Antibody drug conjugates containing STING agonists

Abstract

This disclosure provides a scaffold and an antibody-drug conjugate (ADC) containing interferon gene stimulating factor (STING). This disclosure also provides the use of the ADC in treatment, such as cancer treatment.

Description

[0001] Related applications

[0002] This application is a divisional application of international application PCT / US2021 / 025556, filed on April 2, 2021, and filed in China with application number 202180039592.6, entitled "Antibody-Drug Conjugate Containing a STING Agonist". This application claims priority to U.S. Provisional Application No. 63 / 004,108, filed April 2, 2020; U.S. Provisional Application No. 63 / 040,755, filed June 18, 2020; and U.S. Provisional Application No. 63 / 111,820, filed November 10, 2020. The contents of each of these applications are incorporated herein by reference in their entirety.

[0003] By referencing the incorporated sequence list

[0004] The contents of a 49KB text file named “MRSN-033_001WO_SeqList.txt”, created on March 31, 2021, are incorporated herein by reference in their entirety. Background Technology

[0005] STING (Spiritual Stimulatory Factor) is a receptor in the endoplasmic reticulum that propagates innate immune responses to cytoplasmic pathogens and its own DNA. STING is a 378-amino acid protein containing three main domains: (i) an N-terminal transmembrane domain (aa 1-154); (ii) a central globular domain (aa 155-341); and (iii) a C-terminal tail (aa 342-379). STING can combine with its ligands to form a symmetrical dimer with a V-shaped conformation, without completely covering the bound ligand. STING agonists can bind to the pocket region of STING. However, in some severe disease states, STING activation is easily inhibited, leading to STING pathway inactivation. Therefore, screening and designing effective STING agonists is of great significance for cancer immunotherapy and the treatment of other infectious diseases, including but not limited to obesity, liver injury, glucose and lipid metabolism disorders, and viral infections. Specific targeting of immune pathways offers opportunities for cancer therapy, potentially providing greater specificity than cell population-based treatments.

[0006] Antibody-drug conjugates (ADCs) consist of drug-like small molecules covalently linked to an antibody. The antibody represents a targeting mechanism against a specific site of action. Upon reaching the site, the ADC is designed to release the small molecule (drug) so that it can perform its designed function in a targeted manner, rather than spreading throughout the subject's body. This targeted approach makes it possible to treat with drugs that would otherwise require very high doses, making systemic administration toxic.

[0007] A key feature of the innate immune system is the recognition and elimination of foreign substances. These pathogenic invaders are identified by the host recognizing evolutionarily conserved microbial structures (called pathogen-associated molecular patterns (PAMPs)). Host recognition can occur through various pathways, such as activation of pattern recognition receptors (PRRs), which ultimately leads to downstream signaling events and a resulting immune response.

[0008] The antibody-drug conjugate disclosed herein modulates the activity of STING, and therefore may provide beneficial therapeutic effects in treating diseases, conditions, and / or ailments in which the regulation of STING (interferon gene stimulating factor) is beneficial (including, but not limited to, inflammation, allergic and autoimmune diseases, infectious diseases, cancer, precancerous syndromes, and as a vaccine adjuvant). Novel immunotherapies are still needed to treat diseases, particularly cancer. Summary of the Invention

[0009] In some respects, this disclosure provides conjugates of formula (I) or pharmaceutically acceptable salts or solvates thereof:

[0010] PBRM-[A 1 -(L C ) 0或1 -D] d15 (I)

[0011] in:

[0012] PBRM stands for Protein-Based Recognition Molecules;

[0013] When it exists, L C For connector unit;

[0014] When L C When it exists, A 1 To make PBRM and L C The connected divalent connector portion, or when L C When it does not exist, A 1 The bivalent connector portion for connecting PBRM and D;

[0015] D represents the agonist drug portion of STING; and

[0016] d 15 It is an integer in the range of approximately 1 to approximately 20.

[0017] In some aspects, this disclosure provides a stent for conjugation with PBRM, wherein the stent has the following formula (II) or a pharmaceutically acceptable salt or solvate thereof:

[0018] A 1’ -(L C ) 0或1 -D(II)

[0019] in:

[0020] PBRM stands for Protein-Based Recognition Molecules;

[0021] When it exists, L C For connector unit;

[0022] A 1’ It is a monovalent connector portion containing functional groups that can form covalent bonds with the functional groups of PBRM;

[0023] D represents the agonist drug portion of STING; and

[0024] d 15 It is an integer in the range of approximately 1 to approximately 20.

[0025] In some respects, this disclosure provides pharmaceutical compositions comprising the conjugates described herein and one or more pharmaceutically acceptable carriers or excipients.

[0026] In some respects, this disclosure provides methods for activating or enhancing the activity of interferon gene-stimulating factor (STING) in a subject, including administering the subject the conjugate described herein or a pharmaceutically acceptable salt thereof.

[0027] In some respects, this disclosure provides methods for preventing or treating a disease or ailment in a subject, including administering to the subject a therapeutically effective amount of the conjugate described herein or a pharmaceutically acceptable salt thereof.

[0028] In some respects, this disclosure provides the conjugates described herein or pharmaceutically acceptable salts thereof for activating or enhancing the activity of STING in a subject.

[0029] In some respects, this disclosure provides the conjugates described herein or pharmaceutically acceptable salts thereof for the prevention or treatment of a disease or condition in a subject.

[0030] In some respects, this disclosure provides for the use of the conjugates described herein or pharmaceutically acceptable salts thereof in the manufacture of medicaments for activating or enhancing the activity of STING in a subject.

[0031] In some respects, this disclosure provides for the use of the conjugates described herein or pharmaceutically acceptable salts thereof in the manufacture of medicaments for the prevention or treatment of a subject’s disease or condition.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the specification, the singular form also includes the plural unless the context clearly requires otherwise. Although those methods and materials similar to or equivalent to those described herein may be used in practice or testing of this disclosure, 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 acknowledged as prior art to the claimed invention. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be restrictive. In case of conflict between the chemical structures disclosed herein and the names of compounds, the chemical structures shall prevail.

[0033] Other features and advantages of this disclosure will become apparent from the following detailed description and claims. Attached Figure Description

[0034] Figure 1A Plot the red object blending degree over time for conjugates 8b-1 and 8f (each at 100, 10 and 1 nM) and conjugates 8c-1 and compound 1 (each at 100 and 10 nM) (conjugate concentration based on effective load).

[0035] Figure 1B Plot the red object blending degree over time for conjugate 8l and compound 1 (each at 100, 10 and 1 nM) and conjugate 8m (100 nM) (conjugate concentration based on effective load).

[0036] Figure 2 Displays PBMCs, enriched monocytes, and CD14- / CD3+ cells in a population of CD16-depleted monocytes.

[0037] Figure 3A and Figure 3B Plot the red object confluence of each of conjugates 8a-3, 8j, 8c-2 and compound 1 at 100, 25, 5 and 1 nM (conjugate concentration based on payload) over time and show the killing effect of PBMCs on STING wild-type (sgNT-2) and knockout (sg#3-2) SKBR3 NucRed cells, respectively.

[0038] Figure 4A A graph showing the dose-response relationship of the red objects to the killing activity of STING wild-type SKBR3 cancer cells in SKBR3 cancer cell / PBMC co-culture.

[0039] Figure 4B A graph showing the dose-response relationship of the red objects in SKBR3 cancer cell / PBMC co-culture with the killing activity of conjugate 8a-3, conjugate 8-j, wild-type Fc trastuzumab, and AAG Fc mutant trastuzumab on STING knockout SKBR3 cancer cells.

[0040] Figure 5A Plot the changes in red object fusion of OVCAR3-NucRed cancer cells via PBMC over time for both conjugate 8b-1 and compound 1 at 20 and 4 nM (conjugate concentration based on effective load concentration).

[0041] Figure 5B Plot the changes over time in the red object fusion of OVCAR3-NucRed cancer cells enriched with conjugates 8b-1 and 1 at 20 and 4 nM (conjugate concentrations based on effective load concentrations).

[0042] Figure 5C Plot the changes over time in red object conjugates 8b-1 and 1 in CD16-depleted monocytes at 20 and 4 nM (conjugate concentrations based on effective load concentrations).

[0043] Figure 6 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 given by antibody / load) in a mouse SKOV3 xenograft model.

[0044] Figures 7A-7H The changes in mouse CXCL-10 (IP-10) over 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 / load) in the SKOV3 mouse model. 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 Cytokine levels of 8a-1 and 8c-1 relative to the mediators. The insets in each figure show cytokine levels induced by conjugates 8a-1 and 8c-1.

[0045] Figures 8A-8C The results showed that at 12 h and 72 h 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 written as antibody / effective load) in a mouse SKOV3 xenograft model, mouse CXCL10 ( Figure 8A ), interferon-β ( Figure 8B ) and IL-6 ( Figure 8C mRNA levels.

[0046] Figure 9 Immunohistochemical (IHC) staining of CD45 with rabbit anti-CD45 monoclonal antibody against conjugate 8a-1 (3 / 0.09 mg / kg), conjugate 8c-1 (3 / 0.12 mg / kg), or the mediator at 12 and 72 hours.

[0047] Figure 10 A graph showing the circulating plasma concentrations of total antibody and conjugate drug after administration of CB.17SCID mice to conjugate 8a-1 (3 / 0.1 mg / kg) (dose given as antibody / load).

[0048] Figure 11 This study demonstrates the effects of IFNλ1 (IL29) or IFNλ2 (IL28A) neutralizing antibodies (10, 2, 0.4, 0.08 μg / mL) on the killing activity of conjugate 8a-3 (1 nM or 0.1 nM, based on payload) in co-culture of cancer cells and PBMCs.

[0049] Figure 12 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 given by antibody / load) in mouse OVCAR3 xenograft.

[0050] Figure 13 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 given as antibody / load) in mouse OVCAR3 xenografts.

[0051] Figure 14 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 given as antibody / load) in xenografts of triple-negative breast cancer in mice.

[0052] Figure 15A 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 given as antibody / load) in a mouse model of colorectal cancer homology.

[0053] Figure 15B Kaplan Meier survival curves are shown for conjugate 8d-3 (1 / 0.04 mg / kg) or conjugate 8g (0.88 / 0.04 mg / kg) (all doses are given as antibody / load) in a colon cancer homologous mouse model.

[0054] Figure 16A A graph showing the antitumor efficacy of 8 g (0.9 / 0.04 mg / kg) of the conjugate (all doses are given as antibody / load) in a single mouse in a colon cancer homologous mouse model.

[0055] Figure 16B A graph showing the antitumor efficacy of 8 g (0.9 / 0.04 mg / kg) of the conjugate (all doses are given as antibody / load) when re-excited with homologous mouse colon cancer cells.

[0056] Figure 16C A graph showing the antitumor efficacy of 8 g (0.9 / 0.04 mg / kg) of the conjugate (all doses are given as antibody / load) when re-excited with homologous mouse lung cancer cells.

[0057] Figure 17 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 given as antibody / load) in a homologous mouse embryonic carcinoma model.

[0058] Figure 18 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 given as antibody / load) in mouse SKOV3 xenograft.

[0059] Figure 19 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 given as antibody / load) in mouse SKOV3 xenografts.

[0060] Figure 20 A graph showing the antitumor efficacy of diABZI STING agonist (1.5 mg / kg every three days x3 or 0.128 mg / kg qdx1), compound 30 (1.5 mg / kg every three days x3 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 qdx1) (all doses are given as antibody / load) in mouse SKOV3 xenograft.

[0061] Figure 21A 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 a homologous mouse model.

[0062] Figure 21B shows the antitumor efficacy of conjugate 8d-3 (1 / 0.04 mg / kg) in a single mouse of the homologous mouse model.

[0063] Figure 21C The antitumor efficacy of the conjugate 8k (0.9 / 0.04 mg / kg) was demonstrated in a single mouse of the homologous mouse model.

[0064] Figure 22A 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 given as antibody / load) in mouse SKOV3 xenografts.

[0065] Figure 23 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 IVSTING agonist (0 / 5 mg / kg) (all doses are given as antibody / load) in mouse OVCAR3 xenograft.

[0066] Figure 24 To demonstrate the efficacy of rituximab AF-HPA ADC (0.75 / 0.023 mg / kg) and conjugate 8c-2 (4.0 / 0.126 mg / kg), 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 Charts showing the antitumor efficacy of the combination of ADC (0.75 / 0.023 mg / kg) and conjugate 8b-2 (2.0 / 0.071 mg / kg), the combination of XMT-1535AF-HPA ADC (0.75 / 0.024 mg / kg) and conjugate 8b-2 (4.0 / 0.142 mg / kg), the combination of XMT-1535AF-HPA ADC (0.75 / 0.024 mg / kg) and conjugate 8b-2 (2.0 / 0.071 mg / kg), the combination of XMT-1535AF-HPA ADC (0.75 / 0.024 mg / kg) and XMT-1535 (4.0 / 0 mg / kg), or XMT-1535 (4.75 / 0 mg / kg) in mouse OVCAR3 xenograft.

[0067] Figure 25 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 a mouse SKOV3 xenograft model.

[0068] Figure 26 A graph showing the antitumor efficacy of the mediator, conjugate 28 (0.99 / 0.0325 mg / kg), or conjugate 62 (0.92 / 0.0325 mg / kg) in a mouse SKOV3 xenograft model.

[0069] Figure 27 A graph showing the circulating plasma concentrations of the conjugates after administration of conjugate 28 (3.0 / 0.10 mg / kg) or conjugate 62 (2.84 / 0.10 mg / kg) to CB.17SCID mice (dosage given as antibody / load). Detailed Implementation

[0070] This disclosure provides novel antibody-drug conjugates, synthetic methods for preparing conjugates or scaffolds, pharmaceutical compositions containing them, and various uses of the conjugates.

[0071] definition

[0072] The chemical names provided for the intermediate compounds and / or compounds disclosed herein may refer to any tautomeric representation of such compound (in some cases, such alternative names are provided experimentally). It should be understood that any reference to a specified compound (intermediate compound or compound disclosed herein) or a compound whose structure is described (intermediate compound or compound disclosed herein) is intended to cover all tautomeric forms, including the zwitterionic forms of such compound and any mixtures thereof.

[0073] It should be understood that the terms “in some embodiments,” “in some embodiments of this disclosure,” and “in some embodiments of the compounds disclosed herein” may be used interchangeably where appropriate.

[0074] When used in conjunction with numerical values, the terms "about," "approximately," or "approximately" refer to a set or range of values. 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 X, where X is a numerical value. In some embodiments, the term "about" refers to a range of values ​​that are 5% more or less than a specified value. In some embodiments, the term "about" refers to a range of values ​​that are 2% more or less than a specified value. In some embodiments, the term "about" refers to a range of values ​​that are 1% more or less than a specified value.

[0075] Unless otherwise specified herein, the enumeration of value ranges is intended only as a shorthand for individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually enumerated herein. Unless otherwise specified, the ranges used herein include both limits of the range. In some embodiments, the expressions “x is an integer between 1 and 6” and “x is an integer between 1 and 6” both mean “x is 1, 2, 3, 4, 5, or 6”, that is, the terms “between X and Y” and “within the range of X to Y” include X and Y, as well as integers between them.

[0076] As used herein, the term "antibody" is used in the broadest sense and encompasses a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity. Antibody amino acids are numbered according to 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)).

[0077] The term "antibody fragment" refers to a molecule, other than a complete antibody, that contains a portion of a complete antibody and binds to an antigen that the complete antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies, linear antibodies, single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0078] As used herein, the term "antibody that binds to the same epitope" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. This document provides an exemplary competitive assay.

[0079] The term "class" of antibodies refers to the type of constant domains or constant regions that their heavy chain possesses. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0080] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the individual antibodies comprising that population are identical and / or bind to the same epitope, except for possible variant antibodies, such as those containing naturally occurring mutations or those arising during the production of the monoclonal antibody formulation, which are typically present in small amounts. In contrast to polyclonal antibody formulations, which generally comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used according to the invention can be prepared 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 the human immunoglobulin loci, as well as other exemplary methods for preparing the monoclonal antibodies described herein.

[0081] The term "epitope" refers to a specific site on an antigen molecule where an antibody binds.

[0082] 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-fixing proteins, or proteoglycans. In some embodiments, the PBRM comprises an engineered cysteine ​​residue. Examples of PBRMs include, but are not limited to, antibodies, peptides, lipid carrier proteins, proteins, peptides, or peptide mimics. In addition to targeting and binding to specific cells, tissues, or locations, protein-based recognition molecules may also have therapeutic effects on target cells or pathways, such as antiproliferative (cell-inhibiting and / or cytotoxic) activity. Protein-based recognition molecules comprise or can be engineered to contain at least one chemically reactive group, such as -COOH, primary amine, secondary amine -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 cell surface molecules (such as cell surface receptors or antigens) for a given target cell population. After a ligand specifically binds to or complexes with its receptor, the cell allows uptake of the ligand or ligand-drug conjugate, which is then internalized into the cell. Ligands that "specifically bind or complex" or "target" cell surface molecules, as used herein, are preferentially associated with cell surface molecules by intermolecular forces. In some embodiments, the ligand may be less than about 50 nM, less than about 5 nM, or less than 500 pM K+. d Preferably, association with cell surface molecules is preferred. Techniques for measuring the binding affinity of ligands to cell surface molecules are well known; for example, a suitable technique is called surface plasmon resonance (SPR). In some embodiments, the ligand is used for targeting, and no detectable therapeutic effect is observed when it is separated from the drug it delivers. In some embodiments, the ligand acts as both a targeting moiety and a therapeutic or immunomodulatory agent (e.g., enhancing the activity of an active drug or prodrug). As used herein, the term "PEG unit" refers to a PEG-containing unit. The PEG unit comprises polyethylene glycol subunits. In some embodiments, the PEG unit includes multiple PEG subunits.

[0083] As used herein, the term "alkyl" refers to a saturated, straight, or branched hydrocarbon group having a specified number of carbon atoms. The terms "C1-C6 alkyl" or "C..." 1-6 "Alkyl" refers to a methyl moiety or an orthogonal or branched alkyl moiety containing 2-6 carbon atoms. Exemplary alkyl moieties include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0084] As used herein, the term "halogenated (alkyl)" refers to a saturated, straight, or branched hydrocarbon group having a specified number (n) of carbon atoms and one or more (up to 2n+1) halogen atoms. 1-4Examples of "alkyl" groups include, but are not limited to, -CF3 (trifluoromethyl), -CCl3 (trichloromethyl), 1,1-difluoroethyl, 2,2,2-trifluoroethyl, and hexafluoroisopropyl.

[0085] As used herein, the term "alkenyl" refers to an orthogonal or branched hydrocarbon group having a specified number of carbon atoms and at least one and at most three carbon-carbon double bonds. Examples include vinyl and propenyl groups.

[0086] As used herein, the term "alkynyl" refers to an orthogonal or branched hydrocarbon group having a specified number of carbon atoms and at least one and at most three carbon-carbon triple bonds. Examples include ethynyl and propynyl.

[0087] As used herein, the terms "alkoxy-" or "(alkyl)oxy-" refer to an "alkyl-oxy-" group comprising an alkyl moiety having a specified number of carbon atoms attached by an oxygen linker. An exemplary "C" 1-4 alkoxy-" or "(C 1-4 Alkyl)oxy-" groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, and tert-butoxy.

[0088] As used herein, the term "halogenated (alkoxy)-" refers to a saturated, straight, or branched hydrocarbon group having a specified number (n) of carbon atoms and one or more (up to 2n+1) halogen atoms attached by an oxygen linker. An exemplary "halogenated (C... 1-4 The alkoxy group includes, but is not limited to, -OCHF2 (difluoromethoxy), -OCF3 (trifluoromethoxy), -OCH2CF3 (trifluoroethoxy) and -OCH(CF3)2 (hexafluoroisopropoxy).

[0089] As used herein, the term "amino" refers to a substituent 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 Alkyl)amino or dialkylamino, amide, carbamide, urea and sulfonamide substituents are included in the term "amino".

[0090] As used herein, the term "carbocyclic group or part" refers to a cyclic group or part in which the ring member is a carbon atom, which may be saturated, partially unsaturated (non-aromatic), or fully unsaturated (aromatic).

[0091] As used herein, the term "cycloalkyl" refers to a non-aromatic, saturated hydrocarbon cyclic group containing a specified number of carbon atoms in its ring. For example, the term "C..." 3-6"Cycloalkyl" refers to a cyclic group having 3-6 carbon atoms. An example is "C". 3-6 The "cycloalkyl" group includes cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0092] As used herein, the term "aryl" refers to an aromatic group comprising a "conjugated" or polycyclic system having one or more aromatic rings, the ring structure of which contains no heteroatoms. The term aryl includes both monovalent and divalent types. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, and naphthyl. In some embodiments, the aryl group is phenyl.

[0093] As used herein, the term "heterocyclic group or part" refers to a cyclic group or part having atoms of at least two different elements as ring members, wherein the cyclic group or part may be saturated, partially unsaturated (non-aromatic), or fully unsaturated (aromatic).

[0094] As used in this article, the term "heteroatom" refers to nitrogen, sulfur, or oxygen atoms, such as nitrogen atoms or oxygen atoms.

[0095] As used herein, the term "heterocyclic alkyl" refers to a non-aromatic monocyclic or bicyclic group comprising 3-10 ring atoms and containing one or more (usually one or two) heteroatom ring members independently selected from oxygen, sulfur, and nitrogen. The attachment point of a heterocyclic alkyl group can be through any suitable carbon or nitrogen atom.

[0096] As used herein, the term "heteroaryl" refers to an aromatic monocyclic or bicyclic group comprising 5-10 ring atoms, including 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein at least a portion of the group is aromatic. For example, the term includes bicyclic heterocyclic-aryl groups encompassing a benzene ring fused to a heterocyclic moiety or a heteroaryl ring fused to a carbocyclic moiety. The attachment point of the heteroaryl group can be any suitable carbon or nitrogen atom.

[0097] As used in this article, the terms “halogen” and “halogenated” refer to halogen groups, such as fluorine, chlorine, bromine, or iodine substituents.

[0098] As used in this article, the term "oxo" refers to the oxygen moiety of a double bond; for example, if it is attached directly to a carbon atom, it forms a carbonyl moiety (C=O).

[0099] As used herein, the terms “hydroxyl” or “hydroxyl” are intended to refer to the group -OH.

[0100] As used in this article, the term "cyano" refers to the nitrile group -C≡N.

[0101] As used herein, the term "optionally substituted" means that the group (e.g., alkyl, cycloalkyl, alkoxy, heterocycloalkyl, aryl, or heteroaryl) or ring or part may be unsubstituted, or that the group, ring, or part may be substituted by one or more substituents. Where the group may 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, alkyl carbonyloxy, aryl carbonyloxy, alkoxy carbonyloxy, aryloxy carbonyloxy, carboxylic acid ester, alkyl carbonyl, aryl carbonyl, alkoxy carbonyl, amino carbonyl, alkyl amino carbonyl, dialkyl amino carbonyl, alkyl thiocarbonyl, alkoxy, phosphate ester, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), amide (including alkyl carbonylamino, aryl carbonylamino, carbamoyl and urea), amido, imino, mercapto, alkylthio, arylthio, thiocarboxylic acid ester, sulfate ester, alkyl thionyl, sulfonic acid, aminosulfonyl, sulfonylamino, nitro, trifluoromethyl, cyano, azide, heterocyclic, alkylaryl, or aromatic or heteroaromatic moiety.

[0102] As used herein, the term “independently” means that when more than one substituent is selected from many possible substituents, those substituents may be the same or different.

[0103] As used herein, “pharmaceutically acceptable” refers to compounds, conjugates, materials, compositions, and dosage forms that, to a reasonable extent of medical judgment, are suitable for contact with tissues in humans and animals without excessive toxicity, irritation, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0104] As used herein, the term "treating" or "treatment" describes the management and care of a patient for the purpose of combating a disease, condition, or symptom, and includes administering the compounds of this disclosure or their pharmaceutically acceptable salts, polymorphs, or solvates to alleviate or eliminate symptoms or complications of the disease, condition, or symptom. The term "treatment" may also include treatment in in vitro cell or animal models.

[0105] As used in this article, the terms “preventing,” “preventing,” or “avoiding” describe reducing or eliminating the onset of symptoms or complications of a disease, condition, or ailment.

[0106] The term "subject" refers to an animal, preferably a mammal, and most preferably a human, used as a subject of treatment, observation, or experimentation.

[0107] The term “therapeutic effective amount” refers to the amount of an active compound or agent that elicits a biological or medical response in an tissue system, animal, or human being explored by researchers, veterinarians, physicians, or other clinicians, including the conjugates of this disclosure, which include the reduction or partial reduction of symptoms of the disease, syndrome, condition, or symptom being treated.

[0108] The therapeutic “effective amount” is intended to mean the amount of a conjugate, as defined herein, that is sufficient to effectively treat or prevent a patient requiring such treatment. The amount of a given conjugate corresponding to this dose will depend on factors such as the specific conjugate (e.g., the potency (pIC) of the specific conjugate). 50 ), efficacy (EC) 50 The duration of treatment and the time of administration of the conjugate (time between doses and time of dose, e.g., before / during / after a meal) should vary depending on the mammalian identity (e.g., weight), the specific conjugate and its properties (e.g., pharmacokinetic properties), the disease or condition and its severity, and the specific composition and method used, but can still be determined by those skilled in the art.

[0109] The term "composition" means a product containing a therapeutically effective amount of a specified ingredient, and any product produced directly or indirectly from a combination of specified amounts of the specified ingredient.

[0110] As used herein, the term "pharmaceuticalally acceptable excipient" means an excipient that is generally safe and non-toxic and is not biologically or otherwise undesirable for the preparation of a pharmaceutical composition, and includes excipients acceptable for veterinary and human pharmaceutical use. As used in the specification and claims, "pharmaceuticalally acceptable excipient" includes both one and more such excipients.

[0111] As used herein, the term "STING agonist" refers to a compound or portion capable of interacting with STING, for example, by binding to STING and / or inducing downregulation of signal transduction (e.g., characterized by activation of molecules associated with STING function). This includes direct phosphorylation of STING, IRF3, and / or NF-κB, and may also include STAT6. In some embodiments, STING pathway activation leads to the production of type 1 interferons (primarily IFN-α and IFN-β) and / or increased expression of interferon-stimulated genes.

[0112] As used herein, the term "STING agonist pharmaceutical portion" refers to a portion derived from a STING agonist and capable of interacting with STING. In some embodiments, the STING agonist pharmaceutical portion is a portion derived from a STING agonist such that this portion is attached to the remainder of the conjugate disclosed herein.

[0113] The conjugates disclosed herein are useful in methods for treating or improving viral infections, diseases, syndromes, conditions, or symptoms affected by the agonistic effects of STING. Such methods comprise, consist of, and / or substantially consist of administering a therapeutically effective amount of the conjugate disclosed herein, or its enantiomers, diastereomers, solvates, or pharmaceutically acceptable salts thereof, to a subject (including animals, mammals, and humans) in need of such treatment, improvement, and / or prevention.

[0114] In some embodiments, the conjugates of this disclosure or their enantiomers, diastereomers, solvates or pharmaceutically acceptable salt forms are useful for treating or improving diseases, syndromes, conditions or ailments such as melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma and hepatitis B.

[0115] As used herein, the terms “one or more disclosed conjugates” or “one or more conjugates disclosed herein” mean any form of conjugate as defined herein, namely any tautomer form, any isomer form, any salt or non-salt form (e.g., as a free acid or base, or as a salt, especially a pharmaceutically acceptable salt), and any physical form (e.g., including non-solid forms (e.g., liquid or semi-solid forms) and solid forms (e.g., amorphous or crystalline forms, specific polymorphs, solvate forms, including hydrate forms (e.g., monohydrate, dihydrate, and hemihydrate)) and mixtures of various forms.

[0116] Therefore, this disclosure includes conjugates and mixtures thereof in any salt or non-salt form and any physical form as disclosed herein. Although such conjugates are included within this disclosure, it should be understood that conjugates in any salt or non-salt form and any physical form as disclosed herein may have different levels of activity, different bioavailability, and different therapeutic properties for formulation purposes.

[0117] Unless otherwise specified, the expressions “one or more of A, B or C”, “one or more A, B or C”, “one or more of A, B and C”, “one or more A, B and C”, “selected from A, B and C”, “selected from A, B and C”, etc., used herein are used interchangeably and all refer to selected from A, B and / or C, that is, one or more A, one or more B, one or more C or any combination thereof.

[0118] It should be understood that throughout the description, when a composition is described as having, including, or comprising specific components, it is also considered that the composition is substantially composed of or consists of the listed components. Similarly, when a method or procedure is described as having, including, or comprising specific procedure steps, the procedure is also substantially composed of or consists of the listed procedure steps. Furthermore, it should be understood that the order of the steps or the sequence of certain actions is irrelevant as long as the invention remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0119] Unless otherwise specified, all percentages and ratios used herein are by weight. Other features and advantages of this disclosure will be apparent from the various examples provided. The examples provided illustrate different components and methods for practicing this disclosure. The examples do not limit the scope of the claimed disclosure. Based on this disclosure, those skilled in the art can identify and employ other components and methods that can be used to practice this disclosure.

[0120] All publications and patent documents cited herein are incorporated by reference, as if each such publication or document were specifically and individually indicated to be incorporated herein by reference. References to publications and patent documents are not intended to acknowledge any as relevant prior art, nor do they constitute an admission of their content or dates. The invention has now been described in writing, and those skilled in the art will recognize that the invention can be practiced in a variety of embodiments, and that the foregoing description and the following examples are for illustrative purposes only and not to limit the scope of the appended claims.

[0121] The conjugates and scaffolds disclosed herein

[0122] In some respects, this disclosure provides conjugates of formula (I) or pharmaceutically acceptable salts or solvates thereof:

[0123] PBRM-[A 1 -(L C ) 0或1 -D]d 15 (I)

[0124] in:

[0125] PBRM stands for Protein-Based Recognition Molecules;

[0126] When it exists, L C For connector unit;

[0127] When L C When it exists, A 1 To make PBRM and L C The connected divalent connector portion, or when L C When it does not exist, A 1 The bivalent connector portion for connecting PBRM and D;

[0128] D represents the agonist drug portion of STING; and

[0129] d 15 It is an integer in the range of approximately 1 to approximately 20.

[0130] In some embodiments, the conjugate has the following formula (IA) or a pharmaceutically acceptable salt or solvate thereof:

[0131] PBRM-[A 1 -D]d 15 (IA)

[0132] In some embodiments, the conjugate has the following formula (IB) or a pharmaceutically acceptable salt or solvate thereof:

[0133] PBRM-[A1-LC-D] d15 (IB)

[0134] In some embodiments, the conjugate has formula (I-B') or a pharmaceutically acceptable salt or solvate thereof:

[0135]

[0136] In some aspects, this disclosure provides a stent that can be conjugated with PBRM, wherein the stent has the following formula (II) or a pharmaceutically acceptable salt or solvate thereof:

[0137] A 1’ -(L C ) 0或1 -D(II)

[0138] in:

[0139] PBRM stands for Protein-Based Recognition Molecules;

[0140] When it exists, L C For connector unit;

[0141] A 1’ For a monovalent connector portion containing functional groups capable of forming covalent bonds with functional groups of PBRM; and

[0142] D represents the STING agonist drug component.

[0143] In some embodiments, the stent has the following formula (II-A) or a pharmaceutically acceptable salt or solvate thereof:

[0144] A 1’ -D(II-A)

[0145] In some embodiments, the stent has the following formula (II-B) or a pharmaceutically acceptable salt or solvate thereof:

[0146] A 1’ -L C -D(II-B)

[0147] In some embodiments, the stent has the following formula (II-B') or a pharmaceutically acceptable salt or solvate thereof:

[0148]

[0149] It should be understood that for any conjugate or pharmaceutically acceptable salt or solvate of any of formulas (I), (IA), (IB), (I-B'), (II), (II-A), (II-B), or (II-B'), the variables PBRM, L C A 1 T 1 M A L D D and d 15 Where applicable, each can be selected from the groups described in this paper, and this paper refers to the variables PBRM, L C A 1 T 1 M A L D D and d 15 Any group described in any of these terms may be used in conjunction with the variables PBRM, L, where applicable. C A 1 T 1 M A L D D and d 15 Any combination of one or more groups of descriptions in the remaining part.

[0150] variable d 15

[0151] In some implementation schemes, d 15 It is an integer in the range 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.

[0152] In some implementation schemes, d 15 It is an integer in the range of approximately 2 to approximately 8.

[0153] In some implementation schemes, d 15It is 2, 4, 6, or 8. In some implementations, d 15 It can be 6 or 8.

[0154] In some implementation schemes, d 15 The value is 8. In some implementations, d 15 It is 6.

[0155] VariableA 1 and A 1’

[0156] In some implementations, each A 1 Independently for making PBRM and L C (when L) C (when it exists) or D (when L) C (When not present) the bivalent connector portion of the connection.

[0157] In some implementations, each A 1 Independently

[0158] in:

[0159] R 7 -O-, -NR 8 -(C1-C 10 alkyl)-, -(C1-C 10 alkenyl)-, -(C1-C 10 -(C3-C8 cycloalkyl)-, -aryl-, -O-(C1-C8 alkyl)-, -O-(C1-C8 alkyl)- 10 alkenyl)-, -O-(C1-C 10 ynyl group)-, -(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 (-(C3-C8 cycloalkyl)-, -(C2-C) 10 ynyl)-aryl-,-(C3-C8 cycloalkyl)-(C1-C 10 alkyl)-, aryl-(C1-C 10 alkyl)-, -(C3-C8 cycloalkyl)-(C2-C 10 (-, -aryl)-, -(C2-C 10 alkenyl)-, -(C3-C8 cycloalkyl)-(C2-C 10 ynyl)-, -aryl-(C2-C10 -(alkynyl)-, -(3 to 8-membered heterocyclic alkyl)-, -(5 to 8-membered heteroaryl)-, -(C1-C 10 alkyl)-(3 to 8-membered heterocyclic alkyl)-,-(C1-C 10 alkyl)-(5 to 8-membered heteroaryl)-, -(C2-C 10 alkenyl)-(3 to 8-membered heterocyclic alkyl)-,-(C2-C 10 alkenyl)-(5 to 8-membered heteroaryl)-, -(C2-C 10 ynyl)-(3 to 8-membered heterocyclic alkyl)-,-(C2-C 10 ynyl)-(5 to 8-membered heteroaryl)-, -(3 to 8-membered heterocyclic alkyl)-(C1-C 10 alkyl)-, -(5 to 8-membered heteroaryl)-(C1-C 10 alkyl)-, -(3 to 8-membered heterocyclic alkyl)-(C2-C 10 alkenyl)-, -(5 to 8-membered heteroaryl)-(C2-C 10 alkenyl)-, -(5 to 8-membered heteroaryl)-(C2-C 10 ynyl)-, -(5 to 8-membered heteroaryl)-(C2-C 10 (alkynyl)-, -OC(O)-(CH2CH2O) r -(CH2)2-, -(CH2CH2O) r -or-(CH2CH2O) r -(CH2)2-, wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl or heteroaryl are optionally substituted;

[0160] R 8 H, hydroxyl or C 1-4 alkyl;

[0161] r is an integer in the range of approximately 1 to approximately 12; and

[0162] * indicates attachment to PBRM, while when L C When present, ** indicates attachment to L C , or when L C If it does not exist, ** indicates that it is attached to D.

[0163] In some implementation schemes, 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 10alkyl)-(C3-C8 cycloalkyl)-, -(C3-C8 cycloalkyl)-(C1-C 10 alkyl)-, -(3 to 8-membered heterocyclic alkyl)-, -(5 to 8-membered heteroaryl)-, -(C1-C 10 alkyl)-(3 to 8-membered heterocyclic alkyl)-,-(C1-C 10 alkyl)-(5 to 8-membered heteroaryl)-, -(3 to 8-membered heterocyclic alkyl)-(C1-C 10 alkyl)-, -(5 to 8-membered heteroaryl)-(C1-C 10 Alkyl)-, -OC(O)-(CH2CH2O) r -(CH2)2-, -(CH2CH2O) r -or-(CH2CH2O) r -(CH2)2-.

[0164] In some implementation schemes, R 7 -(C1-C 10 Alkyl)-, -O- (C1-C8 alkyl)-, -(CH2CH2O) r -、-OC(O)-(CH2CH2O) r -(CH2)2- or -(CH2CH2O) r -(CH2)2-.

[0165] In some implementation schemes, R 7 It can be -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-.

[0166] In some implementations, each A 1 Independently:

[0167] Where R 8 H, hydroxyl or C 1-4 Alkyl; r is an integer in the range of about 4 to about 6; and * indicates attachment to PBRM, while L C When present, ** indicates attachment to L C , or when L C If it does not exist, ** indicates that it is attached to D.

[0168] It should be understood that each A 1It corresponds independently to the monovalent part A before being connected to the PBRM. 1’ .

[0169] In some implementations, each A 1’ Independently:

[0170] Where R 7 R 8 And r as described in this article; while when L C When present, ** indicates attachment to L C , or when L C If it does not exist, ** indicates that it is attached to D.

[0171] In some implementations, each A 1’ Independently:

[0172] in:

[0173] r is an integer in the range of approximately 4 to approximately 6; and

[0174] When L C When present, ** indicates attachment to L C , or when L C If it does not exist, ** indicates that it is attached to D.

[0175] variable L C

[0176] In some implementations, when present, each L C Independently:

[0177]

[0178] in:

[0179] # indicates attachment to A 1 And ## indicates attachment to D;

[0180] When it exists, M A A peptide moiety containing at least two amino acids;

[0181] When it exists, T 1 It is a hydrophilic group; and

[0182] When M A When it exists, L D To make D and M A The connected divalent connector portion, or when M A When it does not exist, L D To make D and A1 The connecting bivalent connector portion.

[0183] In some implementations, each L D It contains at least one cleavable bond such that when the bond breaks, D is released in its active form for its intended therapeutic effect.

[0184] In some implementations, when present, each L C Independently

[0185] In some implementations, when present, each L C Independently

[0186]

[0187] variable L D

[0188] In some implementations, each L D Independently for D and M A (when M) A (when it exists) or A 1 (when M) A (When not present) the bivalent connector portion of the connection.

[0189] In some implementations, each L D It contains at least one cleavable bond such that when the bond is cleaved, D is released in its active form for its intended therapeutic effect.

[0190] In some implementations, L D It contains a cleavable bond. In some implementations, L D It contains multiple cleavage sites or bonds.

[0191] It should be understood that each L D It corresponds independently to the monovalent part L before being connected to D. D’ .

[0192] In some implementations, L D’ It contains functional groups capable of forming cleavable bonds. Functional groups capable of forming cleavable bonds may include, for example, a thiol group forming a disulfide bond, an aldehyde, ketone, or hydrazine group forming an hydrazone bond, a hydroxylamine group forming an oxime bond, a carboxyl or amino group forming a peptide bond, a carboxylic acid or hydroxyl group forming an ester bond, and a sugar forming a glycosidic bond.

[0193] In some implementations, each L D It contains a disulfide bond that can be cleaved by disulfide bond exchange, an acid-labile bond that can be cleaved at acidic pH, and / or a bond that can be cleaved by hydrolases. In some embodiments, L DIt contains a carbamate bond (i.e., -OC(O)-NR-, where R is hydrogen or alkyl, etc.).

[0194] In some implementations, L D The structure and sequence of the cleavable bond allow it to be cleaved by an enzyme present at the target site. In some embodiments, the cleavable bond may be cleaved via other mechanisms.

[0195] In some implementations, L D The structure and sequence of cleavable bonds allow these bonds to be cleaved by enzymes present at the target site. In some embodiments, cleavable bonds may be cleaved via other mechanisms.

[0196] In some embodiments, the cleavable bonds may be enzymatically cleaved by one or more enzymes (including tumor-associated proteases) to release the drug unit or D, wherein the conjugate or intermediate of this disclosure or its scaffold is protonated in vivo after release to provide the drug unit or D.

[0197] In some implementations, each L D Independently in:

[0198] When it exists, L E It is -NH-[(CH2CH2O) p -(CH2) 0-2 ] q -C(O)-, -NH-(C1-C6 alkyl)-OC(O)-, or -NH-[(CH2CH2O) p -(CH2) 0-2 ] q -C(O)-NH-(C1-C6 alkyl)-OC(O)-, where p is an integer in the range of about 1 to about 20, and q is an integer in the range of about 1 to about 10;

[0199] Each W is an independent natural or non-natural amino acid unit;

[0200] w is an integer in the range of approximately 0 to approximately 12;

[0201] When M A When present, *** indicates attachment to M. A , or when M A When it does not exist, ** indicates that it is attached to A. 1 ;and

[0202] **** indicates attachment to D.

[0203] In some implementations, each L D Independently In some implementations, each LD Independently In some implementations, each L D Independently

[0204] In some implementations, L E It contains at least one PEG unit.

[0205] In some embodiments, the PEG unit comprises 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 comprises at least four subunits, at least three subunits, at least two subunits, or at least one subunit. In some embodiments, the PEG unit comprises at least one subunit. In some embodiments, the PEG unit comprises at least two subunits.

[0206] In some implementations, p is an integer in the range of about 1 to about 15, about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, or about 1 to about 5.

[0207] In some implementations, p is an integer in the range of approximately 1 to approximately 6. In some implementations, p is an integer in the range of approximately 1 to approximately 4. In some implementations, p is an integer in the range of approximately 1 to approximately 2.

[0208] In some implementations, p is 2.

[0209] In some implementations, q is an integer in the range of about 1 to about 15, about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, or about 1 to about 5.

[0210] In some implementations, q is 1, 2, 3, 4, or 5. In some implementations, q is 2.

[0211] In some implementations, when present, L E for

[0212] -NH-(CH2CH2O) 1-4 -(CH2)2-C(O)-. In some implementations, L, when present... E It is -NH-(CH2CH2O)2-(CH2)2-C(O)-. In some embodiments, L, when present, E It is -NH-(CH2CH2O)3-(CH2) 0-2 -C(O)-. In some implementations, L, when present, EIt is -NH-(CH2CH2O)3-(CH2)1-C(O)-. In some embodiments, when present, L E It is -NH-(CH2CH2O)3-(CH2)2-C(O)-. In some embodiments, L, when present, E It is -NH-CH2CH2O-(CH2) 0-2 -C(O)-. In some implementations, L, when present, E It is -NH-CH2CH2O-C(O)-. In some embodiments, when present, L E It is -NH-(C1-C6 alkyl)-OC(O)-. In some embodiments, L, when present, E It is -NH-CH2-CH(CH3)-OC(O)-. In some embodiments, when present, L E It is -NH-[(CH2CH2O) 1-4 -(CH2)2-C(O)-NH-(C1-C6 alkyl)-OC(O)-. In some embodiments, L, when present... E for

[0213] -NH-CH2CH2O-(CH2)2-C(O)-NH-(CH2)2-OC(O)-.

[0214] In some implementations, w is an integer in the range of about 1 to about 12 (e.g., 1-6, or 1-4, or 1-3, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

[0215] In some implementations, w is 0, 1, 2, 3, 4, or 5.

[0216] In some implementations, w is 1. In some implementations, w is 2. In some implementations, w is 3.

[0217] In some implementations, each W is independently a natural or non-natural amino acid and / or D or L is an isomer.

[0218] In some implementations, each W is independently a natural or non-natural α, β, or γ amino acid.

[0219] 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.

[0220] In some implementation schemes, W w It does not contain natural amino acids. In some implementations, W w It does not contain non-natural amino acids.

[0221] In some implementation schemes, W w It contains natural amino acids linked to non-natural amino acids. In some embodiments, W w Natural amino acids containing D-isomers linked to natural amino acids.

[0222] In some implementation schemes, W w It is a dipeptide, such as -Val-Cit-, -Phe-Lys-, -Val-Ala-, or Glu-Ala.

[0223] In some implementation schemes, W w It can be a single peptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit.

[0224] In some implementation schemes, W w The peptide is a peptide (e.g., a peptide of 1-12 amino acids) that is directly conjugated to D. In some embodiments, the peptide is a single amino acid. In some embodiments, the peptide is a dipeptide. In some embodiments, the peptide is a tripeptide.

[0225] In some implementation schemes, 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, aminoalkyl acid, aminoalkynic acid, aminoalkyl diacid, aminobenzoic acid, amino-heterocyclic alkyl acid, heterocyclic carboxylic acid, citrulline, pepsinic acid, diaminoalkyl acid and their derivatives.

[0226] In some implementation schemes, 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.

[0227] In some implementation schemes, W w Each amino acid in the formula is independently selected from protein and non-protein amino acids.

[0228] In some implementation schemes, W wEach amino acid in the formula is independently selected from the L or D isomers of 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, aminoalkyl diacid, heterocyclic carboxylic acid, citrulline, pepsinic acid, diaminoalkyl acid, valine, citrulline and its derivatives.

[0229] In some implementation schemes, W w Each amino acid in the formula 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.

[0230] In some implementation schemes, W w Each amino acid in the formula is independently selected from the L-isomers of 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 valine.

[0231] In some implementation schemes, W w Each amino acid in the formula is independently selected from the D-isomers of 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 valine.

[0232] In some implementation schemes, W w Each amino acid in it is alanine, β-alanine, glycine, glutamic acid, isoglutamic acid, isoaspartic acid, valine, citrulline, or aspartic acid.

[0233] In some implementation schemes, W w Contains β-alanine. In some embodiments, W contains (β-alanine)-(alanine). In some embodiments, W w It contains (β-alanine) and optionally glutamic acid, isoglutamic acid, aspartic acid, isoaspartic acid, valine, (valine)-(alanine), (alanine)-(alanine) or (valine)-(citrulline).

[0234] In some implementation schemes, W w It contains (glutamic acid)-(alanine).

[0235] In some implementation schemes, W w It contains glutamic acid and optionally alanine, glycine, isoglutamic acid, aspartic acid, isoaspartic acid, valine, (valine)-(alanine), (alanine)-(alanine) or (valine)-(citrulline).

[0236] In some implementation schemes, W w It contains 2,3-diaminopropionic acid. In some embodiments, W w It contains (R)-2,3-diaminopropionic acid. In some embodiments, W w It contains glutamic acid. In some implementations, W w Contains (glutamate)-(alanine). In some implementations, W w It contains (glutamic acid)-(glycine)-(alanine).

[0237] In some implementation schemes, W w It 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).

[0238] In some implementations, W, in addition to one or more amino acids w It also contains carbamate bonds.

[0239] In some implementations, L D (e.g. W) w It exhibits selectivity for enzymatic cleavage (e.g., by a specific enzyme). In some embodiments, the specific enzyme is a tumor-associated protease.

[0240] In some implementations, L D (e.g. W) w It contains a bond whose cleavage is catalyzed by cathepsin B, cathepsin C, cathepsin D, or plasminase.

[0241] In some implementations, L D It contains sugar cleavage sites.

[0242] In some implementations, L D It contains a sugar moiety (Su) that is linked to a self-degrading group via an oxyglycosidic bond.

[0243] In some embodiments, the "self-degrading group" may be a trifunctional chemical moiety capable of integrating three spaced chemical moieties (i.e., the sugar moiety (via glycosidic bond), the pharmaceutical unit (directly or indirectly), and M) A (when M) A When it exists (directly or indirectly) or A 1 (when M) A (When not present) they are covalently linked together.

[0244] In some implementations, the glycosidic bond can be cleaved at the target site to initiate a self-degradation reaction sequence, thereby leading to drug release.

[0245] In some implementations, when present, each L D Independently:

[0246]

[0247]

[0248]

[0249] Where: when M A When present, *** indicates attachment to M. A , or when M A When it does not exist, *** indicates that it is attached to A. 1 ; and **** indicate attachment to D.

[0250] In some implementations, when present, each L D Independently:

[0251]

[0252]

[0253] Where: when M A When present, *** indicates attachment to M. A , or when M A When it does not exist, *** indicates that it is attached to A. 1 ; and **** indicate attachment to D.

[0254] In some implementations, when present, each L D Independently:

[0255]

[0256] Where: *** indicates attachment to M A And **** indicates attachment to D.

[0257] Variable M A

[0258] In some implementation schemes, M A A peptide moiety containing at least two amino acids.

[0259] In some implementations, amino acids are referred to herein as "AA" and amino acid classes as "AA's".

[0260] In some implementation schemes, M A In order to be able to work with -L D -D units form covalent bonds and enable the attachment of various drugs.

[0261] In some implementation schemes, M A It contains a single AA unit or has two or more AA units (e.g., 2-10, 2-6, or 2, 3, 4, 5, or 6), wherein each AA unit is independently a natural or non-natural amino acid, amino alcohol, amino aldehyde, diamine, polyamine, or a combination thereof.

[0262] In some implementations, to achieve the required amount of attachment, at least one AA unit will have a functionalized side chain to provide -L D -D unit attachment. In some embodiments, exemplary functionalized AA units (e.g., amino acids, amino alcohols, or amino aldehydes) include, for example, azido or alkyne functionalized AA units (e.g., amino acids, amino alcohols, or amino aldehydes modified to have azido or alkyne groups).

[0263] In some implementation schemes, M A It contains 2-12 AA units. In some implementations, M A It contains 2-10 AA units. In some implementations, M A It contains 2-6 AA units. In some implementations, M A It contains 2, 3, 4, 5 or 6 AA units.

[0264] In some implementation schemes, M A The peptide moiety has 2 AA units. In some embodiments, the peptide moiety has 3 AA units. In some embodiments, the peptide moiety has 4 AA units. In some embodiments, the peptide moiety has 5 AA units. In some embodiments, the peptide moiety has 6 AA units.

[0265] In some implementation schemes, M A The attachment of components within or to conjugates, intermediates, or other components of the scaffold may be via, for example, amino, carboxyl, or other functional groups.

[0266] In some implementation schemes, M AEach amino acid in the formula may independently be a D or L isomer of a thiol-containing amino acid. In some embodiments, M A Each amino acid in the formula can independently be a D isomer of an amino acid containing a thiol. In some embodiments, M... A Each amino acid in the formula may 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.

[0267] In some implementation schemes, M A Each amino acid in the formula may independently be an L or D isomer 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, aminoalkyl diacid, heterocyclic carboxylic acid, citrulline, pepsinic acid, diaminoalkyl acid, their stereoisomers or derivatives thereof.

[0268] In some implementation schemes, M A Each amino acid in the formula 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 a stereoisomer thereof.

[0269] In some implementation schemes, M A It includes monopeptides, dipeptides, tripeptides, tetrapeptides, or pentapeptides. In some implementations, M A It contains pentapeptides.

[0270] In some implementation schemes, M A It includes monopeptides, dipeptides, tripeptides, tetrapeptides, or pentapeptides. In some implementations, M A It contains pentapeptides.

[0271] In some implementation schemes, M A It contains at least about 5 amino acids (e.g., 5, 6, 7, 8, 9, or 10 amino acids). In some embodiments, M A It contains up to about 10 amino acids.

[0272] In some implementation schemes, M A Each amino acid in it is independently glycine, serine, glutamic acid, lysine, aspartic acid, and cysteine.

[0273] In some implementation schemes, M AIt contains at least 4 glycines and at least 1 glutamic acid, such as (glycine)4 and glutamic acid, wherein the glutamic acid is located at any position along the peptide chain, such as (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).

[0274] In some implementation schemes, M A It contains (glycine)4-(glutamate). In some embodiments, the peptide moiety contains (glutamate)-(glycine)4.

[0275] In some implementation schemes, M A It contains at least 4 glycines and at least 1 serine, such as (glycine)4 and serine, wherein the serine is located at any position along the peptide chain, such as (serine)-(glycine)4, (glycine)-(serine)-(glycine)3, (glycine)2-(serine)-(glycine)2, (glycine)3-(serine)-(glycine) or (glycine)4-(serine).

[0276] In some implementation schemes, M A It contains (glycine)4-(serine). In some embodiments, the peptide moiety contains (serine)-(glycine)4.

[0277] In some implementation schemes, M A It contains (β-alanine)-(glycine)4-(serine), where serine is located at any position along the peptide chain, such as (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).

[0278] In some implementation schemes, M AIt contains (glycine)4-(serine)-(glutamate), where serine is located at any position along the peptide chain, such as (serine)-(glycine)4-(glutamate), (glycine)-(serine)-(glycine)3-(glutamate), (glycine)2-(serine)-(glycine)2-(glutamate), (glycine)3-(serine)-(glycine)-(glutamate) or (glycine)4-(serine)-(glutamate). In some embodiments, the peptide moiety comprises (β-alanine)-(glycine)4-(serine)-(glutamate), wherein the serine is located at any position along the peptide chain, such as (β-alanine)-(serine)-(glycine)4-(glutamate), (β-alanine)-(glycine)-(serine)-(glycine)3-(glutamate), (β-alanine)-(glycine)2-(serine)-(glycine)2-(glutamate), (β-alanine)-(glycine)3-(serine)-(glycine)-(glutamate), or (β-alanine)-(glycine)4-(serine)-(glutamate).

[0279] In some implementation schemes, M A It contains (glycine)4-(serine). In some embodiments, the peptide moiety contains (serine)-(glycine)4.

[0280] In some implementation schemes, M A It contains (β-alanine)-(glycine)4-(serine), where serine is located anywhere along the peptide chain.

[0281] In some implementation schemes, M A It contains (glycine)4-(serine)-(glutamate), where serine is located anywhere along the peptide chain.

[0282] In some implementation schemes, M A It contains (β-alanine)-(glycine)4-(serine)-(glutamate), where serine is located anywhere along the peptide chain.

[0283] In some implementation schemes, M A Contains (glutamic acid)-(glycine) 1-4 , where: M A A glutamate bonded to A 1 M A Glycine attaches to T 1 ; and M A Glutamic acid attaches to L D .

[0284] In some implementation schemes, M A Include

[0285] In some implementation schemes, M A It contains (glutamic acid)-(glycine)4, where: M A Glutamic acid attaches to A 1 M A Glycine attaches to T 1 ; and M A Glutamic acid attaches to L D .

[0286] In some implementation schemes, M A Include

[0287] In some implementation schemes, M A It contains (glutamic acid)-(glycine), where: M A Glutamic acid attaches to A 1 M A Glycine attaches to T 1 ; and M A Glutamic acid attaches to L D .

[0288] In some implementations, the peptide portion comprises

[0289] In some implementation schemes, M A Contains (glycine) 1-4 -(glutamate), where M A A via glycine attachment to A 1 M A Glutamic acid attaches to T 1 ; and M A Glutamic acid attaches to L D .

[0290] In some implementation schemes, M A Include

[0291] In some implementation schemes, M A Contains (glycine) 4-(glutamate), where: M A Glutamic acid attaches to A 1 M A Glycine attaches to T 1 ; and M A Glutamic acid attaches to L D .

[0292] In some implementation schemes, M A Include

[0293] In some implementation schemes, M AContains (glycine)-(glutamic acid), where: M A Glycine attaches to A 1 M A Glutamic acid attaches to T 1 ; and M A Glutamic acid attaches to L D .

[0294] In some implementation schemes, M A Include

[0295] In some implementation schemes, M A Contains (glycine) 1-4 -(serine), where: M A A via glycine attachment to A 1 M A Serine attaches to T 1 ; and M A Serine attaches to L D .

[0296] In some implementation schemes, M A Include

[0297] In some implementation schemes, M A Contains (glycine)-(serine), where: M A Glycine attaches to A 1 M A Serine attaches to T 1 ; and M A Serine attaches to L D .

[0298] In some implementation schemes, M A Include

[0299] In some implementation schemes, M A Contains (glycine) 4-(serine), where: M A A via glycine attachment to A 1 M A Serine attaches to T 1 ; and M A Serine attaches to L D .

[0300] In some implementation schemes, M A Include

[0301] In some implementation schemes, M A Contains (serine)-(glycine) 1-4, where: M A Serine attaches to A 1 M A Glycine attaches to T 1 ; and M A Serine attaches to L D .

[0302] In some implementation schemes, M A Include

[0303] In some implementation schemes, M A Contains (serine)-(glycine)4, where: M A Serine attaches to A 1 M A Glycine attaches to T 1 ; and M A Serine attaches to L D .

[0304] In some implementation schemes, M A Include

[0305] In some implementation schemes, M A Contains (serine)-(glycine), where: M A Serine attaches to A 1 M A Glycine attaches to T 1 ; and M A Serine attaches to L D .

[0306] In some implementation schemes, M A Include

[0307] In some implementation schemes, M A Contains (β-alanine)-(glycine) 1-4 -(serine), where: M A β-alanine attaches to A 1 M A Serine attaches to T 1 ; and M A Serine attaches to L D .

[0308] In some implementation schemes, M A Include

[0309] In some implementation schemes, M A It contains (β-alanine)-(glycine)4-(serine), where: MA β-alanine attaches to A 1 M A Serine attaches to T 1 ; and M A Serine attaches to L D .

[0310] In some implementation schemes, M A Include

[0311] In some embodiments, the peptide moiety comprises (β-alanine)-(glycine)-(glutamate), wherein: the peptide moiety is attached to L 3 (when present) or via β-alanine attachment to L M (when L) 3 (When not present); the peptide portion attaches to T via glutamate. 1 (When present); and the peptide moiety is attached to L via glutamate. D (When it exists).

[0312] In some implementations, the peptide portion comprises

[0313] It should be understood that, for M A The implementation scheme, * indicates attachment to A 1 ** indicates that it is attached to T 1 , and *** indicate attachment to L D .

[0314] Hydrophilic group (variable T) 1 )

[0315] In some embodiments, the hydrophilic groups included in the conjugates or scaffolds of this disclosure are water-soluble and substantially non-antigenic polymers. Examples of hydrophilic groups include, but are not limited to, polyols, polyethers, polyanionic polymers, polycationic polymers, polyphosphoric acids, polyamines, polysaccharides, polyhydroxy compounds, polylysine, and their derivatives. In some embodiments, one end of the hydrophilic group is functionalized such that it is covalently attached to M via an indestructible bond or via a destructible bond. A Connector (e.g., attached to M) A(Amino acids in the linker). In some embodiments, functionalization may be, for example, amine, thiol, NHS ester, maleimide, alkyne, azide, carbonyl, or other functional groups. In some embodiments, the other end (or more ends) of the hydrophilic group will be free and unbound. In some embodiments, "unbound" means that the hydrophilic group will not be attached to another portion, such as the D or pharmaceutical unit, or other components of the conjugate or scaffold disclosed herein. In some embodiments, the free and unbound end of the hydrophilic group may include a methoxy, carboxylic acid, alcohol, or other suitable functional group. In some embodiments, a methoxy, carboxylic acid, alcohol, or other suitable functional group acts as a cap for one or more ends of the hydrophilic group.

[0316] In some embodiments, a cleavable bond is a bond that is substantially insensitive to cleavage during circulation in plasma but sensitive to cleavage in the intracellular or intratumoral environment. In some embodiments, an insoluble bond is a bond that is substantially insensitive 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 bonds. In some embodiments, exemplary attachment of the hydrophilic group is via an amide bond, ether bond, ester bond, hydrazone bond, oxime bond, disulfide bond, peptide bond, or triazole bond. In some embodiments, the hydrophilic group is attached to M... A Connector (e.g. with M) A The attachment of amino acids in the connector is via amide bonds.

[0317] In some embodiments, the conjugate or scaffold of this disclosure comprises more than one hydrophilic group, which may be the same or different chemical moieties (e.g., hydrophilic groups with different molecular weights, numbers of subunits, or chemical structures). In some embodiments, the multiple hydrophilic groups may attach to M at a single attachment site or at different sites. A Connector.

[0318] In some embodiments, the addition of a hydrophilic group may have two potential effects on the pharmacokinetics of the resulting conjugate. In some embodiments, the desired effect is a reduction in clearance (and consequently, an increase in exposure) resulting from reduced nonspecific interactions induced by the drug or drug-connector exposed hydrophobic elements. In some embodiments, the undesirable effect is a reduction in distribution volume and rate, possibly due to an increase in the conjugate's molecular weight. In some embodiments, increasing the molecular weight of the hydrophilic group increases the hydrodynamic radius of the conjugate, leading to a decrease in diffusion rate, which may reduce the conjugate's ability to penetrate into the tumor. Due to these two competing pharmacokinetic effects, it may be desirable to use a sufficiently large hydrophilic group to reduce conjugate clearance, thereby increasing plasma exposure, but not so large as to significantly reduce its diffusion rate, which could reduce the conjugate's ability to reach the intended target cell population.

[0319] In some embodiments, the hydrophilic group includes, but is not limited to, sugar alcohols (also known as polyalcohols, polyhydric alcohols, aldehydes, or glycitol, such as inositol, glycerol, erythritol, threitol, aritol, xylitol, ribitol, galactitol, mannitol, sorbitol, etc.) or their derivatives (e.g., amino polyols), carbohydrates (e.g., sugars), polyvinyl alcohol, carbohydrate-based polymers (e.g., dextran), hydroxypropyl methacrylamide (HPMA), polyepoxides, and / or copolymers thereof.

[0320] In some implementations, T 1 It contains multiple hydroxyl ("-OH") groups, such as those containing monosaccharides, oligosaccharides, polysaccharides, etc.

[0321] In some implementations, T 1 Contains multiple -(CR) 58 OH)- group, wherein R 58 -H or C 1-8 alkyl.

[0322] In some implementations, T 1 -OH or in:

[0323] n1 is an integer from 0 to approximately 6;

[0324] Each R 58 Independently -H or C 1-8 alkyl;

[0325] R 60 For key, C 1-6 Alkyl connector or -CHR 59 -, where R 59 -H, C 1-8 Alkyl, cycloalkyl, or arylalkyl;

[0326] R 61 CH2OR 62 COOR 62 -(CH2) n2 COOR 62 Or a heterocyclic alkyl group substituted with one or more hydroxyl groups;

[0327] R 62 -H or C 1-8 Alkyl; and

[0328] n2 is an integer from 1 to approximately 5.

[0329] In some implementations, T1 For -OH. In some implementations, T 1 for

[0330]

[0331] In some implementation schemes, R 58 -H;R 60 For key or C 1-6 Alkyl connector; n1 is an integer from 1 to approximately 6; and R 61 (CH2OH or COOH).

[0332] In some implementation schemes, R 58 -H;R 60 For -CHR 59 -; n1 is 0; and R 61 A heterocyclic alkyl group substituted with one or more hydroxyl groups, such as a monosaccharide.

[0333] In some implementations, T1 contains glucosamine, diamine, or triamine.

[0334] In some implementations, T1 comprises one or more of the following segments or stereoisomers thereof:

[0335]

[0336]

[0337] Where R 59 -H, C 1-8 Alkyl, cycloalkyl, or arylalkyl; n1 is an integer from 1 to about 6; n2 is an integer from 1 to about 5; and n3 is an integer from about 1 to about 3.

[0338] It should be understood that this article considers all stereochemical forms of the hydrophilic group. For example, in the above formula, the hydrophilic group can be derived from ribose, xylose, glucose, mannose, galactose or other sugars, and retain the stereochemical arrangement of the side hydroxyl groups and alkyl groups present on these molecules.

[0339] It should be understood that various deoxygenated compounds are also considered in the above formulas. Illustratively, where applicable, one or more of the following characteristics are considered for hydrophilic groups.

[0340] In some implementations, n3 is 2 or 3.

[0341] In some implementations, n1 is 1, 2, or 3.

[0342] In some implementations, n2 is 1.

[0343] In some implementation schemes, R 59 It is hydrogen.

[0344] In some implementations, T 1 for In some implementations, T 1 for In some implementations, T 1 for

[0345] In some implementations, T 1 for Where n4 is an integer from 1 to approximately 25;

[0346] Each R 63 Independently -H or C 1-8 alkyl;

[0347] R 64 For key or C 1-8 Alkyl connector;

[0348] R 65 -H, C 1-8 Alkyl group, -(CH2) n2 COOR 62 Or -(CH2) n2 COR 66 ;

[0349] R 62 For H or C 1-8 alkyl;

[0350] R 66 For H, and

[0351] n2 is an integer from 1 to approximately 5.

[0352] In some implementations, T 1 for

[0353] Where R 67 It is: (1)-OH;

[0354]

[0355] Where n4 is an integer from about 2 to about 20, from about 4 to about 16, from about 6 to about 12, and from about 8 to about 12.

[0356] In some implementations, T 1 for

[0357] In some implementations, n4 is an integer from about 2 to about 20, about 4 to about 16, about 6 to about 12, or about 8 to about 12.

[0358] In some implementations, n4 is 6, 7, 8, 9, 10, 11, or 12.

[0359] In some implementations, n4 is 8 or 12.

[0360] In some implementations, T 1 for Where n4 is an integer from about 2 to about 24, about 4 to about 16, about 6 to about 12, or about 8 to about 12.

[0361] In some implementations, n4 is 6, 7, 8, 9, 10, 11, or 12.

[0362] In some implementations, n4 is 8 or 12. In some implementations, n4 is 8.

[0363] In some implementations, T 1 for

[0364] Where n4 is 8.

[0365] In some implementations, T 1 It includes polyethers, such as polyalkylene glycols (PAOs). PAOs include, but are not limited to, polymers of lower epoxides, particularly polymers of ethylene oxide, such as propylene oxide, polypropylene glycol, polyethylene glycol (PEG), polyoxyethylene polyols, their copolymers and block copolymers.

[0366] In some embodiments, the polyalkylene glycol is polyethylene glycol (PEG), including but not limited to polydisperse PEG, monodisperse PEG, and discrete PEG. Polydisperse PEG is a non-homogeneous mixture of sizes and molecular weights, while monodisperse PEG is generally purified from a non-homogeneous mixture and thus provides a single chain length and molecular weight. In some embodiments, the PEG unit is discrete PEG, providing a single molecule with a defined and specified chain length. In some embodiments, the polyethylene glycol is mPEG.

[0367] In some implementations, T 1A PEG unit comprises one or more PEG chains. The PEG chains may be linked together, for example, in a linear, branched, or star configuration. In addition to containing repeating PEG subunits, the PEG unit may also contain non-PEG material (e.g., to promote coupling of 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 -CH2CHO- 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).

[0368] PEG units can react with M via reactive groups. A Connector (e.g. with M) A The amino acids in the linker are covalently bonded. The reactive groups are those that the activated PEG molecule can bind to (e.g., free amino or carboxyl groups). In some embodiments, the N-terminal amino acid and lysine (K) have free amino groups; and the C-terminal amino acid residues have free carboxyl groups. Thiol groups (e.g., those present on cysteine ​​residues) can also be used as reactive groups for attaching PEG.

[0369] In some embodiments, the PEG unit can be attached to M by using methoxylated PEG (“mPEG”) having different reactive moieties including, but not limited to, the following. A Connector (e.g., M) AAmino acids in the linker): succinimide succinate (SS), succinimide carbonate (SC), mPEG-imino ester, p-nitrophenyl carbonate (NPC), succinimide propionate (SPA) and cyanuric chloride. Examples of mPEG include, but are not limited to, mPEG-succinimide succinate (mPEG-SS), mPEG2-succinimide succinate (mPEG2-SS), mPEG-succinimide carbonate (mPEG-SC), mPEG2-succinimide carbonate (mPEG2-SC), mPEG-imino ester, mPEG-p-nitrophenyl carbonate (mPEG-NPC), mPEG-imino ester, mPEG2-p-nitrophenyl carbonate (mPEG2-NPC), mPEG-succinimide propionate (mPEG-SPA), mPEG2-succinimide propionate (mPEG2-SPA), mPEG-N-hydroxy-succinimide (mPEG-NHS), mPEG2-N-hydroxy-succinimide (mPEG2-NHS), mPEG-cyanuric chloride, mPEG2-cyanuric chloride, mPEG2-lysine alcohol-NPC, and mPEG2-Lys-NHS. A wide variety of PEG materials can be used, and virtually any suitable reactive PEG reagent can be used. In some embodiments, the reactive PEG reagent will result in adhesion to multifunctional connectors or M A Connector (e.g., M) A The amino acids in the linker then form urethane or amide bonds. Reactive PEG reagents include, but are not limited to, mPEG2-N-hydroxy-succinimide (mPEG2-NHS), bifunctional PEG-propionaldehyde (mPEG2-ALD), multi-arm PEG, maleimide-containing PEGs (mPEG(MAL)2, mPEG2(MAL)), mPEG-NH2, mPEG-succinimide propionate (mPEG-SPA), mPEG-succinimide butyrate (mPEG-SBA), mPEG-thioester, mPEG-diester, mPEG-BTC, mPEG-ButyrALD, mPEG-acetaldehyde diethyl acetal (mPEG-ACET), heterofunctional PEGs (e.g., NH2-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-vinyl sulfone (NHS-PEG-VS) or NHS-PEG-MAL), PEG acrylates (ACRL-PEG-NHS), PEG-phospholipids (e.g., mPEG-DSPE), and SUNBRITE. TMThe series of multi-arm PEGs (including glycerol-based PEGs activated by chemicals selected by those skilled in the art), any SUNBRITE-activated PEGs (including but not limited to carboxyl-PEG, p-NP-PEG, trifluoroethanesulfonyl chloride-PEG, aldehyde PEG, acetal-PEG, amino-PEG, thiol-PEG, maleimide-PEG, hydroxy-PEG-amine, amino-PEG-COOK hydroxy-PEG-aldehyde, carboxylic anhydride-type PEG, functionalized PEG-phospholipids and other similar and / or suitable reactive PEGs).

[0370] In some embodiments, the PEG unit comprises 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, the PEG unit comprises no more than about 72 subunits.

[0371] In some implementations, the PEG unit comprises 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.

[0372] In some implementations, the PEG unit comprises 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.

[0373] In some implementations, the PEG unit comprises 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.

[0374] In some implementations, the PEG unit comprises at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, or at least 12 subunits.

[0375] In some implementations, the PEG unit comprises at least 6 subunits, at least 7 subunits, or at least 8 subunits.

[0376] In some implementations, the linear PEG unit is:

[0377]

[0378] in:

[0379] Indicates with M A Connector (e.g. with M) A The attachment sites of amino acids in the connector;

[0380] Y 71 For PEG attachment units;

[0381] Y 72 Capping units for PEG;

[0382] Y 73 For PEG coupling units (i.e., used to couple multiple PEG subunit chains together);

[0383] d9 is an integer between 2 and 72;

[0384] Each d 10 Independently, integers from 1 to 72; and

[0385] d 11 It is an integer between 2 and 5.

[0386] In some implementations, d9 is an integer from 2 to 24. In some implementations, d9 is an integer from 4 to 24. In some implementations, d9 is an integer from 6 to 24, 8 to 24, 10 to 24, or 12 to 24.

[0387] In some embodiments, at least six PEG subunits are present in the PEG unit. In some embodiments, at least eight PEG subunits are present in the PEG unit. In some embodiments, at least ten PEG subunits are present in the PEG unit. In some embodiments, at least twelve PEG subunits are present in the PEG unit.

[0388] In some implementations, d9 is 8 or about 8, 12 or about 12, 24 or about 24.

[0389] In some implementation schemes, each Y 72 Independently for -C 1-10 Alkyl, -C 2-10 Alkyl -CO2H, -C 2-10 Alkyl -OH, -C 2-10 Alkyl-NH2, -C2-10 Alkyl-NH(C) 1-3 alkyl) or C 2-10 Alkyl-N(C) 1-3 Alkyl)2.

[0390] In some implementation schemes, Y 72 -C 1-10 Alkyl, -C 2-10 Alkyl -CO2H, -C 2-10 Alkyl -OH or -C 2-10 Alkyl-NH2.

[0391] In some embodiments, the PEG coupling unit is part of a PEG unit and is a non-PEG material used to connect two or more repeating CH2CHO- subunit chains. In some embodiments, the PEG coupling unit Y 73 -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 Alkyl-S-.

[0392] In some implementation schemes, each Y 73 Independently for -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 Alkyl-NH-.

[0393] In some implementations, the PEG attachment unit is part of the PEG unit and is used to attach the PEG unit to the M A Connector (e.g., M) A(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 used to attach the PEG unit to the amino acid include a thiol group forming a disulfide or thioether bond, an aldehyde, ketone, or hydrazine group forming an hydrazone bond, a hydroxylamine group forming an oxime bond, a carboxyl or amino group forming a peptide bond, a carboxyl or hydroxyl group forming an ester bond, a sulfonic acid group forming a sulfonamide bond, an alcohol group forming a carbamate bond, and an amine group forming a sulfonamide, carbamate, or amide bond. In some embodiments, the PEG unit may be attached to the amino acid, for example, via a disulfide, thioether, hydrazone, oxime, peptide, ester, sulfonamide, carbamate, or amide bond. In some embodiments, the reaction used to attach the PEG unit may be a cycloaddition, addition, addition / elimination, or substitution reaction, or a combination thereof (where applicable).

[0394] Examples of linear PEG units include:

[0395]

[0396] in Indicates a multifunctional connector or M A Connector (e.g. with M) A The attachment sites of amino acids in the linker, and each d9 is an integer of 4-24, 6-24, 8-24, 10-24, 12-24, 14-24 or 16-24 independently.

[0397] In some implementations, d9 is about 8, about 12, or about 24. In some implementations, d9 is about 8.

[0398] In some embodiments, the PEG unit is about 300 Da to about 5 kDa, about 300 Da to about 4 kDa, about 300 Da to about 3 kDa, about 300 Da to about 2 kDa, or about 300 Da to about 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 no more than 24 subunits.

[0399] In some embodiments, a suitable polyethylene glycol may have a free hydroxyl group at each end of the polymer molecule, or may have a hydroxyl group etherified with a lower alkyl group, such as a methyl group. In some embodiments, a suitable polyethylene glycol is a polyethylene glycol derivative having an esterifiable carboxyl group. In some embodiments, polyethylene glycol is commercially available under the trade name PEG, typically as a polymer blend characterized by an average molecular weight. In some embodiments, the average molecular weight of the polyethylene glycol is about 300 to about 5000. In some embodiments, the average molecular weight of the polyethylene glycol is about 600 to about 1000.

[0400] In some embodiments, examples of hydrophilic groups suitable for the conjugates, scaffolds and methods disclosed herein can be found, for example, in column 13 of US 8367065, column 6 of US 8524696, WO2015 / 057699 and WO 2014 / 062697, the contents of each of which are incorporated herein by reference in their entirety.

[0401] STING agonist drug component (variable D)

[0402] In some implementations, the STING agonist drug moiety (D) is a compound of formula (A) or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof:

[0403]

[0404] in:

[0405] Y1, Y2, Z1, and Z2 are each independently O, S, C, or N;

[0406] X1, X2, W1, and W2 are each independently C or N;

[0407] X3 and X4 are each independently S or NR. f ;

[0408] X5 is N or CR A2 ;

[0409] X6 is either N or CR A1 ;

[0410] R 3 and R 5 Each independently is -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(R f ) or -CH2N(Rd )CO(R f ) and R 3 and R 5 The other one is H, -COOH, or -CO2(R) C );

[0411] R c C 1-4 alkyl;

[0412] R A2 and R A1 Each is independently H, halogen, hydroxyl, amino, amino(C) 1-4 Alkyl)-, optionally substituted (C) 1-6 Alkyl) or optionally substituted (C 1-6 alkyl)oxy-, wherein the optionally substituted (C 1-6 Alkyl) or optionally substituted (C 1-6 C of alkyl)oxy- 1-6 Alkyl groups consist of 1-4 alkyl groups, each selected from hydroxyl, C... 1-4 Alkoxy, -N(R) e (R) f ), -CO2(R f -CON(R) e (R) f The substituents of -COOH can be optionally substituted;

[0413] Each R d Independently H, hydroxyl or C 1-4 alkyl;

[0414] R e Selected from H, (C 1-4 Alkyl), -CO(C) 1-4 Alkyl), -OCO(C) 1-4 alkyl) and -CO2(C 1-4 alkyl);

[0415] Each R f Independently H, hydroxyl or (C 1-4 alkyl);

[0416] R 14 and R C2 Each independently exists or C 1-4 Alkyl, wherein C 1-4 Alkyl groups are selected from halogens, -OR c -NR c R d -CO2R c -CONR c R d -SO2NR c Rd and -OCONR c R d The substituents may be optionally substituted;

[0417] R 16 and R C1 Each independently represents non-existence, H, or C. 1-4 Alkyl; and

[0418] R 15 R 17 R 18 or R 19 Each independently represents non-existence, H, or C. 1-4 Alkyl, wherein C 1-4 Alkyl groups are selected from halogens, -OR c -NR c R d -CO2R c -CONR c R d -SO2NR c R d and -OCONR c R d The substituents may be optionally substituted;

[0419] Where: (i)R A2 and R A1 At least one of them exists, and where R is... A2 and R A1 At least one of them via R A2 and / or R A1 At least one functional group is connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0420] In some implementations, the STING agonist drug part is a compound of formula (Aa) or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0421]

[0422] in:

[0423] Y1, Y2, Z1, Z2, X1, X2, W1, W2, X3, X4, R 3 R 5 Rc R d R e R f R 14 R C2 R 16 R C1 R 15 R 17 R 18 and R 19 As defined in equation (A);

[0424] X5 is a CR A2 ;and

[0425] R A2 Halogen, hydroxyl, or optionally substituted (C 1-6 Alkyl), substituted (C) 1-6 alkyl)oxy-, optionally substituted (C 1-6 alkyl)amino-or optionally substituted (C 1-6 Alkyl)(C 1-4 alkyl)amino-, wherein the optionally substituted (C 1-6 Alkyl) or substituted (C 1-6 C of alkyl)oxy- 1-6 Alkyl groups consist of 1-4 alkyl groups, each independently selected from hydroxyl, C... 1-4 Alkoxy, -N(R) e (R) f ), -CO2(R f -CON(R) e (R) f The substituents of -COOH can be optionally substituted;

[0426] Where: (i)R A2 via R A2 The functional groups are connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0427] In some implementations, the STING agonist drug part is a compound of formula (Ab) or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0428]

[0429] in:

[0430] 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 As defined in equation (A);

[0431] Where: (i)R A2 and R A1 At least one of them exists, and where R is... A2 and R A1 At least one of them via R A2 and / or R A1 At least one functional group is connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0432] In some implementations, the STING agonist drug portion is a compound of formula (Ac) or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0433]

[0434]

[0435] in:

[0436] 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 16R C1 R 15 R 17 R 18 and R 19 As defined in equation (A);

[0437] One of W1, X1, Y1 and Z1 is N and the other W1, X1, Y1 and Z1 are O, S or C;

[0438] Where: (i)R A2 and R A1 At least one of them exists, and where R is... A2 and R A1 At least one of them via R A2 and / or R A1 At least one functional group is connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0439] In some implementations, the STING agonist drug part is a compound of formula (Ad) or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0440]

[0441] in:

[0442] 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 As defined in equation (A);

[0443] X5 is a CR A2 ;and

[0444] Where: (i)R A2 via RA2 The functional groups are connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0445] In some implementations, the STING agonist drug part is a compound of formula (Ae) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0446]

[0447] in:

[0448] Y1, Y2, Z1, Z2, X1, X2, X3, W1, W2, R A1 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 As defined in equation (A);

[0449] X6 is CR A1 ;and

[0450] Where: (i)R A1 via R A1 The functional groups are connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0451] In some implementations, the STING agonist drug portion is a compound of formula (Af) or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0452]

[0453] in:

[0454] 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 As defined in equation (A); and

[0455] Where: (i)R A2 and R A1 At least one of them exists, and where R is... A2 and R A1 At least one of them via R A2 and / or R A1 At least one functional group is connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0456] In some implementations, the STING agonist drug part is a compound of formula (A-f1) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0457]

[0458] in:

[0459] 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 C1As defined in equation (A);

[0460] X5 is a CR A2 ;and

[0461] Where: (i)R A2 via R A2 The functional groups are connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0462] In some implementations, the STING agonist drug part is a compound of the following formula (A-f2) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0463]

[0464] in:

[0465] X2, X4, X5, X6, W2, Y2, Z2, R 3 R 5 R c R A2 R A1 R d R e R C1 R C2 R 16 R 17 R 18 R 19 and R f As defined in equation (A);

[0466] Where: (i)R A2 and R A1 At least one of them exists, and where R is... A2 and R A1 At least one of them via R A2 and / or R A1 At least one functional group is connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D.

[0467] In some implementations, the STING agonist drug part is a compound of the following formula (A-f3) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0468]

[0469] in:

[0470] 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 As defined in equation (A);

[0471] X5 is a CR A2 ;and

[0472] Where: (i)R A2 via R A2 The functional groups are connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0473] In some implementations, the STING agonist drug moiety is a compound of the following formula (A-f4) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0474]

[0475] in:

[0476] X2, X4, W2, Y2, Z2, R 3 R 5 R c R d R e R f R 16 R A2 RC2 R 16 R 17 R 18 R 19 and R C1 As defined in equation (A);

[0477] X5 is a CR A2 ;and

[0478] Where: (i)R A2 via R A2 The functional groups are connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0479] In some implementations, the STING agonist drug part is a compound of the following formula (A-f5) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0480]

[0481] in:

[0482] 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 As defined in equation (A);

[0483] X5 is a CR A2 ;and

[0484] Where: (i)R A2 via R A2 The functional groups are connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2and / or R C1 At least one functional group is connected to L D .

[0485] In some implementations, the STING agonist drug part is a compound of the following formula (Ag) or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0486]

[0487] in:

[0488] 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 R C1 As defined in equation (A);

[0489] Y2 and Z2 are each independently O, S, C or N;

[0490] X2 and W2 are each independently C or N;

[0491] Where: (i)R A2 and R A1 At least one of them exists, and where R is... A2 and R A1 At least one of them via R A2 and / or R A1 At least one functional group is connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0492] In some implementations, the STING agonist drug moiety is a compound of the following formula (A-g1) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0493]

[0494]

[0495] in:

[0496] 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 As defined in equation (A); where: (i)R A2 via R A2 The functional groups are connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0497] In some implementations, the STING agonist drug moiety is a compound of the following formula (A-g2) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0498]

[0499] in:

[0500] 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 As defined in equation (A);

[0501] Where: (i)R A2 and R A1 At least one of them exists, and where R is... A2 and RA1 At least one of them via R A2 and / or R A1 At least one functional group is connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0502] In some implementations, the STING agonist drug moiety is a compound of the following formula (A-g3) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0503]

[0504] in:

[0505] 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 As defined in equation (A);

[0506] X5 is a CR A2 ;and

[0507] Where: (i)R A2 via R A2 The functional groups are connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D ;and

[0508] Optionally, where R A2 via R A2 The functional groups are connected to L D .

[0509] In some implementations, the STING agonist drug moiety is a compound of the following formula (A-g4) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0510]

[0511] in:

[0512] 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 As defined in equation (A);

[0513] X5 is a CR A2 ;and

[0514] Where: (i)R A2 via R A2 The functional groups are connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0515] In some implementations, the STING agonist drug moiety is a compound of the following formula (A-g5) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0516]

[0517] in:

[0518] 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 R16 and R C1 As defined in equation (A);

[0519] X5 is a CR A2 ;and

[0520] Where: (i)R A2 via R A2 The functional groups are connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0521] In some implementations, the STING agonist drug part is a compound of the following formula (Ah) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0522]

[0523] in:

[0524] X1, W1, Y1, Z1, 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 As defined in equation (A); where: (i)R A2 and R A1 At least one of them exists, and where R is... A2 and R A1 At least one of them via R A2 and / or R A1 At least one functional group is connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D.

[0525] In some implementations, the STING agonist drug part is a compound of the following formula (A-h1) or its prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0526]

[0527]

[0528] in:

[0529] X1, X3, W1, Y1, Z1, 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 As defined in equation (A);

[0530] Where: (i)R A2 and R A1 At least one of them exists, and where R is... A2 and R A1 At least one of them via R A2 and / or R A1 At least one functional group is connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0531] In some implementations, the STING agonist drug moiety is a compound of the following formula (A-h2) or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0532]

[0533] in:

[0534] X1, X3, W1, Y1, Z1, R 3 R 5 Rc R d R e R f R A2 R 14 R 15 R 18 R 19 R 16 and R C1 As defined in equation (A);

[0535] X5 is a CR A2 ;and

[0536] Where: (i)R A2 and R A1 At least one of them exists, and where R is... A2 and R A1 At least one of them via R A2 and / or R A1 At least one functional group is connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0537] In some embodiments, the STING agonist drug moiety (D) is a compound of formula (A), wherein the compound has the following formula (Ai) or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer:

[0538]

[0539] in:

[0540] 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 As defined in equation (A); and

[0541] Where: (i)RA2 and R A1 At least one of them exists, and where R is... A2 and R A1 At least one of them via R A2 and / or R A1 At least one functional group is connected to L D ; or (ii)R C2 and R C1 At least one of them exists, and where R is... C2 and R C1 At least one of them via R C2 and / or R C1 At least one functional group is connected to L D .

[0542] In some implementations, each STING agonist drug fraction (D) is independently:

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555] in:

[0556] R 2 For non-existent, -O-, or -NR4-; R 4 For H or C 1-3 Alkyl; and Indicates attachment to L D .

[0557] In some implementations, each STING agonist drug fraction (D) is independently:

[0558]

[0559]

[0560]

[0561] in:

[0562] R 2 For non-existent, -O-, or -NR4-; R 4 For H or C 1-3 Alkyl; and Indicates attachment to L D .

[0563] In some implementations, each STING agonist drug fraction (D) is independently:

[0564]

[0565] in:

[0566] R 2 For non-existent, -O-, or -NR4-; R 4 For H or C 1-3 Alkyl; and Indicates attachment to L D .

[0567] In some implementations, each STING agonist drug fraction (D) is independently:

[0568]

[0569] in:

[0570] R 2 For non-existent, -O-, or -NR4-; R 4 For H or C 1-3 Alkyl; and Indicates attachment to L D .

[0571] In some implementations, each STING agonist drug fraction (D) is independently:

[0572]

[0573] in:

[0574] R 2 For non-existent, -O-, or -NR4-; R 4 For H or C1-3 Alkyl; and Indicates attachment to L D .

[0575] Protein-based recognition molecules (PBRM)

[0576] In some embodiments, protein-based recognition molecules direct conjugates to specific tissues, cells, or locations within cells. In some embodiments, protein-based recognition molecules may direct conjugates in cultures, throughout the organism, or both. In each case, the protein-based recognition molecule may have a ligand present on the cell surface of the target cell, the ligand binding to the target cell with effective specificity, affinity, and coercivity. 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 may target conjugates to target cells such as cancer cells, such as receptors expressed on cancer cells, stromal tissue, or cancer-associated proteins such as tumor antigens. Alternatively, cells containing the tumor vascular system may be targeted. Protein-based recognition molecules may direct conjugates to specific cell types, such as specific targeting of hepatocytes in the liver rather than Kupffer cells. In some embodiments, protein-based recognition molecules may direct conjugates to cells of the reticuloendothelial or lymphatic system, or specialized phagocytes, such as macrophages or eosinophils. In some implementations, the conjugate itself can also be an effective delivery system without requiring a specific target.

[0577] 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-receptor binding or cytoplasmic translocation to the nucleus and nuclear entry or release from endosomes or other intracellular vesicles.

[0578] In some implementations, the protein-based recognition molecule is an antibody, antibody fragment, protein, peptide, or peptide mimic.

[0579] 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.

[0580] In some embodiments, the antibody or antibody fragment may be 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 replaced by cysteine ​​(e.g., engineered cysteine). In some embodiments, the parent antibody or antibody fragment may be wild-type or mutant.

[0581] In some embodiments, the antibody or antibody fragment may be a mutated antibody or antibody fragment. In some embodiments, monoclonal antibodies known in the art are engineered to form antibodies. In some embodiments, antibody fragments known in the art (e.g., Fab antibody fragments) are engineered to form antibody fragments (e.g., cysteine-engineered Fab antibody fragments). In some embodiments, due to the dimerization of IgG antibodies, a single-site mutation in Fab results in a single residue in Fab, while a single-site mutation in the antibody results in two amino acids in the resulting antibody.

[0582] In some embodiments, the antibody or antibody fragment retains its antigen-binding ability as a corresponding wild-type antibody or antibody fragment. In some embodiments, the antibody or antibody fragment is capable of binding to one or more antigens of its corresponding wild-type antibody or antibody fragment.

[0583] In some implementations, exemplary antibodies or antibodies derived from Fab, Fab2, scFv, or camel antibody heavy chain fragments that are specific to cell surface markers include, but are not limited to, 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, and CD44. v6, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD74, CD79-B, CD80, CD125, CD103, CD138, CD141, CD147, CD152, CD 154, CD326, CEA, CEACAM-5, aggregation 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, adrenergic receptor-β2, Claudine 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, coilin-7, integrins (including α4, α... v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), 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 (aeruginosa), rabies, RANKL, respiratory syncytial virus, rhesus monkey factor, SLAMF7, sphingosine-1-phosphate, TAG-72, T-cell receptor, tendinin C, TGF-1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR2, vimentin, etc.

[0584] In some implementations, one or more antibodies derived from Fab, Fab2, scFv, or camel antibody heavy chain fragments specific to cell surface markers 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, folic acid receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, PTK7, TAG-72, tendinin C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate cancer cells, adrenergic receptor-β2, Claudine 3, mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, coilin-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor and integrins (including α) v β3, α v β5, α v β6, α1β4, α4β1, α5β1, α6β4 integrins), tendinin C, TRAIL-R2, and vimentin.

[0585] In some implementations, the antibody targets the following cell surface markers: 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 (GUCY2C), HGF, and integrin α. v β3, integrin α5β1, IGF-1 receptor, GD3, GPNMB, mucin, LIV1, LY6E, mesothelin, MUC1, MUC13, NaPi2b, PTK7, phosphatidylserine, prostate cancer cells, PDGFRα, TAG-72, tendinin C, TRAIL-R2, VEGF-A, and VEGFR2. In this implementation, the antibodies include, but are not limited to, abagovomab, adecatumumab, alacizumab, attumomab, anatumomab, acitumomab, bavituximab, and bevacizumab. Bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, capromab, cetuximab, citatuzumab, clivatuzumab, conatumumab, dacetuzumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab ab), farletuzumab, figitumumab, gemtuzumab, glembatumumab, ibritumomab, igovomab, intetumumab, inotuzumab, labetuzumab, lexatumumab, lintuzumab, lucarumumab, matuzumab, mitumomab, naaptumomab estafenatox, necitumumab, oportuzumab, oregovomab, panitumumab, pemtumomab, pertuzumab, pritumumab, rituximab Rituximab, Robatumumab, Satumomab, Sibrotuzumab, Taplitumomab, Tenatumomab, Ticalimumab (tremelimumab), Tigatuzumab, Trastuzumab Tositumomab, tremelimumab, tucotuzumab celmoleukin, volociximab, and zalutumumab.

[0586] In some implementations, the antibody against the cell surface marker HER2 is pertuzumab or trastuzumab, and the antibody against EGFR (HER1) is cetuximab or pertumumab; the antibody against CD20 is rituximab; the antibody against VEGF-A is bevacizumab; the antibody against CD-22 is epazizumab or veltuzumab; and the antibody against CEA is labezizumab.

[0587] 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, chloramphenicol-derived peptides, and toad peptides.

[0588] In some implementations, the peptide or peptide mimic is an LHRH receptor-targeting peptide and an ErbB2 (HER2) receptor-targeting peptide.

[0589] Exemplary proteins include insulin, transferrin, fibrinogen γ fragment, platelet-reactive protein, closure protein, apolipoprotein E, affinity molecules such as ABY-025, ankyrin repeat protein, ankyrin-like repeat protein, and synthetic peptides.

[0590] In some implementations, the protein-drug conjugate comprises a combination of a broad-spectrum cytotoxic agent and a cell surface marker, such as pertuzumab or trastuzumab for HER2; cetuximab and pertumumab for EGFR; labezizumab for CEA; rituximab for CD20; bevacizumab for VEGF-A; or epazizumab or vertuzumab for CD-22.

[0591] In some embodiments, the protein-drug conjugates or protein conjugates used in this disclosure comprise combinations of two or more protein-based recognition molecules, such as combinations of bispecific antibodies against the EGF receptor (EGFR) on tumor cells and CD3 and CD28 on T cells; combinations of multiple or one antibodies derived from Fab, Fab2, scFv or camel antibody heavy chain fragments and peptides or peptide mimics; combinations of multiple or one antibodies derived from Fab, Fab2, scFv or camel antibody heavy chain fragments and proteins; and combinations of two bispecific antibodies, such as a CD3-CD19 plus CD28-CD22 bispecific antibody.

[0592] In some embodiments, the protein-drug conjugates or protein conjugates used in this disclosure contain protein-based recognition molecules that are antibodies against antigens, such as trastuzumab, cetuximab, rituximab, bevacizumab, epazolizumab, vetozumab, labezizumab, B7-H4, B7-H3, CD11b, CD103, CA125, CDH6, CD33, CXCR2, CEACAM5, Clec9A, CSFR1, DEC205, EGFR, 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.

[0593] In some embodiments, the protein-drug conjugates or protein conjugates of this disclosure comprise protein-based recognition molecules, such as CSRF1, CD11b, DEC205, clec9A, CD103, B7H4, mesothelin, PTK7, Ly6E, FAP, fibronectin-EDB, Her-2, or NaPi2b antibodies.

[0594] NaPi2b antibody

[0595] In some embodiments, the NaPi2b antibody suitable for conjugation binds to the extracellular region of SLC34A2. In some embodiments, this disclosure provides NaPi2b-targeting monoclonal antibodies that specifically recognize NaPi2b (also known as sodium-dependent phosphate transporter 2B). In some embodiments, the NaPi2b antibodies used in the conjugates disclosed herein are capable of and can be used to modulate, for example, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with at least one biological activity of NaPi2b. In some embodiments, the antibodies disclosed herein also include antibodies that bind to soluble NaPi2b. In some embodiments, the NaPi2b antibody specifically binds to an epitope on the extracellular domain (ECD) of human NaPi2b. These antibodies are collectively referred to herein as "NaPi2b" antibodies.

[0596] In some embodiments, the NaPi2b antibody-drug conjugates provided herein include equilibrium dissociation constants (K0) of ≤1 μM (e.g., ≤100 nM, ≤10 nM, and ≤1 nM). d or K D Antibodies that bind to the NaPi2b epitope. In some embodiments, the NaPi2b antibody used in the antibody-drug conjugates disclosed herein exhibits a K+ level in the range of ≤1 nM to ≤1 pM. d .

[0597] In some embodiments, the NaPi2b antibody-drug conjugates provided herein may include antibodies for modulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with the functional activity of NaPi2b. In some embodiments, the functional activity of NaPi2b includes, for example, participation 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, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with transcellular inorganic phosphate uptake.

[0598] In some embodiments, when the functional activity level of NaPi2b is reduced by at least 95% in the presence of the NaPi2b antibody compared to the functional activity level of NaPi2b in the absence of binding with the NaPi2b antibody described herein, such as 96%, 97%, 98%, 99%, or 100%, the NaPi2b antibody is considered to have completely regulated, blocked, inhibited, reduced, antagonized, neutralized, or otherwise interfered with the functional activity of NaPi2b. In some embodiments, when the activity level of NaPi2b is reduced by less than 95% in the presence of the NaPi2b antibody compared to the activity level of NaPi2b in the absence of binding with the NaPi2b antibody described herein, such as 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%, the NaPi2b antibody is considered to have partially regulated, blocked, inhibited, reduced, antagonized, neutralized, or otherwise interfered with the functional activity of NaPi2b.

[0599] In some embodiments, the exemplary antibodies disclosed herein include the XMT-1535 antibody. These antibodies exhibit specificity for human NaPi2b and have been shown to inhibit NaPi2b activity.

[0600] The NaPi2b human or humanized monoclonal antibody XMT-1535 comprises a heavy chain (HC), a heavy chain variable region (VH), a light chain (LC), and a light chain variable region (VL), as shown in Table I below with the corresponding amino acid sequences. The variable heavy chain and variable light chain regions of each antibody are shaded in the following amino acid sequences. The complementarity-determining regions (CDRs) of the heavy and light chains are highlighted in the following amino acid sequences. The amino acids containing the CDRs of the XMT-1535 antibody are disclosed in U.S. Patent 8,603,474.

[0601] Table I: Sequence of NaPi2b Humanized or Humanized Monoclonal Antibody XMT-1535

[0602] SEQ ID NO: Sequence Description 1 XMT-1535 heavy chain amino acid sequence 2 XMT-1535 light chain amino acid sequence 3 XMT-1535 Heavy Chain Variable Region 4 XMT-1535 Light Chain Variable Zone 5 XMT-1535 CDRH1 6 XMT-1535 CDRH2 7 XMT-1535 CDRH3 8 XMT-1535 CDRL1 9 XMT-1535 CDRL2 10 XMT-1535 CDRL3 11 XMT-1535 IgG1 Heavy Chain Constant Region 12 XMT-1535 Light Chain Constant Zone 13 XMT-1535 Heavy Chain Variable Region Nucleic Acid Sequence 14 XMT-1535 light chain variable region nucleic acid sequence 15 Full-length human NaPi2b sequence

[0603] The antibody disclosed in this article specifically binds to an epitope on the extracellular domain (ECD) of human NaPi2b.

[0604] In some embodiments, those skilled in the art will recognize that, by determining whether a monoclonal antibody prevents the disclosed monoclonal antibody from binding to a natural binding partner or other molecules known to associate with NaPi2b, it is possible to determine, without excessive experimentation, whether the former has the same specificity as the latter (e.g., XMT-1535, 10H1.11.4B). If the test monoclonal antibody competes with the disclosed monoclonal antibody, as indicated by reduced binding of the disclosed monoclonal antibody, then both monoclonal antibodies bind to the same or closely related epitopes.

[0605] An alternative method for determining whether a monoclonal antibody possesses the specificity of the monoclonal antibody disclosed herein is to pre-incubate the monoclonal antibody disclosed herein with soluble NaPi2b (which is generally reactive thereto), and then add the test monoclonal antibody to determine whether the ability of the test monoclonal antibody to bind NaPi2b is inhibited. If the test monoclonal antibody is inhibited, it is highly likely that it has the same or functionally equivalent epitope specificity as the monoclonal antibody disclosed herein.

[0606] For example, the monoclonal antibodies disclosed herein can also be screened by measuring NaPi2b-mediated activity and determining whether the test monoclonal antibody can regulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with NaPi2b activity.

[0607] In some embodiments, the antibody disclosed herein comprises a heavy chain variable region having an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with a sequence selected from SEQ ID NO:3, and a light chain variable region having an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with a sequence selected from SEQ ID NO:4.

[0608] In some embodiments, the antibody disclosed herein comprises a heavy chain amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence of SEQ ID NO:1 and a light chain amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence of SEQ ID NO:2.

[0609] In some embodiments, the antibody disclosed herein comprises the heavy chain variable region amino acid sequence of SEQ ID NO:3 and the light chain variable region amino acid sequence of SEQ ID NO:4.

[0610] In some embodiments, the antibodies disclosed herein comprise the heavy chain amino acid sequence of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:2.

[0611] In some embodiments, the antibodies disclosed herein comprise 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.

[0612] In some embodiments, the antibodies disclosed herein comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence of SEQ ID NO:5; CDRH2 comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence of SEQ ID NO:6; or comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence of SEQ ID NO:5; CDRH3, CDRL1, CDRL2, and CDRL3 contain amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence of SEQ ID NO:7; CDRL3 contains amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence of SEQ ID NO:8; CDRL3 contains amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence of SEQ ID NO:9; and CDRL3 contains amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence of SEQ ID NO:7; and CDRL3 contains amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence of SEQ ID NO:7. The amino acid sequence of NO:10 has 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 CDRL3.

[0613] In some embodiments, the antibodies disclosed herein include one or more conserved amino acid substitutions in a variable domain sequence, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more conserved substitutions in the variable domain sequence. In some embodiments, these conserved amino acid substitutions are located in CDR regions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more conserved substitutions are accumulated in all CDRs, and in some specific embodiments, up to 1, 2, 3 or 4 conserved amino acid substitutions may be present in each CDR sequence, for example, in SEQ ID NO: 5-10.

[0614] In some embodiments, those skilled in the art will recognize that, by determining whether a monoclonal antibody prevents the binding of the monoclonal antibody XMT-1535 to its natural binding partner or other molecules known to associate with NaPi2b, the former and the latter can be determined without excessive experimentation to have the same specificity. If the test monoclonal antibody competes with a monoclonal antibody disclosed herein, as indicated by reduced binding of the monoclonal antibody disclosed herein, then the two monoclonal antibodies bind to the same or closely related epitopes.

[0615] In some embodiments, an alternative method for determining whether a monoclonal antibody possesses the specificity of the monoclonal antibody disclosed herein is to pre-incubate the monoclonal antibody disclosed herein with soluble NaPi2b (which is generally reactive thereto), and then add the test monoclonal antibody to determine whether the ability of the test monoclonal antibody to bind NaPi2b is inhibited. In some embodiments, if the test monoclonal antibody is inhibited, it has the same or functionally equivalent epitope specificity as the monoclonal antibody disclosed herein.

[0616] For example, the monoclonal antibodies disclosed herein can also be screened by measuring NaPi2b-mediated activity and determining whether the test monoclonal antibody can regulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with NaPi2b activity.

[0617] In some implementations, the NaPi2b antibody suitable for conjugation can be generated and purified using well-known techniques, such as WO 2009 / 097128, WO 2017 / 160754 and US16 / 136,706, each of which is incorporated herein by reference in its entirety.

[0618] HER2 antibody

[0619] In some embodiments, the conjugated HER2 antibody is suitable for binding human HER2 in a soluble form or membrane-bound (i.e., when expressed on the cell surface). In some embodiments, this disclosure provides monoclonal antibodies that bind HER2 and are humanized or fully humanized. In some embodiments, this disclosure provides monoclonal antibodies that specifically bind HER2. These antibodies are collectively referred to herein as "HER2 antibodies".

[0620] In some implementations, the suitable conjugated HER2 antibody has an equilibrium dissociation constant (K0) of ≤1 μM (e.g., ≤100 nM, ≤10 nM, ≤1 nM). d or K D This antibody binds to the HER2 epitope. In some embodiments, this disclosure provides monoclonal antibodies that bind to HER2 and are humanized or fully humanized. For example, the HER2 antibodies provided herein exhibit a K+ concentration in the range of ≤1 nM to about 1 pM. d .

[0621] In some embodiments, the HER2 antibodies disclosed herein are used to modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the functional activity of HER2. In some embodiments, the functional activity of HER2 includes, for example, the regulation of PI3K-Akt pathway activity. In some embodiments, the HER2 antibodies completely or partially inhibit HER2 functional activity by partially or completely modulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with PI3K-Akt pathway activity. PI3K-Akt pathway activity is evaluated using any method recognized in the field for detecting PI3K-Akt pathway activity, including but not limited to detecting phosphorylated Akt levels in the presence and absence of the antibodies or antigen-binding fragments disclosed herein.

[0622] In some implementations, when the functional activity level of HER2 is reduced by at least 80% in the presence of the HER2 antibody compared to the functional activity level of HER2 in the absence of binding with the HER2 antibody described herein, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, the HER2 antibody is considered to have completely regulated, blocked, inhibited, reduced, antagonized, neutralized, or otherwise interfered with HER2 functional activity. In some implementations, when the activity level of HER2 is reduced by less than 95% in the presence of the HER2 antibody compared to the activity level in the absence of binding with the HER2 antibody described herein, such as 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%, the HER2 antibody is considered to partially regulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with HER2 functional activity.

[0623] In some embodiments, the exemplary antibodies disclosed herein include the XMT-1519 antibody. This antibody exhibits specificity for human HER2 and has demonstrated in vitro inhibitory activity against HER2.

[0624] The HER2 monoclonal antibody XMT-1519 comprises a heavy chain (HC), a heavy chain variable region (VH), a light chain (LC), and a light chain variable region (VL), as shown in Table II below with their corresponding amino acid sequences. The variable heavy chain and variable light chain regions of each antibody are shaded in the following amino acid sequences. The complementarity-determining regions (CDRs) of the heavy and light chains are highlighted in the amino acid sequences shown below.

[0625] Table II: Sequence of HER2 Humanized or Humanized Monoclonal Antibody XMT-1519

[0626] SEQ ID NO: Sequence Description 16 Full-length human HER2 receptor 17 XMT-1519 Heavy Chain Variable Region 18 XMT-1519IgG1 Heavy Chain Constant Region 19 XMT-1519 heavy chain amino acid sequence 20 XMT-1519 CDRH1 21 XMT-1519 CDRH2 22 XMT-1519 CDRH3 23 XMT-1519 Heavy Chain Variable Region Nucleic Acid Sequence 24 XMT-1519 Light Chain Variable Zone 25 XMT-1519 Light Chain Constant Zone 26 XMT-1519 light chain amino acid sequence 27 XMT-1519CDRL1 28 XMT-1519CDRL2 29 XMT-1519CDRL3 30 XMT-1519 light chain variable region nucleic acid sequence 31 The extracellular domain (ECD) of the human HER2 receptor

[0627] The antibody and its antigen-binding fragment disclosed herein specifically bind to an epitope on the full-length human HER2 receptor containing the amino acid sequence of SEQ ID NO:16.

[0628] The antibody and its antigen-binding fragment disclosed herein specifically bind to an epitope on the extracellular domain (ECD) of the human HER2 receptor containing the amino acid sequence of SEQ ID NO:31.

[0629] 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 bind to different epitopes of HER2 because they cross-block each other but do not block the binding of trastuzumab, pertuzumab, Fab37, or chA21 to HER2. Furthermore, unlike known antibodies, the antibodies disclosed herein can be efficiently internalized into HER2-expressing cells without promoting cell proliferation.

[0630] In some embodiments, the antibodies disclosed herein are fully human monoclonal antibodies that bind to novel epitopes and / or have other advantageous properties for therapeutic use. In some embodiments, exemplary properties include, but are not limited to, advantageous binding to cancer cells expressing human HER2 at high or low levels, specific binding to recombinant human and cynomolgus monkey HER2, efficient internalization upon binding to HER2, highly effective killing of cancer cells expressing high or low levels of HER2 when administered as an antibody-drug conjugate (ADC), no significant agonistic effect on the proliferation of HER2-expressing cancer cells, and / or providing effective antibody-dependent cytotoxicity (ADCC)-mediated killing of HER2-expressing cells, and any combination of the above properties.

[0631] In some embodiments, the antibodies disclosed herein also include an antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor, said epitope including 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.

[0632] In some embodiments, the antibodies disclosed herein comprise antibodies or antigen-binding fragments thereof that bind to at least a portion of the N-terminus of the structural region IV of the human HER2 receptor, but do not cross-compete with antibodies binding to epitope 4D5 of the human HER2 receptor. In some embodiments, the antibodies or antigen-binding fragments thereof described herein do not cross-compete with trastuzumab for binding to the human HER2 receptor because trastuzumab is known to bind to epitope 4D5 of 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 thereof also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0633] In some embodiments, the antibodies disclosed herein also include an antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor, said epitope including 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.

[0634] In some embodiments, the antibodies disclosed herein further include an antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor, said epitope comprising at least one amino acid residue selected from amino acid residues E521, L525, and R530 of the extracellular domain of the human HER2 receptor, such as 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 comprise an antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor, said epitope comprising at least two amino acid residues selected from amino acid residues E521, L525, and R530 of the extracellular domain of the human HER2 receptor. In some embodiments, the antibodies disclosed herein further comprise an antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor, said epitope comprising at least amino acid residues E521, L525, and R530 of the extracellular domain of the human HER2 receptor. In some implementations, any or all of these antibodies or their antigen-binding fragments also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0635] In some embodiments, the antibodies disclosed herein further include antibodies or antigen-binding fragments thereof that bind to at least a portion of structural region III and at least a portion of the N-terminus of structural region IV of the human HER2 receptor, but do not cross-compete with Fab37 monoclonal antibodies or antibodies binding to human HER2 receptor epitope 4D5. In some embodiments, the antibodies or antigen-binding fragments thereof described herein do not cross-compete with Fab37 monoclonal antibodies and / or trastuzumab for binding to the human HER2 receptor. In some embodiments, the antibodies or antigen-binding fragments thereof also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0636] In some embodiments, the antibodies disclosed herein also include an antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor, said epitope including 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.

[0637] In some embodiments, the antibodies disclosed herein also include an antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor, said epitope comprising at least one amino acid residue selected from the extracellular domain of the human HER2 receptor, namely 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 comprise an antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor, said epitope comprising at least two, at least three, at least four, at least five, 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 comprise an antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor, said epitope 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 thereof also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0638] In some embodiments, the antibodies disclosed herein further include an antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor, said epitope comprising at least one amino acid residue selected from the extracellular domain of the human HER2 receptor, namely residues C453, H473, N476, R495, H497, and W499, 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 comprise an antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor, said epitope comprising at least two, at least three, at least four, at least five, or at least six amino acid residues selected from the extracellular domain of the human HER2 receptor, namely residues C453, H473, N476, R495, H497, and W499. In some embodiments, the antibodies disclosed herein comprise antibodies or antigen-binding fragments thereof that specifically bind to epitopes of the extracellular domain of the human HER2 receptor, said epitopes comprising at least 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 thereof also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0639] In some embodiments, these antibodies exhibit specificity for human HER2 and have been shown to modulate, for example, by blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with the PI3K-Akt pathway, which promotes cell survival by reducing phosphorylated Akt levels. In some embodiments, these antibodies are internalized from the cell surface of HER2-expressing cells at a rate that is the same as or substantially similar to that of trastuzumab or its bioanalysts. In some embodiments, the internalization rate of these antibodies and antigen-binding fragments is approximately 50% of the total surface binding at time 0 (4 hours of internalization).

[0640] In some embodiments, the antibody disclosed herein comprises a heavy chain variable region having an amino acid sequence 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 identity with a sequence selected from SEQ ID NO:17, and a light chain variable region having an amino acid sequence 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 identity with a sequence selected from SEQ ID NO:24.

[0641] In some embodiments, the antibody disclosed herein comprises a heavy chain amino acid sequence 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 identity with the amino acid sequence of SEQ ID NO:19, and a light chain amino acid sequence 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 identity with the amino acid sequence of SEQ ID NO:26.

[0642] In some embodiments, the antibodies disclosed herein comprise the heavy chain variable region amino acid sequence of SEQ ID NO:17 and the light chain variable region amino acid sequence of SEQ ID NO:24.

[0643] In some embodiments, the antibodies disclosed herein comprise the heavy chain amino acid sequence of SEQ ID NO:19 and the light chain amino acid sequence of SEQ ID NO:26.

[0644] In some embodiments, the antibodies disclosed herein comprise 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.

[0645] In some embodiments, the antibodies disclosed herein include one or more conserved amino acid substitutions in a variable domain sequence, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more conserved substitutions in the variable domain sequence. In some embodiments, these conserved amino acid substitutions are located in CDR regions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more conserved substitutions are accumulated in all CDRs. In some embodiments, up to 1, 2, 3, or 4 conserved amino acid substitutions may be present in each CDR sequence, such as SEQ ID NO: 20-22 and 27-29.

[0646] Those skilled in the art will recognize that by determining whether a monoclonal antibody prevents the binding of the monoclonal antibody XMT-1519 to a naturally binding partner or other molecules known to associate with HER2, it is possible to determine, without excessive experimentation, whether the former and the latter have the same specificity. In some embodiments, if the test monoclonal antibody competes with a monoclonal antibody disclosed herein, as indicated by reduced binding of the monoclonal antibody disclosed herein, then both monoclonal antibodies bind to the same or closely related epitopes.

[0647] In some embodiments, an alternative method for determining whether a monoclonal antibody possesses the specificity of the monoclonal antibody disclosed herein is to pre-incubate the monoclonal antibody disclosed herein with soluble HER2 (which is generally reactive thereto), and then add the test monoclonal antibody to determine whether the ability of the test monoclonal antibody to bind HER2 is inhibited. If the test monoclonal antibody is inhibited, it is highly likely that it has the same or functionally equivalent epitope specificity as the monoclonal antibody disclosed herein.

[0648] In some embodiments, the monoclonal antibodies disclosed herein may also be screened, for example, by measuring HER2-mediated PI3K-Akt pathway activity and determining whether the test monoclonal antibody can modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with PI3K-Akt pathway activity. In some embodiments, suitable HER2 antibodies for conjugation may be generated and purified using well-known techniques, such as WO 2015 / 195917 and PCT / US2018 / 019873, each of which is incorporated herein by reference in its entirety.

[0649] Conjugate

[0650] In some embodiments, the conjugates of this disclosure contain one or more occurrences of D, wherein D is a STING agonist, and the one or more occurrences of D may be the same or different.

[0651] In some embodiments, one or more occurrences of the PBRM are attached to the linker-drug portion, wherein the one or more occurrences of the PBRM may be the same or different. In some embodiments, one or more linker-drug portions containing one or more occurrences of D are attached to a PBRM (e.g., an antibody).

[0652] In some embodiments, the conjugates disclosed herein comprise PBRM having a molecular weight of about 40 kDa or greater (e.g., about 60 kDa or greater, about 80 kDa or greater, about 100 kDa or greater, about 120 kDa or greater, about 140 kDa or greater, about 160 kDa or greater, about 180 kDa or greater, or about 200 kDa or greater, 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) and having a thiol (i.e., -SH or thiol) group.

[0653] In some implementations, the total number of sulfide bonds (or the total number of attachment points) formed between the connector-drug portion and the PBRM is 10 or less (e.g., 8, 6, 4 or 2).

[0654] In some embodiments, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of about 40 kDa or greater (e.g., about 60 kDa or greater, about 80 kDa or greater, about 100 kDa or greater, about 120 kDa or greater, about 140 kDa or greater, about 160 kDa or greater, or about 180 kDa or greater, 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).

[0655] In some embodiments, the PBRM has a molecular weight of about 40 kDa to about 200 kDa for conjugation with one or more linker-drug moieties. In some embodiments, the PBRM has a molecular weight of about 40 kDa to about 80 kDa for conjugation with one or more linker-drug moieties.

[0656] In some embodiments, the PBRM has a molecular weight of 40 kDa to 200 kDa for conjugation with one or more linker-drug moieties. In some embodiments, the PBRM has a molecular weight of 40 kDa to 80 kDa for conjugation with one or more linker-drug moieties.

[0657] In some implementations, PBRMs within this molecular weight range include, but are not limited to, antibody fragments such as Fab.

[0658] In some implementations, the PBRM has a molecular weight of about 60 kDa to about 120 kDa for attachment to one or more connector-drug moieties.

[0659] In some implementations, PBRM has a molecular weight of 60kDa-120kDa for conjugation with one or more linker-drug moieties.

[0660] In some implementations, PBRMs within this molecular weight range include, but are not limited to, for example, Camelidae, Fab2, scFcFv, etc.

[0661] In some implementations, the PBRM has a molecular weight of about 140 kDa to about 180 kDa for attachment to one or more connector-drug moieties.

[0662] In some implementations, the PBRM has a molecular weight of 140kDa-180kDa for conjugation with one or more linker-drug moieties.

[0663] In some implementations, PBRMs within this molecular weight range include, but are not limited to, full-length antibodies such as IgG and IgM.

[0664] In some embodiments, such as according to the disclosed techniques and methods, the targeting ligands, linkers, and drug or prodrug fragments described herein can be assembled into the disclosed conjugates or scaffolds. The therapeutic and targeting conjugates of this disclosure, as well as methods for producing them, are described below by way of non-limiting examples.

[0665] In some implementations, the total number of sulfide bonds (or the total number of attachment points) formed between the connector-drug portion and the PBRM is 8 or less.

[0666] In some embodiments, the total number of sulfide bonds (or attachment points) formed between the connector-drug portion and the PBRM is 8. In some embodiments, the total number of sulfide bonds (or attachment points) formed between the connector-drug portion and the PBRM is 6. In some embodiments, the total number of sulfide bonds (or attachment points) formed between the connector-drug portion and the PBRM is 5. In some embodiments, the total number of sulfide bonds (or attachment points) formed between the connector-drug portion and the PBRM is 4. In some embodiments, the total number of sulfide bonds (or attachment points) formed between the connector-drug portion and the PBRM is 3. In some embodiments, the total number of sulfide bonds (or attachment points) formed between the connector-drug portion and the PBRM is 2.

[0667] In some embodiments, the ratio between the connector-drug component and the PBRM is between approximately 1:1 and approximately 1:8. In some embodiments, the ratio between the connector-drug component and the PBRM is between approximately 1:1 and approximately 6:1. In some embodiments, the ratio between the connector-drug component and the PBRM is between approximately 1:1 and approximately 4:1. In some embodiments, the ratio between the connector-drug component and the PBRM is between approximately 2:1 and approximately 2:1.

[0668] In some implementations, the ratio between the connector-drug component and the PBRM is between approximately 6:1 and approximately 8:1.

[0669] In some implementations, the ratio between the connector-drug component and the PBRM is approximately 8:1.

[0670] In some implementations, the ratio between the connector-drug component and the PBRM is approximately 6:1.

[0671] In some embodiments, this disclosure also relates to a linker-drug portion comprising at least two parts, each part being capable of conjugating a thiol group in the PBRM to form a protein-linker-drug conjugate.

[0672] In some embodiments, one or more thiol groups of the PBRM are generated by reducing the protein. The one or more thiol groups of the PBRM can then react with one or more linker-drug moieties capable of conjugating to the thiol groups of the PBRM having the linker-drug moieties. In some embodiments, at least two moieties attached to the PBRM are maleimide groups.

[0673] In some embodiments, antibodies can be activated for conjugation to the linker-drug moiety by treatment with a reducing agent such as DTT (Cleland's reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphonic acid hydrochloride). In some embodiments, the full-length monoclonal antibody can be reduced with excess TCEP to reduce disulfide bonds (e.g., between cysteine ​​residues present in the corresponding parent antibody) to produce a reduced form of the antibody. The newly introduced and unpaired cysteine ​​residues remain receptive to react with the linker-drug moiety to form the antibody conjugate of this disclosure. In some embodiments, excess linker-drug moiety is added to influence conjugation and the formation of the antibody-drug conjugate, and the conjugation mixture is purified to remove excess linker-drug intermediates and other impurities.

[0674] In some embodiments, 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) for the linker-drug moiety conjugation. In some embodiments, the ratio of PBRM to each linker-drug moiety is between about 1:1 and about 1:8, about 1:1 and about 1:6, about 1:1 and about 1:5, about 1:1 and about 1:4, about 1:1 and about 1:3, or about 1:1 and about 1:2.

[0675] PBRMs within this molecular weight range include, but are not limited to, full-length antibodies such as IgG and IgM.

[0676] In some embodiments, the PBRM has a molecular weight of 60 kDa to 120 kDa for conjugation with one or more linker-drug moieties. In some embodiments, the ratio of PBRM to each linker-drug moiety is between about 1:1 and about 1:8, about 1:1 and about 1:6, about 1:1 and about 1:5, about 1:1 and about 1:4, about 1:1 and about 1:3, or about 1:1 and about 1:2.

[0677] PBRMs within this molecular weight range include, but are not limited to, antibody fragments such as Fab2, scFcFv, and camelids.

[0678] In some embodiments, the PBRM has a molecular weight of 40 kDa to 80 kDa for conjugation with one or more linker-drug moieties. In some embodiments, the ratio of PBRM to each linker-drug moiety is between about 1:1 and about 1:8, between about 1:1 and about 1:6, between about 1:1 and about 1:5, between 1:1 and about 1:4, between about 1:1 and about 1:3, or between about 1:1 and about 1:2.

[0679] In some implementations, PBRMs within this molecular weight range include, but are not limited to, antibody fragments such as Fab.

[0680] In some embodiments, this disclosure is characterized as a scaffold that can be conjugated with either or both of a protein-based recognition molecule (PBRM) and a STING agonist moiety (D).

[0681] In some implementations, the drug-eluting stents described herein (i.e., not connected to the PBRM) generally have a polydispersity index (PDI) of 1.

[0682] The conjugates and scaffolds disclosed herein can be purified by extensive percolation (i.e., removal of any starting material). Further purification by size exclusion chromatography, if necessary, can be performed to remove any aggregated conjugates. Typically, the purified conjugates generally contain less than 5% (e.g., <2% w / w) of aggregated conjugates as determined by SEC, less than 0.5% (e.g., <0.1% w / w) of free (unconjugated) drug as determined by RP-HPLC, less than 1% of the drug-loaded peptide-containing scaffold as determined by SEC, and less than 2% (e.g., <1% w / w) of unconjugated PBRM as determined by HIC-HPLC.

[0683] In some implementations, the stent is selected from the stents described in Table A1.

[0684] In some implementations, the stent is selected from the stents described in Table A2.

[0685] In some implementations, the conjugate is selected from the conjugates described in Table B1.

[0686] In some implementations, the conjugate is selected from the conjugates described in Table B2.

[0687] Table A1

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695] Where R 14 R 15R 16 R 17 R 18 R 19 R C1 R C2 X3, X4, X6, X1, W1, Y1, Z1, X2, W2, Y2, Z2 are as defined in this article.

[0696] Table A2

[0697]

[0698]

[0699]

[0700]

[0701]

[0702]

[0703]

[0704]

[0705]

[0706]

[0707]

[0708]

[0709]

[0710]

[0711]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721]

[0722]

[0723] Table B1

[0724]

[0725]

[0726]

[0727]

[0728]

[0729]

[0730]

[0731]

[0732]

[0733]

[0734]

[0735]

[0736]

[0737]

[0738]

[0739]

[0740]

[0741]

[0742]

[0743]

[0744]

[0745]

[0746]

[0747]

[0748]

[0749]

[0750]

[0751]

[0752]

[0753]

[0754]

[0755] Where 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, Z2 are as defined in this article.

[0756] Table B2

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

[0763]

[0764]

[0765]

[0766]

[0767]

[0768]

[0769]

[0770]

[0771]

[0772]

[0773]

[0774]

[0775]

[0776]

[0777] In some implementations, the conjugate is:

[0778]

[0779]

[0780]

[0781] Where 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, Z2 are as defined in this article.

[0782] In some implementations, the conjugate is:

[0783]

[0784]

[0785] Where d 15 R 14 R 15 R 16 R 17 R18 R 19 R C1 R C2 X3, X4, X6, X1, W1, Y1, Z1, X2, W2, Y2, Z2 are as defined in this article.

[0786] In some implementations, the conjugate is:

[0787]

[0788]

[0789]

[0790]

[0791] Where d 15 As defined in this article.

[0792] Pharmaceutical Composition

[0793] In one aspect, this disclosure provides pharmaceutical compositions comprising the conjugates described herein and one or more pharmaceutically acceptable carriers or excipients.

[0794] Pharmaceutical compositions containing the conjugates disclosed herein can be manufactured in a manner commonly known, such as by conventional mixing, dissolving, granulation, pelleting, grinding, emulsification, encapsulation, sealing, or lyophilization processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers containing excipients and / or adjuvants that facilitate the formulation of the conjugate into a pharmaceutically usable formulation. Of course, appropriate formulation depends on the chosen route of administration.

[0795] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (when soluble in water) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, and Cremophor EL. TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it is readily injectable. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, appropriate flowability can be maintained by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. The protection against microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, mercuric chloride, etc. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols (e.g., mannitol and sorbitol), and sodium chloride. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption in the composition, such as aluminum monostearate and gelatin.

[0796] Sterile injectable solutions can be prepared by incorporating the desired amount of the conjugate with one or a combination of the aforementioned components (as needed) into a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the conjugate into a sterile medium containing a basic dispersion medium and any other desired components from those described above. In the case of sterile powders used to prepare sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, thereby producing a powder containing the active ingredient plus any other desired components from its previously sterile filtered solution.

[0797] Oral compositions typically include an inert diluent or an edible, pharmaceutically acceptable carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the conjugate may be mixed with excipients and administered in tablet, lozenge, or capsule form. Oral compositions may also be prepared using a fluid carrier used as a mouthwash, wherein the conjugate in the fluid carrier is administered orally and rinsed and coughed up or swallowed. Pharmaceutically compatible adhesives and / or excipients may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds with similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginate, Primogel, or corn starch; lubricants such as magnesium stearate or sterotes; gliding agents such as colloidal silica; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring agents.

[0798] For administration by inhalation, the conjugate is delivered in the form of an aerosol spray from a pressure vessel or dispenser (containing a suitable propellant, such as a gas like carbon dioxide) or a nebulizer.

[0799] Systemic administration can also be administered via mucosal or transdermal routes. For mucosal or transdermal administration, a penetrant suitable for the target permeability barrier is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives used for mucosal administration. Mucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, the conjugate is formulated as an ointment, cream, gel, or cream as generally known in the art.

[0800] The conjugates can be prepared using pharmaceutically acceptable carriers that protect them from rapid elimination from the body, such as sustained-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are readily apparent to those skilled in the art.

[0801] It is particularly advantageous to formulate oral or enteral compositions in dosage units to facilitate administration and uniform dosage. As used herein, dosage units refer to physically discrete units suitable as a single dose for a subject to be treated; each unit containing a predetermined amount of the conjugate can be calculated to produce the desired therapeutic effect in combination with the desired drug carrier. The specifications of the dosage unit forms disclosed herein are determined by and directly depend on the unique properties of the conjugate and the specific therapeutic effect to be achieved.

[0802] In therapeutic applications, the dosage of the pharmaceutical composition used according to this disclosure varies depending on the pharmaceutical agent, the age, weight, and clinical condition of the receiving patient, the experience and judgment of the clinician or practicing physician administering the therapy, and other factors that influence the selected dosage. Generally, the dosage should be sufficient to cause a reduction, and preferably a resolution, of the disease symptoms, and preferably a complete resolution of the disease.

[0803] It should be understood that the pharmaceutical composition may be included in a container, package or dispenser along with the instructions for administration.

[0804] How to use

[0805] In some embodiments, this disclosure provides a method for treating or preventing a disease or condition in a subject who needs it, including administering a therapeutically effective amount of the conjugate disclosed herein to the subject.

[0806] In some embodiments, this disclosure provides a method of treating a disease or condition in a subject who needs it, including administering a therapeutically effective amount of the conjugate disclosed herein to the subject.

[0807] In some embodiments, this disclosure provides methods for activating or enhancing STING activity in a subject, including administering the subject the conjugate disclosed herein.

[0808] In some embodiments, this disclosure relates to a method of treating cancer in a subject who needs it, including administering an effective amount of the conjugate disclosed herein to the subject.

[0809] In some embodiments, this disclosure provides conjugates disclosed herein for use in treating or preventing diseases or conditions in subjects who require them.

[0810] In some embodiments, this disclosure provides conjugates disclosed herein for use in treating a disease or condition in a subject in need of them.

[0811] In some embodiments, this disclosure provides conjugates disclosed herein for treating STING-mediated diseases or conditions in subjects.

[0812] In some embodiments, this disclosure provides the use of the conjugates disclosed herein for treating cancer in subjects who require them.

[0813] In some embodiments, this disclosure provides the use of the conjugates disclosed herein in the manufacture of a medicament for treating a disease or condition in a subject who requires it.

[0814] In some embodiments, this disclosure provides for the use of the conjugates disclosed herein in the manufacture of a medicament for treating or preventing a disease or condition in a subject in need of it.

[0815] In some embodiments, this disclosure provides the use of the conjugates disclosed herein in the manufacture of a medicament for treating a STING-mediated disease or condition in a subject.

[0816] In some embodiments, this disclosure provides the use of the conjugates disclosed herein in the manufacture of a medicament for treating cancer in subjects who require it.

[0817] In some embodiments, this disclosure provides the use of the conjugates disclosed herein for treating or preventing a disease or condition in a subject in need of it.

[0818] In some embodiments, this disclosure provides the use of the conjugates disclosed herein for treating a disease or condition in a subject who requires them.

[0819] In some embodiments, this disclosure provides the use of the conjugates disclosed herein for treating STING-mediated diseases or conditions in subjects.

[0820] In some embodiments, this disclosure provides the use of the conjugates disclosed herein for treating cancer in subjects who require them.

[0821] In some implementations, the conjugates disclosed herein are administered to the subject.

[0822] In some embodiments, this disclosure provides a method for treating or preventing a disease or condition in a subject who needs it, comprising administering to the subject an effective amount of at least one of the conjugates disclosed herein; wherein the conjugate releases one or more therapeutic agents upon biodegradation.

[0823] In some embodiments, this disclosure provides a method of treating a disease or condition in a subject who needs it, comprising administering to the subject an effective amount of at least one of the disclosed conjugates; wherein the conjugate releases one or more therapeutic agents upon biodegradation.

[0824] In some embodiments, the conjugate disclosed herein is an antibody-STING agonist conjugate. In some embodiments, the disease or condition is cancer.

[0825] In some embodiments, this disclosure provides methods for treating or preventing STING-mediated diseases and conditions. Exemplary diseases / conditions include, but are not limited to, cancer, infectious diseases (e.g., HIV, HBV, HCV, HPV, and influenza), and vaccine adjuvants.

[0826] In some implementations, the STING pathway can induce antitumor immunity by upregulating IFNβ and interferon (IFN)-stimulated genes (ISG) in many cell types within tumors in response to agonistic cytoplasmic nucleic acids.

[0827] In some embodiments, this disclosure provides conjugates disclosed herein for use as vaccine adjuvants. Therefore, immunogenic compositions or vaccine adjuvants comprising the conjugates disclosed herein are also provided.

[0828] In some embodiments, a composition comprising the conjugates disclosed herein and one or more immunostimulants is provided.

[0829] In some embodiments, this disclosure provides the use of the conjugates disclosed herein in the manufacture of vaccines. In some embodiments, this disclosure provides the use of the conjugates disclosed herein in the manufacture of immunogenic compositions or vaccine compositions (containing antigens or antigen compositions) for the treatment or prevention of diseases.

[0830] In some embodiments, this disclosure relates to methods of treating or preventing disease, including administering an immunogenic composition or vaccine composition to a human subject who has or is susceptible to a disease, comprising an antigen or antigen composition and a conjugate disclosed herein.

[0831] In some embodiments, the disease or condition is inflammation, autoimmune disease, allergic disease, infectious disease, HIV infection, AIDS infection, HCV infection, influenza, or human papillomavirus (HPV) infection. The scope of the disease is readily recognizable to those skilled in the art. In some embodiments, these diseases are as described in PCT application PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0832] As used herein, the terms “cancer,” “tumor,” and “carcinoma” are used interchangeably and in the singular or plural form, referring to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells can be readily distinguished from non-cancer cells using established techniques, particularly histological examination. The definition of cancer cells as used herein includes not only primary cancer cells but also any cells derived from cancer cell ancestors. This includes metastatic cancer cells as well as in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that typically presents as a solid tumor, a “clinically detectable” tumor is one that is detectable based on the tumor mass; for example, by procedures such as computed tomography (CT) scans, magnetic resonance imaging (MRI), X-rays, ultrasound, or physical examination palpation, and / or by the expression of one or more cancer-specific antigens in samples obtainable from the patient. Tumors can be hematopoietic (or hematologic or hematological or blood-related) cancers, such as cancers originating from blood cells or immune cells, which may be referred to as “liquid tumors.” Specific examples of clinical conditions based on hematologic malignancies include leukemia, 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; lymphomas, such as non-Hodgkin lymphoma and Hodgkin lymphoma; and so on.

[0833] In some implementations, the disease or condition is referred to as a precancerous syndrome.

[0834] The conjugates disclosed herein can be used to treat inflammation of any tissue and organ in the body, including musculoskeletal inflammation, vascular inflammation, neurological inflammation, digestive system inflammation, ocular inflammation, reproductive system inflammation, and other inflammations. The range of diseases is readily apparent to those skilled in the art. In some embodiments, these diseases are described as in PCT application PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0835] Examples of cancers and conditions for which the conjugates disclosed herein may have potentially beneficial antitumor effects include, but are not limited to, cancers of the lungs, bones, pancreas, skin, head, neck, uterus, ovaries, stomach, colon, breast, esophagus, bile duct, small intestine, intestines, endocrine system, thyroid, parathyroid, adrenal glands, urethra, prostate, penis, testes, ureter or urothelial tract, bladder, kidneys or liver; rectal cancer; anal region cancer; fallopian tubes, endometrium, cervix, vagina, vulva, renal pelvis, and kidney cells. Cancer; soft tissue sarcoma; myxoma; rhabdomyosarcoma; fibroma; lipoma; teratoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hemangioma; liver cancer; fibrosarcoma; chondrosarcoma; myeloma; chronic or acute leukemia; lymphocytic lymphoma; primary CNS lymphoma; CNS tumor; spinal cord axis tumor; squamous cell carcinoma; synovial sarcoma; malignant pleural mesothelioma; brainstem glioma; pituitary adenoma; bronchial adenoma; chondromatous hamartoma; mesothelioma; Hodgkin's disease or a combination of one or more of the above cancers.

[0836] In some implementations, the disease or condition is a solid tumor. On one hand, 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 carcinoma, papillary renal cell carcinoma, bile duct cancer, salivary gland duct carcinoma, kidney cancer, cervical cancer, and pancreatic cancer. In some implementations, the human patient has a solid tumor, such as diffuse large B-cell lymphoma (DLBCL), multiple myeloma, chronic lymphocytic leukemia (CLL), follicular lymphoma, acute myeloid leukemia, and chronic myeloid leukemia. In some implementations, the disease or condition is skin cancer (e.g., non-melanoma skin cancer, squamous cell carcinoma, basal cell carcinoma) or actinic keratosis. In addition to clearing the field effect of superficial skin cancer, the conjugates disclosed herein can also prevent the development of secondary skin cancer and pre-malignant actinic keratosis in the treated subject.

[0837] In some implementations, the disease or condition is bladder cancer, breast cancer, colorectal cancer, colon cancer, endometrial cancer, stomach 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.

[0838] In some implementations, the breast cancer is either HER2 amplified / overexpressing breast cancer or HER2-low expressing breast cancer.

[0839] In some implementations, endometrial cancer is defined as serous endometrial cancer.

[0840] The conjugates disclosed herein can also be used to treat one or more diseases afflicting mammals, characterized by cell proliferation in disease regions associated with angiogenesis and / or vascular permeability, fibrotic disorders, and metabolic disorders. The range of diseases is readily apparent to those skilled in the art. In some embodiments, these diseases are as described in PCT application PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0841] In some embodiments, the disease or condition 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 disease is readily recognized by those skilled in the art. In some embodiments, these diseases are described as 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.

[0842] In some embodiments, the disease or symptom is an infectious disease, which is any disease caused by or occurring concurrently with an infection of a pathogen originating from bacteria, a DNA virus family, or an RNA virus family. The scope of the disease is readily apparent to those skilled in the art. In some embodiments, these diseases are as described in PCT application PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0843] The conjugates of this disclosure can be used alone or in combination with other therapeutic agents. As modulators of immune responses, the conjugates of this disclosure can also be used as monotherapy or in combination with another therapeutic agent to treat diseases and conditions in which modulating STING is beneficial. Therefore, combination therapies of this disclosure include administering a conjugate of this disclosure or a pharmaceutically acceptable salt thereof and at least one other therapeutically active agent. In some embodiments, combination therapies of this disclosure include administering at least one conjugate of this disclosure or a pharmaceutically acceptable salt thereof and at least one other therapeutic agent. The (multiple) conjugates of this disclosure and their pharmaceutically acceptable salts and (multiple) other therapeutic agents can be administered together or separately as a single pharmaceutical composition, and when administered separately, this can occur simultaneously or sequentially in any order. The amount of the (multiple) conjugates of this disclosure and their pharmaceutically acceptable salts and (multiple) other therapeutic agents, as well as the relative timing of administration, will be selected to achieve the desired combination therapeutic effect. Therefore, on the other hand, combinations comprising the conjugates of this disclosure or their pharmaceutically acceptable salts together with one or more other therapeutic agents are provided.

[0844] The conjugates disclosed herein and their pharmaceutically acceptable salts may be used in combination with one or more other therapeutic agents that can be used to prevent or treat allergic, inflammatory, or autoimmune diseases, such as antigen immunotherapy, antihistamines, steroids, NSAIDs, bronchodilators, methotrexate, leukotriene modifiers, monoclonal antibody therapy, receptor therapy, or antigen nonspecific immunotherapy.

[0845] The conjugates disclosed herein and their pharmaceutically acceptable salts may be used in combination with radiotherapy and / or surgery and / or at least one other therapeutic agent available for the treatment of cancer and precancerous syndromes. Any antitumor agent, antimicrotubule, antimitotic agent, hormone, hormone analog signal transduction pathway inhibitor, protein tyrosine kinase, or antiangiogenic therapeutic agent may be used in the combination. The range of other therapeutic agents is readily apparent to those skilled in the art. In some embodiments, other therapeutic agents are described as in PCT application PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0846] Agents for immunotherapy regimens, therapeutic agents for apoptosis-promoting regimens, or cell cycle signaling inhibitors may also be used in combination with the conjugates disclosed herein.

[0847] In some embodiments, the combinations of this disclosure comprise the conjugates of this disclosure or salts thereof, particularly pharmaceutically acceptable salts, and at least one antitumor agent, antimicrotubule agent, antimitotic agent, hormone, hormone analog signal transduction pathway inhibitor, protein tyrosine kinase, or antiangiogenic therapeutic agent or combination thereof.

[0848] Further examples of other therapeutic agents (e.g., antitumor agents) used in combination with or co-administered with the conjugates of this disclosure or their pharmaceutically acceptable salts are immunomodulators.

[0849] In some embodiments, the combinations disclosed herein comprise the conjugates of the present disclosure or salts thereof, particularly pharmaceutically acceptable salts, and at least one immunomodulator or at least one immunostimulator.

[0850] As used herein, "immunomodulatory agents" refers to any substance including monoclonal antibodies that affect the immune system. Immunomodulatory agents can be used as antitumor agents for the treatment of cancer. For example, immunomodulatory agents include, but are not limited to, anti-CTLA-4 antibodies and anti-PD-1 antibodies. Other immunomodulatory agents include, but are not limited to, ICOS antibodies, OX-40 antibodies, PD-L1 antibodies, LAG3 antibodies, TIM-3 antibodies, 41BB antibodies, and GITR antibodies.

[0851] Further examples of other therapeutic agents (antitumor agents) used in combination with or co-administered with the conjugates of this disclosure are anti-PD-L1 agents (i.e., anti-PD-L1 antibodies) or PD-1 antagonists.

[0852] Therefore, in some embodiments, a method of treating a person in need of it includes administering the conjugate of this disclosure or a salt thereof and at least one immunomodulatory agent. In some embodiments, the immunomodulatory agent is selected from ICOS agonist antibodies, OX-40 antibodies, and PD-1 antibodies. In some embodiments, the person has cancer. This document also provides the use of a combination of the conjugate of this disclosure or a salt thereof with at least one immunomodulatory agent for treating a person in need of it.

[0853] As used herein, "immunostimulant" refers to any agent that can stimulate the immune system. Immunostimulants as used herein include, but are not limited to, vaccine adjuvants (such as Toll-like receptor agonists), T-cell checkpoint blockers (such as mAbs targeting PD-1 and CTL4), and T-cell checkpoint agonists (such as agonist mAbs targeting OX-40 and ICOS).

[0854] 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 adaptive immune responses. In some embodiments, TLRs activate dendritic cells (DCs). Toll-like receptors (TLRs) are a family of pattern recognition receptors initially identified as sensors of the innate immune system that recognize microbial pathogens. TLRs recognize different structures in microorganisms, often referred to as "PAMPs" (pathogen-associated molecular patterns). Ligands binding to TLRs trigger intracellular signaling pathway cascades, inducing the production of factors involved in inflammation and immunity.

[0855] In some embodiments, the immunostimulant used in combination with the conjugates disclosed herein is a TLR4 agonist.

[0856] Therefore, in some embodiments, a method of treating a person in need of it includes administering the conjugate of this disclosure or a salt thereof and at least one immunostimulant. In some embodiments, the immunostimulant is a TLR4 agonist. In some embodiments, the immunostimulant is AGP. In some embodiments, the person has cancer. This document also provides the use of a combination of the conjugate of this disclosure or a salt thereof with at least one immunostimulant for treating a person in need of it.

[0857] In addition to the immunostimulants described above, the compositions disclosed herein may further comprise other therapeutic agents, which, due to their adjuvant properties, can be used to 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), compositions mediated by (NOD)-like receptors (NLRs) to activate innate immunity, retinoic acid-inducible gene (RIG)-based I-like receptors (RLRs), and / or C-type lectin receptors (CLRs).

[0858] Due to their adjuvant properties, TLR agonists can be used in combination with other vaccines, adjuvants, and / or immunomodulators, and in a variety of combinations. In some embodiments, inactivated tumor cells that bind to STING and induce STING-dependent TBKI activation and express and secrete one or more stimulatory DC-induced, recruited, and / or matured cytokines as described herein can be administered together with one or more TLR agonists for therapeutic purposes.

[0859] Other active ingredients (antitumor agents) used in combination with or co-administered with the conjugates disclosed herein are IDO inhibitors.

[0860] In some embodiments, the conjugates disclosed herein may be combined with at least one other therapeutic agent for the prevention or treatment of infectious diseases such as bacterial infections, viral infections, Kaposi's sarcoma-associated herpesvirus infections, TB infections, chlamydia, malaria infections, staphylococcal infections, amyotrophic lateral sclerosis (ALS), multiple sclerosis, systemic lupus erythematosus and related lupus conditions, psoriasis, or Sjögren's syndrome.

[0861] The conjugates disclosed herein can be administered via any suitable route of administration, including both systemic and local administration. Systemic administration includes oral, enteral, transdermal, rectal, and inhalation administration. Enteral administration refers to routes of administration other than enteral, transdermal, or inhalation, and is generally by injection or infusion. Enteral administration includes intravenous, intramuscular, and subcutaneous injection or infusion. Inhalation refers to administration to the patient's lungs, whether through the mouth or nasal passage. Local administration includes application to the skin.

[0862] In addition to the administration routes suitable for treating tumors described above, the pharmaceutical compositions are also suitable for administration via intratumoral or peritumoral injection. It is anticipated that the conjugates of this disclosure will be injected directly into or near a single solid tumor to elicit an immune response that attacks and destroys cancer cells throughout the body, significantly reducing and, in some cases, permanently eliminating tumors in the affected subject. Activating the immune system in this manner to kill tumors at distant sites is commonly referred to as the remote effect and has been demonstrated in animals using various therapeutic modalities. Another advantage of local, intratumoral, or peritumoral administration is the ability to achieve equivalent potency at much lower doses, thereby minimizing or eliminating adverse events that might be observed at much higher systemic doses.

[0863] The conjugates of this disclosure may be administered once or according to a dosing regimen in which multiple doses are administered at different time intervals within a given period of time. For example, the dose may be administered 1, 2, 3, or 4 times daily. The dose may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. A suitable dosing regimen for the conjugates of this disclosure depends on the pharmacokinetic properties of the conjugate, such as absorption, distribution, and half-life, which may be determined by a person skilled in the art. Furthermore, for the conjugates of this disclosure, a suitable dosing regimen, including the duration of administration of such a regimen, depends on the disease or condition being treated, the severity of the disease or condition being treated, the age and physical condition of the patient being treated, the patient's medical history, the nature of concurrent therapies, the desired therapeutic effect, and similar factors within the knowledge and expertise of a person skilled in the art. Such a person skilled in the art should further understand that a suitable dosing regimen may need to be adjusted over time based on the individual patient's response to the dosing regimen or as the individual patient's needs change. The total daily dose is in the range of 1 mg to 2000 mg, preferably in the range of 1 mg to 250 mg.

[0864] For therapeutic use, the conjugates of this disclosure are typically (but not necessarily) formulated into pharmaceutical compositions before being given to a patient. Therefore, this disclosure also relates to pharmaceutical compositions comprising the conjugates of this disclosure and at least one pharmaceutically acceptable excipient.

[0865] The pharmaceutical compositions disclosed herein can be prepared and packaged in bulk or in unit dosage forms. For oral administration, one or more tablets or capsules may be given, for example. Each dose of the pharmaceutical composition contains at least a therapeutically effective amount of the conjugate disclosed herein (i.e., the conjugate disclosed herein or a salt thereof, particularly a pharmaceutically acceptable salt). When prepared in unit dosage forms, the pharmaceutical composition may contain 1 mg to 1000 mg of the conjugate disclosed herein.

[0866] As provided herein, unit dosage forms (pharmaceutical compositions) containing 1 mg to 1000 mg of the conjugates disclosed herein may be administered once, twice, three or four times daily, preferably once, twice or three times daily, and more preferably once or twice daily, to achieve treatment of STING-mediated diseases or conditions.

[0867] The pharmaceutical compositions disclosed herein generally contain one conjugate of the present disclosure. However, in some embodiments, the pharmaceutical compositions disclosed herein contain more than one conjugate of the present disclosure. Additionally, the pharmaceutical compositions disclosed herein may optionally further comprise one or more other therapeutic agents (e.g., pharmaceutically active conjugates).

[0868] As used herein, "pharmaceuticalally acceptable excipient" means a material, component, or medium that is pharmaceutically acceptable in imparting the form or consistency of a pharmaceutical composition. Each excipient, when mixed, must be compatible with the other components of the pharmaceutical composition to avoid interactions that would significantly reduce the potency of the conjugates disclosed herein and interactions that would lead to a pharmaceutically unacceptable pharmaceutical composition when administered to a patient. Furthermore, each excipient must, of course, have sufficiently high purity to be pharmaceutically acceptable.

[0869] The conjugates and pharmaceutically acceptable excipients disclosed herein are generally formulated into dosage forms suitable for administration to patients via the desired route of administration. Conventional dosage forms include those suitable for: (1) oral administration, such as tablets, capsules, sacs, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and flat sachets; (2) parenteral administration, such as sterile solutions, suspensions, and powders for reconstitution; (3) transdermal administration, such as transdermal patches; (4) rectal administration, such as suppositories; (5) inhalation, such as aerosols and solutions; and (6) topical administration, such as creams, ointments, lotions, solutions, pastes, sprays, foams, and gels.

[0870] Suitable pharmaceutically acceptable excipients will vary depending on the specific dosage form chosen. Additionally, suitable pharmaceutically acceptable excipients may be selected for their specific function in the composition. For example, certain pharmaceutically acceptable excipients may be selected to facilitate the production of a uniform dosage form. Certain pharmaceutically acceptable excipients may be selected to facilitate the production of a stable dosage form. Certain pharmaceutically acceptable excipients may be selected to facilitate the delivery or translocation of one or more conjugates of this disclosure from one organ or part of the body to another organ or part of the body once administered to a patient. Certain pharmaceutically acceptable excipients may be selected to enhance patient compliance.

[0871] Suitable pharmaceutically acceptable excipients include the following types: diluents, fillers, binders, disintegrants, lubricants, glidants, granulators, coating agents, wetting agents, solvents, solubilizers, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, thickeners, antioxidants, preservatives, stabilizers, surfactants, and buffers. Those skilled in the art will recognize that some pharmaceutically acceptable excipients can be used for more than one function and for alternative functions, depending on the amount of excipient present in the formulation and the other ingredients present.

[0872] Those skilled in the art possess the knowledge and skills to select appropriate amounts of suitable pharmaceutically acceptable excipients for use in this disclosure. Furthermore, numerous resources are available to those skilled in the art describing pharmaceutically acceptable excipients and can be used to select 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).

[0873] On the one hand, this disclosure relates to solid oral dosage forms, such as tablets or capsules containing an effective amount of the conjugate of this disclosure and a diluent or filler. Oral solid dosage forms may further contain disintegrants or lubricants.

[0874] It should be understood that the conjugates disclosed herein can also be formulated with vaccines as adjuvants to modulate their activity. Such compositions may contain antibodies (multiple antibodies) or antibody fragments or antigenic components, optionally together with one or more other components having adjuvant activity.

[0875] Certain compounds and / or conjugates disclosed herein may be potent immunomodulators, and therefore should be handled with care. Having described this disclosure, the following examples are provided by way of illustration rather than limitation.

[0876] In some implementations, the conjugate is of the following formula BB

[0877]

[0878] The conjugate contains an XMT-1519 antibody, which includes a variable heavy chain complementarity-determining region 1 (CDRH1) containing the amino acid sequence FTFSSYSMN (SEQ ID NO:20), a variable heavy chain complementarity-determining region 2 (CDRH2) containing the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO:21), a variable heavy chain complementarity-determining region 3 (CDRH3) containing the amino acid sequence GGHGYFDL (SEQ ID NO:22), a variable light chain complementarity-determining region 1 (CDRL1) containing the amino acid sequence RASQSVSSSYLA (SEQ ID NO:27), a variable light chain complementarity-determining region 2 (CDRL2) containing the amino acid sequence GASSRAT (SEQ ID NO:28), and a variable light chain complementarity-determining region 3 (CDRL3) containing the amino acid sequence QQYHHSPLT (SEQ ID NO:29). 15 It is approximately 8.

[0879] In some implementations, the conjugate is of the following formula CC

[0880]

[0881] The conjugate contains the XMT-1535 antibody, which contains CDRH1 containing the amino acid sequence GYTFTGYNIH (SEQ ID NO:5), CDRH2 containing the amino acid sequence AIYPGNGDTSYKQKFRG (SEQ ID NO:6), CDRH3 containing the amino acid sequence GETARATFAY (SEQ ID NO:7), and CDRH3 containing the amino acid sequence...

[0882] CDRL1 of SASQDIGNFLN (SEQ ID NO:8) contains an amino acid sequence.

[0883] CDRL2 containing the amino acid sequence YTSSLYS (SEQ ID NO:9), CDRL3 containing the amino acid sequence QQYSKLPLT (SEQ ID NO:10), and d 15 It is approximately 8.

[0884] In some embodiments, the conjugate of formula BB or formula CC is used to treat a disease or condition in a subject who needs it, including administering a therapeutically effective amount of the conjugate of formula BB or formula CC to the subject. In some embodiments, the disease or condition is cancer.

[0885] In some embodiments, for the BB conjugate, 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, the breast cancer is HER2-amplified / overexpressing breast cancer or HER2-low-expressing breast cancer. In some embodiments, the endometrial cancer is serous endometrial carcinoma.

[0886] In some embodiments, the conjugate of formula CC is used to treat NaPi2b-expressing tumors in subjects who require it. In some embodiments, NaPi2b-expressing tumors are ovarian cancer, non-small cell lung cancer (NSCLC), papillary thyroid cancer, endometrial cancer, cholangiocarcinoma, papillary renal cell carcinoma, clear cell renal cell carcinoma, breast cancer, renal cancer, cervical cancer, or salivary gland duct carcinoma.

[0887] In some implementation schemes, the subjects have epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, platinum-resistant ovarian cancer, non-squamous NSCLC, progressive, radioactive iodine-refractory, locally recurrent or metastatic papillary thyroid cancer, or epithelial endometrial cancer.

[0888] In some embodiments, the BB conjugate can be used to treat a disease or condition in a subject who requires it, including administering a therapeutically effective amount of the BB conjugate in combination with one or more therapeutic agents to the subject. In some embodiments, the therapeutic agent is an immunomodulator or immunostimulator. In some embodiments, the immunomodulator is an anti-CTLA-4 antibody, an anti-PD-1 antibody, an ICOS antibody, an OX-40 antibody, a PD-L1 antibody, a LAG3 antibody, a TIM-3 antibody, a 41BB antibody, or a GITR antibody.

[0889] In some embodiments, the BB conjugate can be used to treat a disease or condition in a subject who requires it, including administering a therapeutically effective amount of the BB conjugate to the subject in combination with one or more HER2 antibodies that bind to a HER2 epitope different from that of the HER2 antibody XMT-1519. In some embodiments, the HER2 antibody that binds to a HER2 epitope different from that of the HER2 antibody XMT-1519 is trastuzumab, pertuzumab, Fab37, or chA21.

[0890] Example

[0891] The following examples illustrate this disclosure. These examples are not intended to limit the scope of this disclosure, but rather to provide guidance to those skilled in the art for preparing and using the compounds, compositions, and methods of this disclosure. Although specific embodiments of this disclosure have been described, those skilled in the art will recognize that various changes and modifications can be made without departing from the spirit and scope of this disclosure.

[0892] It should be understood that some of the compounds disclosed herein may be potent immunomodulators, and therefore caution should be exercised when handling them.

[0893] The reactions described herein are applicable to producing reactions with various substituents as defined herein (e.g., R). 1 R 2 The compounds disclosed herein (e.g., [examples of the compounds]). Those skilled in the art will recognize that if a particular substituent is incompatible with the synthetic methods described herein, the substituent can be protected with a suitable protecting group that is stable to the reaction conditions. Suitable protecting groups and methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples can be found in TW Greene's 'Protective Groups in Organic Synthesis' (4th edition, J. Wiley and Sons, 2006). Unless otherwise stated, all starting materials were obtained from commercial suppliers and were ready for use without further purification.

[0894] abbreviation

[0895] The following abbreviations are used in the following reaction schemes and synthetic examples. This list is not intended to be an all-encompassing list of abbreviations used in this application, as other standard abbreviations readily understood by those skilled in the art of organic synthesis may also be used in the synthetic schemes and examples.

[0896] ACN acetonitrile

[0897] CDI 1,1'-carbonyldiimidazole

[0898] DCC N,N'-Dicyclohexylcarbodiimide

[0899] DCM dichloromethane

[0900] DIPEA N,N-Diisopropylethylamine

[0901] DMA dimethylacetamide

[0902] DMF (dimethylformamide)

[0903] DMPA (dimethylolpropionic acid)

[0904] ESI Electrospray Ionization

[0905] HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate HIC hydrophobic interaction chromatography

[0906] HOBt Hydroxybenzotriazole

[0907] HPLC (High-Performance Liquid Chromatography)

[0908] MeOH (methanol)

[0909] SEC size exclusion chromatography

[0910] TFA (trifluoroacetic acid)

[0911] THF Tetrahydrofuran

[0912] PyBOP (benzotriazol-1-yloxy) tripyrrole alkyl phosphonium hexafluorophosphate

[0913] General information

[0914] Unless otherwise stated, all reagents were purchased from the relevant suppliers.

[0915] diABZI STING agonists were prepared as described by Ramanjulu et al. (Nature, 564(7736):439-443(2018)).

[0916] XMT-1535 (anti-NaPi2b antibody) is disclosed in co-pending application US 15 / 457,574, filed March 13, 2017, the entire contents of which are incorporated herein by reference. XMT-1519 (anti-Her2 antibody) is disclosed in US 9,555,112, published January 31, 2017, and US 9,738,720, published August 22, 2017, the entire contents of which are incorporated herein by reference.

[0917] XMT-1535AF-HPA ADC, DAR 5.9 and rituximab AF-HPA ADC, DAR 5.5 were prepared as described in co-pending applications US 62 / 958,916 filed January 9, 2020 and US 63 / 040,735 filed June 18, 2020, the entire contents of which are incorporated herein by reference.

[0918] HPLC purification was performed on a Phenomenex Gemini 5μm C18 micrometer. Performed on a 250x 10mm semi-preparative column.

[0919] When applicable, the drug content of the conjugate is determined by spectrophotometry; otherwise, the drug content is quantitatively determined by RP-HPLC or LC / MS.

[0920] The protein content of antibody-drug conjugates was determined by spectrophotometry or by ELISA.

[0921] As needed, antibody-drug conjugates, drug-loaded scaffolds, or antibody scaffolds are purified by extensive percolation, CHT chromatography, or HIC (i.e., removal of residual unreacted drug, unconjugated antibody, enzyme, or starting material). If necessary, further purification by SEC or HIC is performed to remove aggregated antibody-drug conjugates. Typically, purified antibody-drug conjugates contain <5% (w / w) (e.g., <2% (w / w)) aggregated antibody-drug conjugate as determined by SEC, 0.5% (w / w) (e.g., <0.1% (w / w)) free (unconjugated) drug as determined by RP-HPLC and / or LC-MS / MS, 1% (w / w) free drug conjugate as determined by SEC and / or RP-HPLC, and <10% (w / w) free drug conjugate as determined by HIC-HPLC and / or RP-HPLC.

[0922] (w / w) (e.g., <1% (w / w)) unconjugated antibody or antibody fragments. 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 concentrations (conjugated and unconjugated) were determined by UV-Vis spectrophotometry or RP-HPLC.

[0923] To determine the concentration of free drug in biological samples, acidified samples were treated with acetonitrile. Free drug was extracted, and the acetonitrile supernatant was analyzed. To determine the concentration of conjugated STING agonists in non-clinical samples, samples were immunocaptured using anti-human Fc antibody magnetic beads, followed by thorough alkaline hydrolysis. The acetonitrile supernatant containing the released drug was analyzed by LC-MS / MS. Total antibodies in non-clinical samples were measured using an MSD ECL immunoassay.

[0924] Free drug was analyzed by RP-HPLC using a C-4 column and acetonitrile gradient. The MRM peak area on integrated tandem mass spectrometry was compared with standards for olistatin F (AF) and olistatin F-hydroxypropylamide (AF-HPA). This method quantifies AF-HPA and AF in plasma and tissue homogenates and is linear in the concentration range of 0.1–150 ng / mL. The dynamic range of total drug released after hydrolysis with NaOH was measured under the same conditions, ranging from 1 ng / mL to 5000 ng / mL. The total antibody standard range was 0.009 μg / mL to 20 μg / mL.

[0925] The drug-antibody ratio (DAR) is determined by measuring the absorbance of the conjugate. The DAR value is calculated using the appropriate molar extinction coefficient of the antibody and the effective load of the STING agonist.

[0926] The tumor was measured twice a week using digital calipers, and the tumor volume was calculated using the following formula: Tumor volume (mm)3 ) = (width) 2 x length) / 2. Weight was recorded daily during the first week and twice weekly thereafter. Animals were maintained in the study until a single tumor volume reached ≥1000 mm. 3 ≥1500mm 3 Or as shown. The percentage change in weight is calculated using the following formula: Weight change (%) = (weight) / (weight) 研究第X天 -weight 研究第1天 ) / weight 研究第1天 *100. Tumor volume is reported as mean ± standard error of the mean (SEM). Tumor growth inhibition (%TGI) is defined as the percentage difference in mean tumor volume (MTV) between treatment and control groups. Tumor size is measured throughout each efficacy study to determine tumor growth inhibition (TGI). The percentage of tumor regression is calculated using the following formula: Regression % = (1 - (mean tumor volume) * 100. 最终 ) / (mean tumor volume) 第1天 Partial response (PR) is defined as a tumor volume of 50% or less of the day 1 volume measured in three consecutive measurements, with at least one of these three measurements being equal to or greater than 13.5 mm. 3 Complete remission (CR) is defined as a tumor volume of less than 13.5 mm in three consecutive measurements. 3 At the end of the study, disease-free survivors (TFS) were classified as having complete remission (CR).

[0927] Example 1: Synthesis of XMT-1519 conjugate 8

[0928]

[0929] Part A: DMF (2 mL) was added to a mixture of compound 1 (prepared as described in WO2017175147A1, 0.5 g, 0.64 mmol), Boc-L-alanine (0.242 g, 1.28 mmol), DMAP (7.8 mg, 0.064 mmol), and DCC (0.264 g, 1.28 mmol). The suspension was stirred overnight at room temperature, then the solution was concentrated, and the residue was purified on silica gel (0-40% MeOH in DCM) to give compound 2 (0.52 g, 85% yield) as a pale yellow solid. 46 H 58 N 13 O 10 [M+H] + The calculated value of ESI-MS m / z is 952.4; the measured value is also 952.4.

[0930] Part B: 4N HCl (2 mL, 8.19 mmol) was added to a suspension of compound 2 (0.52 g, 0.55 mmol) in dioxane (10 mL). The reaction mixture was stirred at room temperature for 2 h, then the suspension was concentrated and used in the next step without further purification. Compound 3 was given as a white solid. C 41 H 50 N 13 The calculated m / z value of O8[M+H] by ESI-MS is 852.4; the measured value is 852.3.

[0931] Part C: To a solution of compound 3 (0.586 g, 0.661 mmol) in DMF (5 mL), 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecane-14-acid (0.202 g, 0.727 mmol) and DIPEA (0.230 mL, 1.322 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 5 min, 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 h. Another aliquot of DIPEA (0.460 mL, 2.6 mmol) was added. After another 1 h, the reaction mixture was concentrated to an oil. The residue was purified by silica gel (0-40% MeOH in DCM) to give compound 4 (0.9 g, >95% yield) as a white solid. 53 H 71 N 14 O 13 [M+H] + The calculated value of ESI-MS m / z is 1111.5; the measured value is 1111.5.

[0932] Part D: 4N HCl (3.04 mL, 12.15 mmol) was added to a suspension of compound 4 (0.9 g, 0.810 mmol) in dioxane (10 mL). The reaction mixture was stirred at room temperature for 1.5 h, and the suspension was concentrated to give compound 5 (0.56 g, 66.0% yield) as a colorless solid. C 48 H 63 N 14 O 11 [M+H] + The calculated ESI-MS value is 1011.5; the measured value is 1011.4.

[0933] 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) and compound 5 (72.7 mg, 0.072 mmol) in DMF (2 mL). The reaction mixture was stirred at room temperature for 2 h, then the solution was concentrated, and the residue was purified by preparative HPLC (0-80% CAN in water) to give compound 7 (109 mg, 64% yield). C 103 H 156 N 24 O 41 [M+2H] 2+ The calculated value of ESI-MS m / z is 1192.5; the measured value is 1192.5.

[0934] Part F: TCEP (0.059 mg, 0.207 μmol) was added to a solution of XMT-1519 (10 mg, 0.069 μmol) in 50 mM HEPES, 1 mM EDTA, pH 7 buffer, and the mixture was shaken at 37 °C for 90 min. Compound 7 (0.987 mg, 0.414 μmol in 200 μL DMA) was added to the reduced antibody. The resulting mixture was shaken at 37 °C for 60 min. The reaction was quenched with cysteine ​​(15 equivalents, 0.125 mg, 1.035 μmol in 125 μL of 50 mM HEPES, 1 mM EDTA, pH 7) and rotated at room temperature for 1 h. The resulting conjugate 8 was purified by ultrafiltration or CHT chromatography. Detailed information on antibody-drug conjugates 8-1 and 8-2 is shown below. Conjugates 8-1 and 8-2 were prepared as described, except that the ratio of TCEP:mAb used in the synthesis of 8-2 was higher than that of 8-1 (4:1 vs. 3:1) and the ratio of compound 7 to mAb was higher (8:1 vs. 6:1).

[0935] Conjugate DAR 8-1 6.1 8-2 6.8

[0936] Example 1a: Synthesis of trastuzumab conjugate 8a

[0937]

[0938] Conjugate 8a was prepared and characterized as described in Example 1, except that trastuzumab was used instead of XMT-1519. Detailed information on antibody-drug conjugates 8a-1, 8a-2, and 8a-3 is shown below.

[0939] Conjugate DAR 8a-1 6.0 8a-2 7.0 8a-3 8.4

[0940] Example 1b: Synthesis of XMT-1535 conjugate 8b, DAR 5.7

[0941]

[0942] 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.

[0943] Conjugate DAR 8b-1 5.7 8b-2 6.6 8b-3 6.4

[0944] Example 1c: Synthesis of palizumab conjugate 8c

[0945]

[0946] Conjugate 8c was prepared and characterized as described in Example 1, except that palizumab was used instead of XMT-1519. Details of antibody-drug conjugates 8c-1 and 8c-2 are shown below.

[0947] Conjugate DAR 8b-1 7.5 8b-2 5.8

[0948] Example 1d: Synthesis of palizumab mIgG2a conjugate 8d

[0949]

[0950] Conjugate 8d was prepared and characterized as described in Example 1, except that palizumab mIgG2a was used instead of XMT-1519. Details of antibody-drug conjugates 8d-1, 8d-2, and 8d-3 are shown below.

[0951]

[0952]

[0953] Example 1e: Synthesis of XMT-1535hIgG1-mIgG2a conjugate 8e, DAR 7.0

[0954] Conjugate 8e was prepared and characterized as described in Example 1, except that XMT-1519 was replaced with XMT-1535mIgG2a. The purified conjugate 8e had a STING agonist to XMT-1535hIgG1-mIgG2a ratio of 7.0.

[0955] Example 1f: Synthesis of XMT-1535AAG conjugate 8f, DAR 7.4

[0956]

[0957] Conjugate 8f was prepared and characterized as described in Example 1, except that XMT-1519 was replaced with XMT-1535AAG. The purified conjugate 8f had a STING agonist to XMT-1535AAG ratio of 7.4.

[0958] Example 1g: Synthesis of 8g of target D mIgG2a conjugate, DAR 7.4

[0959]

[0960] The conjugate 8g was prepared and characterized as described in Example 1, except that the target D mIgG2a was used instead of XMT-1519. The purified conjugate 8g had a STING agonist to target D mIgG2a ratio of 7.4.

[0961] Example 1h: Synthesis of target ChIgG1a conjugate 8h, DAR 6.9

[0962]

[0963] The conjugate 8g was prepared and characterized as described in Example 1, except that XMT-1519 was replaced with the target ChIgG1a. The purified conjugate 8g had a STING agonist to target ChIgG1a ratio of 6.9.

[0964] Example 1i: Synthesis of target E mIgG2a conjugate 8i, DAR 10.0

[0965]

[0966] Conjugate 8i was prepared and characterized as described in Example 1, except that the target E mIgG2a was used instead of XMT-1519. The purified conjugate 8i had a STING agonist to target E mIgG2a ratio of 10.0.

[0967] Example 1j: Synthesis of trastuzumab AAG conjugate 8j, DAR 7.0

[0968]

[0969] Conjugate 8j was prepared and characterized as described in Example 1, except that trastuzumab AAG was used instead of XMT-1519.

[0970] Detailed information on conjugates 8j and 8j-1 is shown below.

[0971] Conjugate DAR 8j 7.0 8j-1 4.2

[0972] Example 1k: Synthesis of trastuzumab mIgG2a conjugate 8k, DAR 8.1

[0973]

[0974] Conjugate 8k was prepared and characterized as described in Example 1, except that trastuzumab mIgG2a was used instead of XMT-1519. The purified conjugate 8k had a STING agonist to trastuzumab mIgG2a ratio of 8.1.

[0975] Example 11: Synthesis of XMT-1535 conjugate 8l, DAR 4.7

[0976]

[0977] Conjugate 8l was prepared and characterized as described in Example 1, except that XMT-1519 was replaced by XMT-1535 and 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetic acid was incorporated into the compound structure to replace (3-(2-(2-aminoethoxy)ethoxy)propionyl)-L-alanine. The purified conjugate 8l had a STING agonist to XMT-1535 ratio of 4.7.

[0978] Example 1m: Synthesis of palizumab conjugate 8m, DAR 4.5

[0979]

[0980] Conjugate 8m was prepared and characterized as described in Example 1, except that palizumab was used instead of XMT-1519 and 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetic acid was incorporated into the compound structure instead of (3-(2-(2-aminoethoxy)ethoxy)propionyl)-L-alanine. The purified conjugate 8m had a STING agonist to palizumab ratio of 4.5.

[0981] Example 2: Synthesis of XMT-1519 conjugate 16, DAR 5.3

[0982]

[0983] Part A: HATU (0.196 g, 0.514 mmol) and HOBt (0.079 g, 0.514 mmol) were added to a solution of compound 9 (0.100 g, 0.429 mmol) in THF (5 mL). The reaction mixture was stirred at 0 °C for 10 min, followed by the addition of 2-(benzyloxy)ethyl-1-amine (0.0648 mg, 0.429 mmol) and DIPEA (0.112 mL, 0.643 mmol). The reaction mixture was stirred overnight at room temperature, the solution was concentrated, and the residue was purified on silica gel (0-10% MeOH in DCM) to give compound 10 (0.2 g, 100% yield). C 19 H 30 N₂O₅Na[(M+Na)] + The calculated value of ESI-MS m / z is 389.2; the measured value is also 389.2.

[0984] Part B: The solution of compound 10 (150 mg, 0.409 mmol) in EtOH (10 mL) was degassed with N2, followed by the addition of Pd-C (43.6 mg, 0.409 mmol). The mixture was then degassed with H2. The reaction mixture was stirred overnight at room temperature and H2 (1 atm). The solids were removed by filtration through a diatomaceous earth pad, and the filtrate was concentrated to give compound 11. Crude compound 11 could be used in the next step without further purification. C 12 H 24 N₂O₅Na[(M+Na)] + The calculated m / z value of ESI-MS for 299.2 is 299.2; the measured value is also 299.2.

[0985] Part C: A 100 mL flask containing the residue of compound 11 (128 mg, 0.463 mmol) and N,N-dimethylpyridin-4-amine (11.32 mg, 0.093 mmol) was rinsed 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 min, then 4-nitrophenyl chloroformate (140 mg, 0.695 mmol) was added, and the resulting mixture was stirred at 20 °C for 2 h, and then concentrated to an oil. The residue was purified by silica gel (0-100% ethyl acetate in hexane) to give compound 12 (50 mg, 24% yield) as a white solid. 14 H 19 N3O7[(M-Boc+H)] + The calculated ESI-MS m / z value is 342.2; the measured value is 342.1.

[0986] Part D: DMAP (1.153 mg, 9.44 μmol) in DMF (500 μL) was added to a solution of compound 12 (50 mg, 0.113 mmol) and compound 1 (36.9 mg, 0.047 mmol). The reaction mixture was heated at 80 °C for 3 h, and then compound 12 (50 mg, 0.113 mmol) was added. The reaction mixture was stirred for another 3 h and then cooled to room temperature. The mixture was concentrated, and the residue was purified by silica gel (0-30% MeOH in DCM) to give compound 13 (20 mg, 39% yield) as a white solid. 51 H 67 N 14 O 13 [(M+H)] + The calculated ESI-MS m / z value is 1083.4; the measured value is 1083.5.

[0987] Part E: To a solution of compound 13 (20 mg, 0.047 mmol) in DCM (0.5 mL), TFA (0.1 mL) was added. The reaction mixture was stirred at room temperature for 4 h and then concentrated to give compound 14 (10 mg, 55% yield) as a solid. 46 H 59 N 14 O 11 [M+H] + The calculated ESI-MS m / z value is 983.4; the measured value is 983.5.

[0988] Part F: HATU (4.64 mg, 0.012 mmol), HOAt (1.881 mg, 0.012 mmol), and DIPEA (0.018 mL, 0.102 mmol) were added to a solution of scaffold 6 (14.15 mg, 10.17 μmol) and compound 14 (10 mg, 10.17 μmol) in DMF (1 mL). The reaction mixture was stirred at room temperature for 2 h and then concentrated. The residue was purified by preparative RP HPLC (0-80% CAN in water) to give scaffold 15 (20 mg, 83% yield).

[0989] C 101 H 152 N 24 O 41 [(M+2H) 2+ The calculated ESI-MS m / z value is 1178.7; the measured value is 1178.59.0.

[0990] Part G: XMT-1519 (10 mg, 0.069 μmol) was conjugated to scaffold 15 (0.823 mg, 0.347 μmol in 200 μL DMA) as described in Example 1. The purified conjugate 16 had a STING agonist to XMT-1519 ratio of 5.3.

[0991] Example 3: Synthesis of trastuzumab conjugate 20

[0992]

[0993] 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 h. Then, compound 1a (prepared as described in WO2017175147A1, 100 mg, 128 μmol) and DIPEA (67 μL, 384 μmol) were added, and the reaction was stirred at room temperature for 23 h. The reaction mixture was concentrated, and the residue was chromatographically separated by silica gel (0-20% MeOH-DCM eluent). The product compound 17 (109 mg, 83% yield) was isolated as a yellow foam. 49 H 64 N 15 O 10 [M+H] + The calculated ESI-MS m / z value is 1022.5; the measured value is 1022.4.

[0994] 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 h. The reaction mixture was concentrated and the residue was dried under high vacuum to give compound 18 (103 mg, quantified) as a milky white foam. C 44 H 56 N 15 The calculated m / z value of O8[M+H] by ESI-MS is 922.4; the measured value is also 922.4.

[0995] 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 min. Then, HATU (42 mg, 109 μmol) was added, and the reaction mixture was stirred at room temperature for 20 h. The reaction mixture was then concentrated and purified by reverse-phase chromatography (10–100% ACN-water w / 0.1% HCOOH eluent). Scaffold 19 (35 mg, 18% yield) was separated as a white, fluffy solid. 99H 149 N 25 O 38 [M+2H] 2+ The calculated ESI-MS m / z value is 1148.0; the measured value is 1148.4.

[0996] Part D: Trastuzumab (10 mg, 0.067 μmol) was conjugated to scaffold 19 (1.237 mg, 0.539 μmol in 200 μL DMA) as described in Example 1, and then purified using CHT type II chromatography to obtain conjugate 20. Detailed information on antibody-drug conjugates 20-1 and 20-2 is shown below.

[0997] Conjugate DAR 20-1 6.0 20-2 6.1

[0998] Example 3a: Synthesis of palizumab conjugate 20a, DAR 5.5

[0999]

[1000] Conjugate 20a was prepared and characterized as described in Example 1, except that palizumab was used instead of XMT-1519. The purified conjugate 20a had a STING agonist to palizumab ratio of 5.5.

[1001] Example 4: Synthesis of trastuzumab conjugate 25,DAR 6.6

[1002]

[1003] Part A: To a mixture of compound 21 (prepared as described in US 62 / 982,935, 38 mg, 0.047 mmol) and tert-butyl(S)-(2-hydroxypropyl)carbamate (9.85 mg, 0.056 mmol) in DMF (2 mL), 3-((ethylimino)methylene)amino)-N,N-dimethylpropyl-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) were added. The suspension was then stirred at room temperature for 2 days. The mixture was concentrated to give a residue, which was then purified on silica gel (0-30% MeOH in DCM) to give compound 22 (20 mg, 44% yield) as a pale yellow solid. 47 H 58 N 11 O 12 [M+H] + The calculated ESI-MS m / z value is 968.4; the measured value is 968.3.

[1004] Part B: 4N HCl (0.52 mL, 8.19 mmol) was added to a suspension of compound 22 (20 mg, 0.021 mmol) in dioxane (4 mL). The reaction mixture was stirred at room temperature for 5 h, concentrated, and used in the next step without purification. Product compound 23 (17 mg, 95% yield) was a white solid. C 42 H 50 N 11 O 10 [M+H] + The calculated ESI-MS m / z value is 868.3; the measured value is 868.4.

[1005] Part C: PyBOP (15.3 mg, 0.03 mmol) and DIPEA (0.034 mL, 0.196 mmol) were added to a solution of compound 23 (17 mg, 0.020 mmol) and scaffold 6 (32.1 mg, 0.023 mmol) in DMF (2 mL). The reaction mixture was stirred at room temperature for 2 h, concentrated, and the residue was then purified by preparative RP HPLC (0-80% ACN in water) to give scaffold 24 (26 mg, 59% yield). 97 H 143 N 21 O 40 [M+2H] 2+ The calculated ESI-MS m / z value is 1120.98; the measured value is 1121.06.

[1006] Part D: As described in Example 1, XMT-1519 antibody (5 mg, 0.0347 μmol) was conjugated to scaffold 24 (0.622 mg, 0.278 μmol). The crude reaction mixture was purified by CHT II column chromatography to give conjugate 25 (3.19 mg, 64% yield). The purified conjugate 25 had a STING agonist to XMT-1519 ratio of 6.6.

[1007] Example 5: Synthesis of XMT-1519 conjugate 28, DAR 6.0

[1008]

[1009] Part A: Compound 26 was prepared as described in Example 1, except that compound 1 was replaced with 26 (prepared as described in US62 / 982,935). Compound 27 (71.0 mg, 40% yield) was obtained as a colorless solid. 103 H 154 N 22 O 43[M+2H] 2+ The calculated ESI-MS m / z value is 1193.52; the measured value is 1193.48.

[1010] Part B: Conjugate 28 was prepared as described in Example 1, yielding conjugate 28. The purified conjugate 28 had a STING agonist to XMT-1519 ratio of 6.0.

[1011] Example 6: Synthesis of XMT-1519 conjugate 29, DAR 5.5

[1012]

[1013] Conjugate 28 (6.5 mg) was prepared in PBS (pH 8) using a 30 kDa MWCO ultrafiltration device, followed by three cycles of concentration and dilution. The reconstituted conjugate was then incubated at 37 °C for 24 h and subsequently prepared as trehalose buffer (pH 5.5). After antibody reduction, ring-opening was confirmed by LCMS analysis of the heavy and light chains. Good resolution was observed across the various light chain species: unconjugated, conjugated with intact succinimidyl, and conjugated with ring-opened succinimidyl. Ring-opening was also observed in the corresponding heavy chain species, but the resolution was poorer between species. Therefore, the degree of ring-opening was estimated by focusing on the light chain species. Using this method, the percentage of ring-opened product relative to intact succinimidyl in conjugate 29 was estimated to be 94%. Conjugate 29 had a STING agonist to XMT-1519 ratio of 5.5.

[1014] Example 7: Synthesis of XMT-1519 conjugate 32-1, DAR 6.5

[1015]

[1016] 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 (9 mg, 8% yield) was obtained as a colorless solid. 101 H 151 N 23 O 42 [M+2H] 2+ The calculated ESI-MS m / z value is 1179.02; the measured value is 1179.21.

[1017] Part B: Conjugates 32-1, 32-2, 32-3, and 32-4 were prepared as described in Example 1 to obtain the title conjugates. The ratios of the STING agonist to XMT-1519 in the purified conjugates 32-1, 32-2, 32-3, 32-4, and 32-5 are shown in the table below. Conjugate 32-5 has a mAb concentration of >10 mg / mL, contains <1% unconjugated mAb, and <1% high molecular weight species.

[1018]

[1019]

[1020] Example 7a: Synthesis of XMT-1535 conjugate 32a, DAR 6.2

[1021]

[1022] 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 ratio of STING agonist to XMT-1535 in the purified conjugates 32a, 32a-1, 32a-2, 32a-3, and 32a-4 is shown in the table below.

[1023] Conjugate DAR 32a 6.2 32a-1 7.4 32a-2 8.0 32a-3 7.5 32a-4 8.0

[1024] Example 7b: Synthesis of palizumab conjugate 32b, DAR 6.8

[1025]

[1026] Conjugates 32b, 32b-1, and 32b-2 were prepared and characterized as described in Example 1, except that palizumab was used instead of XMT-1519. The ratio of STING agonist to palizumab in the purified conjugates 32b, 32b-1, and 32b-2 is shown in the table below.

[1027] Conjugate DAR 32b 6.8 32b-1 5.7 32b-2 7.2

[1028] Example 7c: Synthesis of palizumab mIgG2a conjugate 32c, DAR 9.1

[1029]

[1030] Conjugate 32c was prepared and characterized as described in Example 1 above, except that palizumab mIgG2a was used instead of XMT-1519. The purified conjugate 32c had a STING agonist to palizumab ratio of 9.1.

[1031] Example 7d: Synthesis of XMT-1535mIgG2a conjugate 32d, DAR 8.8

[1032]

[1033] Conjugate 32d was prepared and characterized as described in Example 1 above, except that XMT-1535mIgG2a was used instead of XMT-1519. The purified conjugate 32d had a STING agonist to XMT-1535mIgG2a ratio of 8.8.

[1034] Example 7e: Synthesis of XMT-1519AAG conjugate 32e, DAR 7.4

[1035]

[1036] Conjugate 32e was prepared and characterized as described in Example 1, except that XMT-1519AAG was used instead of XMT-1519. The purified conjugate 32e had a STING agonist to XMT-1519AAG ratio of 7.4.

[1037] Example 7-1: Alternative Synthesis of Compound 31

[1038]

[1039] Part A: Compound 30 (prepared as described in US 62 / 982,935) (500 mg, 0.663 mmol, 1 equivalent), Boc-PEG2-Ala-OH (0.693 g, 1.99 mmol, 3 equivalents), EDC-HCl (381 mg, 1.99 mmol, 3 equivalents), and DMAP (243 mg, 1.99 mmol) were mixed in a vial in DMF (26.5 mL) under air. The reaction was completed within 3 hours. The mixture was quenched with AcOH (0.76 mL, 10 equivalents), concentrated, and purified by silica gel (DCM:MeOH) to give compound 30a as a white solid. C 51 H 66 N 13 O 14 [M+H] + The calculated ESI-MS m / z value is 1084.5; the measured value is 1084.4.

[1040] Part B: Compound 30a (0.663 mmol) was suspended in dioxane (10 mL) in a flask under air. HCl (4 M in dioxane, 6 mL) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated to give a white solid. The solid was dissolved in pure water and purified by reversed-phase chromatography (0-25% ACN in water) to give compound 30b (444 mg, 77%) as a white solid. C 46 H 58 N 13 O 12 [M+H] + The calculated ESI-MS m / z value is 984.4; the measured value is 984.2.

[1041] Part C: PyBOP (185 mg, 0.36 mmol) and triethylamine (0.23 mL, 1.62 mmol) were added to a solution of scaffold 6 (450 mg, 0.32 mmol, prepared as described in PCT / US2018 / 06719) and compound 30b (350 mg, 0.072 mmol) in DMF (6.5 mL). The mixture was stirred at room temperature for 15 min and quenched with AcOH (0.23 mL, 3.99 mmol), and purified by reverse-phase purification (0-40% ACN in water w / 0.1% acetic acid) to give compound 31 (408 mg, 55% yield). 101 H 151 N 23 O 42 [M+2H] 2+ The calculated ESI-MS m / z value is 1179.52; the measured value is 1179.27.

[1042] Example 8: Synthesis of trastuzumab conjugate 34,DAR 6.9

[1043]

[1044] Part A: To a solution of compound 1a (prepared as described in WO2017175147A1, 0.030 g, 0.038 mmol) in DMF (1.5 mL), N-ethyl-N-isopropylpropyl-2-amine (0.067 mL, 0.385 mmol) was added. The solution was stirred at room temperature for 5 minutes, and then 2,5-dioxopyrrolidine-1-yl-1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15,18-hexaoxane-21-ester (0.027 g, 0.050 mmol) was added to DMF (0.5 mL), and the reaction mixture was stirred at room temperature for 15 minutes. Then acetic acid (0.1 mL) was added, followed by purification on a preparative HPLC column (C18, 21.2 mm x 100 mm) with a gradient of 10–100% MeCN (0.1% HOAc) in H₂O (0.1% HOAc) for 20 min, yielding scaffold 33 (0.006 g, 13.05% yield). 57 H 75 N 14 O 15 [M+H] + The calculated ESI-MS m / z value is 1195.55; the measured value is 1195.47.

[1045] Part B: Trastuzumab (10 mg, 0.069 μmol) was conjugated to scaffold 33 (0.658 mg, 0.550 μmol in DMA) as described in Example 1. Crude conjugate 34 was purified by CHT type II chromatography. The purified conjugate 34 had a STING agonist to trastuzumab ratio of 6.9.

[1046] Example 8a: Synthesis of palizumab conjugate 34a, DAR 7.0

[1047]

[1048] Conjugate 34a was prepared and characterized as described in Example 8, except that palizumab was used instead of trastuzumab. The purified conjugate 34a had a STING agonist to palizumab ratio of 7.0.

[1049] Example 9: Synthesis of XMT-1519 conjugate 45, DAR 6.5

[1050]

[1051] Part A: 70% HClO4 (302 mg, 2.11 mmol) was added to a mixture of compound 35 (400 mg, 1688 μmol) and tert-butyl acetate (8 mL). The resulting solution was then stirred at room temperature for 21 h and neutralized with saturated NaHCO3 solution. The aqueous phase was washed with EtOAc (2x) and then the combined organic phases were washed with brine, dried (Na2SO4), filtered, and concentrated to give compound 36 (530 mg, quantified) as an opaque oil. C 16 H 24 N1O4[M+H] + The calculated value of ESI-MS m / z is 294.2; the measured value is also 294.2.

[1052] 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 min, then HATU (416 mg, 1095 μmol) was added, and the reaction was stirred at room temperature for 2.5 h. The reaction mixture was then concentrated, and the residue was chromatographically separated by silica gel (20-100% EtOAc / hexane eluent). Compound 38 (254 mg, 362 μmol, 46% yield) was isolated as a white, fluffy solid. C 39 H 48 N₂O₉[M+H] + The calculated ESI-MS m / z value is 702.3; the measured value is 702.4.

[1053] Part C: A mixture of compound 38 (254 mg, 362 μmol), MeOH (6 mL), and 10% Pd-C catalyst was stirred at room temperature under H2 (1 atm) for 2 h. The reaction mixture was then filtered, and the filtrate was concentrated to give compound 39 (214 mg, 97% yield) as a clear oil. 32 H 42 N3O9[M+H] + The calculated value of ESI-MS m / z is 612.3; the measured value is also 612.3.

[1054] Part D: A mixture of compound 39 (58 mg, 64 μmol), compound 40 (prepared as described in US62 / 982,935, 47 mg, 77 μmol), HOAt (10 mg, 77 μmol), DiPEA (56 μL, 320 μL), and DMF (2 mL) was stirred at room temperature for 5 min. Then HATU (36 mg, 96 μmol) was added, and the reaction mixture was stirred at room temperature for 16 h, followed by concentration to give compound 41 (90 mg, quantified) as a yellow oil. C 70 H 83 N 14 O 16 [M+H] + The calculated ESI-MS m / z value is 1375.6; the measured value is 1375.4.

[1055] Part E: A solution of compound 41 (90 mg, 64 μmol) in 20% piperidine in DMF (2 mL) was stirred at room temperature for 1 h. The reaction mixture was then concentrated and chromatographically purified by silica gel (0-40% MeOH-DCM eluent) to give compound 42 (30 mg, 41% yield) as a yellow oil. C 55 H 73 N 14 O 14 The calculated ESI-MS m / z value for [M+H] is 1153.5; the measured value is 1153.4.

[1056] Part F: A solution of compound 42 (30 mg, 37 μmol), 2,5-dioxopyrrolidone-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrolidone-1-yl) acetate (8 mg, 44 μmol), TEA (7 μL, 74 μmol), and DMF (1 mL) was stirred at room temperature for 17 h. The reaction mixture was then concentrated to give scaffold 43 (40 mg, quantified) as a yellow oil. C 61 H 76 N 15 O 17 [M+H] + The calculated ESI-MS m / z value is 1290.6; the measured value is 1290.3.

[1057] Part G: A solution of scaffold 43 (30 mg, 26 μmol) in 10% TFA-DCM (2 mL) was stirred at room temperature for 1 h. The reaction mixture was then concentrated, and the residue was separated by HPLC (10–100% ACN-water w / 0.1% HCOOH eluent). Scaffold 44 (6 mg, 20% yield) was separated as a light-colored, fluffy solid. 52 H 60 N 15O 15 [M+H] + The calculated ESI-MS m / z value is 1134.4; the measured value is 1134.2.

[1058] Part H: Conjugate 45 was prepared as described in Example 1, except that 4 equivalents of TCEP were used. The purified conjugate 45 had a STING agonist to XMT-1519 ratio of 6.5.

[1059] Example 10: Synthesis of XMT-1519 conjugate 50, DAR 8.2

[1060]

[1061] Part A: A mixture of compound 26 (prepared as described in US 62 / 982,935, 50 mg, 64 μmol), Fmoc-D-glutamic acid-O-tert-butyl (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 h. The reaction mixture was concentrated and used in the next step without purification. Compound 46 (135 mg) was obtained as a yellow oil. C 62 H 68 N 11 O 14 [M+H] + The calculated value of ESI-MS m / z is 1190.5; the measured value is 1190.3.

[1062] Part B: A mixture of compound 46 (135 mg, 64 μmol) and 33% TEA in DMF (2.4 mL) was stirred at room temperature for 4.5 h. The reaction mixture was concentrated, and the residue was purified by reversed-phase HPLC (10-100% ACN-water w / 0.1% HCOOH eluent). Compound 47 (27 mg, 44% yield) was isolated as a yellow powder. C 47 H 58 N 11 O 12 [M+H] + The calculated ESI-MS m / z value is 968.4; the measured value is 968.2.

[1063] Part C: A solution of compound 47 (25 mg, 26 μmol), 2,5-dioxopyrrolidone-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrolidone-1-yl) acetate (8 mg, 31 μmol), TEA (11 μL, 78 μmol), and DMF (1 mL) was stirred at room temperature for 1 h. The reaction mixture was concentrated and used in the next step without purification. Scaffold 48 was a yellow oil (40 mg, quantified). C 53 H 61 N 12 O 15 [M+H] + The calculated ESI-MS m / z value is 1105.4; the measured value is 1105.2.

[1064] Part D: A solution of scaffold 48 (40 mg, 24 μmol) and 15% TFA in DCM (1 mL) was stirred at room temperature for 2 h. The reaction mixture was then concentrated and chromatographically separated by HPLC (using 10–100% ACN-water eluent containing 0.1% HCOOH). Scaffold 49 (5 mg, 20% yield) was separated as a white, fluffy solid. C 49 H 53 N 12 O 15 [M+H] + The calculated ESI-MS m / z value is 1049.4; the measured value is 1049.2.

[1065] Part E: XMT-1519 (10 mg, 0.069 μmol) was conjugated to scaffold 49 as described in Example 1. Conjugate 50 was purified by CHT type II chromatography. The purified conjugate 50 had a STING agonist to XMT-1519 ratio of 8.2.

[1066] Example 11: Synthesis of XMT-1519 conjugate 52, DAR 7.7

[1067] 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 purified conjugate 52 had a STING agonist to XMT-1519 ratio of 7.7.

[1068] Example 12: Synthesis of XMT-1519 conjugate 58, DAR 6.5

[1069]

[1070] Part A: DMF (2 mL) was added to a mixture of compound 26 (prepared as described in US 62 / 982,935, 0.105 g, 0.134 mmol), Boc-L-alanine (50.8 mg, 0.268 mmol), DMAP (50.8 mg, 0.067 mmol), and DCC (0.111 g, 0.537 mmol). The suspension was then stirred at room temperature for 2 days. The mixture was concentrated and purified on silica gel (0-40% MeOH in DCM) to give compound 53 (0.102 g, 80% yield) as a pale yellow solid. 46 H 56 N 11 O 12 [M+H] + The calculated value of ESI-MSm / z is 954.4; the measured value is also 954.4.

[1071] Part B: 4N HCl (0.508 mL, 2.031 mmol) was added to a suspension of compound 53 (0.102 g, 0.107 mmol) in dioxane (10 mL). The reaction mixture was stirred at room temperature for 3 h. The suspension was then concentrated and used in the next step without purification. Compound 54 was obtained as a pale yellow solid. C 41 H 48 N 11 O 10 [M+H] + The calculated value of ESI-MS m / z is 854.3; the measured value is also 854.3.

[1072] Part C: To a solution of compound 54 (0.015 g, 0.017 mmol) in DMF (1 mL), Boc-D-Glu(Otu)-OH (7.67 mg, 0.025 mmol) and DIPEA (0.026 mL, 0.152 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 5 min. Then PyBOP (13.15 mg, 0.025 mmol) was added, and the mixture was stirred at room temperature for 1 h. The mixture was concentrated, and the residue was purified by silica gel (0-30% MeOH in DCM) to give compound 55 (11 mg, 57.3% yield) as a white solid. 55 H 71 N 12 O 15 [M+H] + The calculated value of ESI-MS m / z is 1139.5; the measured value is 1139.5.

[1073] Part D: 4N HCl (0.241 mL, 0.966 mmol) was added to a suspension of compound 55 (11 mg, 0.00966 mmol) in dioxane (3 mL). The reaction mixture was stirred at room temperature for 2 h. The suspension was concentrated and used in the next step without purification. Compound 56 was a white solid. C 46 H 55 N 12 O 13 [M+H] + The calculated value of ESI-MS m / z is 983.4; the measured value is 983.4.

[1074] Part E: To a solution of compound 56 (9.5 mg, 0.00966 mmol) in DMF (3 mL), DIEA (8.44 μL, 0.048 mmol) and 2,5-dioxopyrrolidone-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl) acetate (3.17 mg, 0.013 mmol) were added. The reaction mixture was stirred at room temperature for 1 h, neutralized with HOAc to pH 6–7, and then purified by preparative RP HPLC (0–75% ACN in water) to give scaffold 57 (3.3 mg, 31% yield) as a white solid. C 52 H 58 N 13 O 16 [M+H] + The calculated value of ESI-MS m / z is 1120.4; the measured value is 1120.4.

[1075] Part F: XMT-1519 (10 mg, 0.069 μmol) was conjugated to scaffold 57 (0.700 mg, 0.625 μmol in 200 μL DMA) as described in Example 1. Conjugate 58 was purified by CHT type II chromatography. The purified conjugate 58 had a STING agonist to XMT-1519 ratio of 6.5.

[1076] Example 13: Synthesis of XMT-1519 conjugate 60

[1077]

[1078] Conjugate 60 was prepared from scaffold 59 as described in Example 12, except that Boc-D-Glu-O-tBu was used instead of Boc-L-Glu-O-tBu. Details of antibody-drug conjugates 60-1 and 60-2 are shown below.

[1079] Conjugate DAR 60-1 7.7 60-2 5.5

[1080] Example 14: Synthesis of XMT-1519 conjugate 62, DAR 6.5

[1081]

[1082] 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 purified conjugate 62 had a STING agonist to XMT-1519 ratio of 6.5.

[1083] Example 15: Synthesis of XMT-1519 conjugate 64, DAR 6.4

[1084]

[1085] 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 purified conjugate 64 had a STING agonist to XMT-1519 ratio of 6.4.

[1086] Example 16: Synthesis of XMT-1519 conjugate 66

[1087]

[1088] Conjugate 66 was prepared from scaffold 65 as described in Example 12, except that Boc-2-amino-2-methylpropionic acid was used instead of Boc-L-Ala. Details of antibody-drug conjugates 66-1 and 66-2 are shown below.

[1089] Conjugate DAR 66-1 7.5 66-2 5.3

[1090] Example 17: Synthesis of XMT-1519 conjugate 74, DAR 6.9

[1091]

[1092] Part A: A mixture of compound 26 (prepared as described in US 62 / 982,935, 75 mg, 0.096 mmol), N-Boc-(D)-Ala-OH (91 mg, 0.48 mmol), DCC (99 mg, 0.48 mmol), and DMAP (1.2 mg, 9.58 μmol) in DMF (3 mL) was stirred at room temperature for 1 h and then concentrated under vacuum. Purification by silica gel (DCM:MeOH 60:40 v / v) gave compound 67 (82 mg, 90% yield) as a pale yellow solid. 46 H 56 N11 O 12 [M+H] + The calculated value of ESI-MS m / z is 954.40; the measured value is 954.43.

[1093] Part B: HCl (4M in dioxane, 0.42 mL, 1.68 mmol) was added to a suspension of compound 67 (80 mg, 0.084 mmol) in dioxane (5 mL), and the mixture was stirred at room temperature for 4 h. The mixture was then concentrated under vacuum to give compound 68 (72 mg, 100% yield) as a pale yellow solid. C 41 H 48 N 11 O 10 [M+H] + The calculated value of ESI-MS m / z is 854.35; the measured value is 854.38.

[1094] Part C: DIPEA (0.088 mL, 0.51 mmol) was added to 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) in DMF (3 mL), and the mixture was stirred at room temperature for 2 h. The mixture was concentrated, and the residue was purified by silica gel (DCM:MeOH 60:40 v / v) to give compound 69 (53 mg, 93% yield) as a white solid. 48 H 59 N 12 O 13 [M+H] + The calculated value of ESI-MS m / z is 1011.42; the measured value is 1011.45.

[1095] Part D: HCl (4M in dioxane, 1 mL, 20% v / v) was added to a suspension of compound 69 (50 mg, 0.049 mmol) in dioxane (5 mL), and the mixture was stirred at room temperature for 2 h and then concentrated to give compound 70 (45 mg, 100% yield) as a white solid. C 43 H 51 N 12 O 11 [M+H] + The calculated value of ESI-MS m / z is 911.37; the measured value is 911.39.

[1096] Part E: DIPEA (0.03 mL, 0.22 mmol) was added to a stirred solution of compound 70 (20 mg, 0.022 mmol), N-Boc-(D)-Glu(OtBu)-OH (10 mg, 0.033 mmol), and PyBOP (17 mg, 0.033 mmol) in DMF (2 mL), and the mixture was stirred at room temperature for 2 h. The mixture was concentrated, and the residue was purified by silica gel (DCM:MeOH 60:40 v / v) to give compound 71 (24 mg, 90% yield) as a white solid. 57 H 74 N 13 O 16 [M+H] + The calculated value of ESI-MS m / z is 1196.53; the measured value is 1196.55.

[1097] Part F: TFA (1 mL, 20% v / v) was added to the suspension of compound 71 (24 mg, 0.02 mmol) in DCM (5 mL), and the mixture was stirred at room temperature for 12 h. The mixture was concentrated to give compound 72 (21 mg, 100% yield) as a pale yellow solid.

[1098] C 48 H 58 N 13 O 14 [M+H] + The calculated value of ESI-MS m / z is 1040.41; the measured value is 1040.23.

[1099] Part G: To a stirred solution of compound 72 (21 mg, 0.02 mmol) and 2,5-dioxopyrrolidone-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrolidone-1-yl) acetate (7.6 mg, 0.03 mmol) in DMF (2 mL), DIPEA (0.035 mL, 0.20 mmol) was added, and the mixture was stirred at room temperature for 1 h. The mixture was concentrated and purified by RP HPLC residue to give scaffold 73 (4.5 mg, 19% yield) as a white solid. C 54 H 61 N 14 O 17

[1100] [M+H] + The calculated value of ESI-MS m / z is 1177.43; the measured value is 1177.40.

[1101] Part H: Conjugate 74 was prepared from scaffold 73 as described in Example 12. The purified conjugate 74 had a STING agonist to XMT-1519 ratio of 6.9.

[1102] Example 18: Synthesis of XMT-1519 conjugate 76, DAR 7.5

[1103]

[1104] As described in Example 17, conjugate 76 was prepared from scaffold 75, except that N-Boc-(D)-Ala-OH was used instead of N-Boc-(L)-Ala-OH and N-Boc-(D)-Ala-OH was used.

[1105] N-Boc-(L)-Glu(OtBu)-OH replaces N-Boc-(D)-Glu(OtBu)-OH. The purified conjugate 76 has a STING agonist to XMT-1519 ratio of 7.5.

[1106] Example 19: Synthesis of XMT-1519 conjugate 78, DAR 7.4

[1107]

[1108] Conjugate 78 was prepared from scaffold 70 as described in Example 17, except that N-Boc-(D)-Glu(OtBu)-OH was used instead of N-Boc-(D)-Glu(OtBu)-OH. The purified conjugate 78 had a STING agonist to XMT-1519 ratio of 7.4.

[1109] Example 20: Synthesis of XMT-1519 conjugate 80, DAR 7.5

[1110]

[1111] 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 purified conjugate 80 had a STING agonist to XMT-1519 ratio of 7.5.

[1112] Example 21: Synthesis of XMT-1519 conjugate 82, DAR 5.7

[1113]

[1114] 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 purified conjugate 81 had a STING agonist to XMT-1519 ratio of 5.7.

[1115] Example 22: Synthesis of XMT-1519 conjugate 85, DAR 6.5

[1116]

[1117] 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, 0.5% yield, after 5 steps). 101 H 148 N 22 O 42 S[M+2H] 2+ The calculated ESI-MS m / z value is 1187.49; the measured value is 1187.78.

[1118] Part B: Conjugate 85 was prepared as described in Example 1 to obtain the title conjugate. The purified conjugate 85 had a STING agonist to XMT-1519 ratio of 6.5.

[1119] Example 22a: Synthesis of palizumab conjugate 85a, DAR 7.4

[1120]

[1121] Conjugate 85a was prepared and characterized as described in Example 1, except that palizumab was used instead of XMT-1519. The purified conjugate 85a had a STING agonist to palizumab ratio of 7.4.

[1122] Example 23: Synthesis of XMT-1519 conjugate 88, DAR 6.6

[1123]

[1124] Part A: The 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] + The calculated ESI-MS m / z value is 1091.4; the measured value is 1091.2.

[1125] Part B: Conjugate 88 was prepared as described in Example 12, except that scaffold 87 was used instead of scaffold 57. The purified conjugate had a STING agonist to XMT-1519 ratio of 6.6.

[1126] Example 23a: Synthesis of palizumab conjugate 89, DAR 5.9

[1127]

[1128] Conjugate 89 was prepared and characterized as described in Example 12, except that palizumab was used instead of XMT-1519. The purified conjugate 89 had a STING agonist to palizumab ratio of 5.9.

[1129] Example 23b: Synthesis of CTL-48132_mIgG2a conjugate 89a, DAR 8.8

[1130]

[1131] Conjugate 89a was prepared and characterized as described in Example 12, except that CTL-48132_mIgG2a was used instead of XMT-1519. The purified conjugate 89a had a STING agonist to CTL-48132_mIgG2a ratio of 8.8.

[1132] Example 23c: Synthesis of MFP5_mIgG2a conjugate 89b, DAR 9.0

[1133]

[1134] Conjugate 89b was prepared and characterized as described in Example 12, except that CTL-48132_mIgG2a was used instead of XMT-1519. The purified conjugate 89b had a STING agonist to MFP5_mIgG2a ratio of 9.0.

[1135] Example 24: Synthesis of XMT-1519 conjugate 92, DAR 7.6

[1136]

[1137] 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] + The calculated ESI-MS m / z value is 1136.4; the measured value is 1136.2.

[1138] Part B: Conjugate 88 was prepared as described in Example 12, except that scaffold 87 was used instead of scaffold 57. The purified conjugate had a STING agonist to XMT-1519 ratio of 7.6.

[1139] Example 24a: Synthesis of palizumab conjugate 93, DAR 6.7

[1140]

[1141] Conjugate 93 was prepared and characterized as described in Example 12. Except that palizumab was used instead of XMT-1519, the purified conjugate 93 had a ratio of 6.7 between the STING agonist and palizumab.

[1142] Example 25: Synthesis of XMT-1519 conjugate 100, DAR 7.8

[1143]

[1144] Part A: To a stirred solution of compound 94 (prepared as described in US 62 / 982,935, 45 mg, 0.054 mmol) in DMF (3 mL), (S)-1-(Boc-amino)prop-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 was concentrated under vacuum, and the residue was purified by silica gel (DCM:MeOH 60:40 v / v) to give 95 (49 mg, 92% yield) as a white solid. 47 H 57 N 10 O 12 ESI-MS calculated value of S(M+H): 985.38; measured value: 985.21.

[1145] Part B: TFA (1 mL, 20% v / v DCM) was added to a stirred suspension of compound 95 (49 mg, 0.05 mmol) in DCM (5 mL), and the mixture was stirred at room temperature for 12 hours. The resulting mixture was concentrated to give compound 96 (44 mg, 100% yield) as a pale yellow solid. C 42 H 49 N 10 O 10 The calculated value of S(M+H) by ESI-MS is 885.33, and the measured value is 885.18.

[1146] Part C: To a stirred solution of compound 96 (50 mg, 0.05 mmol) 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 vacuum, and the residue was purified by silica gel (DCM:MeOH 60:40 v / v) to give compound 97 (45 mg, 77% yield) as a white solid. 56 H 72 N 11 O 15 ESI-MS calculated value of S(M+H): 1170.49; measured value: 1170.29.

[1147] Part D: TFA (1 mL, 20% v / v DCM) was added to a stirred suspension of compound 97 (45 mg, 0.038 mmol) in DCM (5 mL), and the mixture was stirred at room temperature for 12 hours. The mixture was concentrated under vacuum to give compound 98 (38 mg, 100% yield) as a pale yellow solid. C 47 H 56 N 11 O 13 The calculated value of S(M+H) by ESI-MS is 1014.37, and the measured value is 1014.20.

[1148] Part E: To a stirred solution of compound 98 (20 mg, 0.02 mmol) in DMF (2 mL), 2,5-dioxopyrrolidone-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrolidone-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 min. The reaction was quenched with acetic acid (0.034 mL, 1:1 v / v DIPEA) and purified directly by HPLC using a C18 stationary phase (water:ACN) to give scaffold 99 (4.7 mg, 20% yield) as a white solid. 53 H 59 N 12 O 16 The calculated ESI-MS value of S(M+H) is 1151.38, and the measured value is 1151.18.

[1149] Part F: Conjugate 100 was prepared as described in Example 12, except that scaffold 99 was used instead of scaffold 57, and the purified conjugate had a ratio of 7.8 for STING agonist to XMT-1519.

[1150] Example 25a: Synthesis of palizumab conjugate 101, DAR 6.5

[1151]

[1152] Conjugate 101 was prepared as described in Example 25, except that palizumab was used instead of XMT-1519. The purified conjugate had a STING agonist to palizumab ratio of 6.5.

[1153] Example 26A: Activity of wild-type or Fc mutant STING-ADC targeting cancer cells in cancer cell / THP1 luciferase reporter cell co-culture

[1154] Generation of NaPi2b Fc Silent Antibodies: NaPi2b mAb and its Fc region (anti-NaPi2b-(AAG)) designed to eliminate Fc effector function were engineered to contain three mutations in the heavy chain constant region: L234A, L235A, and P329G (AAG; Kabat Eu designation), and generated using standard molecular biology procedures. Antibodies were expressed and purified. In short, the heavy chain variable region of a NaPi2b antibody encoding the constant region of human IgG1 carrying the L234A, L235A, and P329G mutations, and the light chain variable region of an anti-NaPi2b antibody containing the human κ light chain, were cloned into a mammalian expression vector. The heavy and light chains of NaPi2b-(AAG) were co-expressed in HEK293 cells, and the antibodies were purified from the cell supernatant using standard protein A affinity chromatography.

[1155] Induction of the STING pathway in immune cells: The induction of the STING pathway in immune cells by a NaPi2b-targeting STING ADC was evaluated by co-culturing cancer cells / THP1-IRF3-luciferase reporter cells. OVCAR3 human ovarian cancer cells were seeded in 96-well CellBind surface tissue culture plates (15,000 / well) and allowed to attach for 6 hours in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin. A series of dilutions of the test conjugates 8b-1, 8f, 8d-1, and compound 1 (based on effective loading of 0.4 nM–100 nM; 3-fold serial dilutions in growth medium) were added to each well, and the plates were incubated at 37°C for 20 min. Then, 30,000 THP1-dual reporter cells were added to each well, and the plates were incubated for another 20 hours at 37°C under 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 Molecular Devices plate reader. EC was determined based on the dose-response curve. 50 Values. Table 1A provides EC values ​​for THP1-Dual cells co-cultured with OVCAR3 cancer cells. 50 value.

[1156] Table 1A

[1157] Test sample Conjugate 8b-1 Conjugate 8f Conjugate 8d-1 Compound 1 <![CDATA[EC 50 (nM)]]> <0.05 43.3 41.8 6.8

[1158] As shown in Table 1A, conjugate 8b-1, possessing the wild-type Fc effector function, exhibited more than 100-fold increased activity compared to the free agonist compound 1, and approximately 1000-fold increased activity compared to conjugates 8f and 8d-1, confirming the role of the Fc receptor in activity. The results shown represent the ECGs from representative experiments. 50 value.

[1159] Example 26B: Activity of wild-type or Fc mutant STING-ADCs targeting cancer cells in cancer cell / THP1 luciferase reporter cell co-culture

[1160] Generation of HER-2 Fc silencing antibody: Trastuzumab mAb designed to eliminate Fc effector function and its Fc region (anti-Her2-(AAG)) were designed with three mutations L234A, L235A and P329G (AAG; KabatEu number) in the heavy chain constant region and generated as described in Example 26A.

[1161] Induction of the STING pathway in immune cells: The induction of the STING pathway in immune cells by a Her2-targeting STING ADC was assessed using SKBR3 human breast cancer cells co-cultured with cancer cells / THP1-IRF3-luciferase reporter cells, as described in Example 26A, in combination with test conjugates 8a-2, 8j, 8c-2, and compound 1. Table 1A provides EC50 data for THP1-Dual cells co-cultured with SKBR3 cancer cells. 50 value.

[1162] Table 1B

[1163] Test sample Conjugate 8a-2 Conjugate 8j Conjugate 8c-2 Compound 1 <![CDATA[EC 50 (nM)]]> 0.35 >200 >200 7.4

[1164] As shown in Table 1B, conjugate 8a-2, possessing the wild-type Fc effector function, exhibited approximately 50-fold increased activity compared to the free agonist compound 1, and approximately 1000-fold increased activity compared to conjugates 8j and 8c-2, confirming the role of the Fc receptor in activity. The results shown represent the ECGs from representative experiments. 50 value.

[1165] Example 27A: Activity of wild-type or Fc mutant STING ADC targeting cancer cells in THP1 luciferase reporter cells cultured on plates coated with tumor cell antigens.

[1166] Human NaPi2b-derived peptides (QINVTVPSTANCTSPSLCWTDGIQNWTMKN) were coated onto the surface of each well of a 96-well plate by incubating overnight at 4°C with the peptide (1 μg / mL in PBS). The wells were then washed 1x with PBS-T and blocked by incubation with BSA (3% in PBS-T) at room temperature for 1 hour. After washing with PBS-T (2x), PBS (1x), and growth medium (RPMI 1640, 10% FBS, 1% penicillin / streptomycin, 1x), a series of dilutions of the test samples (conjugate 8b-1, conjugate 8f, conjugate 8c-1, and compound 1) (based on effective loading of 0.4 nM–100 nM; 3-fold serial dilutions in growth medium) were added to each well, and the plate was incubated at 37°C for 20 min. THP1 dual reporter cells (50,000) were added to each well and incubated at 37°C and 5% CO2 for 20 hours. 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). EC5 was determined based on the dose-response curve. 50 value.

[1167] Table 2A provides EC in THP1 twin cells cultured on plates coated with NaPi2b recombinant peptide. 50 value.

[1168] Table 2A

[1169] Test sample Conjugate 8b-1 Conjugate 8f Conjugate 8c-1 Compound 1 <![CDATA[EC 50 (nM)]]> <0.022 >100 18.99 5.18

[1170] As shown in Table 2, conjugate 8b-1, possessing wild-type Fc, exhibited approximately 100-fold increased activity compared to compound 1. Conjugate 8f was inactive, while conjugate 8c-1 showed approximately 1000-fold lower activity compared to conjugate 8b-1, confirming the role of the Fc receptor in activity. The results shown represent ECGs from representative experiments. 50 value.

[1171] Example 27B: Activity of wild-type or Fc mutant STING ADC targeting cancer cells in THP1 luciferase reporter cells cultured on plates coated with tumor cell antigens.

[1172] Human HER2 / ErbB2 protein (His-labeled, ECD, domain IV, 17.1 kDa) derived peptides were coated onto the surface of each well of a 96-well plate by incubating overnight at 4°C with the peptides (1 μg / mL in PBS) and assayed as described in Example 27A, except that 3-fold serial dilutions (based on effective loading of 0.09 nM–200 nM) of the test conjugates 8a-3, 8j, 8c-2, and compound 1 were used. Table 2B provides EC2 levels in THP1 twin cells cultured on plates coated with the Her2 recombinant protein. 50 value.

[1173] Table 2B

[1174] Test sample Conjugate 8a-3 Conjugate 8j Conjugate 8c-2 Compound 1 <![CDATA[EC 50 (nM)]]> 0.36 >200 >200 8.8

[1175] As shown in Table 2B, conjugate 8a-3, possessing wild-type Fc, exhibited approximately 100-fold increased activity compared to compound 1. Conjugates 8j and 8c-2 were inactive, confirming the role of the Fc receptor in activity. The results shown represent ECGs from representative experiments. 50 value.

[1176] Example 28A: Activity of NaPi2b ADC targeting tumor cells in cancer cell / PBMC co-culture

[1177] By using NucLight red Lentivirus reagent was used to transduce OVCAR3 human ovarian cancer cells that stably expressed nuclear-restricted mKate fluorescent red protein. Stably transduced cells (named OVCAR3-NucRed cells) selected after 2 days in puromycin-containing medium (2 μg / mL) were seeded into 96-well tissue culture plates (8,000 / well) and allowed to attach overnight in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin. The medium was then replaced with fresh medium (50 μL). The test sample (3x concentrate, conjugate 8b-1 (100, 10, and 1 nM), conjugate 8f (100, 10, and 1 nM), conjugate 8c-1 (100 and 10 nM), and compound 1 (100 and 10 nM; conjugate concentrations based on payload) was then added to each well (50 μL), and the plates were incubated at 37°C for 20 minutes. Thaw frozen human peripheral blood mononuclear cells (PBMCs) according to the supplier's instructions and add them to each well (40,000 PBMCs in 50 μL of culture medium), and place the plate in an incubator (37°C, 5% O2). The cells were scanned using a live-cell imaging system, every 4 hours for 2 days. Zoom software quantifies the number of red objects (cancer cells). The red object blending degree in each well is normalized to its own red object blending degree at time T=0.

[1178] Figure 1A A graph showing the degree of red inclusions as a function of time was plotted, demonstrating that PBMC, compared to compound 1, strongly induced cancer cell killing in response to conjugate 8b-1 at a lower effective load concentration of 100x. Conjugate 8f also exhibited activity, although at a lower level compared to conjugate 8b. The activity of conjugate 8c-1 was significantly lower than that of conjugate 8b-1.

[1179] Example 28B: Activity of NaPi2b ADC targeting tumor cells in cancer cell / PBMC co-culture

[1180] OVCAR3-NucRed cells were seeded in 96-well tissue culture plates (20,000 / well) and allowed to adhere for 6 hours in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin. The medium was replaced with fresh medium (50 μL). Then, the test sample (3x concentrate, conjugate 8l and compound 1 (each at 100, 10 and 1 nM) and conjugate 8m (100 nM); conjugate concentration based on effective load) was added to each well (50 μL). Assays were performed as described in Example 28A, except that 30,000 PBMCs were used. Zoom software quantifies the number of red objects (cancer cells). The number of red objects in each well is normalized to the number of red objects at its own time point T=0.

[1181] Figure 1B A graph was plotted showing the change in the number of red objects over time, demonstrating that PBMCs, compared to compound 1, strongly induced cancer cell killing in response to conjugate 8l at a lower effective load concentration of 100x. Conjugate 8m showed no significant activity, and the increase in the number of red objects (cell growth) over time was similar to that of the untreated control. The inset shows that neither the test sample (100 nM) inhibited the growth of OVCAR3-NucRed cells in monoculture.

[1182] Example 29: Flow cytometry analysis of CD14, Fcγ receptor and CD3 expression in PBMCs and isolated monocyte subsets

[1183] Frozen human PBMCs (1x10 8 Thawed cells were aliquoted into three tubes: one aliquot for enrichment of human monocytes [StemCell Technologies] (“CD16-depleted monocytes”), one aliquot for enrichment of human monocytes without CD16 depletion [StemCell Technologies] (“Enriched monocytes”), and one aliquot without enrichment (“PBMCs”). For flow cytometry, cells (50,000) from each group were transferred to U-bottom 96-well plates, repeated four times, washed with PBS, and stained with live / dead fixable Aqua dead cell staining dye (molecular probe), followed by staining with fluorophore-conjugated target-specific (triple) or allotype control antibodies (Pacific Blue anti-human CD14, FITC anti-human CD3, APC / Cy7CD16, PE anti-human CD32, PE / Cy7 anti-human CD64). Cells were fixed and the surface expression of the target protein was determined by flow cytometry on a MACSQuant flow cytometer. Data were analyzed using FlowJo software. Table 3 provides the frequencies (per single cell / living 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.

[1184] Table 3

[1185]

[1186] Gating on a single / living cell

[1187] Table 3 shows the effective depletion of CD3+ cells and the enrichment of monocytes, as well as the decreased level of CD16-positive cells in CD16-depleted monocytes after isolation. CD64 staining results indicate that all CD14-positive cells express CD64 (FcγRI) in PBMCs and in the enriched monocyte subsets.

[1188] Figure 2 The study showed effective depletion of CD3+ cells and enrichment of monocytes, as well as a decrease in the level of CD14-positive cells in CD16-depleted monocytes after isolation.

[1189] Example 30: Cancer cell killing activity of Her2 ADC targeting Fc mutant tumor cells in in vitro co-culture of PBMC and STING wild-type or knockout SKBR3 cells.

[1190] Generation of STING knockout single-cell clones expressing nuclear-restricted mKate fluorescent protein: SKBR3 cells were seeded in 24-well plates (50,000 / well) and processed using the TrueGuide from Thermo Fisher according to the manufacturer's protocol. TM Synthetic gRNA, TrueCut TM Cas9 protein v2 and Lipofectamine TM CRISPRMAX TM Transfection was performed using non-targeting sgRNA and three different sgRNAs targeting the human STING gene. The sgRNA sequences were: sgNT (non-targeting): AAAUGUGAGAUCAGAGUAAU; sg#3: TACTCCCTCCCAAATGCGGT; sg#4:

[1191] CTCGCAGGCACTGAACATCC; and sg#5:

[1192] GTTAAACGGGGTCTGCAGCC. Seven days after transfection, single cells were screened in 96-well plates containing 100 μL of DMEM (containing 20% ​​FBS and 1% penicillin / streptomycin), and clones formed within 2–3 weeks (medium was changed 1–2 times weekly). Multiple clones were trypsinized and amplified by Western blotting using rabbit monoclonal anti-STING (Cell Signaling Technologies) and anti-β-actin (Licor) antibodies to analyze STING expression. Clones without STING protein expression (e.g., as determined by Western blotting) were selected as described in Example 28 to stably express nuclear-restricted mKate fluorescent protein.

[1193] Kill assay: STING wild-type (sgNT-2: non-targeting sgRNA, clone 2) and NucRed-expressing knockout (sg#3-2: sgRNA#3 clone 2) SKBR3 cells were seeded in 96-well plates in RPMI containing 10% FBS and 1% penicillin / streptomycin (15,000 / well), and PBMC kill assays were performed using conjugates 8a-3, 8j, 8c-2, and compound 1 at 100, 25, 5, and 1 nM (conjugate concentration based on payload) as described in Example 29.

[1194] Figure 3A and Figure 3B A graph was plotted showing the degree of red conjugation over time, and the killing effects of co-culturing STING wild-type (sgNT-2) SKBR3 cells / PBMCs and knockout (sg#3-2) SKBR3 cells / PBMCs were displayed separately. At all tested doses, conjugate 8a-3 induced strong killing in both STING wild-type and knockout SKBR3 cell / PBMC co-cultures. In STING wild-type (sgNT-2) SKBR3 cell / PBMC co-culture, conjugate 8a-3 exhibited high activity at 100, 25, and 5 nM, while activity was low in STING knockout (sg#3-2) SKBR3 cell / PBMC co-culture. Compound 1 showed activity only at 100 nM in STING wild-type (sgNT-2) SKBR3 cell / PBMC co-culture, but no activity at all doses in STING knockout (sg#3-2) SKBR3 cell / PBMC co-culture. The conjugate 8c-2 showed no activity in either STING wild-type (sgNT-2) SKBR3 cells / PBMC co-culture or STING knockout (sg#3-2) SKBR3 cells / PBMC co-culture.

[1195] Example 30A: Activity of Her2 ADC and Her2 antibody targeting tumor cells in co-culture of STING wild-type or STING knockout SKBR3 cancer cells / PBMCs

[1196] STING wild-type (sgNT-2: non-targeting sgRNA, clone 2) and NucRed-expressing knockout SKBR3 cells (sgRNA#3-2: sgRNA#3 clone 2) were co-cultured with PBMCs, and killing assays were performed using the dose ranges of conjugate 8a-3 and conjugate 8-j (based on a 200 nM effective load, 4x dilution) as described in Example 30. Unconjugated wild-type Fc trastuzumab and AAG Fc mutant trastuzumab were administered at antibody concentrations corresponding to the concentrations of conjugate 8a-3. Figure 4A and Figure 4BAs shown, conjugate 8a-3 exhibited strong killing activity against both wild-type and knockout SKBR3 cancer cells, while the ADC conjugate 8-j targeting the Fc mutant Her2 showed killing activity only in co-culture with wild-type STING SKBR3 cells, almost disappearing in co-culture with knockout SKBR3 cells. Both unconjugated trastuzumab antibodies against Fc wild-type and AAG mutant cells showed low activity in co-culture with both wild-type and knockout STING cancer cells. These data suggest that the cancer cell killing activity of STING-ADC targeting Fc mutant cancer cells in immune cell co-culture is contributed by the intrinsic STING activation of tumor cells.

[1197] Example 31: Activity of NaPi2bADC targeting tumor cells in the absence of T cells in cancer cell / human monocyte co-culture

[1198] OVCAR3-NucRed cells (generated as described in Example 28) were seeded in 96-well CellBind surface tissue culture plates (Corning) (15,000 / well) and allowed to attach for 6 hours in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin. The medium was replaced with fresh medium (50 μL). Then, the test samples (conjugate 8b-1 and compound 1, each at 20 and 4 nM, conjugate concentration based on effective load concentration) were added to the medium in each well (50 μL), and the plates were incubated at 37°C for 20 minutes. PBMCs, enriched monocytes, and CD16-depleted monocytes were prepared as described in Example 29. Then, live PBMCs (30,000 / well), enriched monocytes (20,000 / well), and CD16-depleted monocytes (20,000 / well) were added to the culture medium (50 μL) in the wells, and the plates were placed in an incubator (37°C, 5% O2). The cells were scanned using a live-cell imaging system, every 4 hours for 2 days. Zoom software quantifies the number of red objects (cancer cells). The red object blending density in each well is normalized to its own red object blending density at time T=0. The change in red object blending density over time is plotted on... Figures 5A-5C Furthermore, it was shown that CD3+ T cells depleted monocyte populations exhibited considerable cancer cell killing activity in response to ADC activity targeting tumor cells. The conjugate 8b-1 induced PBMCs ( ) at effective loading concentrations of 4 nM and 20 nM. Figure 5A ), enriched monocytes ( Figure 5B ) and CD16-depleted monocytes ( Figure 5CCompound 1 exhibits strong killing effects on OVCAR3-NucRed cancer cells. At 20 nM, compound 1 induces cancer cell killing only in co-cultures of enriched monocytes and CD16-depleted monocytes.

[1199] Example 32: In vitro measurement of binding of human CXCL10 and HER2-targeting antibody-drug conjugates to HCC1954 human breast cancer cell line.

[1200] HCC1954 breast cancer cells were grown to approximately 80-95% confluence in RPMI 1640 medium supplemented with FBS (10%) and penicillin / streptomycin (1%). Cells were harvested and added to the wells of 96-well flat-bottom plates (40,000 / well) and incubated overnight at 37°C and 5% CO2. Cells were treated with the HER2 antibody-drug conjugate (20 μL) at a concentration ranging from 1 pM to 10 μM as shown in Table 4 and incubated at 37°C and 5% CO2 for 24 hours. The plates were centrifuged (300 g, 5 min), and the supernatant (100 mL) was collected and analyzed by ELISA for human CXCL10 (human CXCL10 / IP-10). The results were obtained on a SpectraMax M5 plate reader at OD500. 450 Read the developing plate. Plot the values ​​for each treatment and calculate EC using four-parameter curve fitting with GraphPad Prism software. 50 value.

[1201] To determine the cell binding of the HER2 antibody-drug conjugate to HCC1954 cells, HCC1954 cells were grown to approximately 80-95% confluence in RPMI 1640 medium supplemented with FBS (10%) and penicillin / streptomycin (1%). Cells were harvested and added to the wells of a 96-well V-plate (50,000 / well). The cell pellet (300 x g, 5 min) was resuspended in the HER2 antibody-drug conjugate test solution at a concentration ranging from 0.01 nM to 100 nM as shown in Table 4, and incubated on ice for 3 hours. Cells were then washed in ice-cold PBS (3x), precipitated (300 g, 5 min), and incubated with the detection antibody (goat anti-human IgG-Alexa-647 (H+L chain)) at 4°C for 1 h. The cell suspension was precipitated (300 g, 5 min), washed three times in ice-cold PBS, and fixed by resuspending in paraformaldehyde solution (2%). The resuspended cells were then analyzed by flow cytometry on a MACS Quant flow cytometer. Single events (10,000) were collected for analysis. Population gating and mean fluorescence intensity (MFI) analysis were performed using FlowJo software.

[1202] Table 4 summarizes the mean EC50 values ​​induced by cell binding and CXCL10 in HCC1954. 50 value.

[1203] Table 4

[1204] Test sample DAR <![CDATA[CXCL10 ELISA EC 50 (nM)]]> <![CDATA[Cell-binding EC 50 (nM)]]> Conjugate 25 6.6 0.13 ND Conjugate 45 6.5 0.17 ND Conjugate 8-2 6.8 0.51 ND Conjugate 28 6 0.16 1.0 Conjugate 29 5.5 0.18 ND Conjugate 62 6.5 0.20 ND Conjugate 64 6.4 0.22 ND Conjugate 58 6.5 2.3 ND Conjugate 74 6.9 3.4 ND Conjugate 78 7.4 2.7 ND Conjugate 28 6 0.16 1.0 Conjugate 82 5.7 0.17 ND Conjugate 52 7.7 0.12 2.3 Conjugate 50-1 7.7 0.13 ND Conjugate 66-2 5.3 1.38 ND Conjugate 32-1 6.5 1.23 2.4

[1205] ND = Not measured

[1206] As shown in Table 4, treatment of HCC1954 cells with an antibody-drug conjugate targeting Her2 resulted in CXCL10-induced sub-nanomolar to low-nanomolar EC50. 50 Value. Under certain conditions, the EC2 binding of Her2-targeting ADCs to HCC2954 cells. 50 The value is also in the low nanomolar range.

[1207] Example 33: HER2 antibody-drug conjugates targeting tumor cells in cancer cells

[1208] Activity in PBMC co-culture

[1209] Cancer cell killing activity: SKBR3 human breast cancer cells stably expressing nuclear-restricted mKate fluorescent protein were generated as described in Example 28A and named SKBR3NucRed cells. 20,000 SKBR3NucRed cells (per well) were seeded in 96-well tissue culture plates and allowed to attach for 6 hours in 50 μL of RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin. A series of dilutions (based on effective loading of 0.1 nM–200 nM; 4-fold serial dilutions in growth medium) of 50 μL of the test samples (conjugates 8a-2, 8-j, 8c-2, and compound 1) were added to each well, and the plates were incubated at 37°C for 20 min. Then, 50,000 PBMCs or primary human mononuclear cells (isolated from PBMCs as described in Example 29) were added to each well, and assays were performed as described in Example 28. Table 5 shows the IC50 values ​​of the test sample in co-culture of cancer cells / PBMCs and isolated primary human mononuclear cells. 50 Value (killing activity) (cancer cell killing activity).

[1210] Table 5

[1211] Test sample Conjugate 8a-2 Conjugate 8-j Conjugate 8c-2 Compound 1 <![CDATA[IC 50 (nM)-PBMC]]> 0.03 0.91 NA 9.51 <![CDATA[IC 50 (nM)-Monocytes]]> 0.05 1.6 NA 8.32

[1212] As shown in Table 5, conjugate 8a-2 exhibited approximately 300x and 150x higher potency compared to compound 1 in PBMC and monocyte co-culture, respectively. Conjugate 8-j showed approximately 30x and 80x lower potency compared to conjugate 8a-2 in PBMC and monocyte co-culture, respectively. Conjugate 8c-2 exhibited the lowest activity in both PBMC and monocyte co-culture.

[1213] CXCL10 induction: Table 6 shows the EC50 induced by CXCL10 in co-culture of cancer cells / PBMCs and isolated primary human mononuclear cells. 50 value.

[1214] Table 6

[1215] Test sample Conjugate 8a-2 Conjugate 8-j Conjugate 8c-2 Compound 1 <![CDATA[EC 50 (nM)-PBMC]]> 0.06 2.35 NA 26.13 <![CDATA[EC 50 (nM)-Monocytes]]> 0.04 2.05 NA 31.10

[1216] As shown in Table 6, conjugate 8a-2 exhibited approximately 400x and 700x higher CXCL10 induction potency compared to compound 1 in both PBMC and monocyte co-culture, respectively. Conjugate 8-j showed approximately 50x lower potency compared to conjugate 8a-2 in both PBMC and monocyte co-culture. Conjugate 8c-2 exhibited the lowest activity in both PBMC and monocyte co-culture.

[1217] Type III IFN induction: SKBR3 cells and PBMCs were co-cultured as described above to analyze the induction of type III interferon in the supernatant 24 hours after treatment using a human IL29 / IL28b ELISA kit. Table 7 shows the EC50 of IL29 / IL28b induction (IFNλ1 / λ3) in cancer cell / PBMC co-culture. 50 value.

[1218] Table 7

[1219] Test sample Conjugate 8a-2 Conjugate 8-j Conjugate 8c-2 Compound 1 <![CDATA[EC 50 (nM)-PBMC]]> 0.08 0.54 NA 28.30

[1220] As shown in Table 7, conjugate 8a-2 exhibited approximately 400x higher IL29 / IL28b induction potency compared to compound 1 in PBMC co-culture. Conjugate 8-j showed approximately 6x lower potency compared to conjugate 8a-2 in PBMC co-culture. Conjugate 8c-2 exhibited the lowest activity in PBMC co-culture.

[1221] The data in Tables 7-9 indicate that mutations in the Fc region of ADCs that eliminate FcγR interactions reduce but do not eliminate the cancer cell killing activity targeting the ADC, thus demonstrating the Fc-independent contribution of the ADC.

[1222] Example 34: Induction of the STING pathway in immune cells

[1223] The induction of the STING pathway in immune cells by Her2-targeting STINGADCs was evaluated using a co-culture assay of cancer cells / THP1-IRF3-luciferase reporter cells. SKOV3 human ovarian adenocarcinoma cells were seeded in 96-well CellBind surface tissue culture plates (20,000 / well) and allowed to attach for 6 hours in McCoy's 5a medium containing 10% FBS and 1% penicillin / streptomycin. A series of dilutions of the test conjugates 32-5, 32e, and 30 (based on effective loading of 0.01 nM–300 nM; 4-fold serial dilutions in growth medium) were added to each well, and the plates were incubated at 37°C for 20 min. Then, 50,000 THP1-dual reporter cells were added to each well, and the plates were incubated for another 20 hours at 37°C under a humidified atmosphere of 5% CO2. Cell culture supernatant (20 μL) from each incubation sample was added to resuspended QUANTI-Luc (50 μL), and IRF3 luminescence signal was immediately measured using a SpectraMax M5 Molecular Devices plate reader. EC was determined based on dose-response curves. 50 Values. Table 8 provides EC values ​​for THP1 double cells co-cultured with SKOV3 cancer cells. 50 value.

[1224] Table 8

[1225] Test sample Conjugate 32-5 Conjugate 32e Compound 30 <![CDATA[EC 50 (nM)]]> 0.78 NA 38.51

[1226] As shown in Table 8, co-culturing SKOV3 and THP-1 with Her2-targeting ADCs resulted in sub-nanomolar to low-nanomolar ECGs induced by the STING pathway in THP-1 immune cells. 50 value.

[1227] Example 35: Activity of HER2 antibody-drug conjugate targeting tumor cells in cancer cell / PBMC co-culture

[1228] CXCL10 induction: Calu3 human lung adenocarcinoma cells were seeded into 96-well tissue culture plates and allowed to adhere overnight in 100 μL of EMEM medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2. The medium was replaced with fresh medium (100 μL). A series of dilutions (based on effective loading of 0.1 nM–200 nM; 4-fold serial dilutions in growth medium) of 50 μL of the test samples (conjugate 32-4, compound 30, and conjugate 32b-1) were added to each well and assayed as described in Example 33 – CXCL10 induction. Table 9 shows the EC50 induced by CXCL10 in cancer cell / PBMC and cancer cell monocultures. 50value.

[1229] Table 9

[1230] Test sample Conjugate 32-4 Compound 30 Conjugate 32b-1 <![CDATA[EC 50 (nM)-PBMC]]> 0.06 160.20 NA

[1231] As shown in Table 9, conjugate 32-4 exhibited sub-nanomolar potency compared to compound 30 in both cancer cell / PBMC co-culture and monoculture. Conjugate 32b-1 showed the lowest activity in both cancer cell / PBMC co-culture and monoculture.

[1232] Example 36: Activation of the STING pathway in single culture of cancer cells in the presence of conditioned medium from untreated immune cell cultures.

[1233] Conditioned culture medium was prepared by incubating in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% O2 for 1.5 x 10⁻⁶ days. 6 / mL PBMC (two different donors), 1x 10 6 / mL primary mononuclear cells (isolated from PBMCs from two different donors, as described in Example 29) or 1 x 10 6 SKOV3 cells were prepared by culturing THP1 cells at 24 hours per mL. The supernatant was collected and cells were removed by rotating at 2000 rpm for 10 minutes. SKOV3 cells were seeded into 96-well plates (30,000 / well) and allowed to adhere overnight in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin. The medium was replaced with 100 μL / well of fresh medium (control) or conditioned medium 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, CXCL10 production in the supernatant from the 96-well plates was analyzed using a human CXCL10 ELISA kit. Table 10 shows the OD450 values ​​of CXCL10 produced by single culture of SKOV3 cancer cells.

[1234] Table 10

[1235]

[1236] As shown in Table 10, SKOV3 cells in monoculture responded to STING agonist treatment only in the presence of conditioned medium from PBMCs and primary human monocytes, but not from THP1 cells, indicating that PBMCs or primary human monocytes secrete factors that can sensitize cancer cells to STING agonist treatment.

[1237] Example 37: Tumor growth in response to administration of HER2 antibody-drug conjugate in SKOV3

[1238] Female CB.17SCID mice were subcutaneously inoculated with SKOV3 human ovarian cancer cells (10 x 10^10 cells per mouse). 6 (cells). When the tumor volume is 63-75mm... 3 Between (average = 65.4 mm) 3 Animals were randomly assigned to treatment groups (n=10 per group). On day 1, a single intravenous dose of the following mediators was administered: 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) (all doses are given via antibody / load). Transient weight loss within acceptable limits was observed 2–3 days after treatment with diABZI STING agonist (0 / 5 mg / kg) and conjugate 8c-1 (3 / 0.12 mg / kg), with no further clinical observations. Weight loss at later time points was associated with tumor progression, indicating cachexia induced by the tumor model.

[1239] Figure 6 Tumor volume results are provided for SKOV3 tumor-bearing mice treated with trastuzumab, diABZI STING agonist, conjugate 8c-1, conjugate 8a-1, or a mediator. 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, or 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.

[1240] Example 38: Serum cytokines in SKOV3 after administration of HER2 antibody-drug conjugate

[1241] Female CB.17SCID mice were subcutaneously inoculated with SKOV3 human ovarian cancer cells (10 x 10^10 cells per mouse). 6 (cells). When the tumor volume is between 108-172 mm 3 Between (average)

[1242] =128-131.6mm 3Animals were randomly assigned to treatment groups. On day 1, the mediator, diABZISTING agonist (0 / 5 mg / kg), conjugate 8c-1 (3 / 0.12 mg / kg), or conjugate 8a-1 (3 / 0.09 mg / kg) were administered intravenously (all doses were given as antibody / load, n=10 per group). Serum was collected at 6, 12, 24, and 72 hours post-administration (n=5 per group) and flash-frozen on dry ice for serum cytokine analysis. Tumors were collected at 12 and 72 hours post-administration (n=5 at each time point) and processed into formalin-fixed paraffin-embedded (FFPE) blocks.

[1243] 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.

[1244] Figures 7A-7H Provides measurements of cytokines over time. Only cytokines showing a significant increase relative to the mediator control (CXCL-10 (IP-10), IL-6, TNFα, IFNγ, CXCL1 (KC), MIG, MIP-1α, and RANTES) are shown. Insets in each figure show cytokine levels induced by conjugates 8a-1 and 8c-1 relative to the mediator. Intravenously administered diABZI STING agonists induced significantly higher serum cytokine levels than conjugates 8a-1 or 8c-1, with IL-6 showing a fold increase of up to 100-fold, while the difference in CXCL10 was as low as 6-fold. For most cytokines, there were no significant differences between the control and the targeted ADC, and few cytokines showed differences between the mediator control and the ADC. The Y-axis scale in the main figure and insets emphasizes the differences between the diABZI STING agonist and the remaining treatments in this study.

[1245] Example 39A: PD response after administration of HER2 antibody-drug conjugate in SKOV3

[1246] Six-week-old female CB.17SCID mice were subcutaneously inoculated with SKOV3 human ovarian cancer cells (10 x 103 cells per mouse).6 (cells). When the tumor volume is 75-126 mm 3 Between (average)

[1247] =94.5-96.7mm 3 Animals were randomly assigned to treatment groups. On day 1, the mediator, conjugate 8c-1 (3 / 0.12 mg / kg), or 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) were administered intravenously (all doses are given as antibody / load, n=12 per group). Serum was collected at 6, 12, 24, and 72 hours post-administration (n=6 per group) and flash-frozen on dry ice for serum cytokine analysis (data not shown). Tumors were collected at 12 and 72 hours (n=6 per group) and fixed in FFPE blocks for further processing.

[1248] For real-time qPCR analysis, RNA was extracted from FFPE blocks using the Qiagen Rneasy FFPE kit. Based on nanodroplet readings and sample equilibration using cDNA generated with Thermofisher SuperScript IV VILO Master Mix, exDNase enzyme gene expression assays for mouse CXCL10, interferon-β, and IL-6 were established using TaqMan FastAdvanced Master Mix. ABI assays for mouse interferon-β (IFNβ), IL-6, and CXCL10 were performed in conjunction with GAPDH and RPL30 as housekeeping genes. 2 -ΔΔCT The method calculates mRNA levels relative to GAPDH.

[1249] Figures 8A-8C The levels of CXCL10, interferon-β, and IL-6 mRNA in SKOV3 tumors in mice, measured at 12 and 72 hours, are provided. Treatment with conjugate 8a-1 resulted in the highest levels of CXCL10, interferon-β, and IL-6 mRNA at 12 hours relative to either the mediator or conjugate 8c-1. For conjugate 8a-1, the levels of CXCL10, interferon-β, and IL-6 mRNA all decreased at 72 hours.

[1250] Figure 9 Immunohistochemical (IHC) staining of FFPE tumor tissue sections with rabbit anti-CD45 monoclonal antibody at 12 and 72 hours with conjugate 8a-1, conjugate 8c-1, or the mediator is presented. As shown, at 72 hours after treatment with conjugate 8a-1, the infiltration of CD45-positive mouse immune cells into SKOV3 tumors in SCID mice was increased.

[1251] Example 39B: SKOV3 human tumor xenografts in SCID mice respond to STING pathway gene expression in HER2 antibody-drug conjugates.

[1252] Female CB.17SCID mice were subcutaneously inoculated with SKOV3 human ovarian cancer cells and treated with conjugate 8a-1 (3 / 0.09 mg / kg) as described in Example 38. Tumors were harvested and processed into FFPE as described in Example 31. RNA was extracted using the Qiagen Rneasy FFPE kit according to the kit instructions. 150 ng of RNA from each sample was analyzed on the NanoString nCounter Max system using the nCounter pan-cancer human or mouse immunoassay panel and the nCounter StandardMaster kit. Table 11 shows the log2 fold change of selected STING pathway genes at 12 hours.

[1253] Table 11

[1254]

[1255] As shown in Table 11, a single administration of conjugate 8a-1 resulted in significant induction of STING pathway genes in both mice and humans, indicating that tumor-targeting STING agonists ADC can induce tumor-inherent STING pathway activation in vivo.

[1256] Example 40: Pharmacokinetic analysis of Her-2-targeting ADCs in CB17 SCID mice

[1257] Ten-week-old female CB.17SCID mice were administered the medium or conjugate 8a-1 intravenously as a single dose on day 1 (3 / 0.1 mg / kg) (dose given as antibody / load, n=3 per group). Blood was collected serially from all animals at 1, 24, 48, 72, 96, 168, 240, and 336 hours post-treatment (n=3 per group). Whole blood was immediately diluted 1:10 with acidic buffer (0.6% BSA (w / v), 5 mM EDTA in 100 mL PBS + 15.34 mL 10 mg / mL citrate) to a total volume of 0.1 mL. The diluted whole blood was flash-frozen on dry ice and stored at -80°C until analysis of total antibody and conjugate drug.

[1258] Figure 10Results for circulating plasma concentrations of total antibody and conjugated drug are provided. Total antibody and conjugated drug reached plasma concentrations of approximately 25.1 μg / mL and approximately 0.46 μg / mL, respectively, with corresponding clearance rates of approximately 6.71 mL / day / kg for total antibody and approximately 20.2 mL / day / kg for conjugated drug.

[1259] Similarly, conjugate 32-3 (1.14 / 0.04 mg / kg) was administered, and total antibody and conjugate were evaluated at 0.25, 24, 72, 168, 240, and 336 hours. Data are shown in Table 12.

[1260] Table 12

[1261] Cmax (ng / mL) Half-life (days) AUC∞ (days * ng / mL) Cl_obs(mL / day / Kg) Conjugate drugs 562±16 6.43±1.15 2880±468 14.2±2.51 Total antibodies 12200±1160 8.69±1.20 96300±7190 11.9±0.88

[1262] Example 41: STING pathway gene expression in response to Her2-targeted STING agonist ADC therapy in SKBR3 cancer cells / PBMC co-culture.

[1263] Nanostring analysis: Cancer cells (250,000 cells / well) were seeded in duplicate in 0.75 mL of RPMI-1640 medium (containing 10% FBS and 1% penicillin / streptomycin) in 12-well plates and allowed to attach overnight at 37°C in a 5% CO2 incubator. The medium was removed, and conjugate 8a-3 or the medium was added to 0.5 mL of medium in each well to a final concentration of 50 nM (based on payload). 500,000 PBMCs were added to 0.5 mL of medium in each well. After incubation at 37°C for 5 hours, the suspended cells were collected in Eppendorf tubes and briefly twisted. The supernatant was removed, and the tubes were placed on ice. The attached cells were lysed with RNA lysis buffer and transferred to a cell suspension pellet. RNA was extracted using the Qiagen Rneasy mini kit according to the kit instructions. Each samp...

Claims

1. Antibody-drug conjugates (ADCs) or their pharmaceutically acceptable salts, for The stereocenters indicated by * can exhibit different stereochemistry, and the HER2 antibody comprises a variable heavy chain complementarity-determining region 1 (CDRH1) with the amino acid sequence FTFSSYSMN (SEQ ID NO:20), a variable heavy chain complementarity-determining region 2 (CDRH2) with the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO:21), and a variable heavy chain complementarity-determining region 3 (CDRH3) with the amino acid sequence GGHGYFDL (SEQ ID NO:22); a variable light chain complementarity-determining region 1 (CDRL1) with the amino acid sequence RASQSVSSSYLA (SEQ ID NO:27), a variable light chain complementarity-determining region 2 (CDRL2) with the amino acid sequence GASSRAT (SEQ ID NO:28), and a variable light chain complementarity-determining region 3 (CDRL3) with the amino acid sequence QQYHHSPLT (SEQ ID NO:29), and d 15 It represents an integer in the range of 1-20.

2. Antibody-drug conjugates (ADCs) or their pharmaceutically acceptable salts, for ， The HER2 antibody comprises a variable heavy chain complementarity-determining region 1 (CDRH1) with the amino acid sequence FTFSSYSMN (SEQ ID NO:20), a variable heavy chain complementarity-determining region 2 (CDRH2) with the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO:21), and a variable heavy chain complementarity-determining region 3 (CDRH3) with the amino acid sequence GGHGYFDL (SEQ ID NO:22); a variable light chain complementarity-determining region 1 (CDRL1) with the amino acid sequence RASQSVSSSYLA (SEQ ID NO:27), a variable light chain complementarity-determining region 2 (CDRL2) with the amino acid sequence GASSRAT (SEQ ID NO:28), and a variable light chain complementarity-determining region 3 (CDRL3) with the amino acid sequence QQYHHSPLT (SEQ ID NO:29), and d 15 It represents an integer in the range of 1-20.

3. An antibody-drug conjugate having the following formula (I) or a pharmaceutically acceptable salt thereof: PBRM-[A 1 -(L C )-D] d15 (I) in: A 1 for Where * indicates attachment to PBRM and ** indicates attachment to L C ; L C for Where # indicates attachment to A 1 And ## indicates attachment to D; M A for Where * indicates attachment to A 1 ** indicates that it is attached to T 1 , while *** indicates attachment to L D ; L D The formula is ***-NH-(CH2CH2O)2-(CH2)2-C(O)-(alanine)****, where *** indicates attachment to M. A **** represents attachment to D, where L D The stereocenter of alanine can exhibit different stereochemical properties; T 1 for Where n4 is 8; D is Where R 2 It is -O-, and Indicates attachment to L D PBRM is a HER2 antibody containing a variable heavy chain complementarity-determining region (CDRH1) with the amino acid sequence FTFSSYSMN (SEQ ID NO:20), a variable heavy chain complementarity-determining region (CDRH2) with the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO:21), and a variable heavy chain complementarity-determining region (CDRH3) with the amino acid sequence GGHGYFDL (SEQ ID NO:22); a variable light chain complementarity-determining region (CDRL1) with the amino acid sequence RASQSVSSSYLA (SEQ ID NO:27), a variable light chain complementarity-determining region (CDRL2) with the amino acid sequence GASSRAT (SEQ ID NO:28), and a variable light chain complementarity-determining region (CDRL3) with the amino acid sequence QQYHHSPLT (SEQ ID NO:29), and d 15 It represents an integer in the range of 1-20.

4. The conjugate of claim 3 or a pharmaceutically acceptable salt thereof, wherein L D for Where *** indicates attachment to M A ; while *** indicates attachment to D.

5. A conjugate of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, wherein the HER2 antibody comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:17 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:

24.

6. A conjugate of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, wherein the HER2 antibody comprises a heavy chain of the amino acid sequence shown in SEQ ID NO:19 and a light chain of the amino acid sequence shown in SEQ ID NO:

26.

7. The conjugate of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, wherein d 15 It represents an integer in the range of 2-14.

8. The conjugate of claim 7 or a pharmaceutically acceptable salt thereof, wherein d 15 It represents an integer in the range of 2-8.

9. The conjugate of claim 8 or a pharmaceutically acceptable salt thereof, wherein d 15 It can be 2 or 4.

10. The conjugate of claim 7 or a pharmaceutically acceptable salt thereof, wherein d 15 It represents an integer in the range of 6-10.

11. The conjugate of claim 10 or a pharmaceutically acceptable salt thereof, wherein d 15 It is 6.

12. The conjugate of claim 10 or a pharmaceutically acceptable salt thereof, wherein d 15 It is 7.

13. The conjugate of claim 10 or a pharmaceutically acceptable salt thereof, wherein d 15 It is 8.

14. A pharmaceutical composition comprising a conjugate of any one of claims 1-13 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or excipients.

15. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition is administered in combination with at least one immunomodulator or at least one immunostimulant.

16. The use of any conjugate of claims 1-13 or a pharmaceutically acceptable salt thereof, or the use of the pharmaceutical composition of claim 14 or 15 in the manufacture of a medicament for treating a subject’s cancer.

17. The use of claim 16, wherein the cancer is breast cancer, lung cancer, or ovarian cancer.

18. The use of claim 16, wherein the cancer is breast cancer or lung cancer.

19. The use of claim 16, wherein the cancer is breast cancer.

Images