 1’ Independently, The filename is TIFF2026086770000007.tif50128, and in the formula, R 7 , R 8 , and r are as described herein, ** L C When L exists, C It indicates that it is attached to L C If it does not exist, it indicates that it is attached to D.

[0090] In some embodiments, each A 1’ Independently, TIFF2026086770000008.tif55142, and in the formula, r is an integer between approximately 4 and approximately 6. ** L C When L exists, C It indicates that it is attached to L C If it does not exist, it indicates that it is attached to D.

[0091] Variable L C In some aspects, each L C When it exists, it exists independently, The filename is TIFF2026086770000009.tif14128, and in the formula, # is A 1 This indicates that it is attached to, and ## indicates that it is attached to D. M A When present, it is a peptide portion containing at least two amino acids. T 1 When present, it is a hydrophilic group. L D M A When D exists, M A This is the divalent linker portion that connects to M A If it does not exist, D is A 1 This is the divalent linker portion that connects to it.

[0092] In some aspects, each L D It includes at least one cleavable bond such that when the bond is broken, D is released in its active form for its intended therapeutic effect.

[0093] In some aspects, each L C When it exists, it exists independently, The filename is TIFF2026086770000010.tif6128.

[0094] In some aspects, each L C When it exists, it exists independently, The filename is TIFF2026086770000011.tif12128.

[0095] Variable L D In some aspects, each L D M is independent A When D exists, M A This is the divalent linker portion that connects to M A If it does not exist, D is A 1 This is the divalent linker portion that connects to it.

[0096] In some aspects, each L D It includes at least one cleavable bond such that when the bond is broken, D is released in its active form for its intended therapeutic effect.

[0097] In some aspects, L D It includes one cleavable bond. In some embodiments, L D It includes multiple severable parts or joints.

[0098] Each L D Before being connected to D, independently, the single-valent part L D It is understood to correspond to '.

[0099] In some aspects, L D' contains functional groups that can form cleavable bonds. Functional groups that can form cleavable bonds may include, for example, sulfhydryl groups for forming disulfide bonds, aldehyde groups, ketone groups, or hydrazine groups for forming hydrazone bonds, hydroxylamine groups for forming oxime bonds, carboxyl or amino groups for forming peptide bonds, carboxyl or hydroxyl groups for forming ester bonds, and sugars for forming glycosidic bonds.

[0100] In some aspects, each L D This includes a disulfide bond that can be cleaved by disulfide exchange, an acid-unstable bond that can be cleaved at an acidic pH, and / or a bond that can be cleaved by a hydrolase. In some embodiments, L D It contains a carbamate bond (i.e., -OC(O)-NR-, where R is hydrogen or alkyl, etc.).

[0101] In some aspects, L D The structure and sequence of one cleavable bond may be such that the bond is cleaved by the action of an enzyme present at the target site. In some embodiments, the one cleavable bond may be cleavable by other mechanisms.

[0102] In some aspects, L D The structure and arrangement of the multiple cleavable bonds present may be such that the multiple bonds are cleaved by the action of enzymes present at the target site. In some embodiments, the multiple cleavable bonds may be cleavable by other mechanisms.

[0103] In some embodiments, the cleavable bond can be enzymatically cleaved by one or more enzymes, including a tumor-associated protease, to release a drug unit or D, and the conjugate, intermediate, or scaffold of the present disclosure is protonated in vivo to provide a drug unit or D upon release.

[0104] In some aspects, each L D Independently, The filename is TIFF2026086770000012.tif10128.

[0105] L E When present, NH-[(CH2CH2O) p -(CH2) 0~2 ] q -C(O)-, -NH-(C1~C6alkyl)-OC(O)-, or -NH-[(CH2CH2O) p -(CH2) 0~2 ] q The structure is -C(O)-NH-(C1~C6 alkyl)-OC(O)-, where p is an integer from approximately 1 to approximately 20, and q is an integer from approximately 1 to approximately 10. Each W is independently either a natural amino acid unit or a non-natural amino acid unit. w is an integer between approximately 0 and approximately 12. *** M A When M exists, A It indicates that it is attached, or M A When it does not exist, A 1 It indicates that it is attached, **** This indicates that it is attached to D.

[0106] In some aspects, each L D Independently, This is TIFF2026086770000013.tif10128. In some aspects, each L D Independently, This is TIFF2026086770000014.tif11128. In some embodiments, each L D Independently, The filename is TIFF2026086770000015.tif10128.

[0107] In some aspects, L EIt includes at least one PEG unit.

[0108] In some embodiments, the PEG unit includes at least one subunit, at least two subunits, at least three subunits, at least four subunits, at least five subunits, or at least six subunits. In some embodiments, the PEG unit includes at least four subunits, at least three subunits, at least two subunits, or at least one subunit. In some embodiments, the PEG unit includes at least one subunit. In some embodiments, the PEG unit includes at least two subunits.

[0109] In some embodiments, p is an integer between approximately 1 and approximately 15, approximately 1 and approximately 10, approximately 1 and approximately 9, approximately 1 and approximately 8, approximately 1 and approximately 7, approximately 1 and approximately 6, or approximately 1 and approximately 5.

[0110] In some embodiments, p is an integer between approximately 1 and approximately 6. In some embodiments, p is an integer between approximately 1 and approximately 4. In some embodiments, p is an integer between approximately 1 and approximately 2.

[0111] In some embodiments, p is 2.

[0112] In some embodiments, q is an integer between approximately 1 and approximately 15, approximately 1 and approximately 10, approximately 1 and approximately 9, approximately 1 and approximately 8, approximately 1 and approximately 7, approximately 1 and approximately 6, or approximately 1 and approximately 5.

[0113] In some embodiments, q is 1, 2, 3, 4, or 5. In some embodiments, q is 2.

[0114] In some aspects, L E When present, NH-(CH2CH2O) 1~4 It is -(CH2)2-C(O)-. In some embodiments, L E When present, it is NH-(CH2CH2O)2-(CH2)2-C(O)-. In some embodiments, L EWhen present, NH-(CH2CH2O)3-(CH2) 0~2 -C(O)- is -C(O)- in some embodiments, L E When present, it is NH-(CH2CH2O)3-(CH2)1-C(O)-. In some embodiments, L E When present, it is NH-(CH2CH2O)3-(CH2)2-C(O)-. In some embodiments, L E When present, NH-CH2CH2O-(CH2) 0~2 -C(O)- is -C(O)- in some embodiments, L E When present, it is NH-CH2CH2O-C(O)-. In some embodiments, L E When present, it is NH-(C1~C6 alkyl)-OC(O)-. In some embodiments, L E When present, it is NH-CH2-CH(CH3)-OC(O)-. In some embodiments, L E When present, NH-[(CH2CH2O) 1~4 It is -(CH2)2-C(O)-NH-(C1~C6 alkyl)-OC(O)-. In some embodiments, L E When present, it is NH-CH2CH2O-(CH2)2-C(O)-NH-(CH2)2-OC(O)-

[0115] In some embodiments, w is an integer between approximately 1 and approximately 12 (for example, 1 to 6, or 1 to 4, or 1 to 3, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

[0116] In some embodiments, w is 0, 1, 2, 3, 4, or 5.

[0117] In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, w is 3.

[0118] In some embodiments, each W is independently a natural amino acid or a non-natural amino acid and / or a D isomer or an L isomer.

[0119] In some embodiments, each W is independently a natural or unnatural α, β, or γ amino acid.

[0120] In some embodiments, at least one W is a natural amino acid. In some embodiments, at least one W is a non-natural amino acid.

[0121] In some embodiments, W w It does not contain natural amino acids. In some embodiments, W w It does not contain non-natural amino acids.

[0122] In some embodiments, W w It contains natural amino acids linked with non-natural amino acids. In some embodiments, W w It contains natural amino acids linked to D-isomers of natural amino acids.

[0123] In some embodiments, W w These are dipeptides, such as -Val-Cit-, -Phe-Lys-, -Val-Ala-, or Glu-Ala.

[0124] In some embodiments, W w These are monopeptides, dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, undecapeptides, or dodecapeptide units.

[0125] In some embodiments, W w is a peptide (for example, a peptide of 1 to 12 amino acids), which is directly conjugated to D. In some embodiments, this peptide is a single amino acid. In some embodiments, this peptide is a dipeptide. In some embodiments, this peptide is a tripeptide.

[0126] In some embodiments, W w Each amino acid in the formula is independently selected from alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, selenocysteine, ornithine, penicillamine, aminoalkanoic acid, aminoalkynic acid, aminoalkanedioic acid, aminobenzoic acid, aminoheterocycloalkanoic acid, heterocyclocarboxylic acid, citrulline, statin, diaminoalkanoic acid, and their derivatives.

[0127] In some embodiments, W w Each amino acid in the formula is independently selected from alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, citrulline, and their derivatives.

[0128] In some embodiments, W w Each amino acid within is independently selected from proteinaceous and non-proteinaceous amino acids.

[0129] In some embodiments, W w Each amino acid in the formula is independently selected from the following amino acids: alanine, β-alanine, arginine, aspartic acid, asparagine, cysteine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine, penicillamine, aminoalkynic acid, aminoalkanediic acid, heterocyclocarboxylic acid, citrulline, statin, diaminoalkanoic acid, valine, citrulline, and their D-isomers or L-isomers.

[0130] In some embodiments, W w Each of the amino acids within it is, independently, cysteine, homocysteine, penicillamine, ornithine, lysine, serine, threonine, glycine, glutamine, alanine, aspartic acid, glutamic acid, selenocysteine, proline, glycine, isoleucine, leucine, methionine, valine, citrulline, or alanine.

[0131] In some embodiments, W w Each amino acid within is independently selected from the following amino acids: alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, citrulline, and the L-isomer of valine.

[0132] In some embodiments, W w Each amino acid within is independently selected from the following amino acids: alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, citrulline, and the D-isomer of valine.

[0133] In some embodiments, W w The amino acids contained within are alanine, β-alanine, glycine, glutamic acid, isoglutamic acid, isoaspartic acid, valinecitrulline, or aspartic acid.

[0134] In some embodiments, W w W contains β-alanine. In some embodiments, W contains (β-alanine)-(alanine). In some embodiments, W wIt contains (β-alanine), optionally glutamic acid, isoglutamic acid, aspartic acid, isoaspartic acid, valine, (valine)-(alanine), (alanine)-(alanine), or (valine)-(citrulline).

[0135] In some embodiments, W w It contains (glutamic acid)-(alanine).

[0136] In some embodiments, W w It contains glutamic acid, optionally alanine, glycine, isoglutamic acid, aspartic acid, isoaspartic acid, valine, (valine)-(alanine), (alanine)-(alanine), or (valine)-(citrulline).

[0137] In some embodiments, W w It contains 2,3-diaminopropanoic acid. In some embodiments, W w (R)-2,3-diaminopropane is included. In some embodiments, W w It contains glutamic acid. In some embodiments, W w It contains (glutamic acid)-(alanine). In some embodiments, W w It contains (glutamic acid)-(glycine)-(alanine).

[0138] In some embodiments, W w This includes L-glutamic acid, D-glutamic acid, (L-glutamic acid)-(L-alanine), (L-glutamic acid)-(D-alanine), (D-glutamic acid)-(L-alanine), (D-glutamic acid)-(D-alanine), (L-glutamic acid)-(glycine)-(L-alanine), (D-glutamic acid)-(glycine)-(D-alanine), (L-glutamic acid)-(glycine)-(D-alanine), or (D-glutamic acid)-(glycine)-(L-alanine).

[0139] In some embodiments, W w It contains one or more amino acids in addition to a carbamate bond.

[0140] In some aspects, L D (For example, W w ) is selective for enzymatic cleavage (e.g., by a specific enzyme). In some embodiments, the specific enzyme is a tumor-associated protease.

[0141] In some aspects, L D (For example, W w ) includes a bond whose cleavage is catalyzed by cathepsin B, cathepsin C, cathepsin D, or plasmin protease.

[0142] In some aspects, L D It contains a sugar cleavage site.

[0143] In some aspects, L D It contains a sugar moiety (Su) linked to a self-immolative group via an oxygen glycosidic bond.

[0144] In some aspects, the "self-sacrificing base" is M A When present, there are three spaced-apart chemical moieties (i.e., a sugar moiety (via a glycosidic bond), a drug unit (directly or indirectly), and M A It may also be a trifunctional chemical moiety that can be covalently bonded together (directly or indirectly), M A When it does not exist, A 1 But that's fine.

[0145] In some embodiments, glycosidic bonds can be cleaved at the target site to initiate a self-sacrificing reaction sequence that leads to drug release.

[0146] In some aspects, each L D When it exists, it exists independently, TIFF2026086770000016.tif138163TIFF2026086770000017.tif223163TIFF2026086770000018.tif32163, and in the formula,*** M A When M exists, A It indicates that it is attached, or M A When it does not exist, A 1 It indicates that it will be attached, **** This indicates that it is attached to D.

[0147] In some aspects, each L D When it exists, it exists independently, TIFF2026086770000019.tif86163, and in the formula, *** M A When M exists, A It indicates that it is attached, or M A When it does not exist, A 1 It indicates that it is attached, **** This indicates that it is attached to D.

[0148] In some aspects, each L D When it exists, it exists independently, The file is TIFF2026086770000020.tif15128, and in the formula, *** M A It indicates that it is attached, **** This indicates that it is attached to D.

[0149] Variable M A In some embodiments, M A It contains a peptide portion of at least two amino acids.

[0150] In some embodiments, a single amino acid is referred to herein as "AA," and multiple amino acids are referred to as "AA's."

[0151] In some embodiments, M A is, -L D - This is a portion that can form a covalent bond with the D unit, allowing for the attachment of multiple drugs.

[0152] In some embodiments, M A It contains one AA unit or has two or more AA units (e.g., 2-10, 2-6, or 2, 3, 4, 5, or 6), each of which is independently a natural or non-natural amino acid, an amino alcohol, an amino aldehyde, a diamine, a polyamine, or a combination thereof.

[0153] In some embodiments, in order to have the required number of mountings, at least one of the AA units is -L D -Has a functionalized side chain for attaching a D unit. In some embodiments, exemplary functionalized AA units (e.g., amino acids, amino alcohols, or amino aldehydes) include, for example, azide-functionalized or alkyne-functionalized AA units (e.g., amino acids, amino alcohols, or amino aldehydes modified to have an azide group or an alkyne group).

[0154] In some embodiments, M A It includes 2 to 12 AA units. In some embodiments, M A It includes 2 to 10 AA units. In some embodiments, M A It includes 2 to 6 AA units. In some embodiments, M A It includes 2, 3, 4, 5, or 6 AA units.

[0155] In some embodiments, M A It has two AA units. In some embodiments, the peptide portion has three AA units. In some embodiments, the peptide portion has four AA units. In some embodiments, the peptide portion has five AA units. In some embodiments, the peptide portion has six AA units.

[0156] In some embodiments, M AInternal attachment or conjugation, intermediates thereof, or attachment with other components of the scaffold may be via, for example, amino, carboxy, or other functionalities.

[0157] In some embodiments, M A Each amino acid within may independently be a D-isomer or L-isomer of a thiol-containing amino acid. In some embodiments, M A Each amino acid within may independently be a D isomer of a thiol-containing amino acid. In some embodiments, M A Each amino acid in the mixture may independently be an L-isomer of a thiol-containing amino acid. In some embodiments, the thiol-containing amino acid may be, for example, cysteine, homocysteine, or penicillamine.

[0158] In some embodiments, M A Each amino acid within it may independently be one of the following amino acids: alanine (including β-alanine), arginine, aspartic acid, asparagine, cysteine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine, penicillamine, aminoalkynic acid, aminoalkanediic acid, heterocyclocarboxylic acid, citrulline, statin, diaminoalkanoic acid, its stereoisomer, or its derivative, either L-isomer or D-isomer.

[0159] In some embodiments, M A Each of the amino acids within is, independently, cysteine, homocysteine, penicillamine, ornithine, lysine, serine, threonine, glycine, glutamine, alanine, aspartic acid, glutamic acid, selenocysteine, proline, glycine, isoleucine, leucine, methionine, valine, alanine, or their stereoisomers.

[0160] In some embodiments, M AThis includes monopeptides, dipeptides, tripeptides, tetrapeptides, or pentapeptides. In some embodiments, M A It contains pentapeptides.

[0161] In some embodiments, M A It contains at least about 5 amino acids (for example, 5, 6, 7, 8, 9, or 10 amino acids). In some embodiments, M A It contains up to approximately 10 amino acids.

[0162] In some embodiments, M A The individual amino acids within it are, independently, glycine, serine, glutamic acid, lysine, aspartic acid, and cysteine.

[0163] In some embodiments, M A It comprises at least four glycine molecules and at least one glutamic acid molecule, for example, (glycine)4 and glutamic acid, where the glutamic acid molecule is at any position along the peptide chain, for example, (glutamic acid)-(glycine)4; (glycine)-(glutamic acid)-(glycine)3; (glycine)2-(glutamic acid)-(glycine)2; (glycine)3-(glutamic acid)-(glycine); or (glycine)4-(glutamic acid).

[0164] In some embodiments, M A It contains (glycine)4-(glutamic acid). In some embodiments, the peptide portion contains (glutamic acid)-(glycine)4.

[0165] In some embodiments, M A It comprises at least four glycine molecules and at least one serine molecule, for example, (glycine)4 and serine, where the serine molecule is at any position along the peptide chain, for example, (serine)-(glycine)4; (glycine)-(serine)-(glycine)3; (glycine)2-(serine)-(glycine)2; (glycine)3-(serine)-(glycine); or (glycine)4-(serine).

[0166] In some embodiments, M A It contains (glycine)4-(serine). In some embodiments, the peptide portion contains (serine)-(glycine)4.

[0167] In some embodiments, M A It contains (β-alanine)-(glycine)4-(serine), where serine is at any position along the peptide chain, for example, (β-alanine)-(serine)-(glycine)4; (β-alanine)-(glycine)-(serine)-(glycine)3; (β-alanine)-(glycine)2-(serine)-(glycine)2; (β-alanine)-(glycine)3-(serine)-(glycine); or (β-alanine)-(glycine)4-(serine).

[0168] In some embodiments, M A It contains (glycine)4-(serine)-(glutamic acid), and serine is at any position along the peptide chain, for example, (serine)-(glycine)4-(glutamic acid); (glycine)-(serine)-(glycine)3-(glutamic acid); (glycine)2-(serine)-(glycine)2-(glutamic acid); (glycine)3-(serine)-(glycine)-(glutamic acid); or (glycine)4-(serine)-(glutamic acid). In some embodiments, the peptide portion comprises (β-alanine)-(glycine)4-(serine)-(glutamic acid), where serine is at any position along the peptide chain, such as (β-alanine)-(serine)-(glycine)4-(glutamic acid); (β-alanine)-(glycine)-(serine)-(glycine)3-(glutamic acid); (β-alanine)-(glycine)2-(serine)-(glycine)2-(glutamic acid); (β-alanine)-(glycine)3-(serine)-(glycine)-(glutamic acid); or (β-alanine)-(glycine)4-(serine)-(glutamic acid).

[0169] In some embodiments, M A It contains (glycine)4-(serine). In some embodiments, the peptide portion contains (serine)-(glycine)4.

[0170] In some embodiments, M A It contains (β-alanine)-(glycine)4-(serine), where serine is at any position along the peptide chain.

[0171] In some embodiments, M A It contains (glycine)4-(serine)-(glutamic acid), where serine is at any position along the peptide chain.

[0172] In some embodiments, M A It contains (β-alanine)-(glycine)4-(serine)-(glutamic acid), and the serine is at any position along the peptide chain.

[0173] In some embodiments, M A (Glutamic acid)-(glycine) 1~4 Includes M A A is produced via one of the glutamic acids. 1 Attached to M A T via glycine 1 Attached to M A L is mediated by glutamate. D It can be attached.

[0174] In some embodiments, M A teeth, Includes TIFF2026086770000021.tif21128.

[0175] In some embodiments, M A It contains (glutamic acid)-(glycine)4, M A A via glutamate 1 Attached to M A T is via one of the glycine molecules. 1 Attached to M A L is mediated by glutamate. D It can be attached.

[0176] In some embodiments, M A teeth, Includes TIFF2026086770000022.tif22128.

[0177] In some embodiments, M A It contains (glutamic acid)-(glycine), M A A via glutamate 1 Attached to M A T via glycine 1 Attached to M A L is mediated by glutamate. D It can be attached.

[0178] In some embodiments, the peptide portion is Includes TIFF2026086770000023.tif26128.

[0179] In some embodiments, M A (Glycine) 1~4 -(glutamic acid) contains M A A is obtained via one of the glycine molecules. 1 Attached to M A T via glutamate 1 Attached to M A L is mediated by glutamate. D It can be attached.

[0180] In some embodiments, M A teeth, Includes TIFF2026086770000024.tif23128.

[0181] In some embodiments, M A It contains (glycine)4-(glutamic acid), M A A via glutamate 1 Attached to M A T via glycine 1 Attached to M A L is mediated by glutamate. D It can be attached.

[0182] In some embodiments, M A teeth, Includes TIFF2026086770000025.tif23128.

[0183] In some embodiments, M A It contains (glycine)-(glutamic acid), M A A via glycine 1 Attached to M A T via glutamate 1 Attached to M A L is mediated by glutamate. D It can be attached.

[0184] In some embodiments, M A teeth, Includes TIFF2026086770000026.tif21128.

[0185] In some embodiments, M A (Glycine) 1~4 -(Serine) contains M A A is obtained via one of the glycine molecules. 1 Attached to M A T via serine 1 Attached to M A L via serine D It can be attached.

[0186] In some embodiments, M A teeth, Includes TIFF2026086770000027.tif13128.

[0187] In some embodiments, M A It contains (glycine)-(serine), M A A via glycine 1 Attached to M A T via serine 1 Attached to M A L via serine D It can be attached.

[0188] In some embodiments, M A teeth, Includes TIFF2026086770000028.tif15128.

[0189] In some embodiments, M A It contains (glycine)4-(serine), M A A is obtained via one of the glycine molecules. 1 Attached to M A T via serine 1 Attached to M A L via serine D It can be attached.

[0190] In some embodiments, M A teeth, Includes TIFF2026086770000029.tif15128.

[0191] In some embodiments, M A (Serine)-(Glycine) 1~4 Includes M A A via serine 1 Attached to M A T is via one of the glycine molecules. 1 Attached to M A L via serine D It can be attached.

[0192] In some embodiments, M A teeth, Includes TIFF2026086770000030.tif15128.

[0193] In some embodiments, M A It contains (serine)-(glycine)4, M A A via serine 1 Attached to M A T is via one of the glycine molecules. 1 Attached to M A L via serine D It can be attached.

[0194] In some embodiments, M A teeth, Includes TIFF2026086770000031.tif15128.

[0195] In some embodiments, M A It contains (serine)-(glycine), M A A via serine 1 Attached to M A T is via one of the glycine molecules. 1 Attached to M A L via serine D It can be attached.

[0196] In some embodiments, M A teeth, Includes TIFF2026086770000032.tif20128.

[0197] In some embodiments, M A (β-alanine)-(glycine) 1~4 -(Serine) contains M A A via β-alanine 1 Attached to M A T via serine 1 Attached to M A L via serine D It can be attached.

[0198] In some embodiments, M A teeth, Includes TIFF2026086770000033.tif20128.

[0199] In some embodiments, M A It contains (β-alanine)-(glycine)4-(serine), M A A via β-alanine 1 Attached to M A T via serine 1 Attached to M A L via serine D It can be attached.

[0200] In some embodiments, MA teeth, Includes TIFF2026086770000034.tif21128.

[0201] In some embodiments, the peptide portion comprises (β-alanine)-(glycine)-(glutamic acid), and the peptide portion is L 3 When L exists 3 It can be attached to or L 3 When it is not present, L is transmitted via β-alanine. M It is attached to the peptide portion. 1 When present, T is transmitted via glutamate. 1 The peptide portion is attached to L D When present, L is transmitted via glutamate. D It can be attached.

[0202] In some embodiments, the peptide portion is Includes TIFF2026086770000035.tif25128.

[0203] M A Regarding the manner of, * is A 1 It indicates that it is attached, ** is T 1 It indicates that it is attached, *** is L D This is understood to indicate that it is attached to [something].

[0204] Hydrophilic group (variable T 1 ) In some embodiments, the hydrophilic groups contained in the conjugates or scaffolds of the present disclosure are water-soluble and substantially non-antigenic polymers. Examples of hydrophilic groups include, but are not limited to, polyhydric alcohols, polyethers, polyanions, polycations, polyphosphates, polyamines, polysaccharides, polyhydroxy compounds, polylysine, and their derivatives. In some embodiments, one end of the hydrophilic group is connected by an inseparable linkage or via a cleavable linkage, M A Linker (for example, MA The hydrophilic group can be functionalized so that it can be covalently attached to an amino acid in the linker. In some embodiments, the functionalization may be via, for example, an amine, thiol, NHS ester, maleimide, alkyne, azide, carbonyl, or other functional group. In some embodiments, the other end (or more ends) of the hydrophilic group is free and unattached. In some embodiments, "unattached" means that the hydrophilic group is not attached to another part, such as D or a drug unit, or other components of the conjugate or scaffold of this disclosure. In some embodiments, the free and unattached end of the hydrophilic group may contain a methoxy, carboxylic acid, alcohol, or other suitable functional group. In some embodiments, the methoxy, carboxylic acid, alcohol, or other suitable functional group acts as a cap for one or more ends of the hydrophilic group.

[0205] In some embodiments, a cleavable linkage refers to a linkage that is substantially unsensitive to cleavage while circulating in plasma but sensitive to cleavage in the intracellular or intratumoral environment. In some embodiments, an uncleavable linkage is a linkage that is substantially unsensitive to cleavage in any biological environment. In some embodiments, chemical hydrolysis of hydrazones, reduction of disulfides, and enzymatic cleavage of peptide or glycosidic bonds are examples of cleavable linkages. In some embodiments, exemplary attachment of hydrophilic groups is via amide linkages, ether linkages, ester linkages, hydrazone linkages, oxime linkages, disulfide linkages, peptide linkages, or triazole linkages. In some embodiments, M A Linker (for example, M A The attachment of hydrophilic groups to amino acids within the linker occurs via amide linkages.

[0206] In some embodiments, the conjugate or scaffold of the Disclosure comprises multiple hydrophilic groups, which may be the same chemical moiety or different chemical moieties (e.g., hydrophilic groups with different molecular weights, number of subunits, or chemical structures). In some embodiments, the multiple hydrophilic groups are located at only one attachment site. A It may be attached to the linker, and M is located in a different part. A It may be attached to the linker.

[0207] In some embodiments, the addition of a hydrophilic group may have two potential effects on the resulting conjugate pharmacokinetics. In some embodiments, the desirable effect is a decrease in clearance (and the resulting increase in exposure) due to reduced nonspecific interactions induced by the exposed hydrophobic elements of the drug or drug-linker. In some embodiments, the undesirable effect is a decrease in the volume and velocity of distribution, which may result from an increase in the molecular weight of the conjugate. In some embodiments, increasing the molecular weight of the hydrophilic group may increase the hydrodynamic radius of the conjugate, resulting in a decrease in diffusivity, thereby reducing the conjugate's ability to penetrate the tumor. Because of these two competing pharmacokinetic effects, it may be desirable to use a hydrophilic group that is large enough to reduce conjugate clearance and thus increase plasma exposure, but not large enough to significantly reduce its diffusivity and thus not large enough to reduce the conjugate's ability to reach the intended target cell population.

[0208] In some embodiments, the hydrophilic group includes, but is not limited to, sugar alcohols (polyalcohols, polyhydric alcohols, alditols or glycitols, e.g., inositol, glycerol, erythritol, treitol, arabitol, xylitol, ribitol, galactitol, mannitol, sorbitol, etc.) or their derivatives (e.g., aminopolyhydric alcohols), carbohydrates (e.g., sugars), polyvinyl alcohol, carbohydrate-based polymers (e.g., dextran), hydroxypropyl methacrylamide (HPMA), polyalkylene oxides, and / or copolymers thereof.

[0209] In some aspects, T 1 This includes a portion that incorporates multiple hydroxyl ("-OH") groups, such as monosaccharides, oligosaccharides, and polysaccharides.

[0210] In some aspects, T 1 R 58 is -H or C 1~8 Multiple alkyl-(CR 58 Contains an OH)- group.

[0211] In some aspects, T 1 is -OH or The file is TIFF2026086770000036.tif6128, and in the formula, n1 is an integer between 0 and approximately 6. Each R 58 These are independently -H or C 1~8 It is alkyl, R 60 is a bond, C 1~6 Alkyl linker, or -CHR 59 - and R 59 is -H, C 1~8 Alkyl, cycloalkyl, or arylalkyl, R 61 CH2OR 62 COOR 62 ,-(CH2) n2COOR 62 , or heterocycloalkyl substituted with one or more hydroxyls, R 62 is -H or C 1~8 It is alkyl, n² is an integer between 1 and approximately 5.

[0212] In some aspects, T 1 is -OH. In some aspects, T 1 teeth, The filename is TIFF2026086770000037.tif5128.

[0213] In some embodiments, R 58 is -H, and R 60 is a combination or C 1~6 It is an alkyl linker, where n1 is an integer from 1 to approximately 6, and R 61 It is either CH2OH or COOH.

[0214] In some embodiments, R 58 is -H, and R 60 ha-CHR 59 - and n1 is 0, R 61 These are heterocycloalkyl groups, such as monosaccharides, that are substituted with one or more hydroxyls.

[0215] In some aspects, T 1 This includes glucosylamines, diamines, or triamines.

[0216] In some aspects, T 1 These are the following fragments or their stereoisomers: The formula contains one or more TIFF2026086770000038.tif177169, and in the formula, R 59 is -H, C 1~8 It is an alkyl, cycloalkyl, or arylalkyl group, where n1 is an integer from 1 to approximately 6, n2 is an integer from 1 to approximately 5, and n3 is an integer from approximately 1 to approximately 3.

[0217] It is understood that all stereochemical forms of the hydrophilic group are intended herein. For example, in the above formula, the hydrophilic group may be derived from ribose, xylose, glucose, mannose, galactose, or other sugars, and retains the stereochemical configuration of the pendant hydroxyl and alkyl groups present in these molecules.

[0218] In the above formula, it should be understood that various deoxy compounds are also intended. For example, where applicable, one or more of the following characteristics of the hydrophilic group are intended:

[0219] In some embodiments, n3 is 2 or 3.

[0220] In some embodiments, n1 is 1, 2, or 3.

[0221] In some embodiments, n2 is 1.

[0222] In some embodiments, R 59 It is hydrogen.

[0223] In some aspects, T 1 teeth, This is TIFF2026086770000039.tif16128. In some aspects, T 1 teeth, This is TIFF2026086770000040.tif24128. In some aspects, T 1 teeth, The filename is TIFF2026086770000041.tif24128.

[0224] In some aspects, T 1 teeth, TIFF2026086770000042.tif15128, and in the formula, n4 is an integer between 1 and approximately 25. Each R 63 These are independently -H or C 1~8 It is alkyl, R 64is a combination or C 1~8 It is an alkyl linker, R 65 is -H, C 1~8 Alkyl, -(CH2) n2 COOR 62 , or -(CH2) n2 COR 66 And, R 62 is H or C 1~8 It is alkyl, R 66 H, The filename is TIFF2026086770000043.tif48144. n² is an integer between 1 and approximately 5.

[0225] In some aspects, T 1 teeth, TIFF2026086770000044.tif15128, and in the formula, R 67 (1)-OH; The file is TIFF2026086770000045.tif64149, and n4 is an integer between approximately 2 and 20, approximately 4 and 16, approximately 6 and 12, and approximately 8 and 12.

[0226] In some aspects, T 1 teeth, The filename is TIFF2026086770000046.tif12128.

[0227] In some embodiments, n4 is an integer between approximately 2 and 20, approximately 4 and 16, approximately 6 and 12, and approximately 8 and 12.

[0228] In some embodiments, n4 is 6, 7, 8, 9, 10, 11, or 12.

[0229] In some embodiments, n4 is 8 or 12.

[0230] In some aspects, T 1 teeth, The filename is TIFF2026086770000047.tif42128, and n4 is an integer between approximately 2 and 24, approximately 4 and 16, approximately 6 and 12, and approximately 8 and 12.

[0231] In some embodiments, n4 is 6, 7, 8, 9, 10, 11, or 12.

[0232] In some embodiments, n4 is 8 or 12. In some embodiments, n4 is 8.

[0233] In some aspects, T 1 teeth, The filename is TIFF2026086770000048.tif26128, and in the formula, n4 is 8.

[0234] In some aspects, T 1 This includes polyethers, such as polyalkylene glycols (PAOs). PAOs include, but are not limited to, polymers of lower alkylene oxides, particularly polymers of ethylene oxides, such as propylene oxide, polypropylene glycol, polyethylene glycol (PEG), polyoxyethylene-conjugated polyols, their copolymers, and their block copolymers.

[0235] In some embodiments, polyalkylene glycol is polyethylene glycol (PEG) including, but not limited to, polydisperse PEG, monodisperse PEG, and discrete PEG. Polydisperse PEG is a heterogeneous mixture of size and molecular weight, while monodisperse PEG is typically purified from a heterogeneous mixture and therefore exhibits a single chain length and molecular weight. In some embodiments, the PEG unit is discrete PEG representing a single molecule with a defined and specified chain length. In some embodiments, polyethylene glycol is mPEG.

[0236] In some aspects, T 1The PEG unit comprises one or more PEG chains. The PEG chains may be linked together in a linear, branched, or star-shaped configuration, for example. In addition to comprising repeating PEG subunits, the PEG unit may also contain non-PEG material (for example, to facilitate coupling multiple PEG chains to each other or to amino acids). Non-PEG material refers to atoms in the PEG chain that are not part of the repeating -CH2CH2O- subunit. In some embodiments, the PEG chain may comprise two monomeric PEG chains linked to each other via non-PEG elements. In some embodiments, the PEG unit may comprise two linear PEG chains attached to a central core attached to an amino acid (i.e., the PEG unit itself is branched).

[0237] The PEG unit reacts via the M group. A Linker (for example, M A The reactive group may be covalently bonded to an amino acid within the linker. The reactive group is a group to which the activated PEG molecule can be bound (e.g., a free amino group or a carboxyl group). In some embodiments, the N-terminal amino acid and lysine (K) have a free amino group, and the C-terminal amino acid residue has a free carboxyl group. Sulfhydryl groups (e.g., found on cysteine ​​residues) may also be used as reactive groups for attaching PEG.

[0238] In some embodiments, the PEG unit is made possible by using methoxylated PEG ("mPEG") having different reactive moieties, including but not limited to succinimidyl succinate (SS), succinimidyl carbonate (SC), mPEG-imidate, para-nitrophenyl carbonate (NPC), succinimidyl propionate (SPA), and cyanuryl chloride. A Linker (for example, M AIt can be attached to amino acids in the linker. Examples of mPEG include mPEG-succinimidyl succinate (mPEG-SS), mPEG2-succinimidyl succinate (mPEG2-SS), mPEG-succinimidyl carbonate (mPEG-SC), mPEG2-succinimidyl carbonate (mPEG2-SC), mPEG-imidate, mPEG-para-nitrophenyl carbonate (mPEG-NPC), mPEG-imidate, mPEG2-para-nitrophenyl carbonate (mPEG2-NP C) includes, but is not limited to, mPEG-succinimidyl propionate (mPEG-SPA), mPEG2-succinimidyl propionate (mPEG2-SPA), mPEG-N-hydroxysuccinimide (mPEG-NHS), mPEG2-N-hydroxysuccinimide (mPEG2-NHS), mPEG-cyanurate chloride, mPEG2-cyanurate chloride, mPEG2-lysinol-NPC, and mPEG2-Lys-NHS. A wide variety of PEG chemical species can be used, and substantially any suitable reactive PEG reagent can be used. In some embodiments, the reactive PEG reagent is a polyfunctional linker or M A Linker (for example, M AWhen attached to an amino acid in the linker, it forms a carbamate or amide bond. Reactive PEG reagents include mPEG2-N-hydroxysuccinimide (mPEG2-NHS), difunctional PEG propionaldehyde (mPEG2-ALD), multi-armed PEG, maleimide-containing PEG (mPEG(MAL)2, mPEG2(MAL)), mPEG-NH2, mPEG-succinimidyl propionate (mPEG-SPA), succinimide of mPEG butanoic acid (mPEG-SBA), mPEG-thioester, mPEG-double ester, mPEG-BTC, mPEG-ButyrALD, mPEG-acetaldehyde diethyl acetal (mPEG-ACET), heterofunctional PEG (e.g., NH2-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-vinyl sulfone (NHS-PEG-VS), or NH This includes, but is not limited to, multi-armed PEGs in the SUNBRITE® series, which include S-PEG-MAL, PEG acrylate (ACRL-PEG-NHS), PEG-phospholipids (e.g., mPEG-DSPE), glycerin-based PEGs activated by chemical reactions selected by those skilled in the art, and any SUNBRITE activated PEGs (including, but not limited to, carboxyl-PEG, p-NP-PEG, tresyl-PEG, aldehyde-PEG, acetal-PEG, amino-PEG, thiol-PEG, maleimide-PEG, hydroxyl-PEG-amine, amino-PEG-COOK hydroxyl-PEG-aldehyde, carboxylic acid anhydride-type PEG, functionalized PEG-phospholipids, and other similar and / or suitable reactive PEGs).

[0239] In some embodiments, a PEG unit includes at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits, at least 13 subunits, at least 14 subunits, at least 15 subunits, at least 16 subunits, at least 17 subunits, at least 18 subunits, at least 19 subunits, at least 20 subunits, at least 21 subunits, at least 22 subunits, at least 23 subunits, or at least 24 subunits. In some such embodiments, a PEG unit includes about 72 or fewer subunits.

[0240] In some aspects, the PEG unit includes at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits, at least 13 subunits, at least 14 subunits, at least 15 subunits, at least 16 subunits, at least 17 subunits, at least 18 subunits, at least 19 subunits, or at least 20 subunits.

[0241] In some aspects, the PEG unit includes at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits, at least 13 subunits, at least 14 subunits, at least 15 subunits, at least 16 subunits, at least 17 subunits, or at least 18 subunits.

[0242] In some embodiments, the PEG unit includes at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, or at least 12 subunits.

[0243] In some embodiments, the PEG unit includes at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, or at least 12 subunits.

[0244] In some embodiments, the PEG unit includes at least 6 subunits, at least 7 subunits, or at least 8 subunits.

[0245] In some embodiments, the linear PEG unit is The filename is TIFF2026086770000049.tif29147, and in the formula, TIFF2026086770000050.tif9128 is M A Linker (for example, M A This indicates the site to which the amino acids within the linker are attached. Y 71 This is a PEG attachment unit, Y 72 This is a PEG capping unit, Y 73 This is a PEG coupling unit (i.e., a unit for coupling multiple PEG subunit chains together), d9 is an integer between 2 and 72, Each d 10 These are independent integers from 1 to 72. d 11 The integer is between 2 and 5.

[0246] In some embodiments, d9 is an integer between 2 and 24. In some embodiments, d9 is an integer between 4 and 24. In some embodiments, d9 is an integer between 6 and 24, 8 and 24, 10 and 24, or 12 and 24.

[0247] In some embodiments, the PEG unit has at least six PEG subunits. In some embodiments, the PEG unit has at least eight PEG subunits. In some embodiments, the PEG unit has at least ten PEG subunits. In some embodiments, the PEG unit has at least twelve PEG subunits.

[0248] In some embodiments, d9 is 8 or about 8, 12 or about 12, 24 or about 24.

[0249] In some embodiments, each Y 72 Independently, C 1~10 Alkyl, -C 2~10 Alkyl-CO2H, -C 2~10 alkyl-OH, -C 2~10 Alkyl-NH2, -C 2~10 Alkyl-NH(C 1~3 Alkyl), or C 2~10 Alkyl-N(C 1~3 It is alkyl(2).

[0250] In some embodiments, Y 72 is -C 1~10 Alkyl, -C 2~10 Alkyl-CO2H, -C 2~10 alkyl-OH, or -C 2~10 It is alkyl-NH2.

[0251] In some embodiments, the PEG coupling unit is part of the PEG unit and is a non-PEG material that works to connect two or more chains of repeating CH2CH2O- subunits. In some embodiments, the PEG coupling unit Y 73 is -C 2~10 Alkyl-C(O)-NH-, -C 2~10 Alkyl-NH-C(O)-, -C 2~10 Alkyl-NH-,-C 2~10 Alkyl-C(O)-, -C 2~10 Alkyl-O-, or -C 2~10 It is alkyl-S-.

[0252] In some embodiments, each Y 73 Independently, C 1~10 Alkyl-C(O)-NH-, -C 1~10 Alkyl-NH-C(O)-, -C 2~10 Alkyl-NH-,-C 2~10 Alkyl-O-, -C 1~10 Alkyl-S- or -C 1~10 It is alkyl-NH-.

[0253] In some embodiments, the PEG attachment unit is part of the PEG unit, and the PEG unit is M A Linker (for example, M A It works to link to an amino acid (in the linker). In some embodiments, the amino acid has a functional group that forms a bond with the PEG unit. In some embodiments, the functional groups for attaching the PEG unit to the amino acid include a sulfhydryl group for forming a disulfide bond or thioether bond, an aldehyde group, a ketone group, or a hydrazine group for forming a hydrazone bond, a hydroxylamine for forming an oxime bond, a carboxyl group or an amino group for forming a peptide bond, a carboxyl group or a hydroxyl group for forming an ester bond, a sulfonic acid for forming a sulfonamide bond, an alcohol for forming a carbamate bond, and an amine for forming a sulfonamide bond or a carbamate bond or an amide bond. In some embodiments, the PEG unit can be attached to the amino acid via, for example, a disulfide, thioester, hydrazine, oxime, peptide, ester, sulfonamide, carbamate, or amide bond. In some embodiments, the reaction for attaching the PEG unit may be cycloaddition, addition, addition / elimination, or substitution, or a combination thereof, where applicable.

[0254] Examples of linear PEG units include: TIFF2026086770000051.tif38148 is included, and in the formula, TIFF2026086770000052.tif8128 is a polyfunctional linker or M A Linker (for example, M A It indicates the attachment site (to an amino acid within the linker), and each d9 is an integer between 4 and 24, 6 and 24, 8 and 24, 10 and 24, 12 and 24, 14 and 24, or 16 and 24.

[0255] In some embodiments, d9 is about 8, about 12, or about 24. In some embodiments, d9 is about 8.

[0256] In some embodiments, the PEG unit is approximately 300 Da to approximately 5 kDa; approximately 300 Da to approximately 4 kDa; approximately 300 Da to approximately 3 kDa; approximately 300 Da to approximately 2 kDa; or approximately 300 Da to approximately 1 kDa. In some embodiments, the PEG unit has at least 6 subunits or at least 8, 10, or 12 subunits. In some embodiments, the PEG unit has at least 6 subunits or at least 8, 10, or 12 subunits, but has 24 or fewer subunits.

[0257] In some embodiments, a suitable polyethylene glycol may have free hydroxyl groups at each end of the polymer molecule, or it may have one hydroxyl group etherified with a lower alkyl, for example, a methyl group. In some embodiments, a suitable polyethylene glycol is a derivative of polyethylene glycol having an esterifiable carboxyl group. In some embodiments, polyethylene glycol is marketed under the trade name PEG, usually as a polymer mixture characterized by its average molecular weight. In some embodiments, polyethylene glycol having an average molecular weight of about 300 to about 5000. In some embodiments, polyethylene glycol having an average molecular weight of about 600 to about 1000.

[0258] In some embodiments, examples of hydrophilic groups suitable for the conjugates, scaffolds, and methods disclosed herein can be found, for example, in US8,367,065 Column 13; US8524696 Column 6; WO2015 / 057699 and WO2014 / 062697, the contents of which are incorporated herein by reference in their entirety.

[0259] STING agonist drug portion (variable D) In some embodiments, the STING agonist drug portion (D) is formula (A): A compound of TIFF2026086770000053.tif62128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: Y1, Y2, Z1, and Z2 are each independently O, S, C, or N. X1, X2, W1, and W2 are each independently C or N. X3 and X4 are each independently S or NR f And, X5 is N or CR A2 And, X6 is N or CR A1 And, R 3 and R 5 Each of them operates independently, CON(R d )(R f ), -CH2N(R d )(R f ), -N(R d )(R f ), -N(R d )CO(R f ), -CH2N(R d )CO(R f ) or R 3 and R 5 One of them is -CON(R d )(R f ), -CH2N(R d )(R f ), -N(R d )(R f ), -N(R d )CO(Rf ) or -CH2N(R d )CO(R f ) and R 3 and R 5 The other is H, -COOH, or -CO2(R C ) and R c is C 1~4 It is alkyl, R A2 and R A1 Each of these is independently H, halogen, hydroxyl, amino, and amino(C) 1~4 Alkyl)-, may be substituted (C 1~6 Alkyl), or possibly substituted (C 1~6 It is alkyl)oxy- and the aforementioned may be substituted (C 1~6 Alkyl) or may be substituted (C 1~6 C of alkyl)oxy 1~6 Alkyls are each independently hydroxyl, C 1~4 Alkoxyl, -N(R e )(R f ), -CO2(R f ), -CON(R e )(R f ), and may be substituted with 1 to 4 substituents selected from the group including -COOH, Each R d These are independently H, hydroxyl, or C 1~4 It is alkyl, R e H, (C 1~4 Alkyl), -CO(C 1~4 Alkyl), -OCO(C 1~4 Alkyl), and -CO2(C 1~4 Selected from alkyl groups, Each R f These are independently H, hydroxy, or (C 1~4 It is alkyl, R 14 and R C2 Either they do not exist independently, or C 1~4 It is alkyl, C 1~4 Alkyl is a halogen, -ORc , -NR c R d , -CO2R c ,-CONR c R d -SO2NR c R d , and -OCONR c R d They may be substituted with a more selected substituent, R 16 and R C1 Either they do not exist independently, or they are H, or C 1~4 It is alkyl, R 15 , R 17 , R 18 , or R 19 Either they do not exist independently, or they are H, or C 1~4 It is alkyl, C 1~4 Alkyl is a halogen, -OR c , -NR c R d , -CO2R c ,-CONR c R d -SO2NR c R d , and -OCONR c R d They may be substituted with a more selected substituent, (i)R A2 and R A1 At least one of the following exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0260] In some embodiments, the STING agonist drug portion is of formula (Aa): A compound of TIFF2026086770000054.tif75128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: Y1, Y2, Z1, Z2, X1, X2, W1, W2, X3, X4, R 3 , R 5 , R c , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 , and R 19 This is defined in equation (A), X5 is CR A2 And, R A2 (C) may be substituted with halogens, hydroxyls, or other elements. 1~6 Alkyl), substituted (C 1~6 Alkyl)oxy-, may be substituted (C 1~6 Alkyl)amino-, or possibly substituted (C 1~6 Alkyl)(C 1~4 It is alkyl)amino-, and the aforementioned may be substituted (C 1~6 Alkyl) or substituted (C 1~6 C of alkyl)oxy 1~6 Alkyls are each independently hydroxyl, C 1~4 Alkoxyl, -N(R e )(R f ), -CO2(R f ), -CON(R e )(R f ), and may be substituted with 1 to 4 substituents selected from the group including -COOH, (i)R A2 is R A2 L via the functional group D (ii)R C2 and R C1At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0261] In some embodiments, the STING agonist drug portion is of formula (Ab): A compound of TIFF2026086770000055.tif75128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: Y1, Y2, Z1, Z2, X1, X2, W1, W2, X4, X5, X6, R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 , and R 19 This is defined in equation (A), (i)R A2 and R A1 At least one of the following exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0262] In some embodiments, the STING agonist drug portion is of formula (Ac): A compound of TIFF2026086770000056.tif75128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: Y2, Z2, X2, W2, X3, X4, X5, X6, R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 , and R 19 This is defined in equation (A), One of W1, X1, Y1, and Z1 is N, and any further W1, X1, Y1, and Z1 is O, S, or C. (i)R A2 and R A1 At least one of the following exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0263] In some embodiments, the STING agonist drug portion is of formula (Ad): The compound TIFF2026086770000057.tif75128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, Y1, Y2, Z1, Z2, X1, X2, W1, W2, X3, R 3 , R 5 , R c , R d , R e , R f , R 14 , R A2 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 , and R 19 This is defined in equation (A), X5 is CR A2 And, (i)R A2 is R A2 L via the functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0264] In some embodiments, the STING agonist drug portion is of formula (Ae): A compound of TIFF2026086770000058.tif76128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: Y1, Y2, Z1, Z2, X1, X2, X3, W1, W2, R A1 , R3, R 5 , R c , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 , and R 19 This is defined in equation (A), X6 is CRA1 And, (i)R A1 is R A1 L via the functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0265] In some embodiments, the STING agonist drug portion is of formula (Af): A compound of TIFF2026086770000059.tif71128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: X2, X3, X4, X5, X6, W2, Y2, Z2, R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 16 , R 17 , R 18 , R 19 , R C2 , and R C1 This is defined in equation (A), (i)R A2 and R A1 At least one of the following exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1L via at least one functional group D It connects to the network.

[0266] In some embodiments, the STING agonist drug portion is formula (A-f1): A compound of TIFF2026086770000060.tif71128, or its prodrug, solvated compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: X2, X3, X4, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R 16 , R A2 , R 17 , R 18 , R 19 , R C2 , and R C1 This is defined in equation (A), X5 is CR A2 And, (i)R A2 is R A2 L via the functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0267] In some embodiments, the STING agonist drug portion is formula (A-f2): A compound of TIFF2026086770000061.tif71128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: X2, X4, X5, X6, W2, Y2, Z2, R 3 , R 5 , R c , R A2 , R A1 , Rd , R e , R C1 , R C2 , R 16 , R 17 , R 18 , R 19 , and R f are as defined by formula (A), (i) At least one of R A2 and R A1 exists, and at least one of R A2 and R A1 is connected to L A2 and / or R A1 through at least one functional group of R D , or (ii) at least one of R C2 and R C1 exists, and at least one of R C2 and R C1 is connected to L C2 and / or R C1 through at least one functional group of R D .

[0268] In some embodiments, the STING agonist drug moiety is a compound of formula (A-f3): TIFF2026086770000062.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R 16 , R A2 , R C2 , R 16 , R 17 , R 18 , R 19 , and R C1 are as defined by formula (A), X5 is CR A2 , (i) R A2 is through the functional group of R A2 connected to LD (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0269] In some embodiments, the STING agonist drug portion is formula (A-f4): A compound of TIFF2026086770000063.tif71128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R 16 , R A2 , R C2 , R 16 , R 17 , R 18 , R 19 , and R C1 This is defined in equation (A), X5 is CR A2 And, (i)R A2 is R A2 L via the functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0270] In some embodiments, the STING agonist drug portion is formula (A-f5): The compound of TIFF2026086770000064, or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer, wherein, X2, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R 16 , R A2 , R C2 , R 16 , R 17 , R 18 , R 19 , and R C1 are as defined by formula (A), X5 is CR A2 , and (i) R A2 is connected to L A2 via the functional group of R D , or (ii) at least one of R C2 and R C1 is present, and at least one of R C2 and R C1 is connected to L C2 and / or R C1 via at least one functional group of R D .

[0271] In some embodiments, the STING agonist drug moiety is of formula (A-g): The compound of TIFF2026086770000065, or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer, wherein, X3, X4, X5, X6, R 3 , R 5 , R c , R A2 , R A1 , R C2 , R 17 , R 18 , R 19 , R d , R e , R f , R 16 , and RC1 This is defined in equation (A), Y2 and Z2 are each independently O, S, C, or N. X2 and W2 are independently either C or N. (i)R A2 and R A1 At least one of the following exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0272] In some embodiments, the STING agonist drug portion is formula (A-g1): A compound of TIFF2026086770000066.tif71128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: X2, W2, Y2, Z2, X3, X4, X5, R 3 , R 5 , R c , R d , R e , R f , R A2 , R C2 , R 17 , R 18 , R 19 , R 16 , and R C1 This is defined in equation (A), (i)R A2 is R A2 L via the functional group D (ii)R C2 and R C1 At least one of the following exists, R C2and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0273] In some embodiments, the STING agonist drug portion is formula (A-g2): A compound of TIFF2026086770000067.tif71128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: X2, X4, X5, X6, W2, Y2, Z2, R 3 , R 5 , R c , R A2 , R A1 , R C2 , R 17 , R 18 , R 19 , R d , R e , R f , R 16 , and R C1 This is defined in equation (A), (i)R A2 and R A1 At least one of the following exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0274] In some embodiments, the STING agonist drug portion is of formula (A-g3): A compound of TIFF2026086770000068.tif71128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R A2 , R C2 , R 17 , R 18 , R 19 , R 16 , and R C1 This is defined in equation (A), X5 is CR A2 And, (i)R A2 is R A2 L via the functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D Connected to Optionally, R A2 R A2 L via the functional group D It connects to the network.

[0275] In some embodiments, the STING agonist drug portion is formula (A-g4): A compound of TIFF2026086770000069.tif71128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein in the formula, X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R A2 , R C2 , R 17 , R 18 , R 19 , R 16, and R C1 This is defined in equation (A), X5 is CR A2 And, (i)R A2 is R A2 L via the functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0276] In some embodiments, the STING agonist drug portion is of formula (A-g5): The compound TIFF2026086770000070.tif71128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, X2, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R A2 , R C2 , R 17 , R 18 , R 19 , R 16 , and R C1 This is defined in equation (A), X5 is CR A2 And, (i)R A2 is R A2 L via the functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0277] In some embodiments, the STING agonist drug portion is of formula (Ah): A compound of TIFF2026086770000071.tif78128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein in the formula, X1, W1, Y1, Z1, X3, X4, X5, X6, R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 14 , R 15 , R 18 , R 19 , R 16 , and R C1 This is defined in equation (A), (i)R A2 and R A1 At least one of the following exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0278] In some embodiments, the STING agonist drug portion is formula (A-h1): A compound of TIFF2026086770000072.tif78128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, wherein the formula is: X1, X3, W1, Y1, Z1, X5, X6, R 3 , R 5 , R c , RA2 , R A1 , R d , R e , R f , R 14 , R 15 , R 18 , R 19 , R 16 , and R C1 This is defined in equation (A), (i)R A2 and R A1 At least one of the following exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0279] In some embodiments, the STING agonist drug portion is of formula (A-h2): A compound of TIFF2026086770000073.tif81128, or its prodrug, solvated compound, pharmaceutically acceptable salt, or tautomer, wherein in the formula, X1, X3, W1, Y1, Z1, R 3 , R 5 , R c , R d , R e , R f , R A2 , R 14 , R 15 , R 18 , R 19 , R 16 , and R C1 This is defined in equation (A), X5 is CR A2 And, (i)R A2 and RA1 At least one of the following exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0280] In some embodiments, the STING agonist drug portion (D) is a compound of formula (A), wherein the compound is of formula (Ai): The compound TIFF2026086770000074.tif57128, or its prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer, Y1, Y2, Z1, Z2, X1, X2, X3, X6, W1, W2, R A1 , R A2 , R c , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 , and R 19 This is defined in equation (A), (i)R A2 and R A1 At least one of the following exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of the following exists, R C2 and RC1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0281] In some embodiments, each STING agonist drug portion (D) is independently TIFF2026086770000075.tif202131TIFF2026086770000076.tif215128TIFF2026086770000077.tif 218128TIFF2026086770000078.tif215126TIFF2026086770000079.tif218128TIFF20260867700000 80.tif214128TIFF2026086770000081.tif219131TIFF2026086770000082.tif208126TIFF2026086770000083.tif197126TIFF2026086770000084.tif200126TIFF2026086770000085.tif96128, and in the formula, R 2 It either does not exist, is -O-, or -NR 4 - and R 4 is H or C 1~3 It is alkyl, TIFF2026086770000086.tif4128 is L D This indicates that it is attached.

[0282] In some embodiments, each STING agonist drug portion (D) is independently TIFF2026086770000087.tif52128TIFF2026086770000088.tif216130TIFF2026086770000089.tif168130TIFF2026086770000090.tif103128, and in the formula, R 2 It either does not exist, is -O-, or -NR 4 - and R 4is H or C 1~3 It is alkyl, TIFF2026086770000091.tif4128 is L D This indicates that it is attached.

[0283] In some embodiments, each STING agonist drug portion (D) is independently TIFF2026086770000092.tif162128, and in the formula, R 2 It either does not exist, is -O-, or -NR 4 - and R 4 is H or C 1~3 It is alkyl, TIFF2026086770000093.tif4128 is L D This indicates that it is attached.

[0284] In some embodiments, each STING agonist drug portion (D) is independently TIFF2026086770000094.tif153139, and in the formula, R 2 It either does not exist, is -O-, or -NR 4 - and R 4 is H or C 1~3 It is alkyl, TIFF2026086770000095.tif4128 is L D This indicates that it is attached.

[0285] In some embodiments, each STING agonist drug portion (D) is independently TIFF2026086770000096.tif109139, and in the formula, R 2 It either does not exist, is -O-, or -NR 4 - and R 4 is H or C 1~3 It is alkyl, TIFF2026086770000097.tif4128 is LD This indicates that it is attached.

[0286] Protein-based recognition molecules (PBRMs) In some embodiments, protein-based recognition molecules direct conjugates to specific tissues, cells, or intracellular locations. In some embodiments, protein-based recognition molecules can direct conjugates in culture, throughout the organism, or both. In each case, the protein-based recognition molecule may have ligands present on the cell surface of target cells to which the protein-based recognition molecule binds with effective specificity, affinity, and avidity. In some embodiments, protein-based recognition molecules target conjugates to tissues other than the liver. In some embodiments, protein-based recognition molecules target conjugates to specific tissues such as the liver, kidney, lung, or pancreas. Protein-based recognition molecules can target conjugates to target cells, such as cancer cells, receptors expressed on such cells, matrix tissues, or cancer-associated proteins, such as tumor antigens. Alternatively, cells containing tumor vascular structures may be targeted. Protein-based recognition molecules can direct conjugates to specific types of cells, such as specific targeting to hepatocytes within the liver, as opposed to Kupffer cells. In some embodiments, the protein-based recognition molecule can direct the conjugate to cells of the reticuloendothelial system or lymphoid system, or to professional phagocytic cells such as macrophages or eosinophils. In some embodiments, the conjugate itself may be an effective delivery system that does not require specific targeting.

[0287] In some embodiments, protein-based recognition molecules can target conjugates to intracellular locations such as the nucleus, cytoplasm, or endosomes. In some embodiments, protein-based recognition molecules can enhance cell binding to receptors, cytoplasmic transport to and nuclear translocation, or release from endosomes or other intracellular vesicles.

[0288] In some embodiments, the protein-based recognition molecule is an antibody, antibody fragment, protein, peptide, or peptide mimic.

[0289] In some embodiments, the protein-based recognition molecule is an antibody. In some embodiments, the protein-based recognition molecule is an antibody fragment. In some embodiments, the protein-based recognition molecule is a protein. In some embodiments, the protein-based recognition molecule is a peptide. In some embodiments, the protein-based recognition molecule is a peptide mimic.

[0290] In some embodiments, the antibody or antibody fragment is an antibody or antibody fragment in which one or more amino acids of the corresponding parent antibody or antibody fragment (e.g., the corresponding wild-type antibody or antibody fragment) are substituted with cysteine ​​(e.g., engineered cysteine). In some embodiments, the parent antibody or antibody fragment may be wild-type or mutated.

[0291] In some embodiments, the antibody or antibody fragment may be a mutated antibody or antibody fragment. In some embodiments, a monoclonal antibody known in the art is manipulated to form an antibody. In some embodiments, an antibody fragment known in the art (e.g., a Fab antibody fragment) is manipulated to form an antibody fragment (e.g., a cysteine-manipulated Fab antibody fragment). In some embodiments, a one-site mutation in Fab results in one residue in Fab, whereas because IgG antibodies are dimeric, a one-site mutation in an antibody results in two amino acids in the resulting antibody.

[0292] In some embodiments, the antibody or antibody fragment retains the antigen-binding ability of its corresponding wild-type antibody or antibody fragment. In some embodiments, the antibody or antibody fragment can bind to one or more antigens of its corresponding wild-type antibody or antibody fragment.

[0293] In some embodiments, the exemplary antibodies specific to cell surface markers, or antibodies derived from Fab, Fab2, scFv, or camel antibody heavy chain fragments, include 5T4, AOC3, ALK, AXL, B7-H4, C242, C4.4a, CA-125, CCL11, CCR5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD18, CD19, CA19-9, CDH6, CD20, CD22, CD23, CD25, CD28, CD30, CD31, CD33, CD37, CD38, CD40, CD41, CD44, CD44v6, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, C D70, CD74, CD79-B, CD80, CD125, CD103, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM-5, clamping factor, Clec9A, CSFR1, CTLA-4, CXCR2, DEC205, EGFR (HER1), ErbB1, ErbB2, ErbB3, EpCAM, EPHA2, EPHB2, EPHB4, FAP, FGFR (i.e., FGFR1, FGFR2, FGFR3, FGFR4), FLT3, fibronectin-EDB, folate receptor, GD2, GD3, GPNMB, GCC (GUCY2C), HGF, HER2, HER3, HMI.24, ICAM, ICOS-L, IGF-1 receptor, VEGFR1, EphA2, TRPV1, CFTR, gpNMB, CA9, Cripto, c-KIT, c-MET, ACE, APP, Adrenaline receptor-β2, Clozin 3, LIV1, LY6E, Mesothelin, MUC1, MUC13, NaPi2b, NOTCH1, NOTCH2, NOTCH3, NOTCH4, RON, ROR1, PD-L1, PD-L2, PTK7, B7-H3, B7-B4, IL-2 receptor, IL-4 receptor, IL-13 receptor, TROP-2, frizzled-7, Integrin (Including α4, αvβ3, αvβ5, αvβ6, α1β4, α4β1, α4β7, α5β1, α6β4, αIIbβ3 integrins), IFN-α, IFN-γ, IgE, IgE, IGF-1 receptor, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, ITGB2 (CD18), LFA-1 (CD11a), CD11b, L-selectin (CD62L), mucin, myostatin, NCA-90, NGF, PDGFRα, phosphatidylserine, prostate cancer cells, Pseudomonas aeruginosa This includes, but is not limited to, Aeruginosa, rabies, RANKL, respiratory syncytial virus, Rh factor, SLAMF7, sphingosine-1-phosphate, TAG-72, T cell receptor, tenascin C, TGF-1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR2, vimentin, etc.

[0294] In some embodiments, antibodies specific to cell surface markers, or antibodies derived from Fab, Fab2, scFv, or camel antibody heavy chain fragments, include CA-125, C242, CD3, CD11b, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD103, CD138, CD141, CD326, CEA, Clec9A, CSFR1, CTLA-4, DEC205, EGFR (HER1), ErbB2, ErbB3, FAP, fibronectin-EDB, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEG This product contains FR2, VEGFR1, EphA2, EpCAM, 5T4, PTK7, TAG-72, tenascin C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate cancer cells, adrenergic receptor-β2, clodin 3, mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, frizzled-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, and integrins (including αvβ3, αvβ5, αvβ6, α1β4, α4β1, α5β1, α6β4 inertins), tenascin C, TRAIL-R2, and vimentin.

[0295] In some embodiments, the antibodies are 5T4, CA-125, CEA, CDH6, CD3, CD11b, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD51, CD-103, CTLA-4, CEACAM5, Clec9A, CSFR1, DEC205, EpCAM, HER2, EGFR (HER1), FAP, fibronectin-EDB, folate receptor, GCC (GU) It has been developed for the cell surface markers of CY2C, HGF, integrin αvβ3, integrin α5β1, IGF-1 receptor, GD3, GPNMB, mucin, LIV1, LY6E, mesothelin, MUC1, MUC13, NaPi2b, PTK7, phosphatidylserine, prostate cancer cells, PDGFRα, TAG-72, tenascin C, TRAIL-R2, VEGF-A, and VEGFR2. In this embodiment, the antibodies include avagovomab, adecatumumab, aracizumab, artumomab, anatumomab, artumomab, bavituximab, bevacizumab (AVASTIN®), vibatuzumab, blinatumomab, brentuximab, cantuzumab, catumakisomab, capromab, cetuximab, sitatuzumab, cribatuzumab, conatumumab, dacetuzumab, edrecolomab, epratuzumab, erzumakisomab, etalacizumab, faretuzumab, figtumumab, gemtuzumab, grembatumumab, ibritumomab, igovomab, intetumumab, inotuzumab, rabetuzumab, lexatumumab, This includes, but is not limited to, lintuzumab, lucatumumab, matsuzumab, mitumomab, naptumomab estafenatox, nesitumumab, oportuzumab, olegobomab, panitumumab, pemtumomab, pertuzumab, pritumumab, rituximab (RITUXAN®), rilotumumab, lobatumumab, satumomab, sibrotuzumab, tapritumomab, tenatumomab, tenatumomab, tisilimmab (tremelimumab), tigatuzumab, trastuzumab (HERCEPTIN®), tositumomab, tremelimumab, tucotzumab sermoloukin, borosiximab, and zaltumumab.

[0296] In some embodiments, the antibody produced against the cell surface marker is pertuzumab or trastuzumab for HER2, cetuximab or panitumumab for EGFR (HER1), rituximab for CD20, bevacizumab for VEGF-A, epratuzumab or vertuzumab for CD-22, and rabetuzumab for CEA.

[0297] Exemplary peptides or peptide mimics include integrin-targeting peptides (RGD peptides), LHRH receptor-targeting peptides, ErbB2 (HER2) receptor-targeting peptides, prostate-specific membrane-bound antigen (PSMA)-targeting peptides, lipoprotein receptor LRP1 targeting peptides, ApoE protein-derived peptides, ApoA protein peptides, somatostatin receptor-targeting peptides, chlorotoxin-derived peptides, and bombesin.

[0298] In some embodiments, the peptide or peptide mimic is an LHRH receptor targeting peptide and an ErbB2 (HER2) receptor targeting peptide.

[0299] Exemplary proteins include insulin, transferrin, fibrinogen-γ fragments, thrombospondin, claudin, apolipoprotein E, aphibody molecules such as ABY-025, ankyrin repeat proteins, ankyrin-like repeat proteins, and synthetic peptides.

[0300] In some embodiments, the protein-drug conjugate includes broad-spectrum cytotoxics combined with cell surface markers, such as pertuzumab or trastuzumab for HER2, cetuximab and panitumumab for EGFR, rabetuzumab for CEA, rituximab for CD20, bevacizumab for VEGF-A, or epratuzumab or vertuzumab for CD-22.

[0301] In some embodiments, the protein-drug conjugates or protein conjugates used in this disclosure include combinations of two or more protein-based recognition molecules, for example, a combination of an EGF receptor (EGFR) on tumor cells and a bispecific antibody made against CD3 and CD28 on T cells; a combination of an antibody or an antibody derived from Fab, Fab2, scFv, or a camel antibody heavy chain fragment and a peptide or peptidomimetic; a combination of an antibody or an antibody derived from Fab, Fab2, scFv, or a camel antibody heavy chain fragment and a protein; and a combination of two bispecific antibodies, for example, a CD3-CD19+CD28-CD22 bispecific antibody.

[0302] In some embodiments, the protein-drug conjugate or protein conjugate used in this disclosure is, for example, trastuzumab, cetuximab, rituximab, bevacizumab, epratuzumab, vertuzumab, rabetuzumab, B7-H4, B7-H3, CD11b, CD103, CA125, CDH6, CD33, CXCR2, CEACAM5, Clec9A, CSFR1, DEC205, E This includes protein-based recognition molecules that are antibodies against antigens, such as GFR, FAP, fibronectin-EDB, FGFR1, FGFR2, FGFR3, FGFR4, GCC (GUCY2C), HER2, LIV1, LY6E, NaPi2b, c-Met, mesothelin, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PD-L1, PTK7, c-Kit, MUC1, MUC13, and 5T4.

[0303] In some embodiments, the protein-drug conjugate or protein conjugate of the present disclosure comprises a protein-based recognition molecule which is CSRF1, CD11b, DEC205, clec9A, CD103, B7H4, mesothelin, PTK7, Ly6E, FAP, fibronectin-EDB, Her-2, or NaPi2b antibody.

[0304] NaPi2b antibody In some embodiments, NaPi2b antibodies suitable for conjugation bind to the extracellular domain of SLC34A2. In some embodiments, the disclosure provides NaPi2b target-directed monoclonal antibodies that specifically recognize NaPi2b, also known as sodium-dependent phosphate transport protein 2B. In some embodiments, NaPi2b antibodies used in the conjugates disclosed herein can and can modulate at least one biological activity of NaPi2b, for example, by blocking, inhibiting, reducing, antagonistizing, neutralizing, or otherwise interfering, and are useful in modulating, for example, by blocking, inhibiting, reducing, antagonistizing, neutralizing, or otherwise interfering. In some embodiments, the antibodies disclosed herein also include antibodies that bind to soluble NaPi2b. In some embodiments, NaPi2b antibodies specifically bind to an epitope on the extracellular domain (ECD) of human NaPi2b. These antibodies are collectively referred to herein as “NaPi2b” antibodies.

[0305] In some embodiments, the NaPi2b antibody-drug conjugate provided herein has an equilibrium dissociation constant (K) of ≤1 μM (e.g., ≤100 nM; ≤10 nM; ≤1 nM) to the NaPi2b epitope. d or K D The antibody conjugate contains an antibody that binds via ) . In some embodiments, the NaPi2b antibody used in the antibody-drug conjugate disclosed herein has a K in the range of approximately ≤1 nM to approximately 1 pM d This indicates.

[0306] In some embodiments, the NaPi2b antibody-drug conjugates provided herein may include an antibody that acts to modulate, block, inhibit, reduce, antagonistize, neutralize, or otherwise interfere with the functional activity of NaPi2b. In some embodiments, the functional activity of NaPi2b includes, for example, involvement in transcellular inorganic phosphate (Pi) uptake, thereby contributing to the maintenance of phosphate homeostasis in the body. In some embodiments, the NaPi2b antibody completely or partially inhibits the functional activity of NaPi2b by partially or completely modulating, block, inhibit, reduce, antagonistize, neutralize, or otherwise interfering with transcellular inorganic phosphate uptake.

[0307] In some embodiments, a NaPi2b antibody is considered to completely modulate, block, inhibit, reduce, antagonistize, neutralize, or otherwise interfere with NaPi2b functional activity when the level of NaPi2b functional activity in the presence of the NaPi2b antibody is reduced by at least 95%, for example, 96%, 97%, 98%, 99%, or 100%, compared to the level of NaPi2b functional activity in the absence of binding to the NaPi2b antibody described herein. In some embodiments, a NaPi2b antibody is considered to partially modulate, block, inhibit, reduce, antagonistize, neutralize, or otherwise interfere with NaPi2b functional activity when the level of NaPi2b activity in the presence of a NaPi2b antibody is reduced by less than 95%, for example, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%, compared to the level of NaPi2b activity in the absence of binding to the NaPi2b antibody described herein.

[0308] In some embodiments, the exemplary antibodies disclosed herein include the XMT-1535 antibody. These antibodies have been shown to exhibit specificity for human NaPi2b and to inhibit NaPi2b activity.

[0309] The NaPi2b human or humanized monoclonal antibody, XMT-1535, contains a heavy chain (HC), heavy chain variable region (VH), light chain (LC), and light chain variable region (VL), as shown in the amino acid sequence and corresponding nucleic acid sequence in Table I below. In the following amino acid sequences, the variable heavy chain region and variable light chain region of each antibody are shaded. In the following amino acid sequences, the complementarity-determining regions (CDRs) of the heavy and light chains are underlined. The amino acids containing the complementarity-determining regions (CDRs) of the XMT-1535 antibody are disclosed in U.S. Patent No. 8,603,474.

[0310] (Table I) Sequence of NaPi2b human or humanized monoclonal antibody XMT-1535 TIFF2026086770000098.tif76161

[0311] The antibodies disclosed herein specifically bind to epitopes located on the extracellular domain (ECD) of human NaPi2b.

[0312] In some embodiments, those skilled in the art will recognize that, without excessive experimentation, it is possible to determine whether a monoclonal antibody has the same specificity as the monoclonal antibodies disclosed herein (e.g., XMT-1535, 10H1.11.4B) by examining whether the former prevents the latter from binding to a natural binding partner or other molecules known to bind to NaPi2b. If the monoclonal antibody being tested competes with the monoclonal antibody disclosed herein, as indicated by the reduced binding by the monoclonal antibodies disclosed herein, the two monoclonal antibodies will bind to the same or closely related epitopes.

[0313] An alternative method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody disclosed herein is to pre-incubate the monoclonal antibody disclosed herein with soluble NaPi2b (which typically reacts with the monoclonal antibody disclosed herein), and then add the monoclonal antibody under test to determine whether the monoclonal antibody under test is inhibited in terms of its ability to bind to NaPi2b. If the monoclonal antibody under test is inhibited, it likely has the same or functionally equivalent epitope specificity as the monoclonal antibody disclosed herein.

[0314] Screening of monoclonal antibodies disclosed herein can also be performed, for example, by measuring NaPi2b-mediated activity to determine whether the test monoclonal antibody can modulate, block, inhibit, reduce, antagonistize, neutralize, or otherwise interfere with NaPi2b activity.

[0315] In some embodiments, the antibodies disclosed herein include a heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical amino acid sequences to a sequence selected from SEQ ID NO:3, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical amino acid sequences to a sequence selected from SEQ ID NO:4.

[0316] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence that is identical to the amino acid sequence of SEQ ID NO:1 for at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and a light chain amino acid sequence that is identical to the amino acid sequence of SEQ ID NO:2 for at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0317] In some embodiments, the antibodies disclosed herein include a heavy chain variable region amino acid sequence of SEQ ID NO:3 and a light chain variable region amino acid sequence of SEQ ID NO:4.

[0318] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence of SEQ ID NO:1 and a light chain amino acid sequence of SEQ ID NO:2.

[0319] In some embodiments, the antibodies disclosed herein include the CDRH1 amino acid sequence of SEQ ID NO:5, the CDRH2 amino acid sequence of SEQ ID NO:6, the CDRH3 amino acid sequence of SEQ ID NO:7, the CDRL1 amino acid sequence of SEQ ID NO:8, the CDRL2 amino acid sequence of SEQ ID NO:9, and the CDRL3 amino acid sequence of SEQ ID NO:10.

[0320] In some embodiments, the antibodies disclosed herein contain at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the same amino acid sequence as SEQ ID NO:5; CDRH2 contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the same amino acid sequence as SEQ ID NO:6; SEQ ID CDRH3 contains the same amino acid sequence as NO:7 for at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more; CDRL1 contains the same amino acid sequence as SEQ ID NO:8 for at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more; CDRL2 contains the same amino acid sequence as SEQ ID NO:9 for at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more; and SEQ ID CDRL3 containing the same amino acid sequence as NO:10 for at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0321] In some embodiments, the antibodies disclosed herein contain one or more conserved amino acid substitutions in the variable domain sequence, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more conserved substitutions in the variable domain sequence. In some embodiments, these conserved amino acid substitutions are located in the CDR region, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more conserved substitutions are cumulative across all CDRs, and in some specific embodiments, each CDR sequence may contain up to one, two, three, or four conserved amino acid substitutions, for example, in SEQ ID NO: 5-10.

[0322] In some embodiments, those skilled in the art will recognize that, without excessive experimentation, it is possible to determine whether a monoclonal antibody has the same specificity as the monoclonal antibody XMT-1535 by examining whether the former prevents the latter from binding to a natural binding partner or other molecule known to bind to NaPi2b. If the monoclonal antibody being tested competes with the monoclonal antibody disclosed herein, as indicated by the reduced binding by the monoclonal antibody disclosed herein, the two monoclonal antibodies will bind to the same or closely related epitopes.

[0323] In some embodiments, an alternative method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody disclosed herein is to pre-incubate the monoclonal antibody disclosed herein with soluble NaPi2b (which typically reacts with the monoclonal antibody disclosed herein), and then add the monoclonal antibody under test to determine whether the monoclonal antibody under test is inhibited in terms of its ability to bind to NaPi2b. In some embodiments, if the monoclonal antibody under test is inhibited, it has the same or functionally equivalent epitope specificity as the monoclonal antibody disclosed herein.

[0324] Screening of monoclonal antibodies disclosed herein can also be carried out, for example, by measuring NaPi2b-mediated activity and determining whether the test monoclonal antibody can modulate, block, inhibit, reduce, antagonistize, neutralize, or otherwise interfere with NaPi2b activity.

[0325] In some embodiments, NaPi2b antibodies suitable for conjugation can be prepared and purified by well-known techniques, e.g., WO2009 / 097128, WO2017 / 160754, and US16 / 136,706. Each of WO2009 / 097128, WO2017 / 160754, and US16 / 136,706, in whole, is incorporated herein by reference.

[0326] HER2 antibody In some embodiments, HER2 antibodies suitable for conjugation bind to human HER2 in a soluble or membrane-bound form (i.e., when expressed on the cell surface). In some embodiments, the disclosure provides monoclonal antibodies that bind to HER2 and are humanized or fully human. In some embodiments, the disclosure provides monoclonal antibodies that specifically bind to HER2. These antibodies are collectively referred to herein as “HER2” antibodies.

[0327] In some embodiments, a HER2 antibody suitable for conjugation has an equilibrium dissociation constant (K) of ≤1 μM (e.g., ≤100 nM; ≤10 nM; ≤1 nM) for the HER2 epitope. d or K D ) binds. In some embodiments, the disclosure provides monoclonal antibodies that bind to HER2 and are humanized or fully human. For example, the HER2 antibodies provided herein have a K in the range of about ≤1 nM to about 1 pM. d This indicates.

[0328] In some embodiments, the HER2 antibodies disclosed herein act to modulate, block, inhibit, reduce, antagonistize, neutralize, or otherwise interfere with HER2 functional activity. In some embodiments, HER2 functional activity includes, for example, the modification of PI3K-Akt pathway activity. In some embodiments, HER2 antibodies completely or partially inhibit HER2 functional activity by partially or completely modulating, block, inhibit, reduce, antagonistize, neutralize, or otherwise interfering with PI3K-Akt pathway activity. PI3K-Akt pathway activity is evaluated using any method accepted in the art for detecting PI3K-Akt pathway activity, including, but not limited to, detecting the level of phosphorylated Akt in the presence and absence of the antibodies or antigen-binding fragments disclosed herein.

[0329] In some embodiments, the HER2 antibody is considered to completely modulate, block, inhibit, reduce, antagonistize, neutralize, or otherwise interfere with HER2 functional activity when the level of HER2 functional activity in the presence of the HER2 antibody is reduced by at least 80%, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, compared to the level of HER2 functional activity in the absence of binding to the HER2 antibody described herein. In some embodiments, a HER2 antibody is considered to partially modulate, block, inhibit, reduce, antagonistize, neutralize, or otherwise interfere with HER2 functional activity when the level of HER2 activity in the presence of a HER2 antibody is reduced by less than 95%, for example, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%, compared to the level of HER2 activity in the absence of binding to the HER2 antibody described herein.

[0330] In some embodiments, the exemplary antibodies disclosed herein include the XMT-1519 antibody, which exhibits specificity for human HER2 and has been shown to inhibit HER2 functional activity in vitro.

[0331] The HER-2 monoclonal antibody XMT-1519 contains a heavy chain (HC), a variable heavy chain region (VH), a light chain (LC), and a variable light chain region (VL), as shown in the amino acid sequence and corresponding nucleic acid sequence in Table II below. The variable heavy chain region and variable light chain region of each antibody are shaded in the following amino acid sequence. The complementarity-determining regions (CDRs) of the heavy and light chains are underlined in the following amino acid sequence.

[0332] (Table II) Sequence of HER2 human or humanized monoclonal antibody XMT-1519 TIFF2026086770000099.tif80161

[0333] The antibodies and antigen-binding fragments disclosed herein specifically bind to an epitope on the full-length human HER2 receptor, comprising the amino acid sequence of SEQ ID NO:16.

[0334] The antibodies and antigen-binding fragments disclosed herein specifically bind to an epitope on the extracellular domain (ECD) of the human HER2 receptor, comprising the amino acid sequence SEQ ID NO:31.

[0335] In some embodiments, the antibodies disclosed herein exhibit HER2-binding properties different from those of antibodies described in the Art. In some embodiments, the antibodies disclosed herein cross-block each other, but do not cross-block trastuzumab, pertuzumab, Fab37, or chA21 in terms of binding to HER2, thus binding to different HER2 epitopes. Furthermore, in contrast to known antibodies, the antibodies disclosed herein can efficiently translocate into HER2-expressing cells without promoting cell proliferation.

[0336] In some embodiments, the antibodies disclosed herein are fully human monoclonal antibodies that bind to novel epitopes and / or possess other desirable properties for therapeutic use. In some embodiments, exemplary properties include, but are not limited to, desirable binding properties to cancer cells expressing high or low levels of human HER2, specific binding to recombinant human and cynomolgus monkey HER2, efficient internal transfer when bound to HER2, high ability to kill cancer cells expressing high or low levels of HER2 when administered as an antibody-drug conjugate (ADC), lack of significant agonist activity against the proliferation of HER2-expressing cancer cells, and / or resulting in effective antibody-dependent cell-mediated death of HER2-expressing cells, and any combination of the aforementioned properties.

[0337] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor, comprising residues 452-531 of the extracellular domain of the human HER2 receptor, residues 474-553 of SEQ ID NO:16, or residues 452-531 of SEQ ID NO:31.

[0338] In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments that bind to at least a portion of the N-terminus of domain IV of the human HER2 receptor but do not cross-compete with antibodies that bind to epitope 4D5 of the human HER2 receptor. In some embodiments, since trastuzumab is known to bind to epitope 4D5 of the human HER2 receptor, the antibodies or antigen-binding fragments described herein do not cross-compete with trastuzumab in binding to the human HER2 receptor. As used herein, the term epitope 4D5 of the human HER2 receptor refers to amino acid residues 529-627 of the extracellular domain of the human HER2 receptor, residues 551-649 of SEQ ID NO:16, or residues 529-627 of SEQ ID NO:31. In some embodiments, the antibodies or antigen-binding fragments also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0339] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor, comprising residues 452-500 of the extracellular domain of the human HER2 receptor, residues 474-522 of SEQ ID NO:16, or residues 452-500 of SEQ ID NO:31.

[0340] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that specifically bind to an epitope of the human HER2 receptor, comprising at least one amino acid residue selected from the extracellular domain of the human HER2 receptor, for example, residues 543, 547, and 552 of SEQ ID NO: 16 and residues 521, 525, and 530 of SEQ ID NO: 31. In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that specifically bind to an epitope of the extracellular domain of the human HER2 receptor, comprising at least two amino acid residues selected from the extracellular domain of the human HER2 receptor, E521, L525, and R530. In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that specifically bind to an epitope of the human HER2 receptor, comprising at least one amino acid residue selected from the extracellular domain of the human HER2 receptor, E521, L525, and R530. In some embodiments, any or all of these antibodies or their antigen-binding fragments also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0341] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that bind to at least a portion of domain III and at least a portion of the N-terminus of domain IV of the human HER2 receptor, but do not cross-compete with Fab37 monoclonal antibodies or antibodies that bind to epitope 4D5 of the human HER2 receptor. In some embodiments, the antibodies or antigen-binding fragments described herein do not cross-compete with Fab37 monoclonal antibodies and / or trastuzumab in binding to the human HER2 receptor. In some embodiments, the antibodies or antigen-binding fragments also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0342] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor, comprising residues 520-531 of the extracellular domain of the human HER2 receptor, residues 542-553 of SEQ ID NO:16, or residues 520-531 of SEQ ID NO:31.

[0343] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor containing at least one amino acid residue selected from the extracellular domain residues C453, H456, H473, N476, R495, G496, H497, and W499 of the human HER2 receptor, for example, residues 475, 478, 495, 498, 517, 518, 519, and 521 of SEQ ID NO:16, or residues 453, 456, 473, 476, 495, 496, 497, and 499 of SEQ ID NO:31. In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments that specifically bind to an epitope in the extracellular domain of the human HER2 receptor, comprising at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, or at least six amino acid residues selected from the amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HER2 receptor. In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments that specifically bind to an epitope in the extracellular domain of the human HER2 receptor, comprising at least the amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HER2 receptor. In some embodiments, any or all of these antibodies or antigen-binding fragments also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0344] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor containing at least one amino acid residue selected from the extracellular domain residues C453, H473, N476, R495, H497, and W499 of the human HER2 receptor, for example, residues 475, 495, 498, 517, 519, and 521 of SEQ ID NO:16, or residues 453, 473, 476, 495, 497, and 499 of SEQ ID NO:31. In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments that specifically bind to an epitope in the extracellular domain of the human HER2 receptor, comprising at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, or at least six amino acid residues selected from the amino acid residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HER2 receptor. In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments that specifically bind to an epitope in the extracellular domain of the human HER2 receptor, comprising at least the amino acid residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HER2 receptor. In some embodiments, any or all of these antibodies or antigen-binding fragments also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0345] In some embodiments, these antibodies have been shown to exhibit specificity for human HER2 and modulate the PI3K-Akt pathway, which promotes cell survival by reducing phosphorylated AKT levels, for example, by blocking, inhibiting, reducing, antagonistizing, neutralizing, or otherwise interfering with it. In some embodiments, these antibodies internalize from the cell surface of HER2-expressing cells at the same or substantially similar rate as trastuzumab or its biosimilars internalize. In some embodiments, these antibodies and antigen-binding fragments have an internalization rate that, by 4 hours, internalize to about 50% of the total surface bound at time 0.

[0346] In some embodiments, the antibodies disclosed herein include a heavy chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical amino acid sequences to a sequence selected from SEQ ID NO:17, and a light chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical amino acid sequences to a sequence selected from SEQ ID NO:24.

[0347] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence that is identical to or more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the amino acid sequence of SEQ ID NO:19, and a light chain amino acid sequence that is identical to or more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the amino acid sequence of SEQ ID NO:26.

[0348] In some embodiments, the antibodies disclosed herein include a heavy chain variable region amino acid sequence of SEQ ID NO:17 and a light chain variable region amino acid sequence of SEQ ID NO:24.

[0349] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence of SEQ ID NO:19 and a light chain amino acid sequence of SEQ ID NO:26.

[0350] In some embodiments, the antibodies disclosed herein include the CDRH1 amino acid sequence of SEQ ID NO:20, the CDRH2 amino acid sequence of SEQ ID NO:21, the CDRH3 amino acid sequence of SEQ ID NO:22, the CDRL1 amino acid sequence of SEQ ID NO:27, the CDRL2 amino acid sequence of SEQ ID NO:28, and the CDRL3 amino acid sequence of SEQ ID NO:29.

[0351] In some embodiments, the antibodies disclosed herein contain one or more conserved amino acid substitutions in the variable domain sequence, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more conserved substitutions in the variable domain sequence. In some embodiments, these conserved amino acid substitutions are located in the CDR region, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more conserved substitutions are cumulative across all CDRs. In some specific embodiments, each CDR sequence may contain, for example, one, two, three, or up to four conserved amino acid substitutions in SEQ ID NO: 20-22 and 27-29.

[0352] Those skilled in the art will recognize that, without excessive experimentation, it is possible to determine whether a monoclonal antibody has the same specificity as the monoclonal antibody XMT-1519 by examining whether the former prevents the latter from binding to a natural binding partner or other molecules known to bind to HER2. In some embodiments, if the monoclonal antibody being tested competes with the monoclonal antibody disclosed herein, as indicated by the reduced binding by the monoclonal antibody disclosed herein, the two monoclonal antibodies will bind to the same or closely related epitopes.

[0353] In some embodiments, an alternative method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody disclosed herein is to pre-incubate the monoclonal antibody disclosed herein with soluble HER2 (which typically reacts with the monoclonal antibody disclosed herein), and then add the monoclonal antibody under test to determine whether the monoclonal antibody under test is inhibited in terms of its ability to bind to HER2. If the monoclonal antibody under test is inhibited, it likely has the same or functionally equivalent epitope specificity as the monoclonal antibody disclosed herein.

[0354] In some embodiments, screening of monoclonal antibodies disclosed herein may also be carried out, for example, by measuring HER2-mediated PI3K-Akt pathway activity to determine whether the test monoclonal antibody can modulate, block, inhibit, reduce, antagonistize, neutralize, or otherwise interfere with PI3K-Akt pathway activity. In some embodiments, HER2 antibodies suitable for conjugation can be prepared and purified by well-known techniques, e.g., WO2015 / 195917 and PCT / US2018 / 019873. WO2015 / 195917 and PCT / US2018 / 019873, each in whole, are incorporated herein by reference.

[0355] Conjugate In some embodiments, the conjugate of the present disclosure comprises one or more entities of D, where D is a STING agonist, and the one or more entities of D may be the same or different.

[0356] In some embodiments, the presence of one or more PBRMs is attached to the linker-drug moiety, and the presence of one or more PBRMs may be the same or different. In some embodiments, one or more linker-drug moieties containing one or more D are connected to one PBRM (e.g., an antibody).

[0357] In some embodiments, the conjugate of the Disclosure is approximately 40 kDa or greater (e.g., approximately 60 kDa or greater; approximately 80 kDa or greater; approximately 100 kDa or greater; approximately 120 kDa or greater; approximately 140 kDa or greater; approximately 160 kDa or greater; approximately 180 kDa or greater; or approximately 200 kDa or greater; or approximately 40-200 kDa, approximately 40-180 kDa, approximately 40-140 kDa) a. Contains PBRM having a molecular weight of approximately 60-200 kDa, approximately 60-180 kDa, approximately 60-140 kDa, approximately 80-200 kDa, approximately 80-180 kDa, approximately 80-140 kDa, approximately 100-200 kDa, approximately 100-180 kDa, or approximately 100-140 kDa) and having a sulfhydryl (i.e., -SH or thiol) group.

[0358] In some embodiments, the total number of sulfide bonds (or attachment points) formed between the linker-drug portion and the PBRM is 10 or less (e.g., 8, 6, 4, or 2).

[0359] In some embodiments, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of about 40 kDa or more (e.g., about 60 kDa or more, about 80 kDa or more, about 100 kDa or more, about 120 kDa or more, about 140 kDa or more, about 160 kDa or more, or about 180 kDa or more; or about 40-200 kDa, about 40-180 kDa, about 40-140 kDa, about 60-200 kDa, about 60-180 kDa, about 60-140 kDa, about 80-200 kDa, about 80-180 kDa, about 80-140 kDa, about 100-200 kDa, about 100-180 kDa, or about 100-140 kDa).

[0360] In some embodiments, the PBRM has a molecular weight of approximately 40 kDa to approximately 200 kDa for conjugation with one or more linker-drug moieties. In some embodiments, the PBRM has a molecular weight of approximately 40 kDa to approximately 80 kDa for conjugation with one or more linker-drug moieties.

[0361] In some embodiments, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 40 kDa to 200 kDa. In some embodiments, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 40 kDa to 80 kDa.

[0362] In some embodiments, PBRMs within this molecular weight range include, but are not limited to, antibody fragments such as Fab.

[0363] In some embodiments, the PBRM has a molecular weight of approximately 60 kDa to approximately 120 kDa for conjugation with one or more linker-drug moieties.

[0364] In some embodiments, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 60 kDa to 120 kDa.

[0365] In some embodiments, PBRMs within this molecular weight range include, but are not limited to, camelid animals, Fab2, scFvFc, and the like.

[0366] In some embodiments, the PBRM has a molecular weight of approximately 140 kDa to approximately 180 kDa for conjugation with one or more linker-drug moieties.

[0367] In some embodiments, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 140 kDa to 180 kDa.

[0368] In some embodiments, PBRMs within this molecular weight range include, but are not limited to, full-length antibodies such as IgG and IgM.

[0369] In some embodiments, for example, the targeting ligands, linkers, and drug or prodrug fragments described herein can be assembled according to the techniques and methods disclosed herein to construct the conjugates or scaffolds of this disclosure. The therapeutic and targeting conjugates of this disclosure and methods for generating them are described below as non-limiting examples.

[0370] In some embodiments, the total number of sulfide bonds (or attachment points) formed between the linker-drug portion and the PBRM is 8 or less.

[0371] In some embodiments, the total number of sulfide bonds (or attachment points) formed between the linker-drug portion and the PBRM is 8. In some embodiments, the total number of sulfide bonds (or attachment points) formed between the linker-drug portion and the PBRM is 6. In some embodiments, the total number of sulfide bonds (or attachment points) formed between the linker-drug portion and the PBRM is 5. In some embodiments, the total number of sulfide bonds (or attachment points) formed between the linker-drug portion and the PBRM is 4. In some embodiments, the total number of sulfide bonds (or attachment points) formed between the linker-drug portion and the PBRM is 3. In some embodiments, the total number of sulfide bonds (or attachment points) formed between the linker-drug portion and the PBRM is 2.

[0372] In some embodiments, the linker-drug portion to PBRM ratio is approximately 1:1 to approximately 8:1. In some embodiments, the linker-drug portion to PBRM ratio is approximately 1:1 to approximately 6:1. In some embodiments, the linker-drug portion to PBRM ratio is approximately 1:1 to approximately 4:1. In some embodiments, the linker-drug portion to PBRM ratio is approximately 2:1 to approximately 2:1.

[0373] In some embodiments, the linker-to-drug ratio to the PBRM is approximately 6:1 to 8:1.

[0374] In some embodiments, the linker-drug portion to PBRM ratio is approximately 8:1.

[0375] In some embodiments, the linker-drug portion to PBRM ratio is approximately 6:1.

[0376] In some embodiments, the disclosure also relates to a linker-drug moiety comprising at least two parts, each of which can be conjugated to a thiol group in the PBRM to form a protein-linker-drug conjugate.

[0377] In some embodiments, one or more thiol groups of the PBRM are generated by reducing a protein. These one or more thiol groups of the PBRM can then react with one or more linker-drug moieties capable of conjugating the thiol groups from the PBRM with a linker-drug moiety. In some embodiments, at least two of the moieties connected to the PBRM are maleimide groups.

[0378] In some embodiments, the antibody may be activated for conjugation with a linker-drug moiety by treatment with a reducing agent, such as DTT (Clealand's Reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride). In some embodiments, a reduced antibody can be obtained by reducing a full-length monoclonal antibody with an excess amount of TCEP to reduce a disulfide bond (e.g., between cysteine ​​present in the corresponding parent antibody). The newly introduced, unpaired cysteine ​​may still be available for reaction with the linker-drug moiety to form the antibody conjugate of the present disclosure. In some embodiments, an excess amount of linker-drug moiety is added to induce conjugation and form an antibody-drug conjugate, and the conjugation mixture is purified to remove excess linker-drug intermediates and other impurities.

[0379] In some embodiments, for linker-drug moiety conjugation, the PBRM has a molecular weight of 40 kDa or greater (e.g., 60 kDa or greater; 80 kDa or greater; or 100 kDa or greater; 120 kDa or greater; 140 kDa or greater; 160 kDa or greater; or 180 kDa or greater). In some embodiments, the ratio of the PBRM to the linker-drug moiety is approximately 1:1 to approximately 1:8; approximately 1:1 and approximately 1:6; approximately 1:1 to approximately 1:5; approximately 1:1 to approximately 1:4; approximately 1:1 to approximately 1:3; or approximately 1:1 to approximately 1:2.

[0380] PBRMs within this molecular weight range include, but are not limited to, full-length antibodies such as IgG and IgM.

[0381] In some embodiments, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 60 kDa to 120 kDa. In some embodiments, the ratio of the PBRM to the linker-drug moiety is approximately 1:1 and approximately 1:8; approximately 1:1 to approximately 1:6; approximately 1:1 to approximately 1:5; approximately 1:1 to approximately 1:4; approximately 1:1 to approximately 1:3; or approximately 1:1 to approximately 1:2.

[0382] PBRMs within this molecular weight range include, but are not limited to, antibody fragments such as Fab2, scFcFv, and camelid antibodies.

[0383] In some embodiments, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 40 kDa to 80 kDa. In some embodiments, the ratio of the PBRM to the linker-drug moiety is approximately 1:1 and approximately 1:8; approximately 1:1 to approximately 1:6; approximately 1:1 to approximately 1:5; 1:1 to approximately 1:4; approximately 1:1 to approximately 1:3; or approximately 1:1 to approximately 1:2.

[0384] In some embodiments, PBRMs within this molecular weight range include, but are not limited to, antibody fragments, such as Fab.

[0385] In some embodiments, the disclosure features a scaffold useful for conjugating either or both of a protein-based recognition molecule (PBRM) and a STING agonist moiety (D).

[0386] In some embodiments, the drug transport scaffolds described herein (i.e., those not connected to a PBRM) each typically have a polyvariance index (PDI) of 1.

[0387] The conjugates and scaffolds disclosed herein can be purified by large-scale diafiltration (i.e., removal of all starting materials). If necessary, further purification can be performed by size exclusion chromatography to remove all aggregated conjugates. Generally, when purified, the conjugates typically contain less than 5% (e.g., <2% w / w) of aggregated conjugate as confirmed by SEC; less than 0.5% (e.g., <0.1% w / w) of free (unconjugated) drug as confirmed by RP-HPLC; less than 1% of drug-carrying peptide-containing scaffold as confirmed by SEC; and less than 2% (e.g., <1% w / w) of unconjugated PBRM as confirmed by HIC-HPLC.

[0388] In some embodiments, the scaffold is selected from the scaffolds listed in Table A1.

[0389] In some embodiments, the scaffold is selected from the scaffolds listed in Table A2.

[0390] In some embodiments, the conjugate is selected from the conjugates listed in Table B1.

[0391] In some embodiments, the conjugate is selected from the conjugates listed in Table B2.

[0392] [Table A1] TIFF2026086770000101.tif176161TIFF2026086770000102.tif199161TIFF2026086770000103.tif183161TI FF2026086770000104.tif197161TIFF2026086770000105.tif186161TIFF2026086770000106.tif175161In formula, R 14 , R 15 , R16 , R 17 , R 18 , R 19 , R C1 , R C2 X3, X4, X6, X1, W1, Y1, Z1, X2, W2, Y2, and Z2 are as defined herein.

[0393] [Table A2] TIFF2026086770000108.tif150170TIFF2026086770000109.tif165170TIFF2026086770000110.tif15717 0TIFF2026086770000111.tif206170TIFF2026086770000112.tif148170TIFF2026086770000113.tif19517 0TIFF2026086770000114.tif197170TIFF2026086770000115.tif199170TIFF2026086770000116.tif19817 0TIFF2026086770000117.tif186170TIFF2026086770000118.tif214170TIFF2026086770000119.tif17517 0TIFF2026086770000120.tif200170TIFF2026086770000121.tif177170TIFF2026086770000122.tif18317 0TIFF2026086770000123.tif190170TIFF2026086770000124.tif176170TIFF2026086770000125.tif19517 0TIFF2026086770000126.tif200170TIFF2026086770000127.tif204170TIFF2026086770000128.tif20417 0TIFF2026086770000129.tif204170TIFF2026086770000130.tif204170TIFF2026086770000131.tif74170

[0394]

Table B1

[0395] [Table B2] TIFF2026086770000164.tif217170TIFF2026086770000165.tif198170TIFF2026086770000166.tif202170TIFF2026086770000167.tif182170TIFF202 6086770000168.tif151170TIFF2026086770000169.tif185170TIFF2026086 770000170.tif205170TIFF2026086770000171.tif211170TIFF20260867700 00172.tif210170TIFF2026086770000173.tif180170TIFF2026086770000174.tif215170TIFF2026086770000175.tif178170TIFF2026086770000176.t if192170TIFF2026086770000177.tif211170TIFF2026086770000178.tif179170TIFF2026086770000179.tif182170TIFF2026086770000180.tif200170

[0396] In some embodiments, the conjugate is TIFF2026086770000181.tif198156TIFF2026086770000182.tif157131, and in the formula, R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , RC1 , R C2 X3, X4, X6, X1, W1, Y1, Z1, X2, W2, Y2, and Z2 are as defined herein.

[0397] In some embodiments, the conjugate is TIFF2026086770000183.tif123144, and in the formula, d 15 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R C1 , R C2 X3, X4, X6, X1, W1, Y1, Z1, X2, W2, Y2, and Z2 are as defined herein.

[0398] In some embodiments, the conjugate is TIFF2026086770000184.tif161138TIFF2026086770000185.tif201156TIFF2026086770000186.tif38167, In the formula, d 15 This is defined as described herein.

[0399] Pharmaceutical composition In some aspects, this disclosure provides pharmaceutical compositions comprising a conjugate described herein and one or more pharmaceutically acceptable carriers or excipients.

[0400] Pharmaceutical compositions containing the conjugates of this disclosure may be prepared in commonly known ways, for example, by conventional mixing, dissolution, granulation, sugar preparation, wet grinding, emulsification, encapsulation, encapsulation, or lyophilization processes. The pharmaceutical compositions may be formulated in conventional ways using one or more pharmaceutically acceptable carriers, including excipients and / or adjuvants, to facilitate processing the conjugates into pharmaceutically usable preparations. Of course, the appropriate formulation will depend on the chosen route of administration.

[0401] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to allow for simple syringeability. The composition must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or a suitable mixture thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using a surfactant. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols, such as mannitol and sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0402] Sterile injectable solutions can be prepared by filtration sterilization after incorporating the required amount of conjugate, along with one or a combination of the components listed above as needed, into a suitable solvent. Generally, dispersions are prepared by incorporating the conjugate into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which yield a powder of the active ingredient and any further desired components from a pre-filtered sterile solution.

[0403] Oral compositions generally contain an inert diluent or a pharmaceutically acceptable food-grade carrier. Oral compositions may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, conjugates may be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier for use as an oral rinse, in which the conjugate dissolved in the fluid carrier is applied orally, moved quickly, and either spat out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain the following ingredients: binders, e.g., crystalline cellulose, tragacanth gum, or gelatin; excipients, e.g., starch or lactose; disintegrants, e.g., alginic acid, Primogel, or corn starch; lubricants, e.g., magnesium stearate or Sterotes; fluidizers, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose or saccharin; or flavorings, e.g., peppermint, methyl salicylate, or orange flavoring, or compounds of similar properties.

[0404] When administered by inhalation, the conjugate is delivered in the form of an aerosol spray from a suitable nebulizer, such as a pressurized container or dispenser containing a gas like carbon dioxide, or from a nebulizer.

[0405] Systemic administration may also be performed via mucosal or transdermal means. In the case of mucosal or transdermal administration, a permeabilizing agent suitable for the barrier to be penetrated is used in the formulation. Such permeabilizing agents are generally known in the art and, for example, in the case of mucosal administration, include surfactants, bile salts, and fusidic acid derivatives. Mucosal administration can be achieved using nasal drops or suppositories. In the case of transdermal administration, the conjugate is formulated into an ointment, plaster, gel, or cream, which are generally known in the art.

[0406] The conjugate can be prepared using a sustained-release formulation comprising a pharmaceutically acceptable carrier, such as a graft and a microencapsulation delivery system, to protect the conjugate from rapid elimination from the body. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are obvious to those skilled in the art.

[0407] Formulating oral or parenteral compositions into unit dosage forms can be particularly advantageous for ease of administration and uniformity of dosage. As used herein, a unit dosage form refers to a physically distinct unit suitable for administration alone to the target of treatment. Each unit, containing a predetermined amount of conjugate, can be calculated to produce a desired therapeutic effect in conjunction with the required pharmaceutical carrier. The specifications of the unit dosage forms in this disclosure are determined and directly depend on the unique characteristics of the conjugate and the specific therapeutic effect to be achieved.

[0408] In therapeutic applications, the dosage of a pharmaceutical composition used in accordance with this disclosure will vary depending on factors influencing the selected dosage, including the drug, the age, weight, and clinical condition of the recipient patient, as well as the experience and judgment of the clinician or practitioner administering the therapy. Generally, the dose should be sufficient to slow, preferably regress, the symptoms of the disease, and preferably also sufficient to cause complete regression of the disease.

[0409] It is understood that pharmaceutical compositions may be placed in containers, packs, or dispensers along with instructions for administration.

[0410] How to use In some embodiments, the Disclosure provides a method for treating or preventing a disease or disorder in a subject requiring treatment or prevention of the disease or disorder, the method comprising the step of administering a therapeutically effective amount of a conjugate disclosed herein to the subject.

[0411] In some embodiments, the Disclosure provides a method for treating a disease or disorder in a subject requiring treatment of the disease or disorder, the method comprising the step of administering a therapeutically effective amount of a conjugate disclosed herein to the subject.

[0412] In some embodiments, the Disclosure provides a method for activating or enhancing the activity of STING in a subject, the method comprising administering a conjugate disclosed herein to the subject.

[0413] In some embodiments, the Disclosure relates to a method for treating cancer in a subject requiring treatment, the method comprising the step of administering an effective amount of the conjugate disclosed herein to the subject.

[0414] In some embodiments, the Disclosure provides conjugates disclosed herein for use in treating or preventing diseases or disorders in subjects requiring treatment or prevention of diseases or disorders.

[0415] In some embodiments, the Disclosure provides conjugates disclosed herein for use in treating a disease or disorder in a subject requiring treatment of the disease or disorder.

[0416] In some embodiments, the Disclosure provides conjugates disclosed herein for treating STING-mediated diseases or disorders in a subject.

[0417] In some embodiments, this disclosure provides the use of the conjugates disclosed herein for treating cancer in subjects requiring treatment of cancer.

[0418] In some embodiments, the Disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for treating a disease or disorder in a subject requiring treatment of the disease or disorder.

[0419] In some embodiments, the Disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for treating or preventing a disease or disorder in a subject requiring treatment or prevention of the disease or disorder.

[0420] In some embodiments, this disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for treating a disease or disorder mediated by STING in a subject.

[0421] In some embodiments, the Disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for treating cancer in subjects requiring treatment of cancer.

[0422] In some embodiments, this disclosure provides the use of the conjugates disclosed herein for treating or preventing a disease or disorder in a subject requiring treatment or prevention of the disease or disorder.

[0423] In some embodiments, the Disclosure provides the use of the conjugates disclosed herein for treating a disease or disorder in a subject requiring treatment of the disease or disorder.

[0424] In some embodiments, this disclosure provides the use of the conjugates disclosed herein for treating STING-mediated diseases or disorders in a subject.

[0425] In some embodiments, this disclosure provides the use of the conjugates disclosed herein for treating cancer in subjects requiring treatment of cancer.

[0426] In some embodiments, the conjugates disclosed herein are administered to a subject.

[0427] In some embodiments, the Disclosure provides a method for treating or preventing a disease or disorder in a subject requiring treatment or prevention of the disease or disorder, comprising the step of administering a sufficient amount of at least one conjugate of the Disclosure to the subject, wherein the conjugate releases one or more therapeutic agents upon biodegradation.

[0428] In some embodiments, the Disclosure provides a method for treating a disease or disorder in a subject requiring treatment of the disease or disorder, comprising the step of administering a sufficient amount of at least one conjugate of the Disclosure to the subject, wherein the conjugate releases one or more therapeutic agents upon biodegradation.

[0429] In some embodiments, the present disclosure, the conjugate is an antibody-STING agonist conjugate. In some embodiments, the disease or disorder is cancer.

[0430] In certain embodiments, this disclosure provides methods for treating or preventing STING-mediated diseases and disorders. Exemplary diseases / disorders include, but are not limited to, cancer, infectious diseases (e.g., HIV, HBV, HCV, HPV, and influenza), and vaccine adjuvants.

[0431] In some embodiments, the STING pathway may induce antitumor immunity by upregulating IFNβ and interferon (IFN) activating genes (ISGs) in many cell types within tumors in response to agonist cytosolic nucleic acids.

[0432] In some embodiments, the Disclosure provides conjugates disclosed herein for use as vaccine adjuvants. Accordingly, immunogenic compositions or vaccine adjuvants comprising conjugates disclosed herein are provided.

[0433] In some embodiments, compositions are provided that comprise a conjugate disclosed herein and one or more immunostimulants.

[0434] In some embodiments, the Disclosure provides the use of the conjugates disclosed herein in vaccine manufacturing. In some embodiments, the Disclosure provides the use of the conjugates disclosed herein for manufacturing immunogenic compositions or vaccine compositions comprising an antigen or antigenic composition for treating or preventing a disease.

[0435] In some embodiments, the Disclosure relates to a method for treating or preventing a disease, comprising the step of administering to a human subject who is suffering from or susceptible to the disease an immunogenic composition or vaccine composition comprising an antigen or antigenic composition and a conjugate disclosed herein.

[0436] In some embodiments, the disease or disorder is inflammation, autoimmune disease, allergic disease, infection, HIV infection, AIDS infection, HCV infection, influenza, or human papillomavirus (HPV) infection. The scope of the disease will be readily apparent to those skilled in the art. In some embodiments, these diseases are as described in PCT application number PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0437] As used herein, the terms “cancer,” “neoplasm,” and “tumor” are synonymous and, in both singular and plural forms, refer to cells that have undergone malignant transformation that makes them pathological to the host organism. Primary cancer cells can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells but also any cells derived from the ancestors of cancer cells. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that typically manifest as solid tumors, a “clinically detectable” tumor is one that can be detected based on tumor volume, for example, by procedure, such as computed tomography (CT) scans, magnetic resonance imaging (MRI), X-rays, ultrasound or palpation during a physical examination, and / or due to the expression of one or more cancer-specific antigens in a sample available from the patient. Tumors may also be hematopoietic (or hematologic, hematological, or blood-related) cancers, such as cancers originating from blood cells or immune cells, which are sometimes called “liquid tumors.” Specific examples of clinical conditions based on hematological malignancies include leukemias, such as chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and acute lymphoblastic leukemia; plasma cell malignancies, such as multiple myeloma, MGUS, and Waldenström macroglobulinemia; and lymphomas, such as non-Hodgkin lymphoma and Hodgkin lymphoma.

[0438] In some embodiments, the disease or disorder is a precancerous syndrome.

[0439] The conjugates of this disclosure may be used to treat inflammation of any tissue and organ of the body, including musculoskeletal inflammation, vasculitis, neuroinflammation, gastrointestinal inflammation, ocular inflammation, genital inflammation, and other inflammations. The scope of diseases will be readily apparent to those skilled in the art. In some embodiments, these diseases are as described in PCT application number PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0440] Examples of cancerous diseases and conditions in which the conjugates of this disclosure may potentially have beneficial antitumor effects include: cancers of the lung, bone, pancreas, skin, head, neck, uterus, ovaries, stomach, colon, breast, esophagus, biliary tract, small intestine, intestine, endocrine system, thyroid, parathyroid, adrenal gland, urethra, prostate, penis, testis, ureter or urothelium, bladder, kidney or liver; rectal cancer; anal cancer; cancers of the fallopian tube, endometrium, cervix, vagina, vulva, renal pelvis, or renal cell carcinoma; soft tissue sarcomas; myxomas; rhabdomyomas; fibromas; lipomas; teratomas; cholangiocarcinomas; hepatoblastomas; angiosarcomas; hemangiomas; hepatomas; fibrosarcomas; chondrosarcomas; myelomas; chronic or acute leukemia; lymphocytic lymphomas; primary CNS lymphomas; CNS neoplasms; spinal axis Axis tumors; squamous cell carcinoma; synovial sarcoma; malignant pleural mesothelioma; brainstem glioma; pituitary adenoma; bronchial adenoma; chondromatoid hamartoma; mesothelioma; Hodgkin's disease; or any combination of one or more of the aforementioned cancers.

[0441] In some embodiments, the disease or disorder is a solid tumor. In one aspect, the tumor is selected from head and neck cancer, gastric cancer, melanoma, renal cell carcinoma (RCC), esophageal cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), prostate cancer, colorectal cancer (CRC), colon cancer, ovarian cancer, endometrial cancer, urothelial carcinoma, cervical cancer, bladder cancer, papillary thyroid cancer, papillary renal cell carcinoma, bile duct cancer, salivary duct cancer, kidney cancer, cervical cancer, and pancreatic cancer. In some embodiments, the human has humoral tumors, such as diffuse large B-cell lymphoma (DLBCL), multiple myeloma, chronic lymphoblastic leukemia (CLL), follicular lymphoma, acute myeloid leukemia, and chronic myeloid leukemia. In some embodiments, the disease or disorder is skin cancer (e.g., non-melanoma skin cancer, squamous cell carcinoma, basal cell carcinoma) or actinic keratosis. In addition to the electric field effect for removing superficial skin cancer, the conjugates of this disclosure may prevent the development of subsequent skin cancer and premalignant actinic keratosis in the treated subject.

[0442] In some embodiments, the disease or disorder is bladder cancer, breast cancer, colorectal cancer, colon cancer, endometrial cancer, gastric cancer, esophageal cancer, biliary tract cancer, urothelial carcinoma, head and neck squamous cell carcinoma, melanoma, non-small cell lung cancer, ovarian cancer, or pancreatic cancer. In some embodiments, the disease or disorder is breast cancer, gastric cancer, colorectal cancer, colon cancer, esophageal cancer, biliary tract cancer, endometrial cancer, urothelial carcinoma, or non-small cell lung cancer.

[0443] In some embodiments, breast cancer is HER2-amplified / overexpressing breast cancer or HER2-low breast cancer.

[0444] In some aspects, endometrial cancer is serous endometrial cancer.

[0445] The conjugates of this disclosure may also be useful in treating one or more diseases affecting mammals characterized by cell proliferation in the areas of neoangiogenesis and / or vascular permeability disorders, fibrous disorders, and metabolic disorders. The scope of diseases will be readily apparent to those skilled in the art. In some embodiments, these diseases are as described in PCT application number PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0446] In some embodiments, the disease or disorder is a neurodegenerative disease. Exemplary neurodegenerative diseases include, but are not limited to, multiple sclerosis, Huntington's disease, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). The scope of the diseases will be readily apparent to those skilled in the art. In some embodiments, these diseases are as described in U.S. Provisional Applications Nos. 62 / 882,081, 62 / 944,643, and 62 / 982,935, the contents of which are incorporated herein by reference in their entirety.

[0447] In some embodiments, a disease or disorder is an infectious disease which is triggered by or occurs concurrently with an infectious disease caused by a pathogen derived from bacteria of the DNA viridae or RNA viridae families. The scope of diseases will be readily apparent to those skilled in the art. In some embodiments, these diseases are as described in PCT application number PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0448] The conjugates of this disclosure may be used alone or in combination with other therapeutic agents. In the treatment of diseases and conditions where modification of STING is beneficial, the conjugates of this disclosure may also be used as monotherapy or in combination with other therapeutic agents as modulators of the immune response. Accordingly, combination therapy according to this disclosure includes the administration of the conjugate of this disclosure or a pharmaceutically acceptable salt thereof and at least one other therapeutic agent. In some embodiments, combination therapy according to this disclosure includes the administration of at least one conjugate of this disclosure or a pharmaceutically acceptable salt thereof and at least one other therapeutic agent. The conjugates of this disclosure and a pharmaceutically acceptable salt thereof and the other therapeutic agents may be administered together in a single pharmaceutical composition or separately. When administered separately, this may be done simultaneously or sequentially in any order. The amounts and relative timing of administration of the conjugates of this disclosure and a pharmaceutically acceptable salt thereof and the other therapeutic agents are selected to achieve the desired combination therapeutic effect. Accordingly, in further contexts, combinations are provided that include the conjugate of the present disclosure or a pharmaceutically acceptable salt thereof together with one or more other therapeutic agents.

[0449] The conjugates of this disclosure and their pharmaceutically acceptable salts may be used in combination with one or more other therapeutic agents that may be useful in the prevention or treatment of allergic, inflammatory, or autoimmune diseases, such as antigen immunotherapy, antihistamines, steroids, NSAIDs, bronchodilators, methotrexate, leukotriene modulators, monoclonal antibody therapy, receptor therapy, or antigen-nonspecific immunotherapy.

[0450] The conjugates of this disclosure and their pharmaceutically acceptable salts may be used in combination with at least one other therapeutic agent that may be useful in radiotherapy and / or surgery and / or treatment of cancer and precancerous syndromes. Any anti-cancer agent, microtubule inhibitor, mitotic inhibitor, hormone, hormone-analogous signaling pathway inhibitor, protein tyrosine kinase, or anti-angiogenic therapeutic agent may be used in combination. The range of other therapeutic agents will be readily apparent to those skilled in the art. In some embodiments, the other therapeutic agents are as described in PCT application number PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0451] Drugs used in immunotherapy regimens, therapeutic agents used in pro-apoptotic regimens, or cell cycle signaling inhibitors may also be useful in combination with the conjugates of this disclosure.

[0452] In some embodiments, the combinations of the Disclosure include the conjugate or a salt thereof of the Disclosure, in particular a pharmaceutically acceptable salt, and at least one anti-cancer agent, microtubule inhibitor, mitotic inhibitor, hormone, hormone analog signaling pathway inhibitor, protein tyrosine kinase, or anti-angiogenic therapeutic agent or a combination thereof.

[0453] Further examples of other therapeutic agents (e.g., anti-cancer agents) for use in combination with or concurrently with the conjugate or pharmaceutically acceptable salt thereof of the present disclosure are immunomodulators.

[0454] In some embodiments, the combinations of the Disclosure include a conjugate or salt thereof of the Disclosure, in particular a pharmaceutically acceptable salt, and at least one immunomodulator or at least one immunostimulant.

[0455] As used herein, “immunomodulator” refers to any substance containing a monoclonal antibody that affects the immune system. Immunomodulators can be used as anti-cancer agents to treat cancer. For example, immunomodulators include, but are not limited to, anti-CTLA-4 antibodies and anti-PD-1 antibodies. Other immunomodulators include, but are not limited to, ICOS antibodies, OX-40 antibodies, PD-L1 antibodies, LAG3 antibodies, TIM-3 antibodies, 41BB antibodies, and GITR antibodies.

[0456] Further examples of other therapeutic agents (antineoplastic agents) for use in combination with or concurrently with the conjugates of the Disclosure are anti-PD-L1 agents (i.e., anti-PD-L1 antibodies) or PD-1 antagonists.

[0457] Accordingly, in some embodiments, a method is provided for treating a person in need of treatment, comprising the step of administering the conjugate or a salt thereof of the present disclosure and at least one immunomodulator. In some embodiments, the immunomodulator is selected from ICOS agonist antibodies, OX-40 antibodies, and PD-1 antibodies. In some embodiments, the person has cancer. The use of the conjugate or a salt thereof of the present disclosure in combination with at least one immunomodulator for treating a person in need of treatment is also provided herein.

[0458] As used herein, “immunostimulant” refers to any agent capable of stimulating the immune system. Immunostimulants as used herein include, but are not limited to, vaccine adjuvants, e.g., Toll-like receptor agonists; T-cell checkpoint blockers, e.g., mAbs against PD-1 and CTL4; and T-cell checkpoint agonists, e.g., agonist mAbs against OX-40 and ICOS. As used herein, “immunostimulant” refers to any agent capable of stimulating the immune system. Immunostimulants as used herein include, but are not limited to, vaccine adjuvants.

[0459] As used herein, the term “Toll-like receptor” (or “TLR”) refers to a member or fragment of the Toll-like receptor family of proteins that sense microbial products and / or initiate an adaptive immune response. In some embodiments, TLRs activate dendritic cells (DCs). Toll-like receptors (TLRs) are a family of pattern recognition receptors initially identified as innate immune system sensors that recognize microbial pathogens. TLRs recognize distinctive structures found in microorganisms, often referred to as “PAMPs” (pathogen-associated molecular patterns). When a ligand binds to a TLR, it triggers an intracellular signaling pathway cascade that induces the production of factors involved in inflammation and immunity.

[0460] In some embodiments, the immunostimulant for use in combination with the conjugates of this disclosure is a TLR4 agonist.

[0461] Accordingly, in some embodiments, a method is provided for treating a person in need of treatment, comprising the step of administering the conjugate or a salt thereof of the present disclosure and at least one immunostimulant. In some embodiments, the immunostimulant is a TLR4 agonist. In some embodiments, the immunostimulant is an AGP. In some embodiments, the person has cancer. The use of the conjugate or a salt thereof of the present disclosure in combination with at least one immunostimulant for treating a person in need of treatment is also provided herein.

[0462] In addition to the immunostimulants described above, the compositions of this disclosure may further include other therapeutic agents that, due to their adjuvant properties, can stimulate the immune system to respond to cancer antigens present on inactivated tumor cells. Such adjuvants include, but are not limited to, lipids, liposomes, inactivated bacteria that induce innate immunity (e.g., inactivated or attenuated Listeria monocytogenes), (NOD)-like receptors (NLRs), retinoic acid-inducible gene-based (RIG)-I-like receptors (RLRs), and / or compositions that mediate innate immune activation via C-type lectin receptors (CLRs).

[0463] Because TLR agonists possess adjuvant properties, they may be used in combination with other vaccines, adjuvants, and / or immune modulators, and may be combined in a variety of combinations. In some embodiments, the conjugates of this disclosure bind to STING and induce STING-dependent TBKI activation. For therapeutic purposes, inactivated tumor cells expressing and secreting one or more cytokines that stimulate DC induction, recruitment, and / or maturation as described herein may be administered together with one or more TLR agonists.

[0464] Further active ingredients (antineoplastic agents) for use in combination with or concurrently administered with the conjugates of the present disclosure are IDO inhibitors.

[0465] In some embodiments, the conjugates of the Disclosure may be used in combination with at least one other therapeutic agent useful in the prevention or treatment of infections, bacterial infections, viral infections, Kaposi's sarcoma-associated herpesvirus infections, TB infections, Chlamydia species, Plasmodium infections, Staphylococcus infections, amyotrophic lateral sclerosis (ALS), multiple sclerosis, systemic lupus erythematosus and related lupus disorders, psoriasis, or Sjögren's syndrome.

[0466] The conjugates of this disclosure may be administered by any suitable route of administration, including both systemic and topical administration. Systemic administration includes oral, parenteral, transdermal, rectal, and inhalation administration. Parenteral administration refers to routes of administration other than enteral, transdermal, or inhalation administration, and is typically administered by injection or infusion. Parenteral administration includes intravenous, intramuscular, and subcutaneous injection or infusion. Inhalation refers to administration to the patient's lungs, whether inhaled through the mouth or through the nasal cavity. Topical administration includes application to the skin.

[0467] In addition to the above-mentioned routes of administration suitable for oncological treatment, the pharmaceutical composition may be adapted for administration by intratumoral or peritumoral injection. Intratumoral or peritumoral injection of the conjugate of the Disclosure directly or adjacent to a solid tumor is expected to induce an immune response capable of attacking and destroying cancer cells throughout the body, significantly reducing, and in some cases permanently eliminating, the tumor from the diseased subject. This activation of the immune system to kill tumors in distant sites is commonly known as the abscopal effect and has been demonstrated in animals with several therapeutic modalities. A further advantage of local, intratumoral, or peritumoral administration is that comparable efficacy can be achieved at considerably lower doses, thus minimizing or eliminating adverse events that may be observed at considerably higher systemic doses.

[0468] The conjugates of this disclosure may be administered as a single dose or according to a dosage regimen, in which case multiple doses may be administered at various intervals over a predetermined period. For example, doses may be administered once, twice, three times, or four times daily. Dose administration may continue indefinitely until the desired therapeutic effect is achieved or to maintain the desired therapeutic effect. A suitable dosage regimen for the conjugates of this disclosure will depend on the pharmacokinetic properties of the conjugate, e.g., absorption, distribution, and half-life, which can be determined by those skilled in the art. Furthermore, a suitable dosage regimen for the conjugates of this disclosure, including the duration for which such a regimen is administered, will depend on the disease or disorder being treated, the severity of the disease or disorder being treated, the age and physical condition of the patient being treated, the patient's medical history, the nature of the concomitant therapy, the desired therapeutic effect, and similar factors within the scope of the knowledge and expertise of those skilled in the art. It will be further understood by those skilled in the art that an appropriate dosage regimen may need to be adjusted over time, taking into account the individual patient's response to the dosage regimen or as individual patients require changes. The total daily dose is 1 mg to 2000 mg, preferably 1 mg to 250 mg.

[0469] For use in therapy, the conjugates of this disclosure are typically formulated into a pharmaceutical composition before being administered to a patient, but are not necessarily formulated into a pharmaceutical composition. Accordingly, this disclosure also relates to a pharmaceutical composition comprising the conjugates of this disclosure and at least one pharmaceutically acceptable excipient.

[0470] The pharmaceutical compositions of this disclosure may be prepared and packaged in bulk form or in unit dosage forms. For oral use, for example, one or more tablets or capsules may be administered. The dose of the pharmaceutical composition contains at least a therapeutically effective amount of the conjugate of this disclosure (i.e., the conjugate of this disclosure or a salt thereof, in particular a pharmaceutically acceptable salt). When prepared in unit dosage forms, the pharmaceutical composition may contain 1 mg to 1000 mg of the conjugate of this disclosure.

[0471] When provided herein, a unit dosage form (pharmaceutical composition) containing 1 mg to 1000 mg of the conjugate of this disclosure may be administered once, twice, three, or four times daily, preferably once, twice, or three times daily, more preferably once or twice daily, to treat a disease or disorder mediated by STING.

[0472] The pharmaceutical compositions of this disclosure typically contain one type of conjugate of this disclosure. However, in certain embodiments, the pharmaceutical compositions of this disclosure contain multiple types of conjugates of this disclosure. Furthermore, the pharmaceutical compositions of this disclosure may optionally further contain one or more additional therapeutic agents (e.g., pharmaceutically active conjugates).

[0473] As used herein, “pharmaceutically acceptable excipients” means pharmaceutically acceptable materials, compositions, or vehicles that contribute to giving form or consistency to a pharmaceutical composition. Each excipient must be compatible with the other components of the pharmaceutical composition when mixed so as to avoid interactions that would significantly reduce the potency of the conjugates of this disclosure when administered to a patient, and interactions that would result in a pharmaceutically unacceptable pharmaceutical composition. Furthermore, each excipient must, of course, be of sufficiently high purity to be pharmaceutically acceptable.

[0474] The conjugates and pharmaceutically acceptable excipients of this disclosure are typically formulated into dosage forms tailored to administration to a patient via a preferred route of administration. Conventional dosage forms include: (1) dosage forms tailored for oral administration, e.g., tablets, capsules, caplets, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets; (2) dosage forms tailored for parenteral administration, e.g., sterile solutions, suspensions, and powders for reconstitution; (3) dosage forms tailored for transdermal administration, e.g., transdermal patches; (4) dosage forms tailored for rectal administration, e.g., suppositories; (5) dosage forms tailored for inhalation, e.g., aerosols and solutions; and (6) dosage forms tailored for topical administration, e.g., creams, ointments, lotions, solutions, pastes, sprays, foams, and gels.

[0475] Appropriate pharmaceutically acceptable excipients vary depending on the specific dosage form selected. Furthermore, appropriate pharmaceutically acceptable excipients may be selected for specific functions that may be useful in the composition. For example, a particular pharmaceutically acceptable excipient may be selected for its ability to facilitate the production of a uniform dosage form. A particular pharmaceutically acceptable excipient may be selected for its ability to facilitate the production of a stable dosage form. A particular pharmaceutically acceptable excipient may be selected for its ability to facilitate the transport or delivery of the conjugate of this disclosure to a patient, once administered from one organ or part of the body to another organ or part of the body. A particular pharmaceutically acceptable excipient may be selected for its ability to enhance patient adherence to medication.

[0476] Appropriate pharmaceutically acceptable excipients include the following types of excipients: diluents, bulking agents, binders, disintegrants, lubricants, fluidizers, granulators, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, colorants, anti-caking agents, wetting agents, chelating agents, plasticizers, thickeners, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. Those skilled in the art will understand that a particular pharmaceutically acceptable excipient may perform multiple functions and may perform alternative functions depending on the amount of excipient present in the formulation and what other components are present in the formulation.

[0477] Those skilled in the art will possess the knowledge and skills in the art that will enable them to select appropriate amounts of suitable pharmaceutically acceptable excipients for use in this disclosure. Furthermore, there are numerous sources available to those skilled in the art that describe pharmaceutically acceptable excipients and may be useful in selecting appropriate pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).

[0478] In one aspect, the present disclosure relates to a solid oral dosage form, such as a tablet or capsule, comprising an effective amount of the conjugate of the present disclosure and a diluent or expander. The oral solid dosage form may further comprise a disintegrant or lubricant.

[0479] The conjugates of this disclosure are also understood to be formulated together with the vaccine as adjuvants to modulate the vaccine's activity. Such compositions may optionally contain one or more antibodies or antibody fragments or antigenic components together with one or more other components having adjuvant activity.

[0480] Certain compounds and / or conjugates in this disclosure may be potent immunomodulators and therefore require careful handling. Although this disclosure has been described, the following examples are provided as illustrations and not as limitations.

[0481] In some embodiments, the conjugate is of formula BB The filename is TIFF2026086770000187.tif69138, and the conjugate contains the amino acid sequence FTFSSYSMN (SEQ ID NO:20) and the variable heavy chain complementarity determination region 1 (CDRH1); amino acid sequence Variable heavy chain complementarity determination region 2 (CDRH2) containing TIFF2026086770000188.tif4128; Variable heavy chain complementarity determination region 3 (CDRH3) containing amino acid sequence GGHGYFDL (SEQ ID NO:22); and amino acid sequence The XMT-1519 antibody contains variable light chain complementarity determination region 1 (CDRL1) containing TIFF2026086770000189.tif4128; variable light chain complementarity determination region 2 (CDRL2) containing amino acid sequence GASSRAT (SEQ ID NO:28); and variable light chain complementarity determination region 3 (CDRL3) containing amino acid sequence QQYHHSPLT (SEQ ID NO:29), d 15 It is approximately 8.

[0482] In some embodiments, the conjugate is of formula CC The filename is TIFF2026086770000190.tif79160, and the conjugate is an amino acid sequence. CDRH1 containing TIFF2026086770000191.tif4128; amino acid sequence CDRH2 containing TIFF2026086770000192.tif4128; amino acid sequence CDRH3 containing TIFF2026086770000193.tif4128; amino acid sequence The XMT-1519 antibody contains CDRL1 containing TIFF2026086770000194.tif4128; CDRL2 containing amino acid sequence YTSSLYS (SEQ ID NO:9); and CDRL3 containing amino acid sequence QQYSKLPLT (SEQ ID NO:10). 15 It is approximately 8.

[0483] In some embodiments, a conjugate of formula BB or formula CC is useful for treating a disease or disorder in a subject requiring treatment of the disease or disorder, comprising the step of administering a therapeutically effective amount of the conjugate of formula BB or formula CC to a subject. In some embodiments, the disease or disorder is cancer.

[0484] In some embodiments, for the conjugate of formula BB, the cancer is breast cancer, gastric cancer, colorectal cancer, esophageal cancer, biliary tract cancer, endometrial cancer, urothelial carcinoma, or non-small cell lung cancer. In some embodiments, breast cancer is HER2-amplified / overexpressing breast cancer or HER2-low breast cancer. In some embodiments, endometrial cancer is serous endometrial cancer.

[0485] In some embodiments, the conjugate of formula CC is useful for treating NaPi2b-expressing tumors in subjects requiring treatment of NaPi2b-expressing tumors. In some embodiments, NaPi2b-expressing tumors include ovarian cancer, non-small cell lung cancer (NSCLC), papillary thyroid cancer, endometrial cancer, cholangiocarcinoma, papillary renal cell carcinoma, clear cell kidney cancer, breast cancer, kidney cancer, cervical cancer, or salivary duct cancer.

[0486] In some embodiments, the subjects are those with epithelial ovarian cancer, ductal fallopian carcinoma, primary peritoneal cancer, platinum-resistant ovarian cancer, non-squamous NSCLC cancer, advanced, radioactive iodine-refractory, locally recurrent, or metastatic disease, papillary thyroid cancer, or epithelial endometrial cancer.

[0487] In some embodiments, the conjugate of formula BB is useful for treating a disease or disorder in a subject requiring treatment of the disease or disorder, comprising the step of administering a therapeutically effective amount of the conjugate of formula BB in combination with one or more therapeutic agents to the subject. In some embodiments, the therapeutic agent is an immunomodulator or immunostimulant. In some embodiments, the immunomodulator is an anti-CTLA-4 antibody, an anti-PD-1 antibody, an ICOS antibody, an OX-40 antibody, an PD-L1 antibody, an LAG3 antibody, an TIM-3 antibody, an 41BB antibody, or an GITR antibody.

[0488] In some embodiments, the formula BB conjugate is useful for treating a disease or disorder in a subject requiring treatment, comprising the step of administering a therapeutically effective amount of the formula BB conjugate to the subject in combination with one or more HER2 antibodies that bind to a HER2 epitope different from the HER2 antibody XMT-1519. In some embodiments, the HER2 antibody that binds to a HER2 different from the HER2 antibody XMT-1519 is trastuzumab, pertuzumab, Fab37, or chA21. [Examples]

[0489] The following examples illustrate the present disclosure. These examples are intended to provide guidance for those skilled in the art to prepare and use the compounds, compositions, and methods of the present disclosure, rather than limiting the scope of the present disclosure. While specific aspects of the present disclosure have been described, those skilled in the art will understand that various changes and modifications can be made without departing from the spirit and scope of the present disclosure.

[0490] Certain compounds in this disclosure may be potent immunomodulators and therefore require careful handling.

[0491] The reactions described herein involve various different substituents (e.g., R) as defined herein. 1 , R 2 This method can be applied to produce compounds of the present disclosure having (etc.). Those skilled in the art will understand that if a particular substituent is not compatible with the synthetic method described herein, the substituent may be protected with a suitable protecting group that is stable to the reaction conditions. Suitable protecting groups and methods for protecting and deprotecting various substituents with such suitable protecting groups are well known to those skilled in the art. Examples of these can be found in TW Greene, "Protective Groups in Organic Synthesis" (4th edition, J. Wiley and Sons, 2006). Unless otherwise specified, all starting materials were obtained from commercial suppliers and used without further purification.

[0492] Abbreviation The following abbreviations are used in the reaction schemes and synthesis examples below. This list is not intended to be a comprehensive list of additional standard abbreviations used in this application that are readily understood by those skilled in the art of organic synthesis, and may also be used in the synthesis schemes and examples. TIFF2026086770000195.tif139154

[0493] General information Unless otherwise specified, all reagents were purchased from the relevant suppliers.

[0494] The diABZI STING agonist was prepared as described in Ramanjulu et al (Nature, 564(7736):439-443 (2018)).

[0495] XMT-1535 (anti-NaPi2b antibody) is disclosed in concurrently pending application US15 / 457,574, filed on 13 March 2017, the contents of which are incorporated herein by reference. XMT-1519 (anti-HER2 antibody) is disclosed in US9,555,112, issued on 31 January 2017, and US9,738,720, issued on 22 August 2017, the contents of which are incorporated herein by reference.

[0496] XMT-1535 AF-HPA ADC, DAR 5.9 and rituximab AF-HPA ADC, DAR 5.5 were prepared as described in concurrently pending applications US62 / 958,916 filed on 9 January 2020 and US63 / 040,735 filed on 18 June 2020, the entire contents of which are incorporated herein by reference.

[0497] HPLC purification was performed using a Phenomenex Gemini 5μm C18 110Å, 250x10mm semi-preparative column.

[0498] Where applicable, the drug content of the conjugate was determined spectrophotometrically; otherwise, it was determined quantitatively by RP-HPLC or LC / MS.

[0499] The protein content of the antibody-drug conjugate was determined spectrophotometrically or by ELISA.

[0500] If necessary, the antibody-drug conjugate, drug-carrying scaffold, or antibody scaffold was purified by large-scale diafiltration, CHT chromatography, or HIC (i.e., removal of any remaining unreacted drug, non-conjugate antibody, enzyme, or starting material). If necessary, further purification was performed by SEC or HIC to remove aggregated antibody-drug conjugate. Generally, when purified, antibody-drug conjugates contained <5% (w / w) (e.g., <2% (w / w)) of aggregated antibody-drug conjugate when confirmed by SEC, <0.5% (w / w) (e.g., <0.1% (w / w)) of free (unconjugated) drug when confirmed by RP-HPLC and / or LC-MS / MS, <1% (w / w) of free drug conjugate when confirmed by SEC and / or RP-HPLC, and <10% (w / w) (e.g., <1% (w / w)) of unconjugated antibody or antibody fragment when confirmed by HIC-HPLC and / or RP-HPLC. Reduced or partially reduced antibodies were prepared using procedures described in the literature. See, for example, Francisco et al., Blood 102 (4):1458-1465 (2003). Total drug (conjugate and unconjugate) concentrations were determined by UV-Vis spectrophotometric analysis or RP-HPLC.

[0501] To determine the concentration of free drugs in biological samples, acidified samples were treated with acetonitrile. Free drugs were extracted, and the acetonitrile supernatant was analyzed. To determine the concentration of conjugated STING agonists in nonclinical samples, samples were subjected to immunocapture using anti-human Fc antibody magnetic beads, followed by thorough basic hydrolysis. The acetonitrile supernatant containing the released drugs was analyzed by LC-MS / MS. Total antibodies in nonclinical samples were measured using an MSD ECL immunoassay.

[0502] Free drug analysis was performed by RP-HPLC using a C-4 column and acetonitrile gradient. The MRM peak area obtained by tandem mass spectrometry was integrated and compared with auristatin F (AF) and auristatin F hydroxypropylamide (AF-HPA) standards. This method quantifies AF-HPA and AF in plasma and tissue homogenates, showing a linear relationship in the concentration range of 0.1–150 ng / mL. All drugs released after NaOH hydrolysis were measured under the same conditions. The dynamic range was 1 ng / mL–5000 ng / mL. The total antibody standard range was 0.009 μg / mL–20 μg / mL.

[0503] The drug-to-antibody ratio (DAR) was determined by measuring the absorption of the conjugate. The DAR value was calculated using the appropriate molar extinction coefficients of the antibody and STING agonist payload.

[0504] The tumor was measured twice a week using a digital caliper, and the tumor volume was calculated using the formula: Tumor volume (mm³). 3 )=(width 2 The calculation was performed using x (length) / 2. Body weight was recorded daily during the first week, and twice a week thereafter. The tumor volume of each animal was ≥1000 mm. 3 Until it reaches ≥1500mm 3 The test continued until it reached or was as indicated. Weight percentage change is calculated using the formula: Weight change (%) = ((Weight 試験X日目 -body weight 試験1日目 ) / body weight 試験1日目 ) * Calculations were performed using 100. Tumor volume was reported as mean ± standard error of mean (SEM). Tumor growth inhibition (%TGI) was defined as the percentage difference in mean tumor volume (MTV) between the treatment group and the control group. Tumor size was measured throughout each efficacy trial to determine tumor growth inhibition (TGI). Percent tumor regression was calculated using the formula: % regression = (1 - (mean tumor volume) 最終 ) / (Average tumor volume 1日目 )) *Calculations were performed using 100. Partial remission (PR) is defined as the tumor volume being 50% or less of the volume on day 1 for three consecutive measurements, or the tumor volume being 13.5 mm in at least one of those three measurements. 3 It is defined as becoming equal to or greater than . Complete remission (CR) is defined as a tumor volume of 13.5 mm² over three consecutive measurements. 3 This is defined as being less than 100%. Tumor-free survivors (TFS) are classified as CR at the end of the trial.

[0505] Example 1: Synthesis of XMT-1519 conjugate 8 TIFF2026086770000196.tif180160

[0506] Part A: Compound 1 (0.5 g, 0.64 mmol, prepared as described in WO2017175147A1), Boc-L-alanine (0.242 g, 1.28 mmol), DMAP (7.8 mg, 0.064 mmol), and DCC (0.264 g, 1.28 mmol) were mixed with DMF (2 mL). The suspension was stirred overnight at room temperature, then the solution was concentrated, and the residue was purified over silica gel (0-40% MeOH dissolved in DCM) to obtain Compound 2 as a pale yellow solid (0.52 g, 85% yield). 46 H 58 N 13 O 10 [M+H] + ESI-MS m / z calculated value: 952.4; measured value: 952.4.

[0507] Part B: A suspension of compound 2 (0.52 g, 0.55 mmol) dissolved in dioxane (10 mL) was mixed with 4N HCl (2 mL, 8.19 mmol). The reaction mixture was stirred at room temperature for 2 hours, and then the suspension was concentrated and used in the next step without further purification. Compound 3 was obtained as a white solid. 41 H 50 N 13 ESI-MS m / z calculated value for O8[M+H]: 852.4; measured value: 852.3.

[0508] Part C: To a solution of compound 3 (0.586 g, 0.661 mmol) dissolved in DMF (5 mL), 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecano-14-acid (0.202 g, 0.727 mmol) was added, followed by the addition of DIPEA (0.230 mL, 1.322 mmol). The reaction mixture was stirred at room temperature for 5 minutes, and then HATU (0.376 g, 0.992 mmol) and HOBt (0.153 g, 0.992 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. Further aliquots of DIPEA (0.460 mL, 2.6 mmol) were added. After another 1 hour, the reaction mixture was concentrated to obtain an oil. The residue was purified over silica gel (0-40% MeOH dissolved in DCM) to obtain compound 4 (0.9 g, >95% yield) as a white solid. 53 H 71 N 14 O 13 [M+H] + ESI-MS m / z calculated value: 1111.5; measured value: 1111.5.

[0509] Part D: A suspension of compound 4 (0.9 g, 0.810 mmol) dissolved in dioxane (10 mL) was mixed with 4N HCl (3.04 mL, 12.15 mmol). The reaction mixture was stirred at room temperature for 1.5 hours. The suspension was concentrated to obtain compound 5 as a colorless solid (0.56 g, 66.0% yield). 48 H 63 N 14 O 11 [M+H] + ESI-MS calculated value: 1011.5; measured value: 1011.4.

[0510] Part E: PyBOP (37.5 mg, 0.072 mmol) and DIPEA (0.075 mL, 0.431 mmol) were added to a solution of Scaffold 6 (100 mg, 0.072 mmol, prepared as described in PCT / US2018 / 06719) dissolved in DMF (2 mL) and Compound 5 (72.7 mg, 0.072 mmol). The reaction mixture was stirred at room temperature for 2 hours, then the solution was concentrated, and the residue was purified by preparative HPLC (0-80% ACN dissolved in water) to obtain Compound 7 (109 mg, 64% yield). 103 H 156 N 24 O 41 [M+2H] 2+ ESI-MS m / z calculated value: 1192.5; measured value: 1192.5.

[0511] Part F: To a solution of XMT-1519 (10 mg, 0.069 μmol) dissolved in 50 mM HEPES, 1 mM EDTA, and pH 7 buffer, TCEP (0.059 mg, 0.207 μmol) was added, and the mixture was shaken at 37°C for 90 minutes. Compound 7 (0.987 mg, 0.414 μmol dissolved in 200 μL DMA) was added to the reduced antibody. The resulting mixture was shaken at 37°C for 60 minutes. The reaction was quenched with cysteine ​​(15 equivalents, 0.125 mg, 1.035 μmol dissolved in 125 μL of 50 mM HEPES, 1 mM EDTA, and pH 7), and the mixture was rotated at room temperature for 1 hour. The resulting conjugate 8 was purified by ultrafiltration or CHT chromatography. Details of antibody-drug conjugates 8-1 and 8-2 are shown below. Conjugates 8-1 and 8-2 were prepared as described, except that a higher TCEP:mAb ratio (4:1 compared to 3:1) and a higher compound 7:mAb ratio (8:1 compared to 6:1) were used for the synthesis of 8-2 compared to 8-1. TIFF2026086770000197.tif16128

[0512] Example 1a: Synthesis of trastuzumab conjugate 8a TIFF2026086770000198.tif74137

[0513] Conjugate 8a was prepared and characterized as described in Example 1, except that trastuzumab was used instead of XMT-1519. Details of antibody-drug conjugates 8a-1, 8a-2, and 8a-3 are shown below. TIFF2026086770000199.tif20128

[0514] Example 1b: Synthesis of XMT-1535 conjugate 8b and DAR 5.7 TIFF2026086770000200.tif74132

[0515] Conjugate 8b was prepared and characterized as described in Example 1, except that XMT-1535 was used instead of XMT-1519. Details of antibody-drug conjugates 8b-1, 8b-2, and 8b-3 are shown below. TIFF2026086770000201.tif20128

[0516] Example 1c: Synthesis of palivizumab conjugate 8c TIFF2026086770000202.tif71136

[0517] Conjugate 8c was prepared and characterized as described in Example 1, except that palivizumab was used instead of XMT-1519. Details of antibody-drug conjugates 8c-1 and 8c-2 are shown below. TIFF2026086770000203.tif16128

[0518] Example 1d: Synthesis of palivizumab mIgG2a conjugate 8d TIFF2026086770000204.tif74150

[0519] Conjugate 8d was prepared and characterized as described in Example 1, except that palivizumab mIgG2a was used instead of XMT-1519. Details of antibody-drug conjugates 8d-1, 8d-2, and 8d-3 are shown below. TIFF2026086770000205.tif20128

[0520] Example 1e: Synthesis of XMT-1535 hIgG1-mIgG2a conjugate 8e, DAR 7.0 TIFF2026086770000206.tif73158

[0521] Conjugate 8e was prepared and characterized as described in Example 1, except that XMT-1535 mIgG2a was used instead of XMT-1519. The STING agonist:XMT-1535 hIgG1-mIgG2a ratio of the purified conjugate 8e was 7.0.

[0522] Example 1f: Synthesis of XMT-1535 AAG conjugate 8f and DAR 7.4 TIFF2026086770000207.tif73142

[0523] Conjugate 8f was prepared and characterized as described in Example 1, except that XMT-1535 AAG was used instead of XMT-1519. The STING agonist:XMT-1535 AAG ratio of the purified conjugate 8f was 7.4.

[0524] Example 1g: Synthesis of target D mIgG2a conjugate 8g, DAR 7.4 TIFF2026086770000208.tif76141

[0525] Eight g of conjugates were prepared and characterized as described in Example 1, except that target D mIgG2a was used instead of XMT-1519. The STING agonist:target D_mIgG2a ratio of the purified eight g of conjugates was 7.4.

[0526] Example 1h: Synthesis of target C hIgG1a conjugate 8h and DAR 6.9 TIFF2026086770000209.tif74139

[0527] A conjugate 8 g was prepared and characterized as described in Example 1, except that targeted C hIgG1a was used instead of XMT-1519. The STING agonist:target C hIgG1a ratio of the purified conjugate 8 g was 6.9.

[0528] Example 1i: Synthesis of target E mIgG2a conjugate 8i and DAR 10.0 TIFF2026086770000210.tif75140

[0529] Conjugate 8i was prepared and characterized as described in Example 1, except that targeted E mIgG2a was used instead of XMT-1519. The STING agonist:targeted E mIgG2a ratio of the purified conjugate 8g was 10.0.

[0530] Example 1j: Synthesis of trastuzumab AAG conjugate 8j and DAR 7.0 TIFF2026086770000211.tif76148

[0531] Conjugate 8j was prepared and characterized as described in Example 1, except that trastuzumab AAG was used instead of XMT-1519.

[0532] Details of Conjugate 8j and 8j-1 are shown below. TIFF2026086770000212.tif15128

[0533] Example 1k: Synthesis of trastuzumab mIgG2a conjugate 8k, DAR 8.1 TIFF2026086770000213.tif75154

[0534] Conjugate 8k was prepared and characterized as described in Example 1, except that trastuzumab mIgG2a was used instead of XMT-1519. The STING agonist:trastuzumab mIgG2a ratio of the purified conjugate 8k was 8.1.

[0535] Example 1: Synthesis of XMT-1535 conjugate 8L, DAR 4.7 TIFF2026086770000214.tif86160

[0536] Conjugate 8L was prepared and characterized as described in Example 1, except that XMT-1535 was used instead of XMT-1519. 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetic acid was incorporated into the compound structure instead of (3-(2-(2-aminoethoxy)ethoxy)propanoyl)-L-alanine. The STING agonist:XMT-1535 ratio of the purified conjugate 8L was 4.7.

[0537] Example 1m: Synthesis of palivizumab conjugate 8m, DAR 4.5 TIFF2026086770000215.tif84154

[0538] Conjugate 8m was prepared and characterized as described in Example 1, except that palivizumab was used instead of XMT-1519. 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetic acid was incorporated into the compound structure instead of (3-(2-(2-aminoethoxy)ethoxy)propanoyl)-L-alanine. The STING agonist:palivizumab ratio of the purified conjugate 8m was 4.5.

[0539] Example 2: Synthesis of XMT-1519 conjugate 16 and DAR 5.3 TIFF2026086770000216.tif93160

[0540] Part A: Compound 9 (0.100 g, 0.429 mmol) dissolved in THF (5 mL) was mixed with HATU (0.196 g, 0.514 mmol) and HOBt (0.079 g, 0.514 mmol). The reaction mixture was stirred at 0°C for 10 minutes, and then 2-(benzyloxy)ethane-1-amine (0.0648 mg, 0.429 mmol) and DIPEA (0.112 mL, 0.643 mmol) were added. The reaction mixture was stirred at room temperature overnight, the solution was concentrated, and the residue was purified with silica gel (0-10% MeOH dissolved in DCM) to obtain compound 10 (0.2 g, 100% yield). 19 H 30 N2O5Na[(M+Na)] + ESI-MS m / z calculated value: Calculated value 389.2; Measured value 389.2.

[0541] Part B: A solution of compound 10 (150 mg, 0.409 mmol) dissolved in EtOH (10 mL) was degassed with N2, and then Pd-C (43.6 mg, 0.409 mmol) was added. The mixture was then degassed with H2. The reaction mixture was stirred overnight at room temperature under H2 (1 atm). The solid was filtered through a Celite pad, and the filtrate was concentrated to obtain compound 11. Crude compound 11 was used in the next step without further purification. 12 H 24 N2O5Na[(M+Na)] + ) ESI-MS m / z calculated value: Calculated value 299.2; Measured value 299.2.

[0542] Part C: A 100 mL flask containing the residue of compound 11 (128 mg, 0.463 mmol) and N,N-dimethylpyridine-4-amine (11.32 mg, 0.093 mmol) was washed with argon, and then triethylamine (129 μL, 0.926 mmol), acetonitrile (1.544 mL), and DMF (0.772 mL) were added. The reaction mixture was cooled to 0°C and stirred for 5 minutes, after which 4-nitrophenyl carbonochloride (140 mg, 0.695 mmol) was added, and the resulting mixture was stirred at 20°C for 2 hours, and then concentrated to obtain oil. The residue was purified with silica gel (0-100% ethyl acetate dissolved in hexane) to obtain compound 12 (50 mg, 24% yield) as a white solid. 14 H 19 N3O7[(M-Boc+H] + ESI-MS m / z calculated value: Calculated value 342.2; Measured value 342.1.

[0543] Part D: To a solution of compound 12 (50 mg, 0.113 mmol) and compound 1 (36.9 mg, 0.047 mmol), DMAP (1.153 mg, 9.44 μmol) dissolved in DMF (500 μL) was added. The reaction mixture was heated at 80°C for 3 hours, and then compound 12 (50 mg, 0.113 mmol) was added. The reaction mixture was stirred for a further 3 hours and then cooled to room temperature. The mixture was concentrated, and the residue was purified with silica gel (0-30% MeOH dissolved in DCM) to obtain compound 13 (20 mg, 39% yield) as a white solid. 51 H 67 N 14 O 13 [(M+H)] + ESI-MS m / z calculated value: Calculated value 1083.4; Measured value 1083.5.

[0544] Part E: To a solution of compound 13 (20 mg, 0.047 mmol) dissolved in DCM (0.5 mL), TFA (0.1 mL) was added. The reaction mixture was stirred at room temperature for 4 hours, and then concentrated to obtain compound 14 (10 mg, 55% yield) as a solid. 46 H59 N 14 O 11 [M+H] + ESI-MS m / z calculated value: Calculated value 983.4; Measured value 983.5.

[0545] Part F: To a solution of scaffold 6 (14.15 mg, 10.17 μmol) dissolved in DMF (1 mL) and compound 14 (10 mg, 10.17 μmol), HATU (4.64 mg, 0.012 mmol), HOAt (1.881 mg, 0.012 mmol), and DIPEA (0.018 mL, 0.102 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, then concentrated, and the residue was purified by preparative RP HPLC (0-80% ACN dissolved in water) to obtain scaffold 15 (20 mg, 83% yield). 101 H 152 N 24 O 41 [(M+2H)] 2+ ESI-MS m / z calculated value: Calculated value 1178.7; Measured value 1178.59.0.

[0546] Part G: A solution of XMT-1519 (10 mg, 0.069 μmol) was conjugated with scaffold 15 (0.823 mg, 0.347 μmol dissolved in 200 μL DMA) as described in Example 1. The STING agonist:XMT-1519 ratio of the purified conjugate 16 was 5.3.

[0547] Example 3: Synthesis of trastuzumab conjugate 20 TIFF2026086770000217.tif180160

[0548] Part A: A solution of Boc-ala-ala-OH (67 mg, 256 μmol), CDI (70 mg, 435 μmol), and DMF (2 mL) was stirred at room temperature for 23 hours. Then, compound 1a (100 mg, 128 μmol, prepared as described in WO2017175147A1) and DIPEA (67 μL, 384 μmol) were added, and the reaction mixture was stirred at room temperature for 23 hours. The reaction mixture was concentrated, and the residue was subjected to chromatography using silica gel (0-20% MeOH-DCM eluent). The product, compound 17, was isolated as a yellow foam (109 mg, 83% yield). 49 H 64 N 15 O 10 [M+H] + ESI-MS m / zi calculated value: 1022.5; measured value: 1022.4.

[0549] Part B: A mixture of compound 17 (108 mg, 106 μmol) and 2 M HCl-dioxane (6 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated, and the residue was dried under high pressure to obtain compound 18, an off-white foam (103 mg, quant.). 44 H 56 N 15 ESI-MS m / z calculated value for O8[M+H]: 922.4; measured value: 922.4.

[0550] Part C: A mixture of compound 18 (80 mg, 84 μmol), scaffold 6 (117 mg, 84 μmol), HOAt (12 mg, 84 μmol), DiPEA (59 μL, 336 μmol), and DMF (3 mL) was stirred at room temperature for 5 minutes. Then, HATU (42 mg, 109 μmol) was added, and the reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was then concentrated and chromatographed using reversed phase (10-100% ACN-water w / 0.1% HCOOH eluent). Scaffold 19 was isolated as a white, fluffy solid (35 mg, 18% yield). 99 H 149 N 25 O 38 [M+2H] 2+ESI-MS m / z calculated value: Calculated value 1148.0; Measured value 1148.4.

[0551] Part D: Trastuzumab (10 mg, 0.067 μmol) was conjugated with scaffold 19 (1.237 mg, 0.539 μmol dissolved in 200 μL DMA) as described in Example 1, and then purified using CHT type II chromatography to obtain conjugate 20. Details of antibody-drug conjugates 20-1 and 20-2 are shown below. TIFF2026086770000218.tif16128

[0552] Example 3a: Synthesis of palivizumab conjugate 20a and DAR 5.5 TIFF2026086770000219.tif71160

[0553] Conjugate 20a was prepared and characterized as described in Example 1, except that palivizumab was used instead of XMT-1519. The STING agonist:palivizumab ratio of the purified conjugate 20a was 5.5.

[0554] Example 4: Synthesis of trastuzumab conjugate 25 and DAR 6.6 TIFF2026086770000220.tif142157

[0555] Part A: Compound 21 (38 mg, 0.047 mmol, prepared as described in US62 / 982,935, dissolved in DMF (2 mL)) and a mixture of tert-butyl(S)-(2-hydroxypropyl)carbamate (9.85 mg, 0.056 mmol) were mixed with 3-((ethylamino)methylene)amino)-N,N-dimethylpropane-1-amine hydrochloride (13.48 mg, 0.070 mmol), HOBt (10.77 mg, 0.070 mmol), DIPEA (0.016 mL, 0.094 mmol), and DMAP (5.73 mg, 0.047 mmol). The suspension was then stirred at room temperature for 2 days. The mixture was concentrated to obtain a residue, which was then purified over silica gel (0-30% MeOH dissolved in DCM) to obtain compound 22 as a pale yellow solid (20 mg, 44% yield). 47 H 58 N 11 O 12 [M+H] + ESI-MS m / z calculated value: Calculated value 968.4; Measured value 968.3.

[0556] Part B: A suspension of compound 22 (20 mg, 0.021 mmol) dissolved in dioxane (4 mL) was to which 4N HCl (0.52 mL, 8.19 mmol) was added. The reaction mixture was stirred at room temperature for 5 hours, concentrated, and used in the next step without purification. The product, compound 23 (17 mg, 95% yield), was a white solid. 42 H 50 N 11 O 10 [M+H] + ESI-MS m / z calculated value: 868.3; measured value: 868.4.

[0557] Part C: Compound 23 (17 mg, 0.020 mmol) dissolved in DMF (2 mL) and scaffold 6 (32.1 mg, 0.023 mmol) were mixed, to which PyBOP (15.3 mg, 0.03 mmol) and DIPEA (0.034 mL, 0.196 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, concentrated to obtain a residue, and then purified by preparative RP HPLC (0-80% ACN dissolved in water) to obtain scaffold 24 (26 mg, 59% yield). 97 H 143 N 21 O 40 [M+2H] 2+ ESI-MS m / z calculated value: 1120.98; measured value: 1121.06.

[0558] Part D: XMT-1519 antibody (5 mg, 0.0347 μmol) was conjugated with scaffold 24 (0.622 mg, 0.278 μmol) as described in Example 1. The crude reaction mixture was purified by CHT column chromatography to obtain conjugate 25 (3.19 mg, 64% yield). The STING agonist:XMT-1519 ratio of the purified conjugate 25 was 6.6.

[0559] Example 5: Synthesis of XMT-1519 conjugate 28 and DAR 6.0 TIFF2026086770000221.tif130160

[0560] Part A: Compound 26 was prepared in the same manner as in Example 1, except that compound 26 (prepared as described in US62 / 982,935) was used instead of compound 1. Compound 27 was obtained as a colorless solid (71.0 mg, 40% yield). 103 H 154 N 22 O 43 [M+2H] 2+ ESI-MS m / z calculated value: 1193.52; measured value: 1193.48.

[0561] Part B: Conjugate 28 was prepared as described in Example 1 to obtain conjugate 28. The STING agonist:XMT-1519 ratio of the purified conjugate 28 was 6.0.

[0562] Example 6: Synthesis of XMT-1519 conjugate 29 and DAR 5.5 TIFF2026086770000222.tif61165

[0563] Conjugate 28 (6.5 mg) was formulated by dissolving it in PBS, pH 8, through three cycles of concentration and dilution using a 30 kDa MWCO ultrafiltration unit. The reformulated conjugate was then incubated at 37°C for 24 hours and subsequently reformulated in trehalose buffer, pH 5.5. Ring opening was confirmed by LC-MS analysis of the heavy and light chains after antibody reduction. Good degradation was observed among various light chain species, i.e., between the unconjugated, intact succinimide-conjugated, and ring-opened succinimide-conjugated. Ring opening was also observed in the corresponding heavy chain species, but degradation among different species was poor. Therefore, the degree of ring opening was estimated by focusing on the light chain species. Using this approach, the percentage of ring-opened products in conjugate 29 compared to intact succinimide was estimated to be 94%. The ratio of the STING agonist XMT-1519 to Conjugate 29 was 5.5.

[0564] Example 7: Synthesis of XMT-1519 conjugate 32-1 and DAR 6.5 TIFF2026086770000223.tif130160

[0565] Part A: Scaffold 31 was prepared as described in Example 1, except that compound 30 (prepared as described in US62 / 982,935) was used instead of compound 1. Scaffold 31 was obtained as a colorless solid (9 mg, 8% yield). 101 H 151 N 23 O42 [M+2H] 2+ ESI-MS m / z calculated value: 1179.02; measured value: 1179.21.

[0566] Part B: Conjugates 32-1, 32-2, 32-3, and 32-4 were prepared as described in Example 1 to obtain the title conjugate. The STING agonist:XMT-1519 ratios of the purified conjugates 32-1, 32-2, 32-3, 32-4, and 32-5 are shown in the table below. The mAb concentration of conjugate 32-5 was >10 mg / mL and contained <1% unconjugated mAbs and <1% high molecular weight species. TIFF2026086770000224.tif30128

[0567] Example 7a: Synthesis of XMT-1535 conjugate 32a and DAR 6.2 TIFF2026086770000225.tif68139

[0568] Conjugates 32a, 32a-1, 32a-2, 32a-3, and 32a-4 were prepared and characterized as described in Example 1, except that XMT-1535 was used instead of XMT-1519. The STING agonist:XMT-1535 ratios of the purified conjugates 32a, 32a-1, 32a-2, 32a-3, and 32a-4 are shown in the table below. TIFF2026086770000226.tif30128

[0569] Example 7b: Synthesis of palivizumab conjugate 32b and DAR 6.8 TIFF2026086770000227.tif68143

[0570] Conjugates 32b, 32b-1, and 32b-2 were prepared and characterized as described in Example 1, except that palivizumab was used instead of XMT-1519. The STING agonist:palivizumab ratios of the purified conjugates 32b, 32b-1, and 32b-2 were as shown in the table below. TIFF2026086770000228.tif20128

[0571] Example 7c: Synthesis of palivizumab mIgG2a conjugate 32c, DAR 9.1 TIFF2026086770000229.tif68152

[0572] Conjugate 32c was prepared and characterized as described above in Example 1, except that palivizumab mIgG2a was used instead of XMT-1519. The STING agonist:palivizumab ratio of the purified conjugate 32c was 9.1.

[0573] Example 7d: Synthesis of XMT-1535 mIgG2a conjugate 32d and DAR 8.8 TIFF2026086770000230.tif63139

[0574] Conjugate 32d was prepared and characterized as described above in Example 1, except that XMT-1535 mIgG2a was used instead of XMT-1519. The STING agonist:XMT-1535 mIgG2a ratio of the purified conjugate 32d was 8.8.

[0575] Example 7e: Synthesis of XMT-1519 AAG conjugate 32e and DAR 7.4 TIFF2026086770000231.tif75143

[0576] Conjugate 32e was prepared and characterized as described in Example 1, except that XMT-1519 AAG was used instead of XMT-1519. The STING agonist:XMT-1519 AAG ratio of the purified conjugate 32e was 7.4.

[0577] Example 7-1: Alternative synthesis of compound 31 TIFF2026086770000232.tif94159

[0578] Part A: The mixture was placed in a vial and dissolved in DMF (26.5 mL) under air. Compound 30 (prepared as described in US62 / 982,935) (500 mg, 0.663 mmol, 1 eq), Boc-PEG2-Ala-OH (0.693 g, 1.99 mmol, 3 eq), EDC-HCl (381 mg, 1.99 mmol, 3 eq), and DMAP (243 mg, 1.99 mmol) were mixed. The reaction was completed in 3 hours. The mixture was quenched with AcOH (0.76 mL, 10 eq), concentrated, and purified over silica gel (DCM:MeOH) to obtain compound 30a as a white solid. 51 H 66 N 13 O 14 [M+H] + ESI-MS m / z calculated value: 1084.5; measured value: 1084.4.

[0579] Part B: Compound 30a (0.663 mmol) was suspended in dioxane (10 mL) in a flask under air. HCl (dissolved in 6 mL of dioxane to form 4 M) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated to obtain a white solid. The solid was dissolved in pure water and purified by reverse-phase chromatography (dissolved in water to form 0-25% ACN) to obtain compound 30b (444 mg, 77%) as a white solid. 46 H 58 N 13 O 12 [M+H] + ESI-MS m / z calculated value: 984.4; measured value: 984.2.

[0580] Part C: A solution of scaffold 6 (450 mg, 0.32 mmol, prepared as described in PCT / US2018 / 06719) dissolved in DMF (6.5 mL) and compound 30b (350 mg, 0.072 mmol) was prepared, to which PyBOP (185 mg, 0.36 mmol) and triethylamine (0.23 mL, 1.62 mmol) were added. The mixture was stirred at RT for 15 minutes, then quenched with AcOH (0.23 mL, 3.99 mmol), and purified by reverse-phase purification (0-40% ACN dissolved in water w / 0.1% acetic acid) to obtain compound 31 (408 mg, 55% yield). 101 H 151 N 23 O 42 [M+2H] 2+ ESI-MS m / z calculated value: 1179.52; measured value: 1179.27.

[0581] Example 8: Synthesis of trastuzumab conjugate 34 and DAR 6.9 TIFF2026086770000233.tif77160

[0582] Part A: To a solution of compound 1a (0.030 g, 0.038 mmol, prepared as described in WO2017175147A1) dissolved in DMF (1.5 mL), N-ethyl-N-isopropylpropan-2-amine (0.067 mL, 0.385 mmol) was added. After stirring the solution at room temperature for 5 minutes, 2,5-dioxopyrrolidine-1-yl-1-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oate (0.027 g, 0.050 mmol) dissolved in DMF (0.5 mL) was added, and the reaction mixture was stirred at room temperature for 15 minutes. Next, after adding acetic acid (0.1 mL), the solution was purified using a preparative HPLC column (C18, 21.2 mm x 100 mm) with 10-100% MeCN (0.1% HOAc) dissolved in H2O (0.1% HOAc, gradient for 20 minutes) to obtain scaffold 33 (0.006 g, 13.05% yield). 57 H 75N 14 O 15 [M+H] + ESI-MS m / z calculated value: Calculated value 1195.55; Measured value 1195.47.

[0583] Part B: Trastuzumab (10 mg, 0.069 μmol) was conjugated with scaffold 33 (0.658 mg, 0.550 μmol dissolved in DMA) as described in Example 1. Crude conjugate 34 was purified by CHT type II chromatography. The STING agonist:trastuzumab ratio of the purified conjugate 34 was 6.9.

[0584] Example 8a: Synthesis of palivizumab conjugate 34a and DAR 7.0 TIFF2026086770000234.tif45152

[0585] Conjugate 34a was prepared and characterized as described in Example 8, except that palivizumab was used instead of trastuzumab. The STING agonist:palivizumab ratio of the purified conjugate 34a was 7.0.

[0586] Example 9: Synthesis of XMT-1519 conjugate 45 and DAR 6.5 TIFF2026086770000235.tif145160

[0587] Part A: Compound 35 (400 mg, 1688 μmol) and t-butyl acetate (8 mL) were mixed with 70% HClO4 (302 mg, 2.11 mmol). The resulting solution was then stirred at room temperature for 21 hours and neutralized with saturated NaHCO3 solution. The aqueous phase was washed with RINKAN (2x), and the mixed organic solvent was washed with brine, dried with (Na2SO4), filtered, and concentrated to obtain compound 36, an opaque oil (530 mg, quant.). 16 H 24 N1O4[M+H] +ESI-MS m / z calculated value: 294.2; measured value: 294.2.

[0588] Part B: A mixture of compound 36 (279 mg, 782 μmol), compound 37 (370 mg, 938 μmol), HOAt (128 mg, 938 μmol), DIPEA (409 μL, 2.35 mmol), and DMF (4 mL) was stirred at room temperature for 5 minutes. Then, HATU (416 mg, 1095 μmol) was added, and the reaction mixture was stirred at room temperature for 2.5 hours. The reaction mixture was then concentrated, and the residue was subjected to chromatography using silica gel (20-100% HCl / hexane eluent). Compound 38 was isolated as a white, fluffy solid (254 mg, 362 μmol, 46% yield). 39 H 48 N2O9[M+H] + ESI-MS m / z calculated value: 702.3; measured value: 702.4.

[0589] Part C: A mixture of compound 38 (254 mg, 362 μmol), MeOH (6 mL), and 10% Pd-C catalyst (50 mg) was stirred at room temperature under H2 (1 atm) for 2 hours. The reaction mixture was then filtered, and the filtrate was concentrated to obtain compound 39, a clear oil (214 mg, 97% yield). 32 H 42 N3O9[M+H] + ESI-MS m / z calculated value: 612.3; measured value: 612.3.

[0590] Part D: A mixture of compound 39 (58 mg, 64 μmol), compound 40 (47 mg, 77 μmol, prepared as described in US62 / 982, 935), HOAt (10 mg, 77 μmol), DiPEA (56 μL, 320 μL), and DMF (2 mL) was stirred at room temperature for 5 minutes. Then, HATU (36 mg, 96 μmol) was added, and the reaction mixture was stirred at room temperature for 16 hours, and then concentrated to obtain compound 41, a yellow oil (90 mg, quant.). 70 H 83 N 14 O16 [M+H] + ESI-MS m / z calculated value: 1375.6; measured value: 1375.4.

[0591] Part E: A solution of compound 41 (90 mg, 64 μmol) dissolved in 20% piperidine dissolved in DMF (2 mL) was stirred at room temperature for 1 hour. The reaction mixture was then concentrated and subjected to chromatography using silica gel (0-40% MeOH-DCM eluent) to obtain compound 42, a yellow oil (30 mg, 41% yield). 55 H 73 N 14 O 14 [M+H] ESI-MS m / z calculated value: 1153.5; measured value: 1153.4.

[0592] Part F: A solution of compound 42 (30 mg, 37 μmol), 2,5-dioxopyrrolidine-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) acetate (8 mg, 44 μmol), TEA (7 μL, 74 μmol), and DMF (1 mL) was stirred at room temperature for 17 hours. The reaction mixture was then concentrated to obtain scaffold 43 as a yellow oil (40 mg, quant.). 61 H 76 N 15 O 17 [M+H] + ESI-MS m / z calculated value: 1290.6; measured value: 1290.3.

[0593] Part G: A solution of scaffold 43 (30 mg, 26 μmol) dissolved in 10% TFA-DCM (2 mL) was stirred at room temperature for 1 hour. The reaction mixture was then concentrated, and the residue was subjected to chromatography by HPLC (10-100% ACN-water w / 0.1% HCOOH eluent). Scaffold 44 was isolated as an off-white, fluffy solid (6 mg, 20% yield). 52 H 60 N 15 O 15 [M+H] +ESI-MS m / z calculated value: 1134.4; measured value: 1134.2.

[0594] Part H: Conjugate 45 was prepared as described in Example 1, except that 4 equivalents of TCEP were used. The STING agonist:XMT-1519 ratio of the purified conjugate 45 was 6.5.

[0595] Example 10: Synthesis of XMT-1519 conjugate 50 and DAR 8.2 TIFF2026086770000236.tif67160

[0596] Part A: A mixture of compound 26 (50 mg, 64 μmol, prepared as described in US62 / 982, 935), Fmoc-D-glutamic acid-O-tBu (54 mg, 128 μmol), DCC (26 mg, 128 μmol), DMAP (1 mg, 6 μmol), and DMF (2 mL) was stirred at room temperature for 17 hours. The reaction mixture was concentrated and used in the next step without purification. Compound 46 was obtained as a yellow oil (135 mg). 62 H 68 N 11 O 14 [M+H] + ESI-MS m / z calculated value: 1190.5; measured value: 1190.3.

[0597] Part B: Compound 46 (135 mg, 64 μmol) dissolved in DMF (2.4 mL) and a mixture of 33% TEA were stirred at room temperature for 4.5 hours. The reaction mixture was concentrated, and the residue was purified by reverse-phase HPLC chromatography (10-100% ACN-water w / 0.1% HCOOH eluent). Compound 47 was isolated as a yellow powder (27 mg, 44% yield). 47 H 58 N 11 O 12 [M+H] + ESI-MS m / z calculated value: 968.4; measured value: 968.2.

[0598] Part C: A solution of compound 47 (25 mg, 26 μmol), 2,5-dioxopyrrolidine-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) acetate (8 mg, 31 μmol), TEA (11 μL, 78 μmol), and DMF (1 mL) was stirred at room temperature for 1 hour. The reaction mixture was concentrated and used in the next step without purification. Scaffold 48 is a yellow oil (40 mg, quant.). 53 H 61 N 12 O 15 [M+H] + ESI-MS m / z calculated value: 1105.4; measured value: 1105.2.

[0599] Part D: A solution of scaffold 48 (40 mg, 24 μmol) dissolved in DCM (1 mL) and 15% TFA was stirred at room temperature for 2 hours. The reaction mixture was then concentrated and subjected to chromatography by HPLC (10-100% ACN-water with 0.1% HCOOH eluent). Scaffold 49 was isolated as a white, fluffy solid (5 mg, 20% yield). 49 H 53 N 12 O 15 [M+H] + ESI-MS m / z calculated value: 1049.4; measured value: 1049.2.

[0600] Part E: As described in Example 1, XMT-1519 (10 mg, 0.069 μmol) was conjugated with scaffold 49. Conjugate 50 was purified by CHT type II chromatography. The STING agonist:XMT-1519 ratio of the purified conjugate 50 was 8.2.

[0601] Example 11: Synthesis of XMT-1519 conjugate 52 and DAR 7.7 TIFF2026086770000237.tif48160

[0602] Conjugate 52 was prepared from scaffold 51 as described in Example 10, except that Fmoc-L-Glu(O-tBu) was used instead of Fmoc-D-Glu(O-tBu). The STING agonist:XMT-1519 ratio of the purified conjugate 52 was 7.7.

[0603] Example 12: Synthesis of XMT-1519 conjugate 58 and DAR 6.5 TIFF2026086770000238.tif120160

[0604] Part A: Compound 26 (0.105 g, 0.134 mmol, prepared as described in US62 / 982, 935), Boc-L-alanine (50.8 mg, 0.268 mmol), DMAP (50.8 mg, 0.067 mmol), and DCC (0.111 g, 0.537 mmol) were mixed with DMF (2 mL). The suspension was then stirred at room temperature for 2 days. The mixture was concentrated and purified over silica gel (0-40% MeOH dissolved in DCM) to obtain compound 53 as a pale yellow solid (0.102 g, 80% yield). 46 H 56 N 11 O 12 [M+H] + ESI-MS m / z calculated value: 954.4; measured value: 954.4.

[0605] Part B: A suspension of compound 53 (0.102 g, 0.107 mmol) dissolved in dioxane (10 mL) was mixed with 4N HCl (0.508 mL, 2.031 mmol). The reaction mixture was stirred at room temperature for 3 hours. The suspension was then concentrated and used in the next step without purification. Compound 54 was obtained as a pale yellow solid. 41 H 48 N 11 O 10 [M+H] + ESI-MS m / z calculated value: 854.3; measured value: 854.3.

[0606] Part C: A solution of compound 54 (0.015 g, 0.017 mmol) dissolved in DMF (1 mL) was mixed with Boc-D-Glu(Otu)-OH (7.67 mg, 0.025 mmol), followed by the addition of DIPEA (0.026 mL, 0.152 mmol). The reaction mixture was stirred at room temperature for 5 minutes. Then, PyBOP (13.15 mg, 0.025 mmol) was added, the mixture was stirred at room temperature for 1 hour, concentrated, and the residue was purified with silica gel (0-30% MeOH dissolved in DCM) to obtain compound 55 (11 mg, 57.3% yield) as a white solid. 55 H 71 N 12 O 15 [M+H] + ESI-MS m / z calculated value: 1139.5; measured value: 1139.5.

[0607] Part D: A suspension of compound 55 (11 mg, 0.00966 mmol) dissolved in dioxane (3 mL) was to which 4N HCl (0.241 mL, 0.966 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The suspension was concentrated and used in the next step without purification. Compound 56 was a white solid. 46 H 55 N 12 O 13 [M+H] + ESI-MS m / z calculated value: 983.4; measured value: 983.4.

[0608] Part E: To a solution of compound 56 (9.5 mg, 0.00966 mmol) dissolved in DMF (3 mL), DIEA (8.44 μL, 0.048 mmol) and 2,5-dioxopyrrolidine-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) acetate (3.17 mg, 0.013 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, neutralized to pH 6-7 with HOAc, and then purified by preparative RP HPLC (0-75% ACN dissolved in water) to obtain scaffold 57 (3.3 mg, 31% yield) as a white solid. 52 H 58 N 13 O 16[M+H] + ESI-MS m / z calculated value: 1120.4; measured value: 1120.4.

[0609] Part F: As described in Example 1, XMT-1519 (10 mg, 0.069 μmol) was conjugated with scaffold 57 (0.700 mg, 0.625 μmol dissolved in 200 μL DMA). Conjugate 58 was purified by CHT type II chromatography. The STING agonist:XMT-1519 ratio of the purified conjugate 58 was 6.5.

[0610] Example 13: Synthesis of XMT-1519 conjugate 60 TIFF2026086770000239.tif41160

[0611] Conjugate 60 was prepared from 59 as described in Example 12, except that Boc-L-Glu(Otu)-OH was used instead of Boc-D-Glu-O-tBu. Details of antibody-drug conjugates 60-1 and 60-2 are shown below. TIFF2026086770000240.tif16128

[0612] Example 14: Synthesis of XMT-1519 conjugate 62 and DAR 6.5 TIFF2026086770000241.tif41160

[0613] Conjugate 62 was prepared from scaffold 61 as described in Example 12, except that Boc-L-Glu(Otu)-OH was used instead of Boc-D-Glu-O-tBu and Boc-D-Ala was used instead of Boc-L-Ala. The STING agonist:XMT-1519 ratio of the purified conjugate 62 was 6.5.

[0614] Example 15: Synthesis of XMT-1519 conjugate 64 and DAR 6.4 TIFF2026086770000242.tif41159

[0615] Conjugate 64 was prepared from scaffold 63 as described in Example 12, except that Boc-D-Ala was used instead of Boc-L-Ala. The STING agonist:XMT-1519 ratio of the purified conjugate 64 was 6.4.

[0616] Example 16: Synthesis of XMT-1519 conjugate 66 TIFF2026086770000243.tif41160

[0617] Conjugate 66 was prepared from scaffold 65 as described in Example 12, except that Boc-2-amino-2-methylpropanoic acid was used instead of Boc-L-Ala. Details of antibody-drug conjugates 66-1 and 66-2 are shown below. TIFF2026086770000244.tif16128

[0618] Example 17: Synthesis of XMT-1519 conjugate 74 and DAR 6.9 TIFF2026086770000245.tif84160

[0619] Part A: Compound 26 (75 mg, 0.096 mmol, prepared as described in US62 / 982,935, 3 mL) dissolved in DMF, N-Boc-(D)-Ala-OH (91 mg, 0.48 mmol), DCC (99 mg, 0.48 mmol), and DMAP (1.2 mg, 9.58 μmol) were mixed and stirred at room temperature for 1 hour, then concentrated under reduced pressure. Purification with silica gel (DCM:MeOH 60:40 v / v) yielded compound 67 (82 mg, 90% yield) as a pale yellow solid. 46 H 56 N 11 O 12 [M+H] + The calculated ESI-MS m / z value was 954.40, and the measured value was 954.43.

[0620] Part B: To a suspension of compound 67 (80 mg, 0.084 mmol) dissolved in dioxane (5 mL), HCl (dissolved in dioxane, 0.42 mL, 1.68 mmol, and prepared to 4 M) was added, and the mixture was stirred at room temperature for 4 hours. The mixture was concentrated under reduced pressure to obtain compound 68 (72 mg, 100% yield) as a pale yellow solid. 41 H 48 N 11 O 10 [M+H] + The calculated ESI-MS m / z value was 854.35, and the measured value was 854.38.

[0621] Part C: A stirred solution of compound 68 (48 mg, 0.056 mmol), N-Boc-glycine (15 mg, 0.084 mmol), and PyBOP (44 mg, 0.084 mmol) dissolved in DMF (3 mL) was mixed with DIPEA (0.088 mL, 0.51 mmol), and the mixture was stirred at room temperature for 2 hours. The mixture was concentrated, and the residue was purified over silica gel (DCM:MeOH 60:40 v / v) to obtain compound 69 (53 mg, 93% yield) as a white solid. 48 H 59 N 12 O 13 [M+H] + The calculated ESI-MS m / z value was 1011.42, and the measured value was 1011.45.

[0622] Part D: To a suspension of compound 69 (50 mg, 0.049 mmol) dissolved in dioxane (5 mL), HCl (dissolved in dioxane, 1 mL, 20% v / v, to 4 M) was added, the mixture was stirred at room temperature for 2 hours, and then concentrated to obtain compound 70 (45 mg, 100% yield) as a white solid. 43 H 51 N 12 O 11 [M+H] + The calculated ESI-MS m / z value was 911.37, and the measured value was 911.39.

[0623] Part E: Compound 70 (20 mg, 0.022 mmol) dissolved in DMF (2 mL), N-Boc-(D)-Glu(OtBu)-OH (10 mg, 0.033 mmol), and PyBOP (17 mg, 0.033 mmol) were stirred together. DIPEA (0.03 mL, 0.22 mmol) was added to the mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was concentrated, and the residue was purified over silica gel (DCM:MeOH 60:40 v / v) to obtain compound 71 (24 mg, 90% yield) as a white solid. 57 H 74 N 13 O 16 [M+H] + ESI-MS m / z calculated value: 1196.53; measured value: 1196.55.

[0624] Part F: A suspension of compound 71 (24 mg, 0.02 mmol) dissolved in DCM (5 mL) was mixed with TFA (1 mL, 20% v / v), and the mixture was stirred at room temperature for 12 hours. The mixture was concentrated to obtain compound 72 (21 mg, 100% yield) as a pale yellow solid. 48 H 58 N 13 O 14 [M+H] + ESI-MS m / z calculated value: 1040.41; measured value: 1040.23.

[0625] Part G: Compound 72 (21 mg, 0.02 mmol) dissolved in DMF (2 mL) and 2,5-dioxopyrrolidine-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) acetate (7.6 mg, 0.03 mmol) were stirred together. DIPEA (0.035 mL, 0.20 mmol) was added to the mixture, and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated, and the residue was purified by RP HPLC to obtain scaffold 73 (4.5 mg, 19% yield) as a white solid. 54 H 61 N 14 O 17 [M+H] + ESI-MS m / z calculated value: 1177.43; measured value: 1177.40.

[0626] Part H: As described in Example 12, conjugate 74 was prepared from scaffold 73. The STING agonist:XMT-1519 ratio of the purified conjugate 74 was 6.9.

[0627] Example 18: Synthesis of XMT-1519 conjugate 76 and DAR 7.5 TIFF2026086770000246.tif36160

[0628] Conjugate 76 was prepared from scaffold 75 as described in Example 17, except that N-Boc-(L)-Ala-OH was used instead of N-Boc-(D)-Ala-OH, and N-Boc-(L)-Glu(OtBu)-OH was used instead of N-Boc-(D)-Glu(OtBu)-OH. The STING agonist:XMT-1519 ratio of the purified conjugate 76 was 7.5.

[0629] Example 19: Synthesis of XMT-1519 conjugate 78 and DAR 7.4 TIFF2026086770000247.tif36160

[0630] Conjugate 78 was prepared from scaffold 70 as described in Example 17, except that N-Boc-(L)-Glu(OtBu)-OH was used instead of N-Boc-(D)-Glu(OtBu)-OH. The STING agonist:XMT-1519 ratio of the purified conjugate 78 was 7.4.

[0631] Example 20: Synthesis of XMT-1519 conjugate 80 and DAR 7.5 TIFF2026086770000248.tif36160

[0632] Conjugate 80 was prepared from scaffold 79 as described in Example 17, except that N-Boc-(L)-Ala-OH was used instead of N-Boc-(D)-Ala-OH. The STING agonist:XMT-1519 ratio of the purified conjugate 80 was 7.5.

[0633] Example 21: Synthesis of XMT-1519 conjugate 82 and DAR 5.7 TIFF2026086770000249.tif41160

[0634] Conjugate 82 was prepared from scaffold 81 as described in Example 12, except that Boc-glycine was used instead of Boc-(L)-Ala-OH. The STING agonist:XMT-1519 ratio of the purified conjugate 81 was 5.7.

[0635] Example 22: Synthesis of XMT-1519 conjugate 85 and DAR 6.5 TIFF2026086770000250.tif130160

[0636] Part A: Scaffold 84 was prepared as described in Example 1, except that compound 83 (prepared as described in US62 / 982,935) was used instead of compound 1. Scaffold 84 was obtained as a white, fluffy solid (3.3 mg in 5 steps, 0.5% yield). 101 H 148 N 22 O 42 S[M+2H] 2+ ESI-MS m / z calculated value: 1187.49; measured value: 1187.78.

[0637] Part B: Conjugate 85 was prepared as described in Example 1 to obtain the title conjugate. The STING agonist:XMT-1519 ratio of the purified conjugate 85 was 6.5.

[0638] Example 22a: Synthesis of palivizumab conjugate 85a and DAR 7.4 TIFF2026086770000251.tif66141

[0639] Conjugate 85a was prepared and characterized as described in Example 1, except that palivizumab was used instead of XMT-1519. The STING agonist:palivizumab ratio of the purified conjugate 85a was 7.4.

[0640] Example 23: Synthesis of XMT-1519 conjugate 88 and DAR 6.6 TIFF2026086770000252.tif81128

[0641] Part A: Scaffold 87 was prepared as described in Example 12, except that compound 86 (prepared as described in US62 / 982,935) was used instead of compound 26. 50 H 55 N 14 O 15 [M+H] + ESI-MS m / z calculated value: 1091.4; measured value: 1091.2.

[0642] Part B: Conjugate 88 was prepared as described in Example 12, except that scaffold 87 was used instead of scaffold 57. The STING agonist:XMT-1519 ratio of the purified conjugate was 6.6.

[0643] Example 23a: Synthesis of palivizumab conjugate 89 and DAR5.9 TIFF2026086770000253.tif54128

[0644] Conjugate 89 was prepared and characterized as described in Example 12, except that palivizumab was used instead of XMT-1519. The STING agonist:palivizumab ratio of the purified conjugate 89 was 5.9.

[0645] Example 23b: Synthesis of CTL-48132_mIgG2a conjugate 89a and DAR 8.8 TIFF2026086770000254.tif47128

[0646] Conjugate 89a was prepared and characterized as described in Example 12, except that CTL-48132_mIgG2a was used instead of XMT-1519. The STING agonist:CTL-48132_mIgG2a ratio of the purified conjugate 89a was 8.8.

[0647] Example 23c: Synthesis of MFP5_mIgG2a conjugate 89b and DAR 9.0 TIFF2026086770000255.tif47128

[0648] Conjugate 89b was prepared and characterized as described in Example 12, except that CTL-48132_mIgG2a was used instead of XMT-1519. The STING agonist:MFP5_mIgG2a ratio of the purified conjugate 89b was 9.0.

[0649] Example 24: Synthesis of XMT-1519 conjugate 92 and DAR 7.6 TIFF2026086770000256.tif79128

[0650] Part A: Scaffold 91 was prepared as described in Example 12, except that compound 90 (prepared as described in US62 / 982,935) was used instead of compound 26. 52 H 58 N 13 O 15 S[M+H] + ESI-MS m / z calculated value: 1136.4; measured value: 1136.2.

[0651] Part B: Conjugate 88 was prepared as described in Example 12, except that scaffold 87 was used instead of scaffold 57. The STING agonist:XMT-1519 ratio of the purified conjugate was 7.6.

[0652] Example 24a: Synthesis of palivizumab conjugate 93 and DAR6.7 TIFF2026086770000257.tif51128

[0653] Conjugate 93 was prepared and characterized as described in Example 12, except that palivizumab was used instead of XMT-1519. The STING agonist:palivizumab ratio of the purified conjugate 93 was 6.7.

[0654] Example 25: Synthesis of XMT-1519 conjugate 100 and DAR 7.8 TIFF2026086770000258.tif109170

[0655] Part A: Compound 94 (45 mg, 0.054 mmol, prepared as described in US62 / 982, 935) dissolved in DMF (3 mL) was stirred, to which (S)-1-(Boc-amino)propan-2-ol (19 mg, 0.11 mmol), EDC (17 mg, 0.109 mmol), and DMAP (3.3 mg, 0.027 mmol) were added, and the mixture was stirred at room temperature for 12 hours. The reaction product was concentrated under reduced pressure, and the residue was purified over silica gel (DCM:MeOH 60:40 v / v) to obtain compound 95 (49 mg, 92% yield) as a white solid. ESI-MS:C 47 H 57 N 10 O 12 S(M+H): Calculated value 985.38, Measured value: 985.21.

[0656] Part B: A stirred suspension of compound 95 (49 mg, 0.05 mmol) dissolved in DCM (5 mL) was mixed with TFA (1 mL, 20% v / v DCM), and the mixture was stirred at room temperature for 12 hours. The resulting mixture was concentrated to obtain compound 96 (44 mg, 100% yield) as a pale yellow solid. ESI-MS:C 42 H 49 N 10 O 10 S(M+H): Calculated value: 885.33, Measured value: 885.18.

[0657] Part C: To a stirred solution of compound 96 (50 mg, 0.05 mmol) dissolved in DMF (2 mL), Boc-Glu-OtBu (86 mg, 0.28 mmol), PyBOP (118 mg, 0.23 mmol), and DIPEA (0.12 mL, 0.68 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. The mixture was concentrated under reduced pressure, and the residue was purified with silica gel (DCM:MeOH 60:40 v / v) to obtain compound 97 (45 mg, 77% yield) as a white solid. ESI-MS:C 56 H 72 N 11 O 15 S(M+H): Calculated value: 1170.49, Measured value: 1170.29.

[0658] Part D: A stirred suspension of compound 97 (45 mg, 0.038 mmol) dissolved in DCM (5 mL) was mixed with TFA (1 mL, 20% v / v DCM), and the mixture was stirred at room temperature for 12 hours. The mixture was concentrated under reduced pressure to obtain compound 98 (38 mg, 100% yield) as a pale yellow solid. ESI-MS:C 47 H 56 N 11 O 13 S(M+H): Calculated value 1014.37, Measured value: 1014.20.

[0659] Part E: To a stirred solution of compound 98 (20 mg, 0.02 mmol) dissolved in DMF (2 mL), 2,5-dioxopyrrolidine-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) acetate (5 mg, 0.02 mmol) and DIPEA (0.034 mL, 0.20 mmol) were added, and the mixture was stirred at room temperature for 15 minutes. The reaction was quenched with acetic acid (0.034 mL, 1:1 v / v DIPEA), and direct purification was performed by HPLC using a C18 stationary phase (water: ACN) to obtain scaffold 99 (4.7 mg, 20% yield) as a white solid. ESI-MS:C 53 H 59 N 12 O 16 S(M+H): Calculated value 1151.38, Measured value: 1151.18.

[0660] Part F: Conjugate 100 was prepared as described in Example 12, except that scaffold 99 was used instead of scaffold 57. The STING agonist:XMT-1519 ratio of the purified conjugate was 7.8.

[0661] Example 25a: Synthesis of palivizumab conjugate 101 and DAR6.5 Conjugate 101 was prepared as described in Example 25, except that palivizumab was used instead of TIFF2026086770000259.tif50128XMT-1519. The STING agonist:palivizumab ratio of the purified conjugate was 6.5.

[0662] Example 26A: Cancer cell-targeted wild-type or Fc-mutated STING-ADC activity in co-culture of cancer cells / THP1 luciferase reporter cells Preparation of NaPi2b Fc silent antibody: NaPi2b mAb (anti-NaPi2b-(AAG)), in which the Fc region is manipulated to eliminate Fc effector function, was designed to have three mutations in the heavy chain constant region: L234A, L235A, and P329G (AAG; Kabat Eu numbering), and was constructed using standard molecular biology procedures. The antibody was expressed and purified. Briefly, the variable region of the heavy chain of the NaPi2b antibody, the constant region of human IgG1 with L234A, L235A, and P329G mutations, and the DNA encoding the variable region of the light chain of the anti-NaPi2b antibody and the human κ light chain were cloned and placed into a mammalian expression vector. The heavy and light chains of NaPi2b-(AAG) were co-expressed in HEK293 cells, and the antibody was purified from the cell supernatant by standard protein A affinity chromatography.

[0663] Induction of the STING pathway in immune cells: The induction of the STING pathway in immune cells by NaPi2b-targeted STING ADCs was evaluated using a cancer cell / THP1-IRF3-luciferase reporter cell co-culture assay. OVCAR3 human ovarian cancer cells were seeded into 96-well CellBind surface tissue culture plates (15,000 cells / well) and incubated in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin for 6 hours. Various dilutions of the test articles, conjugate 8b-1, conjugate 8f, conjugate 8d-1, and compound 1 (0.4 nM to 100 nM based on payload; 3-fold serial dilution in growth medium), were added to each well, and the plates were incubated at 37°C for 20 minutes. Then, THP1-dual reporter cells (30,000 cells) were added to each well, and incubation was continued at 37°C for 20 hours in a humidified atmosphere of 5% CO2. Cell culture supernatant (20 μl) from each incubation sample was added to resuspended QUANTI-Luc (50 μl), and the luminescence signal was immediately measured using a SpectraMax M5 plate reader (Molecular Devices). EC 50The values ​​were determined from the dose-response curve. Table 1A shows the EC levels in THP1-Dual cells co-cultured with OVCAR3 cancer cells. 50 Show the value.

[0664] [Table 1A]

[0665] As shown in Table 1A, conjugate 8b-1, which possesses wild-type Fc effector function, exhibits more than 100 times the activity compared to the free agonist and compound 1, and approximately 1000 times the activity compared to conjugate 8f and conjugate 8d-1. This confirms the role of the Fc receptor in obtaining activity. The results shown are representative of the EC of a typical experiment. 50 It is a value.

[0666] Example 26B: Cancer cell-targeted wild-type or Fc-mutated STING-ADC activity in co-culture of cancer cells / THP1 luciferase reporter cells Production of HER-2 Fc silent antibodies: Trastuzumab mAb (anti-Her2-(AAG)) in which the Fc region is manipulated to eliminate the Fc effector function was designed to have three mutations in the heavy chain constant region: L234A, L235A, and P329G (AAG; Kabat Eu numbering), and was prepared as described in Example 26A.

[0667] Induction of the STING pathway in immune cells: The induction of the STING pathway in immune cells by HER2-targeted STING ADCs was evaluated using a cancer cell / THP1-IRF3-luciferase reporter cell co-culture assay with SKBR3 human breast cancer cells and the test articles conjugate 8a-2, conjugate 8j, conjugate 8c-2, and compound 1), as described in Example 26A. Table 1A shows the EC in THP1-Dual cells co-cultured with SKBR3 cancer cells. 50 Show the value.

[0668] [Table 1B]

[0669] As shown in Table 1B, conjugate 8a-2, which possesses wild-type Fc effector function, exhibits approximately 50 times higher activity compared to the free agonist and compound 1, and approximately 1000 times higher activity compared to conjugate 8j and conjugate 8c-2. This confirms the role of the Fc receptor in obtaining activity. The results shown are representative of the EC of a typical experiment. 50 It is a value.

[0670] Example 27A: Cancer cell targeting wild-type or Fc-mutated STING ADC activity in THP1 luciferase reporter cells cultured on tumor cell antigen-coated plates. Human NaPi2b-derived peptide TIFF2026086770000262.tif4128 was coated onto the surface of each well of a 96-well plate by incubating it overnight at 4°C with this peptide (1 μg / mL dissolved in PBS). The wells were then washed 1x with PBS-T and blocked by incubating with BSA (3% dissolved in PBS-T) at room temperature for 1 hour. After washing with PBS-T (2x), PBS (1x), and growth medium (RPMI1640, 10% FBS, 1% penicillin / streptomycin, 1x), test articles (conjugate 8b-1, conjugate 8f, conjugate 8c-1, and compound 1) at various dilutions (0.4 nM to 100 nM based on payload; 3-fold serial dilution in growth medium) were added to each well, and the plate was incubated at 37°C for 20 minutes. THP1-dual reporter cells (50,000 cells) were added to each well and incubated at 37°C for 20 hours in a humidified atmosphere of 5% CO2. Cell culture supernatant (20 μl) from each incubated sample was added to resuspended QUANTI-Luc (50 μl), and the luminescence signal was immediately measured using a SpectraMax M5 plate reader (Molecular Devices). EC 50The values ​​were determined from the dose-response curve. Table 2A shows the EC levels of THP1-Dual cells cultured on NaPi2b recombinant peptide-coated plates. 50 Show the value.

[0671] [Table 2A]

[0672] As shown in Table 2, conjugate 8b-1, which possesses wild-type Fc, exhibits approximately 100 times the activity compared to compound 1. Conjugate 8f shows no activity, and conjugate 8c-1 has approximately 1 / 1000th the activity of conjugate 8b-1. This confirms the role of the Fc receptor in obtaining activity. The results shown are representative of the EC of a typical experiment. 50 It is a value.

[0673] Example 27B: Cancer cell targeting wild-type or Fc-mutated STING ADC activity in THP1 luciferase reporter cells cultured on tumor cell antigen-coated plates. Peptides derived from human HER2 / ErbB2 protein (His-tagged, ECD, domain IV, 17.1 kDa) were coated onto the surface of each well of a 96-well plate by incubating the peptide (dissolved in PBS at 1 μg / mL) overnight at 4°C. The assay was performed as described in Example 27A, except that 3-fold serial dilutions (0.09 nM to 200 nM based on payload) of test articles, conjugate 8a-3, conjugate 8j, conjugate 8c-2, and compound 1, were used. Table 2B shows the EC2 levels in THP1-Dual cells cultured on Her2 recombinant protein-coated plates. 50 Show the value.

[0674] [Table 2B]

[0675] As shown in Table 2B, conjugate 8a-3, which possesses wild-type Fc, exhibits approximately 100 times greater activity compared to compound 1. Conjugates 8j and 8c-2 show no activity. This confirms the role of the Fc receptor in obtaining activity. The results shown are representative of the EC of a typical experiment. 50 It is a value.

[0676] Example 28A: Activity of tumor cell-targeting NaPi2b ADC in cancer cell / PBMC co-culture Human OVCAR3 ovarian cancer cells stably expressing the nuclear restriction mKate fluorescent red protein were generated by transduction using the IncuCyte (copyright) NucLight Red Lentivirus reagent. Selected stable transduction cells (named OVCAR3-NucRed cells) were seeded in puromycin-containing medium (2 μg / mL) for 2 days and seeded into 96-well tissue culture plates (8,000 cells / well). They were then incubated overnight in RPMI1640 medium containing 10% FBS and 1% penicillin / streptomycin. The culture medium was then replaced with fresh medium (50 μL). Next, the test materials (3x concentrates, conjugate 8b-1 (100nM, 10nM, and 1nM), conjugate 8f (100nM, 10nM, and 1nM), conjugate 8c-1 (100nM and 10nM), and compound 1 (100nM and 10nM); conjugate concentrations were based on the payload) were added to each well containing medium (50 μL), and the plates were incubated at 37°C for 20 minutes. Frozen human peripheral blood mononuclear cells (PBMCs) were thawed according to the supplier's instructions and added to each well (40,000 PBMCs dissolved in 50 μL of medium), and the plates were placed in an IncuCyte (copyright) live cell imaging instrument in an incubator (37°C, 5% O2) and scanned every 4 hours for 2 days. The number of red objects (cancer cells) was quantified using IncuCyte (copyright) Zoom software. The red object confluence in each well was normalized relative to its own red object confluence at time T=0.

[0677] Figure 1A plots the red object confluence as a function of time, showing the rapid induction of cancer cell death by PBMCs in response to conjugate 8b-1 at 1 / 100th the payload concentration compared to compound 1. Conjugate 8f also showed activity, but at a lower level compared to conjugate 8b. The activity of conjugate 8c-1 was significantly lower than that of conjugate 8b-1.

[0678] Example 28B: Activity of tumor cell-targeting NaPi2b ADC in cancer cell / PBMC co-culture OVCAR3-NucRed cells were seeded in 96-well tissue culture plates (20,000 cells / well) and incubated in RPMI1640 medium containing 10% FBS and 1% penicillin / streptomycin for 6 hours. The culture medium was replaced with fresh medium (50 μL). The test products (3x concentrates, 8l conjugate and compound 1 (100 nM, 10 nM, and 1 nM, respectively) and 8m conjugate (100 nM); conjugate concentration was based on the payload) were then added to each well containing medium (50 μL). The assay was performed as described in Example 28A, except that 30,000 PBMCs were used. The number of red objects (cancer cells) was quantified using IncuCyte (copyright) Zoom software. The number of red objects in each well was normalized to its own number of red objects at time T=0.

[0679] Figure 1B plots the number of red objects as a function of time, showing the rapid induction of cancer cell death by PBMCs in response to conjugate 8l at 1 / 100th the payload concentration compared to compound 1. Conjugate 8m showed no significant activity, and the increase in the number of red objects (cell proliferation) over time was similar to that of the untreated control. The inset shows that none of the test items (100 nM) inhibited the proliferation of OVCAR3-NucRed cells in monoculture.

[0680] Example 29: Flow cytometry analysis of CD14, Fcγ receptor, and CD3 expression in PBMCs and isolated monocyte subpopulations. Frozen human PBMC (1x10 8 The cells were thawed and divided equally into three tubes. One aliquot was used for human monocyte enrichment [StemCell Technologies] ("CD16-depleted monocytes"), one aliquot for human monocyte enrichment without CD16 depletion [StemCell Technologies] ("enriched monocytes"), and one aliquot was not enriched ("PBMC"). For flow cytometry, cells (50,000) from each group were transferred four times to a U-bottom 96-well plate, washed with PBS, stained with live / dead fixable Aqua dead cell staining dye (Molecular Probes), and then stained with fluorophore-conjugated target-specific (triplicate) or isotype-controlled antibodies (Pacific Blue anti-human CD14, FITC anti-human CD3, APC / Cy7 CD16, PE anti-human CD32, PE / Cy7 anti-human CD64). Cells were fixed, and surface expression of proteins of interest was determined by flow cytometry analysis using a MACSQuant flow cytometer. Data analysis was performed using FlowJo software. Table 3 shows the frequency (per single cell / per live cell) of CD14- / CD16+, CD14+ / CD16+, CD14- / CD32+, CD14+ / CD32+, CD14- / CD64+, CD14+ / CD64+, and CD14- / CD3+ cells in PBMCs, enriched monocytes, and CD16-depleted monocyte populations.

[0681] [Table 3] * Single cell / living cell gating

[0682] Table 3 shows efficient CD3+ cell depletion and monocyte enrichment, as well as low levels of CD16-positive cells in isolated CD16-depleted monocytes. CD64 staining results indicate that all CD14-positive cells in PBMCs and enriched monocyte subpopulations express CD64 (FcγRI).

[0683] Figure 2 demonstrates efficient CD3+ cell depletion and monocyte proliferation, as well as low levels of CD14-positive cells in isolated CD16-depleted monocytes.

[0684] Example 30: Cancer cell death activity of Fc-mutated tumor cell-targeting Her2 ADC in in vitro co-culture of PBMC and STING wild-type or knockout SKBR3 cells Generation of STING knockout single-cell clones expressing nuclear restriction mKate fluorescent protein: SKBR3 cells were seeded in 24-well plates (50,000 cells / well) and transfected with three different sgRNAs targeting the human STING gene and a non-targeting sgRNA using Thermo Fisher's TrueGuide® Synthetic gRNA, TrueCut® Cas9 Protein v2, and Lipofectamine® CRISPRMAX® Transfection Reagent, according to the manufacturer's protocol. The sgRNA sequences were sgNT (non-targeting): The transfection code was TIFF2026086770000266.tif18161. Seven days after transfection, single cells were selected and placed in 96-well plates containing 100 μL of DMEM with 20% FBS and 1% penicillin / streptomycin, and clones were formed in 2-3 weeks (the medium was refreshed once or twice a week). Multiple clones were trypsin-treated and grown, and STING expression was analyzed by Western blotting using rabbit monoclonal anti-STING (Cell Signaling Technologies) and anti-β-actin (Licor) antibodies. To ensure stable expression of nuclear restriction mKate fluorescent protein as described in Example 28, clones without STING protein expression (confirmed by Western blotting) were selected.

[0685] Death assay: STING wild-type (sgNT-2: non-targeting sgRNA, clone 2) and knockout (sg#3-2: sgRNA#3 clone 2) SKBR3 cells expressing NucRed were seeded into 96-well plates (15,000 cells / well) containing RPMI with 10% FBS and 1% penicillin / streptomycin, and PBMC death assays were performed using 100 nM, 25 nM, 5 nM, and 1 nM conjugates 8a-3, 8j, 8c-2, and compound 1, respectively, as described in Example 29 (conjugate concentrations were based on payload).

[0686] Figures 3A and 3B plot the red object confluence as a function of time, showing the death of STING wild-type (sgNT-2) SKBR3 cell / PBMC co-cultures and knockout (sg#3-2) SKBR3 cell / PBMC co-cultures, respectively. Conjugate 8a-3 induced rapid death of STING wild-type and knockout SKBR3 cell / PBMC co-cultures at all doses tested. Conjugate 8j showed high activity at 100 nM, 25 nM, and 5 nM in STING wild-type (sgNT-2) SKBR3 cell / PBMC co-cultures and low activity in STING knockout (sg#3-2) SKBR3 cell / PBMC co-cultures. Compound 1 showed activity only at 100 nM in STING wild-type (sgNT-2) SKBR3 cell / PBMC co-culture, and showed no activity at any dose in STING knockout (sg#3-2) SKBR3 cell / PBMC co-culture. Conjugate 8c-2 showed no activity at all in either STING wild-type (sgNT-2) SKBR3 cell / PBMC co-culture or STING knockout (sg#3-2) SKBR3 cell / PBMC co-culture.

[0687] Example 30A: Activity of tumor cell-targeting Her2 ADCs and Her2 antibodies in STING wild-type or STING knockout SKBR3 cancer cell / PBMC co-cultures. STING wild-type (sgNT-2: non-targeting sgRNA, clone 2) and knockout (sg#3-2: sgRNA#3 clone 2) SKBR3 cells expressing NucRed were co-cultured with PBMCs, and cell death assays were performed using dose-range conjugate 8a-3 and conjugate 8-j (200 nM, 4x dilution based on payload) as described in Example 30. Unconjugate wild-type Fc trastuzumab and AAG Fc mutant trastuzumab were administered based on antibody concentrations corresponding to the conjugate 8a-3 antibody concentration. As shown in Figures 4A and 4B, conjugate 8a-3 showed vigorous cell death of STING wild-type and knockout SKBR3 cancer cells. In contrast, conjugate 8-j, an Fc mutant Her2 targeting ADC, showed cell death activity only in STING wild-type SKBR3 co-culture and was almost reduced in STING knockout SKBR3 co-culture. Both Fc wild-type and AAG mutant non-conjugate trastuzumab antibodies showed low activity in both STING wild-type and knockout cancer cell co-cultures. These data demonstrate that the cancer cell killing activity of Fc mutant cancer cell-targeted STING-ADC activity in immune cell co-cultures is derived from intrinsic STING activation within tumor cells.

[0688] Example 31: Activity of tumor cell-targeting NaPi2b ADC in cancer cell / human monocyte co-culture in the absence of T cells OVCAR3-NucRed cells (prepared as described in Example 28) were seeded (15,000 cells / well) into a 96-well CellBind surface tissue culture plate (Corning) and incubated for 6 hours in RPMI1640 medium containing 10% FBS and 1% penicillin / streptomycin. The culture medium was replaced with fresh medium (50 μL). The test products (conjugate 8b-1 and compound 1, 20 nM and 4 nM respectively; conjugate concentrations were based on payload concentrations) were then added to each well containing medium (50 μL). The plate was incubated at 37°C for 20 minutes. PBMCs, enriched monocytes, and CD16-depleted monocytes were prepared as described in Example 29. Next, live PBMCs (30,000 cells / well), enriched monocytes (20,000 cells / well), and CD16-depleted monocytes (20,000 cells / well) were added to wells containing culture medium (50 μL). The plates were placed in an IncuCyte (copyright) live cell imaging instrument in an incubator (37°C, 5% O2) and scanned every 4 hours for two days. The number of red objects (cancer cells) was quantified using IncuCyte (copyright) Zoom software. The red object confluence in each well was normalized to its own red object confluence at time T=0. The red object confluence was plotted as a function of time in Figures 5A-5C. From this, it can be seen that the CD3+ T cell-depleted monocyte population has equivalent cancer cell killing activity in response to tumor cell-targeting ADC activity. Conjugate 8b-1 at 4nM and 20nM payload concentrations induced rapid death of OVCAR3-NucRed cancer cells in PBMCs (Figure 5A), enriched monocytes (Figure 5B), and CD16-depleted monocytes (Figure 5C). Compound 1 at 20nM induced cancer cell death only in enriched monocyte co-cultures and CD16-depleted monocyte co-cultures.

[0689] Example 32: In vitro measurement of human CXCL10 and cell conjugation of HER2-targeted antibody-drug conjugate with HCC1954 human breast cancer cell line HCC1954 breast cancer cells were grown to approximately 80-95% confluence in RPMI1640 medium supplemented with FBS (10%) and penicillin / streptomycin (1%). Cells were collected and added to the wells of a 96-well flat-bottom plate (40,000 cells / well) and incubated overnight at 37°C and 5% CO2. The cells were then treated with 1 pM-10 μM test HER2 antibody-drug conjugates (20 μL) as shown in Table 4 and incubated at 37°C and 5% CO2 for 24 hours. The plates were centrifuged (300 g, 5 min), the supernatant was collected (100 mL), and subjected to ELISA analysis for human CXCL10 (human CXCL10 / IP-10). The color-developed plates were chromosomally analyzed using a SpectraMax M5 plate reader. 450 This was read. The values ​​for each process were plotted, and EC 50 The values ​​were calculated using 4-parameter curve fitting with GraphPad Prism software.

[0690] To confirm cell binding between HER2 antibody-drug conjugates and HCC1954 cells, HCC1954 cells were grown to approximately 80–95% confluence in RPMI1640 medium supplemented with FBS (10%) and penicillin / streptomycin (1%). Cells were collected and added to wells of a 96-well V-bottom plate (50,000 cells / well). Cells were pelleted (300xg, 5 min), resuspended in solutions of test HER2 antibody-drug conjugates at concentrations of 0.01 nM–100 nM as shown in Table 4, and incubated on ice for 3 hours. Next, the cells were washed with ice-cold PBS (3x), pelletized (300g, 5 min), and incubated with detection antibody (goat anti-human IgG-Alexa-647 (H+L chain)) at 4°C for 1 hour. The cell suspension was pelletized (300g, 5 min), washed three times with ice-cold PBS, and fixed by resuspending in a paraformaldehyde (2%) solution. The resuspended cells were then subjected to flow cytometry analysis using MACS Quant Flow Cytometry. Single events (10,000) were collected for analysis. Population gating and mean fluorescence intensity (MFI) analysis were performed using FlowJo software.

[0691] Table 4 shows the mean EC2 of cell binding and CXCL10 induction in HCC1954. 50 This is a compilation of the information.

[0692] [Table 4] ND=Not determined

[0693] As shown in Table 4, treatment of HCC1954 cells with Her2-targeted antibody-drug conjugates resulted in CXCL10 induction of EC levels ranging from less than nM to low nM. 50 Values ​​were obtained. EC of the binding of Her2 target-directed ADCs to HCC1954 cells. 50 If the value was determined, it was in the low nM range.

[0694] Example 33: Activity of tumor cell-targeted HER2 antibody-drug conjugate in cancer cell / PBMC co-culture Cancer cell death activity: Human breast cancer cells SKBR3 stably expressing nuclear restriction mKate fluorescent protein were prepared as described in Example 28A and named SKBR3 NucRed cells. 20,000 SKBR3 NucRed cells (per well) were seeded into a 96-well tissue culture plate and incubated for 6 hours in 50 μL of RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin. 50 μL of test articles (conjugate 8a-2, conjugate 8-j, conjugate 8c-2, and compound 1) at various dilutions (0.0.1 nM to 200 nM based on payload; 4-fold serial dilution with growth medium) were added to each well, and the plate was incubated at 37°C for 20 minutes. Subsequently, PBMCs or primary human monocytes (isolated from PBMCs as described in Example 29) (50,000 cells) were added to each well, and the assay was performed as described in Example 28. Table 5 shows the IC values ​​of the test articles in co-cultures of cancer cells / PBMCs and isolated primary human monocytes (cancer cell death activity). 50 The value (death activity) is shown.

[0695] [Table 5]

[0696] As shown in Table 5, conjugate 8a-2 showed approximately 300x and 150x efficacy compared to compound 1 in PBMC co-cultures and monocyte co-cultures, respectively. The efficacy of conjugate 8-j was approximately 1 / 30 and 1 / 80 compared to conjugate 8a-2 in PBMC co-cultures and monocyte co-cultures, respectively. The activity of conjugate 8c-2 was minimal in both PBMC co-cultures and monocyte co-cultures.

[0697] CXCL10 induction: Table 6 shows the EC of test articles in cancer cell / PBMC and isolated primary human monocyte cocultures for CXCL10 induction.50 Show the value.

[0698] [Table 6]

[0699] As shown in Table 6, conjugate 8a-2 exhibited approximately 400x and 700x CXCL10 induction efficacy compared to compound 1 in PBMC co-cultures and monocyte co-cultures, respectively. The efficacy of conjugate 8-j was approximately 1 / 50th that of conjugate 8a-2 in both PBMC co-cultures and monocyte co-cultures. The activity of conjugate 8c-2 was minimal in both PBMC co-cultures and monocyte co-cultures.

[0700] Type III IFN induction: To analyze type III interferon induction in the supernatant 24 hours after treatment with the human IL29 / IL28b ELISA kit, SKBR3 cells and PBMCs were co-cultured as described above. Table 7 shows the EC of test items in cancer cell / PBMC co-cultures targeting IL29 / IL28b induction (IFNλ1 / λ3). 50 Show the value.

[0701] [Table 7]

[0702] As shown in Table 7, in PBMC co-culture, conjugate 8a-2 showed approximately 400x IL29 / IL28b induction activity compared to compound 1. In PBMC co-culture, the activity of conjugate 8-j was approximately 1 / 6 that of conjugate 8a-2. In PBMC co-culture, the activity of conjugate 8c-2 was minimal.

[0703] The data in Tables 7-9 demonstrates that mutations in the Fc region of ADCs that suppress FcγR interaction reduce, but do not eliminate, the cancer cell killing activity of targeted ADCs. This suggests an Fc-independent contribution to ADC activity.

[0704] Example 34: Induction of the STING pathway in immune cells The induction of the STING pathway in immune cells by Her2-targeted STING ADCs was evaluated using a cancer cell / THP1-IRF3-luciferase reporter cell co-culture assay. SKOV3 human ovarian adenocarcinoma cells were seeded into 96-well CellBind surface tissue culture plates (20,000 cells / well) and deposited in McCoy's 5a medium containing 10% FBS and 1% penicillin / streptomycin for 6 hours. Conjugate 32-5, conjugate 32e, and compound 30, test articles at various dilutions (0.01 nM to 300 nM based on payload; 4-fold serial dilution in growth medium), were added to each well, and the plates were incubated at 37°C for 20 minutes. Subsequently, 50,000 THP1-dual reporter cells were added to each well, and incubation was continued at 37°C for 20 hours in a humidified atmosphere of 5% CO2. Cell culture supernatant (20 μL) from each incubated sample was added to resuspended QUANTI-Luc (50 μL), and the luminescence signal of IRF3 was immediately measured using a SpectraMax M5 plate reader (Molecular Devices). EC 50 The values ​​were determined from the dose-response curve. Table 8 shows the EC levels in THP1-Dual cells co-cultured with SKOV3 cancer cells. 50 Show the value.

[0705] [Table 8]

[0706] As shown in Table 8, treatment of co-cultures of SKOV3 and THP-1 with HER2-targeted ADCs resulted in STING pathway induction in THP-1 immune cells from sub-nM to low nM EC1. 50 A value was obtained.

[0707] Example 35: Activity of tumor cell-targeted HER2 antibody drug conjugate in cancer cell / PBMC co-culture CXCL10 induction: Calu3 human lung adenocarcinoma cells were seeded in 96-well tissue culture plates and incubated overnight at 37°C and 5% CO2 in 100 μL of EMEM medium containing 10% FBS and 1% penicillin / streptomycin. The culture medium was replaced with fresh medium (100 μL). 50 μL of test articles (conjugate 32-4, compound 30, and conjugate 32b-1) at various dilutions (0.0.1 nM to 200 nM based on payload; 4-fold serial dilution in growth medium) were added to each well, and the assay was performed as described in Example 33 - CXCL10 induction. Table 9 shows the EC of test articles in cancer cell / PBMC and cancer cell monoculture for CXCL10 induction. 50 Show the value.

[0708] [Table 9]

[0709] As shown in Table 9, conjugate 32-4 showed efficacy of less than nM compared to compound 30 in both cancer cell / PBMC co-culture and monoculture. In both cancer cell / PBMC co-culture and monoculture, the activity of conjugate 32b-1 was only minimal.

[0710] Example 36: Activation of the STING pathway in cancer cell monoculture in the presence of conditional medium from untreated immune cell cultures. The conditioning medium was RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin, in an incubator, with 1.5 x 10 units. 6 / mL PBMC (two different donors), 1x10 6 / mL primary monocytes (isolated from PBMCs derived from two different donors, as described in Example 29), or 1x10 6THP1 cells were prepared by culturing them at 37°C and 5% O2 for 24 hours. The supernatant was collected and centrifuged at 2000 rpm for 10 minutes to remove all cells. SKOV3 cells were seeded into 96-well plates (30,000 cells / well) and incubated overnight in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin. The culture medium was replaced with 100 μL / well of fresh medium (control) or conditional medium derived from untreated immune cells, and then compound 1 (50 μL / well, 100 nM final concentration) or control medium (untreated) was added to each well. After incubation at 37°C and 5% O2 for 24 hours, the supernatant from the 96-well plates was analyzed using a human CXCL10 ELISA kit to determine whether CXCL10 was produced. Table 10 shows the OD450 values ​​of CXCL10 produced by SKOV3 cancer cell monoculture.

[0711] [Table 10]

[0712] As shown in Table 10, SKOV3 cells responded to STING agonist treatment in monoculture alone in the presence of conditioning media derived from PBMCs and primary human monocytes, but did not respond in the presence of conditioning media derived from THP1 cells. This suggests that PBMCs or primary human monocytes secrete factors that can enhance the sensitivity of cancer cells to STING agonist treatment.

[0713] Example 37: Tumor growth response to administration of HER2 antibody-drug conjugate in SKOV3 Female CB.17 SCID mice with SKOV3 human ovarian cancer cells (10x10 6 Individual cells (per mouse) were subcutaneously inoculated. Tumor volume was 63-75 mm. 3 (Average=65.4mm 3When the number of animals reached n=10 / group, they were randomized to the treatment group. Vehicle, trastuzumab (3 / 0 mg / kg), diABZI STING agonist (0 / 5 mg / kg), conjugate 8c-1 (1 / 0.04 mg / kg or 3 / 0.12 mg / kg), or conjugate 8a-1 (1 / 0.03 mg / kg or 3 / 0.09 mg / kg) were administered intravenously as a single dose on day 1 (all doses are indicated by antibody / payload). For diABZI STING agonist (0 / 5 mg / kg) and conjugate 8c-1 (3 / 0.12 mg / kg), transient weight loss within acceptable limits was observed 2-3 days after treatment, but there were no further clinical observations. Weight loss at later time points correlated with tumor progression. This indicates tumor-induced cachexia.

[0714] Figure 6 shows the tumor volume results for SKOV3 tumor mice treated with trastuzumab, diABZI STING agonist, conjugate 8c-1, conjugate 8a-1, or vehicle. Treatment with trastuzumab (3 / 0 mg / kg), diABZI STING agonist (0 / 5 mg / kg), or conjugate 8c-1 (1 / 0.04 mg / kg or 3 / 0.12 mg / kg) resulted in TGI of 47.5%, 36.9%, 13.5%, and 25.5%, respectively. Treatment with conjugate 8a-1 (1 / 0.03 mg / kg or 3 / 0.09 mg / kg) resulted in tumor regression of 86.5% and 100%, respectively.

[0715] Example 38: Serum cytokines after administration of HER2 antibody-drug conjugate in SKOV3 Female CB.17 SCID mice with SKOV3 human ovarian cancer cells (10x10 6 Individual cells (per mouse) were subcutaneously inoculated. Tumor volume was 108-172 mm². 3 (Average=128~131.6mm 3When the animals reached the specified time point, they were randomized to a treatment group. On day 1, they received intravenous administration of either a vehicle, diABZI STING agonist (0 / 5 mg / kg), conjugate 8c-1 (3 / 0.12 mg / kg), or conjugate 8a-1 (3 / 0.09 mg / kg) (all doses are shown as antibody / payload, n=10 for each group). Serum was collected 6, 12, 24, and 72 hours after administration (n=5 / group) and flash-frozen with dry ice for serum cytokine analysis. Tumors were collected 12 and 72 hours after administration (n=5 for each time point) and processed into formalin-fixed paraffin-embedded (FFPE) blocks.

[0716] Serum cytokines (eotaxin, G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17, CXCL10(IP-10), CXCL1(KC), LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα, and VEGF) were analyzed using a mouse cytokine / chemokine magnetic bead panel on a FlexMap3D Luminex instrument. Belysa Immunoassay Curve Fitting Software was used for data analysis.

[0717] Figures 7A–7H show cytokine measurements as a function of time. Only cytokines with measurable increases compared to the vehicle control are shown (CXCL-10 (IP-10), IL-6, TNFα, IFNγ, CXCL1 (KC), MIG, MIP-1α, and RANTES). The insets in each plot show cytokine levels induced by conjugate 8a-1 and conjugate 8c-1 compared to the vehicle. Intravenously administered diABZI STING agonists induced significantly higher levels of serum cytokines than conjugate 8a-1 or conjugate 8c-1, with fold differences being as high as 100-fold for IL-6 and as low as 6-fold for CXCL10. There were no significant differences between the control and targeted ADCs for most cytokines, and there were few cytokines that showed differences b...

Claims

1. Equation (I): PBRM-[A 1 -(L C ) 0または1 -D]d 15 (I) An antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt or solvation compound thereof, wherein, PBRM represents a protein-based recognition molecule. L C When it exists, it is a linker unit. A 1 is a divalent linker moiety that connects PBRM to L when L C is present, or connects PBRM to D when L C is not present, C and D is the STING agonist drug portion, d 15 These are integers ranging from approximately 1 to approximately 20. The aforementioned antibody-drug conjugate (ADC), or a pharmaceutically acceptable salt or solvation compound thereof.

2. Equation (II) is useful for conjugating with PBRM: HAS 1’ -(L C ) 0または1 -D (II) A scaffold of, or a pharmaceutically acceptable salt or solvated compound thereof, wherein, L C When it exists, it is a linker unit. A 1’ This is a monovalent linker moiety containing a functional group that can form a covalent bond with the functional group of PBRM, D is the STING agonist drug portion. The aforementioned scaffold, or a pharmaceutically acceptable salt or solvated compound thereof.

3. Each L C However, when they exist, they exist independently. And in the formula, # is A 1 This indicates that it is attached to, and ## indicates that it is attached to D. M A When present, it is a peptide portion containing at least two amino acids. T 1 When present, it is a hydrophilic group. L D M A When D exists, M A This is the divalent linker portion that connects to M A If it does not exist, D is A 1 This is the divalent linker portion that connects to it. The conjugate or scaffold according to any one of the above claims.

4. Formula (I-B'): The conjugate according to any one of the claims, which is a conjugate of or a pharmaceutically acceptable salt or solvation compound thereof.

5. Each A 1 However, they became independent, And in the formula, R 7 -O-, -NR 8 ,-(C 1 ~C 10 Alkyl)-,-(C 1 ~C 10 Alkenil)-,-(C 1 ~C 10 Alkinyl)-,-(C 3 ~C 8 Cycloalkyl)-,-aryl-,-O-(C 1 ~C 8 Alkyl)-,-O-(C 1 ~C 10 Alkenyl)-, -O-(C 1 ~C 10 Alkinyl)-,-(C 1 ~C 10 Alkyl)-(C 3 ~C 8 Cycloalkyl)-,-(C 1 ~C 10 Alkyl)-aryl-,-(C 2 ~C 10 Alkenil)-(C 3 ~C 8 Cycloalkyl)-,-(C 2 ~C 10 Alkenyl)-aryl-,-(C 2 ~C 10 Alkinyl)-(C 3 ~C 8 Cycloalkyl)-,-(C 2 ~C 10 Alkinyl)-aryl-,-(C 3 ~C 8 Cycloalkyl)-(C 1 ~C 10 Alki)-, -Aryl-(C 1 ~C 10 Alki)-,-(C 3 ~C 8 Cycloalkyl)-(C 2 ~C 10 Alkenyl)-, -aryl-(C 2 ~C 10 Alkenil)-,-(C 3 ~C 8 Cycloalkyl)-(C 2 ~C 10 alkynyl)-, -aryl-(C 2 ~C 10 alkynyl)-, -(3- to 8-membered heterocycloalkyl)-, -(5- to 8-membered heteroaryl)-, -(C 1 ~C 10 alkyl)-(3- to 8-membered heterocycloalkyl)-, -(C 1 ~C 10 alkyl)-(5- to 8-membered heteroaryl)-, -(C 2 ~C 10 alkenyl)-(3- to 8-membered heterocycloalkyl)-, -(C 2 ~C 10 alkenyl)-(5- to 8-membered heteroaryl)-, -(C 2 ~C 10 alkynyl)-(3- to 8-membered heterocycloalkyl)-, -(C 2 ~C 10 alkynyl)-(5- to 8-membered heteroaryl)-, -(3- to 8-membered heterocycloalkyl)-(C 1 ~C 10 alkyl)-, -(5- to 8-membered heteroaryl)-(C 1 ~C 10 alkyl)-, -(3- to 8-membered heterocycloalkyl)-(C 2 ~C 10 alkenyl)-, -(5- to 8-membered heteroaryl)-(C 2 ~C 10 alkenyl)-, -(5- to 8-membered heteroaryl)-(C 2 ~C 10 alkynyl)-, -(5- to 8-membered heteroaryl)-(C 2 ~C 10 alkynyl)-, -O-C(O)-(CH 2 CH 2 O) r -(CH 2 ) 2 -、-(CH 2 CH 2 O) r -、または-(CH 2 CH 2 O) r -(CH 2 ) 2 - and the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl may be substituted. R 8 is H, hydroxy, or C 1～4 It is alkyl, r is an integer between approximately 1 and approximately 12. * This indicates that it is attached to the PBRM. ** is L C When L exists, C It indicates that it is attached, or L C If it does not exist, it indicates that it is attached to D. The conjugate or scaffold according to any one of the above claims.

6. Each A 1’ However, they became independent, And in the formula, r is an integer between approximately 4 and approximately 6. ** is L C When L exists, C It indicates that it is attached, or L C If it does not exist, it indicates that it is attached to D. The conjugate or scaffold according to any one of the above claims.

7. Each L D However, they became independent, And in the formula, L E When present, -NH-[(CH 2 CH 2 O) p -(CH 2 ) 0～2 ] q -C(O)-, -NH-(C 1 ~C 6 Alkyl)-OC(O)-, or -NH-[(CH 2 CH 2 O) p -(CH 2 ) 0～2 ] q -C(O)-NH-(C 1 ~C 6 The molecule is alkyl)-OC(O)-, where p is an integer from approximately 1 to approximately 20, q is an integer from approximately 1 to approximately 10, and each W is independently either a natural amino acid unit or a non-natural amino acid unit. w is an integer between approximately 0 and approximately 12. *** M A When M exists, A It indicates that it is attached, or M A When it does not exist, A 1 It indicates that it is attached, **** This indicates that it is attached to D. The conjugate or scaffold according to any one of the above claims.

8. A conjugate or scaffold according to any one of the claims, wherein w is 0, 1, 2, 3, 4, or 5.

9. L E However, when present, -NH-(CH 2 CH 2 O) 2 -(CH 2 ) 2 -C(O)-, -NH-CH 2 -CH(CH 3 )-OC(O)-, or -NH-[(CH 2 CH 2 O) 1～4 -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 A conjugate or scaffold according to any one of the claims, wherein the conjugate or scaffold is -OC(O)-.

10. Each L D However, when they exist, they exist independently. And in the formula, *** M A When M exists, A It indicates that it is attached, or M A When it does not exist, A 1 It indicates that it is attached, **** This indicates that it is attached to D. The conjugate or scaffold according to any one of the above claims.

11. L D but, And in the formula, *** M A When M exists, A It indicates that it is attached, or M A When it does not exist, A 1 It indicates that it is attached, **** This indicates that it is attached to D. The conjugate or scaffold according to any one of the above claims.

12. L D but, And in the formula, *** M A When M exists, A It indicates that it is attached, or M A When it does not exist, A 1 It indicates that it is attached, **** The conjugate or scaffold according to any one of the claims, indicating that it is attached to D.

13. M A but, And in the formula, * is, A 1 It indicates that it is attached, ** is, T 1 It indicates that it is attached, *** is L D A conjugate or scaffold according to any one of the claims, indicating that it is attached to a

14. T 1 However, -OH or And in the formula, n 1 This is an integer from 0 to approximately 6. Each R 58 These are independently -H or C 1～8 It is alkyl, R 60 is a bond, C 1～6 Alkyl linker, or -CHR 59 - and R 59 is -H, C 1～8 Alkyl, cycloalkyl, or arylalkyl, R 61 CH 2 Ure 62 COOR 62 ,-(CH 2 ) n2 COOR 62 , or heterocycloalkyls substituted with one or more hydroxyls, R 62 is -H or C 1～8 It is alkyl, n 2 These are integers from 1 to approximately 5. The conjugate or scaffold according to any one of the above claims.

15. T 1 but, A conjugate or scaffold according to any one of the claims.

16. T 1 but, And, n 4 This is an integer between 1 and approximately 25. Each R 63 These are independently hydrogen or C 1～8 It is alkyl, R 64 is a combination or C 1～8 It is an alkyl linker, R 65 H, C 1～8 Alkyl, -(CH 2 ) n2 COOR 62 , or -(CH 2 ) n2 COR 66 And, R 62 is H or C 1～8 It is alkyl, R 66 H, And, n 2 These are integers from 1 to approximately 5. The conjugate or scaffold according to any one of the above claims.

17. T 1 but, And in the formula, R 67 (1)-OH; And, n 4 These are integers ranging from approximately 2 to approximately 20, approximately 4 to approximately 16, approximately 6 to approximately 12, and approximately 8 to approximately 12. The conjugate or scaffold according to any one of the above claims.

18. n 4 A conjugate or scaffold according to any one of the claims, wherein is 6, 7, 8, 9, 10, 11, or 12.

19. d 13 A conjugate or scaffold according to any one of the claims, wherein is an integer between approximately 2 and approximately 8.

20. d 13 A conjugate or scaffold according to any one of the claims, wherein is 6 or 8.

21. Each D is independent of equation (A): or its prodrug, solvated compound, pharmaceutically acceptable salt, or tautomer, in which, Y 1 , Y 2 , Z 1 , and Z 2 Each of these is independently O, S, C, or N. X 1 , X 2 , W 1 , and W 2 Each is independently either C or N, X 3 and X 4 Each is independently S or NR f And, X 5 is N or CR A2 And, X 6 is N or CR A1 And, R 3 and R 5 Each of them is independent of -CON(R d )(R f ), -CH 2 N(R d )(R f ), -N(R d )(R f ), -N(R d )CO(R f ), -CH 2 N(R d )CO(R f ) or R 3 and R 5 One of them is -CON(R d )(R f ), -CH 2 N(R d )(R f ), -N(R d )(R f ), -N(R d )CO(R f ), or -CH 2 N(R d )CO(R f ) and R 3 and R 5 The other of these is H, -COOH, or -CO 2 (R C ) and R c is C 1～4 It is alkyl, R A2 and R A1 These are independently H, halogen, hydroxyl, amino, and amino(C) 1～4 Alkyl)-, may be substituted (C 1～6 Alkyl), or possibly substituted (C 1～6 It is alkyl)oxy-, and the substituted (C) is also alkyl. 1～6 Alkyl) or may be substituted (C 1～6 C of alkyl)oxy 1～6 Each alkyl group is independently hydroxyl, C 1～4 Alkoxyl, -N(R e )(R f ), -CO 2 (R f ), -CON(R e )(R f ), and may be substituted with 1 to 4 substituents selected from the group including -COOH, Each R d These are independently H, hydroxyl, or C 1～4 It is alkyl, R e H, (C 1～4 Alkyl), -CO(C 1～4 Alkyl), -OCO(C 1～4 Alkyl, and -CO 2 (C 1～4 Selected from alkyl groups, Each R f These are independently H, hydroxy, or (C 1～4 It is alkyl, R 14 and R C2 They either do not exist independently of each other, or C 1～4 It is alkyl, C 1～4 Alkyl is a halogen, -OR c , -NR c R d , -CO 2 R c ,-CONR c R d , -SO 2 NR c R d , and -OCONR c R d They may be substituted with a more selected substituent, R 16 and R C1 Either they do not exist independently, or they are H, or C 1～4 It is alkyl, R 15 , R 17 , R 18 , or R 19 Either they do not exist independently, or they are H, or C 1～4 It is alkyl, C 1～4 Alkyl is a halogen, -OR c , -NR c R d , -CO 2 R c ,-CONR c R d , -SO 2 NR c R d , and -OCONR c R d They may be substituted with a more selected substituent, (i)R A2 and R A1 At least one of the following exists, and R A2 and R A1 At least one of L C When L exists C Directly or indirectly connected to, or L C When R does not exist A2 and / or R A1 A via at least one functional group 1 (ii)R C2 and R C1 At least one of the following exists, and R C2 and R C1 At least one of L C When L exists C Directly or indirectly connected to, or L C When R does not exist C2 and / or R C1 A via at least one functional group 1 Connected to The conjugate or scaffold according to any one of the above claims.

22. Each D independently corresponds to the equations (Aa), (Ab), (Ac), (Ad), (Ae), (Af), (A-f1), (A-f2), (Af)3, (A-f4), (A-f5), (Ag), (A-g1), (A-g2), (A-g3), (A-g4), (A-g5), (Ah), (A-h1), (A-h2), or (Ai): A conjugate or scaffold according to any one of the claims, which is either a prodrug, solvation compound, pharmaceutically acceptable salt, or tautomer thereof.

23. Each D is independent, And in the formula, R 2 It either does not exist, is -O-, or -NR 4 -and, R 4 is H or C 1～3 It is alkyl, is L C When L exists, C It indicates that it is attached, or L C When it does not exist, A 1 This indicates that it is attached. The conjugate or scaffold according to any one of the above claims.

24. Each D is independent, And in the formula, R 2 It either does not exist, is -O-, or -NR 4 -and, R 4 is H or C 1～4 It is alkyl, is L C When L exists, C It indicates that it is attached, or L C When it does not exist, A 1 This indicates that it is attached. The conjugate or scaffold according to any one of the above claims.

25. A scaffold according to any one of the claims, selected from the scaffolds listed in Table A1.

26. A scaffold according to any one of the claims, selected from the scaffolds listed in Table A2.

27. A conjugate according to any one of the claims, selected from the conjugates listed in Table B1.

28. A conjugate selected from the conjugates listed in Table B2, according to any one of the claims.

29. below The conjugate according to any one of the above claims.

30. below The conjugate according to any one of the above claims.

31. below The conjugate according to any one of the above claims.

32. A pharmaceutical composition comprising the conjugate according to any one of the above claims and one or more pharmaceutically acceptable carriers or excipients.

33. A pharmaceutical composition comprising the conjugate according to any one of the claims, further comprising at least one immunomodulator or at least one immunostimulant.

34. A method for activating or enhancing the activity of interferon gene stimulator (STING) in a subject, comprising the step of administering the conjugate or a pharmaceutically acceptable salt thereof according to any one of the preceding claims to the subject.

35. A method for preventing or treating a disease or disorder in a subject, comprising the step of administering a therapeutically effective amount of the conjugate or a pharmaceutically acceptable salt thereof according to any one of the preceding claims to the subject.

36. A conjugate according to any one of the claims or a pharmaceutically acceptable salt thereof for activating or enhancing the activity of interferon gene stimulating factor (STING) in a subject.

37. A conjugate according to any one of the claims or a pharmaceutically acceptable salt thereof for preventing or treating a disease or disorder in a subject.

38. Use of the conjugate or a pharmaceutically acceptable salt thereof according to any one of the claims in the manufacture of a pharmaceutical product for activating or enhancing the activity of interferon gene stimulating factor (STING) in a subject.

39. Use of the conjugate or a pharmaceutically acceptable salt thereof according to any one of the claims in the manufacture of a medicament for preventing or treating a disease or disorder in a subject.

40. The method, conjugate for use, or use according to any one of the claims, wherein the disease or disorder is associated with STING agonism.

41. The method, conjugate for use, or use according to any one of the claims, wherein the disease or disorder is cancer.

42. The method, conjugate for use, or use according to any one of the preceding claims, wherein the disease or disorder is bladder cancer, breast cancer, colorectal cancer, colon cancer, endometrial cancer, gastric cancer, head and neck squamous cell carcinoma, melanoma, lung cancer, ovarian cancer, esophageal cancer, biliary tract cancer, urothelial carcinoma, cervical cancer, papillary thyroid cancer, papillary renal cell carcinoma, bile duct cancer, salivary duct cancer, kidney cancer, or pancreatic cancer.

43. BB formula A conjugate or a pharmaceutically acceptable salt thereof The conjugate contains the XMT-1519 antibody, which includes a variable heavy chain complementarity determination region 1 (CDRH1) containing the amino acid sequence FTFSSYSMN (SEQ ID NO: 20); a variable heavy chain complementarity determination region 2 (CDRH2) containing the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 21); a variable heavy chain complementarity determination region 3 (CDRH3) containing the amino acid sequence GGHGYFDL (SEQ ID NO: 22); a variable light chain complementarity determination region 1 (CDRL1) containing the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 27); a variable light chain complementarity determination region 2 (CDRL2) containing the amino acid sequence GASSRAT (SEQ ID NO: 28); and a variable light chain complementarity determination region 3 (CDRL3) containing the amino acid sequence QQYHHSPLT (SEQ ID NO: 29). 15 It is approximately 8. The aforementioned conjugate or a pharmaceutically acceptable salt thereof.

44. A method for preventing or treating a disease or disorder in a subject, comprising the step of administering a therapeutically effective amount of the conjugate or a pharmaceutically acceptable salt thereof according to claim 43 to the subject.

45. The method according to claim 44, wherein the disease or disorder is cancer.

46. The method according to claim 45, wherein the cancer is breast cancer, gastric cancer, colorectal cancer, colon cancer, esophageal cancer, biliary tract cancer, endometrial cancer, urothelial carcinoma, or non-small cell lung cancer.

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