B7H4 targeting antibody-drug conjugate and method of use

Abstract

This provides a therapeutic approach that targets the biological activity of B7-H4. [Solution] This disclosure generally relates to antibody-drug conjugates comprising monoclonal antibodies that specifically bind to human B7-H4 in a soluble form or a membrane-bound state (i.e., when expressed on the cell surface), and to methods for using these conjugates as therapeutic and / or diagnostic agents.

Description

[Technical Field]

[0001] Related applications This application claims priority and benefits of U.S. Provisional Application No. 63 / 133,707, filed on 4 January 2021, and U.S. Provisional Application No. 63 / 172,968, filed on 9 April 2021. The contents of each of these applications are incorporated herein by reference in their entirety.

[0002] Inclusion by referencing the sequence list The contents of the text file named "MRSN-034_001WO_SeqList.txt", created on January 3, 2022, with a size of 118KB, are incorporated in their entirety into this specification by reference. [Background technology]

[0003] background B7-H4, also known as B7x, B7S1, B7S1, and VTCN1, is a type I transmembrane protein and a member of the B7 superfamily of proteins that provides co-signaling in conjunction with T cell receptor antigenic signaling. B7-H4 is a negative regulator of T cell function; T cell ligation inhibits their proliferation, cytokine secretion, and cytotoxicity. In mice, elimination of B7-H4 does not affect immune cell homeostasis and does not result in signs of autoimmunity. The receptor for B7-H4 is unknown and has not been identified.

[0004] Human B7-H4 is mapped to chromosome 1 and consists of 66kb of six exons and five introns, of which exon 6 is used for alternative splicing to generate two different transcripts. Human B7-H4 is a 282-amino acid protein (including an amino-terminal signal sequence), of which 227 amino acids are predicted to be in extracellular space after cleavage of the amino-terminal signal sequence. B7-H4 contains an Ig-like V domain, an Ig-like C domain, a transmembrane domain, and a short cytoplasmic tail.

[0005] While B7-H4 expression is relatively limited at the protein level in healthy tissues, it is consistently overexpressed in several solid tumors, including gynecological cancers of the breast, ovary, and endometrium. B7-H4 expression within tumors tends to correlate with poor prognosis. The receptor for B7-H4 is unknown, but it is thought to be expressed on T cells. B7-H4 is thought to directly inhibit T cell activity.

[0006] A wide variety of treatments are available for advanced cancer, including radiotherapy, conventional chemotherapy with cytotoxic antitumor agents, hormone therapy (aromatase inhibitors, luteinizing hormone-releasing hormone analogs), bisphosphonates, and signaling inhibitors. Unfortunately, however, many patients do not respond well to any of these treatments, or do not respond at all. Therefore, there is a need to identify new therapeutic agents that target the biological activity of B7-H4.

[0007] Therefore, therapeutic approaches that target the biological activity of B7-H4 are needed. [Overview of the project]

[0008] In some aspects, this disclosure includes a variable heavy chain complementarity determination region 1 (CDRH1) containing the amino acid sequence GFIVSRNY (SEQ ID NO:2), a variable heavy chain complementarity determination region 2 (CDRH2) containing the amino acid sequence IYGSGRT (SEQ ID NO:3), and an amino acid sequence TIFF2026082984000002.tif4128 or amino acid sequence Variable heavy chain complementarity determination region 3 (CDRH3) containing TIFF2026082984000003.tif4128, variable light chain complementarity determination region 1 (CDRL1) containing amino acid sequence QSVSSSY (SEQ ID NO: 53), variable light chain complementarity determination region 2 (CDRL2) containing amino acid sequence GAS (SEQ ID NO: 54), amino acid sequence This provides an isolated antibody that specifically binds to B7-H4 and contains a variable light chain complementarity determination region 3 (CDRL3) including TIFF2026082984000004.tif4128.

[0009] In some cases, the isolated antibody contains a heavy chain variable sequence containing the amino acid sequence of SEQ ID NO: 44 and a light chain variable sequence containing the amino acid sequence of SEQ ID NO: 50. In some cases, the isolated antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 45 and a light chain containing the amino acid sequence of SEQ ID NO: 52. In some cases, the isolated antibody contains a heavy chain variable sequence containing the amino acid sequence of SEQ ID NO: 22 and a light chain variable sequence containing the amino acid sequence of SEQ ID NO: 50. In some cases, the isolated antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 23 and a light chain containing the amino acid sequence of SEQ ID NO: 52.

[0010] In some cases, the isolated antibody is a monoclonal antibody. In some cases, the isolated antibody is a rabbit monoclonal antibody, a mouse monoclonal antibody, a chimeric monoclonal antibody, a humanized monoclonal antibody, or a fully human monoclonal antibody. In some cases, the isolated antibody is an IgG isotype. In some cases, the isolated antibody is an IgG1 isotype.

[0011] In some aspects, the isolated antibody contains variable heavy chain complementarity determination region 1 (CDRH1) containing the amino acid sequence GFIVSRNY (SEQ ID NO:2), variable heavy chain complementarity determination region 2 (CDRH2) containing the amino acid sequence IYGSGRT (SEQ ID NO:3), and amino acid sequence Variable heavy chain complementarity determination region 3 (CDRH3) containing TIFF2026082984000005.tif4128, variable light chain complementarity determination region 1 (CDRL1) containing amino acid sequence QSVSSSY (SEQ ID NO: 53), variable light chain complementarity determination region 2 (CDRL2) containing amino acid sequence GAS (SEQ ID NO: 54), amino acid sequence The isolated antibody containing variable light chain complementarity determination region 3 (CDRL3), including TIFF2026082984000006.tif4128, competes for specific binding to human B7-H4. In some aspects, the isolated antibody competes for specific binding to human B7-H4 with the isolated antibody containing a heavy chain variable sequence containing the amino acid sequence of SEQ ID NO:44 and a light chain variable sequence containing the amino acid sequence of SEQ ID NO:50, or with the isolated antibody containing a heavy chain containing the amino acid sequence of SEQ ID NO:45 and a light chain containing the amino acid sequence of SEQ ID NO:52.

[0012] In some aspects, the present disclosure provides a B7-H4 antibody-drug conjugate comprising the isolated antibody of the present disclosure.

[0013] In some cases, one or more linker-drug portions are covalently bonded to the targeting portion. Each linker-drug portion, A polyfunctional linker that connects a targeting portion to one or more drug units (e.g., one or more therapeutic agents (D)) via a releaseable assembly unit for each drug unit, and connects hydrophilic groups to drug units of each linker-drug portion. Includes, The releaseable assembly unit is capable of releasing free drug in close proximity to the target site targeted by the targeting portion, and The polyfunctional linker includes a peptide moiety between the targeting moiety and the hydrophilic group, and the peptide moiety contains at least two amino acids.

[0014] In some aspects, this disclosure provides a conjugate selected from either one of the conjugates in Table A1 or Table A2.

[0015] In some aspects, this disclosure provides a conjugate that can be selected from either the conjugates in Table B1 or Table B2.

[0016] In some aspects, the Disclosure provides a method for treating or preventing a disease or disorder in a subject, comprising the step of administering a therapeutically effective dose of the conjugate of the Disclosure to a subject in need.

[0017] In some aspects, this disclosure provides a conjugate for use in treating or preventing a disease or disorder in which such treatment is necessary.

[0018] In some contexts, this disclosure provides the use of the conjugates of this disclosure in the manufacture of a pharmaceutical product for treating a disease or disorder in which there is a need. In some contexts, this disclosure provides the use of the conjugates of this disclosure for treating or preventing a disease or disorder in which there is a need.

[0019] In some aspects, in any one of the embodiments of this disclosure, the method, conjugate, or use, the conjugate releases one or more therapeutic agents upon biodegradation.

[0020] In some aspects, in any one method, conjugate, or use of any aspect of this disclosure, the disease or disorder is cancer. In some aspects, in any one method, conjugate, or use of any aspect of this disclosure, the cancer is B7-H4 positive cancer.

[0021] In some aspects, in any one of the embodiments of this disclosure, by method, conjugate, or use, B7-H4 positive cancers are selected from the group consisting of bile duct carcinoma, breast cancer, endometrial cancer, ovarian cancer, non-small cell lung cancer, small cell lung cancer, uterine cancer, thyroid cancer, renal cancer, head and neck cancer, gastric cancer, melanoma, bile duct carcinoma, cholangial carcinoma, pancreatic cancer, colon cancer, and bladder cancer.

[0022] In some situations, the subject is human beings.

[0023] In some aspects, this disclosure further includes the administration of a therapeutic agent to a subject. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 is a graph showing different glycoforms of antibody glycans (G0, G1, G2, G0F, GIF, G2F, and M5). [Figure 2] Figure 2 is a scheme showing the deglycosylation of a mixture of glycoforms G0, G1, G2, G0F, GIF, G2F, and M5 in the presence of endoglycosidase. [Figure 3] Figure 3 is a scheme illustrating the process for preparing an azide-modified antibody, in which an intermediate antibody containing a terminal GlcNAc moiety is reacted with an azide-modified UDP-GalNAc derivative molecule in the presence of glycosyltransferase. [Figure 4] Figure 4 is a scheme illustrating one aspect of the process for preparing azide-modified antibodies. [Figure 5] Figure 5 is a scheme illustrating one aspect of the process for preparing an antibody-drug conjugate in which an azide-modified antibody is conjugated to a linker-drug moiety containing a strained cycloalkynyl group. [Figure 6] Figure 6 is a graph showing modified antibodies. [Figure 7] Figure 7 shows the MX-1 This graph shows the antitumor effects of B7-H4_2F9 cytotoxic drug conjugates in a TNBC xenograft mouse model: Conjugate 9-1 (2.46 / 0.075, or 4.92 / 0.150 mg / kg), Conjugate 11 (2.28 / 0.075, or 4.56 / 0.150 mg / kg), Conjugate 3 (2.30 / 0.075, or 4.60 / 0.150 mg / kg), Conjugate 6 (2.30 / 0.075, or 4.61 / 0.150 mg / kg), Conjugate 7 (2.30 / 0.075, or 4.60 / 0.150 mg / kg), and Conjugate 1-1 (2.30 / 0.075, or 4.60 / 0.150 mg / kg) (all doses are given by antibody / payload). [Figure 8]Figure 8 shows B7-H4_2F9 cytotoxic drug conjugates in the MX-1 TNBC xenograft mouse model: Conjugate 14 (2.57 / 0.150 mg / kg), Conjugate 9-2 (4.56 / 0.150 mg / kg), Conjugate 10 (14.37 / 0.150 mg / kg), Conjugate 12 (2.26 / 0.150, or 0.75 / 0.050 mg / kg), Conjugate 1-2 (5.37 / 0.177, 2.33 / 0.077, or 1.79 / 0.059 mg / kg), Conjugate 2-1 (13.45 / 0.150, 4.60 / 0.050, or 2.30 / 0.025 mg / kg) and XMT-1604. This graph shows the antitumor effect of B7-H4_2F9V18 (13.80 / 0 mg / kg) (the dose is given by the antibody / payload). [Figure 9] Figure 9 is a graph showing the antitumor effects of B7-H4_2F9 cytotoxic drug conjugates in an HBCx-19 patient-derived xenograft model: conjugate 1-2 (1.79 / 0.059, or 5.37 / 0.177 mg / kg), conjugate 9-2 (4.56 / 0.150 mg / kg), conjugate 2-1 (4.60 / 0.050, or 13.45 / 0.150 mg / kg), and conjugate 10 (14.37 / 0.150 mg / kg) (all doses are given by antibody / payload). [Figure 10] Figure 10 shows B7-H4_2F9 cytotoxic drug conjugates in an HBCx-24 patient-derived xenograft model: Conjugate 14 (2.57 / 0.150 mg / kg), Conjugate 9-2 (4.56 / 0.150 mg / kg), Conjugate 10 (14.37 / 0.150 mg / kg), Conjugate 12 (2.26 / 0.150, or 0.75 / 0.050 mg / kg). This graph shows the antitumor effects of Conjugate 1-2 (4.56 / 0.150, 2.30 / 0.076, or 1.52 / 0.05 mg / kg), Conjugate 2-1 (13.45 / 0.150, 4.60 / 0.050, or 2.30 / 0.025 mg / kg), and XMT-1604 (13.80 / 0 mg / kg) (all doses are given by antibody / payload). [Figure 11] Figure 11 is a graph showing the antitumor effects of B7-H4_2F9 STING agonist drug conjugates in an MX-1 xenograft mouse model: conjugate 17 (0.085 / 0.030, or 2.84 / 0.100 mg / kg), conjugate 16 (0.89 / 0.030, or 2.97 / 0.100 mg / kg), conjugate 15 (0.85 / 0.030, or 2.83 / 0.100 mg / kg), and diABZI IV STING agonist (5 mg / kg) (all doses are given by antibody / payload). [Figure 12] Figure 12 is a graph showing the antitumor effects of B7-H4_2F9 STING agonist drug conjugates in an MX-1 TNBC xenograft model: Conjugate 14 (2.57 / 0.150 mg / kg), Conjugate 9-2 (4.56 / 0.150 mg / kg), Conjugate 12 (2.26 / 0.150, or 1.13 / 0.075 mg / kg), Conjugate 1-3 (4.68 / 0.150, or 2.34 / 0.075 mg / kg), and Conjugate 2-2 (13.81 / 0.150, or 6.90 / 0.075 mg / kg) (all doses are expressed as antibody / payload). [Figure 13] Figure 13 shows the effects of conjugate 1-3, ordered by median best response (MBR), classified by receptor status, for a set of unselected xenograft models derived from TNBC and ER-positive breast cancer patients. The Y-axis shows the MBR achieved by each model, and the X-axis identifies the model ID. [Figure 14] Figure 14 shows protein expression in a subset of xenograft models derived from TNBC and ER-positive breast cancer patients in Example 44, as evaluated by the TPS score. [Modes for carrying out the invention]

[0025] Detailed explanation The present invention provides monoclonal antibodies that specifically bind to human B7-H4 in a soluble form or a membrane-bound state (i.e., when expressed on the cell surface). The present invention further provides monoclonal antibodies that specifically bind to B7-H4. These antibodies are collectively referred to herein as "B7-H4" antibodies.

[0026] definition The chemical names provided herein for intermediate compounds and / or compounds of the Disclosure may refer to any one of the tautomeristic expressions of such compounds (in some examples, such alternative names are provided experimentally). Any reference to a named compound (intermediate compound, or compound of the Disclosure) or a structurally represented compound (intermediate compound, or compound of the Disclosure) is intended to encompass all tautomeristic forms, including the zwitterionic form of such compound and any mixture thereof.

[0027] It should be understood that the terms “in some aspects,” “in some aspects of this disclosure,” and “in some aspects of the compounds of this disclosure” may be used interchangeably where appropriate.

[0028] The terms “about,” “approximately,” or “nearly,” when used in relation to a number, mean that a set or range of values ​​is included. In some aspects, “about X” includes a range of values ​​that are ±25%, ±20%, ±15%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where X is a number. In some aspects, the term “about” refers to a range of values ​​that are 5% higher or lower than a given value. In some aspects, the term “about” refers to a range of values ​​that are 2% higher or lower than a given value. In some aspects, the term “about” refers to a range of values ​​that are 1% higher or lower than a given value.

[0029] The enumeration of value ranges is intended solely as a convenient way to refer individually to each distinct value that falls within that range, unless otherwise specified herein, and each distinct value is incorporated herein as if it were individually enumerated herein. The ranges used herein, unless otherwise specified, include two limits to that range. In some aspects, the expressions “x being an integer between 1 and 6” and “x being an integer of 1 to 6” both mean “x is 1, 2, 3, 4, 5, or 6,” that is, the terms “X~Y” and “the range of X~Y” include X and Y as well as integers between them.

[0030] When used in accordance with this disclosure, the following terms shall be understood to have the following meanings unless otherwise specified:

[0031] As used herein, the terms “anti-B7-H4 antibody,” “B7-H4 antibody,” and “antibody that binds to B7-H4” refer to an antibody that can bind to B7-H4 with sufficient affinity to be useful as a diagnostic and / or therapeutic agent in that the antibody targets B7-H4.

[0032] As used herein, the term "B7-H4" refers to any naturally occurring mature B7-H4 resulting from the processing of B7-H4 precursor proteins within cells. Unless otherwise specified, the term includes B7-H4 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring variants of B7-H4, such as splice variants or allele variants.

[0033] The term "B7-H4 positive cancer" refers to cancers that contain cells expressing B7-H4 on their surface. In some embodiments, B7-H4 expression on the cell surface is determined using antibodies against B7-H4 by methods such as immunohistochemical testing or FACS.

[0034] Alternatively, B7-H4 mRNA expression is thought to correlate with B7-H4 expression on the cell surface and can be determined by a method selected from in-situ hybridization and RT-PCR (including quantitative RT-PCR).

[0035] The term "B7-H4 positive cell" refers to a cell that expresses B7-H4 on its surface.

[0036] As used herein, the term “antibody” is used in its broadest sense and encompasses a wide range of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, without limitation, as long as they exhibit the desired antigen-binding activity. Various methods for numbering the amino acid sequences of antibodies and identifying complementarity-determining regions are known in the art. For example, the Kabat numbering system (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)) or the IMGT numbering system (IMGT®, international ImMunoGeneTics information system®; available online: http: / / www.imgt.org / ). The IMGT numbering system is commonly used and accepted in the art as a reliable and accurate system for determining amino acid positions in coding sequences, allele alignments, and for easily comparing sequences in immunoglobulins (IG) and T cell receptors (TR) from all vertebrate species. The accuracy and consistency of IMGT data are based on the IMGT-ONTOLOGY, the first and so far unique ontology for immunogenetics and immunoinformatics (see Lefranc.MP et al., Biomolecules, 2014 Dec;4(4), 1102-1139). IMGT tools and databases are run against the IMGT reference directory, which is built from a large repository of sequences. In the IMGT system, IG V-DOMAIN and IG C-DOMAIN are delimited, taking exon delimiters into account, whenever appropriate. Therefore, since many more sequences are available in the IMGT database, the IMGT exon numbering system can and should be reliably used by those skilled in the art to determine amino acid positions within coding sequences and align alleles.Furthermore, correspondences between IMGT-specific numbering and other numbering systems (i.e., Kabat) are available in the IMGT Scientific chart (see Lefranc.MP et al., Biomolecules, 2014 Dec;4(4), 1102-1139).

[0037] The term "antibody fragment" refers to a molecule other than the intact antibody that contains a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0038] As used herein, the term "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to the antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to the antigen by 50% or more in a competitive assay. An example of a competitive assay is provided herein.

[0039] The term "class" of an antibody refers to the type of constant domain or constant region in its heavy chain. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0040] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, except for, for example, naturally occurring mutations or mutant antibodies that may arise during the production of the monoclonal antibody preparation, and such mutations are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation targets a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0041] The term "naked antibody" refers to an antibody that is not conjugated to a heterogeneous moiety (e.g., a cytotoxic moiety or a STING agonist drug moiety). Naked antibodies can be present in pharmaceutical formulations.

[0042] The term "natural antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical disulfide-linked light chains and two identical heavy chains. Each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, extending from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain, extending from the terminal to the C-terminus. Based on the amino acid sequence of its constant domain, the light chains of an antibody can be assigned to one of two types called kappa (κ) and lambda (λ).

[0043] "Isolated antibodies" are those separated from components of their natural environment. In some embodiments, antibodies are purified to a purity of 95% or more than 99%, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion-exchange HPLC or reverse-phase HPLC). For an overview of antibody purity assessment methods, see, for example, Flatman et al., J.Chromatogr.B 848:79-87 (2007).

[0044] The term "epitope" refers to a specific site on an antigen molecule to which an antibody binds.

[0045] The term "humanized antibody" refers to an antibody derived from a non-human antibody (e.g., mouse) that retains or substantially retains the antigen-binding properties of the parent antibody but has low immunogenicity in humans. As used herein, "humanization" is intended to include deimmunized antibodies.

[0046] The term "humanized" for antibodies, such as non-human antibodies, refers to antibodies that have undergone humanization.

[0047] As used herein in the context of two or more antibodies, the terms "competes with" or "cross-competes with" indicate that two or more antibodies compete for binding to B7-H4, e.g., compete for B7-H4 binding. When an antibody competes with one or more other antibodies by 25% or more, the antibody "blocks" or "cross-blocks" the binding of the one or more other antibodies to B7-H4, where 25% to 74% represents "partial block" and 75% to 400% represents "complete block". Unless otherwise defined or negated by context, as used herein, the terms "competes with", "cross-competes with", "blocks" or "cross-blocks" are also intended to encompass such pairs of antibodies.

[0048] As used herein, an antibody that "specifically binds to human B7-H4" is intended to refer to an antibody that binds to human B7-H4 with a K -7 of, more typically, 5×10 -8 M or less, more typically 3×10 -8 M or less, more typically 1×10 -9 M or less, and even more typically 5×10 -9 M or less. D

[0049] As used herein, the term "does not substantially bind to" a protein or cell means that it does not bind to or binds to the protein or cell with low affinity, i.e., with a K -8 of 1×10 -5 M or more, more preferably 1×10 -4 M or more, more preferably 1×10 -3 M or more, and even more preferably 1×10 -2 M or more. D

[0050] The term “cytotoxic agent” or “cytotoxic drug portion” refers, without limitation, to compounds that induce cell death or inhibit or interfere with cell myosis, primarily by directly interfering with cellular function, including alkylating agents, tumor necrosis factors, interferants, microtubulin inhibitors, and topoisomerase inhibitors.

[0051] As used herein, the term “STING agonist” refers to a compound or moiety that can interact with STING, for example, by binding to STING and / or by inducing downstream signaling (e.g., characterized by the activation of molecules related to STING function). This includes direct phosphorylation of STING, IRF3 and / or NF-κB, and may also include STAT6. In some embodiments, STING pathway activation increases the production of type 1 interferons (primarily IFN-α and IFN-β) and / or the expression of interferon-stimulated genes.

[0052] As used herein, the term “STING agonist drug portion” refers to a portion derived from a STING agonist that is capable of interacting with STING. In some embodiments, the STING agonist drug portion is a STING agonist modified to allow that portion to be conjugated to the remainder of the conjugate of this disclosure.

[0053] The term "sugar" refers to monosaccharides, such as glucose (Glc), galactose (Gal), mannose (Man), and fucose (Fuc). The term "sugar derivative" refers to a derivative of a monosaccharide, i.e., a monosaccharide containing substituents and / or functional groups. Examples of sugar derivatives include, but are not limited to, amino sugars and sugar acids. Other examples of sugar derivatives include S'(F') X1 Compounds represented as shown are also examples, where S' is a sugar or sugar derivative, F' is a functional group, and x1 indicates the number of functional groups.

[0054] As used herein, the term “core-GlcNAc moiety” refers to a monosaccharide, polysaccharide, or oligosaccharide moiety containing GlcNAc (e.g., core-GlcNAc) bound to an antibody (e.g., via the C1 position of GlcNAc). In some embodiments, GlcNAc is bound to the antibody via an N-glycosidic bond to the amide nitrogen atom of the side chain of the asparagine amino acid of the antibody. In some embodiments, the core-GlcNAc moiety is present at the native glycosylation site of the antibody or is introduced at a different site of the antibody. In some embodiments, the core-GlcNAc moiety is a monosaccharide (e.g., the core-GlcNAc moiety is also the terminal GlcNAc moiety). In some embodiments, the core-GlcNAc moiety further contains fucose, for example, the core-GlcNAc moiety is a disaccharide core-GlcNAc-(α1-6-Fuc) moiety (which may be called GlcNAc(Fuc)). Therefore, if an antibody contains a core-GlcNAc moiety, the antibody may contain a monosaccharide core-GlcNAc moiety or a disaccharide core-GlcNAc moiety, and the core-GlcNAc moiety may further contain fucose (e.g., a disaccharide core-GlcNAc(Fuc) moiety). If the core-GlcNAc moiety further contains fucose, the fucose may be bound to the α-1,6 to O-6 of the core-GlcNAc moiety. A core-GlcNAc moiety further containing fucose may be called core-GlcNAc(Fuc).

[0055] The term "core-GlcNAc" refers to internal GlcNAc, which is part of a polysaccharide or oligosaccharide, and which binds to the antibody via the internal GlcNAc.

[0056] As used herein, the term “terminal GlcNAc moiety” refers to a portion comprising GlcNAc that binds to an antibody and has terminal functional groups available for further modification (e.g., by a P''-S''-A'' compound). In some embodiments, the terminal GlcNAc moiety further comprises fucose. In some embodiments, the terminal GlcNAc moiety is formed by reacting a core-GlcNAc moiety of a glycoprotein (e.g., antibody glycan) with an endoglycosidase.

[0057] The term "nucleotide," used in its ordinary scientific sense, refers to a molecule composed of a nucleic acid base, a five-carbon sugar (either ribose or 2-deoxyribose), and one, two, or three phosphate groups. Without the phosphate groups, the nucleic acid base and sugar form a nucleoside. Therefore, a nucleotide can also be called a nucleoside monophosphate, nucleoside diphosphate, or nucleoside triphosphate. The nucleic acid base can be adenine, guanine, cytosine, uracil, or thymine.

[0058] The term "protein," used in its usual scientific sense, refers to polypeptides containing approximately 10 or more amino acids. Proteins may contain either naturally occurring or unnatural amino acids.

[0059] The term “glycoprotein” is used herein in its ordinary scientific sense and refers to a protein containing one or more monosaccharides or oligosaccharides ("glycans") covalently bonded to the protein. Glycans may be bonded to a hydroxyl group on the protein (O-linked glycans), to an amide function on the protein (N-glycoproteins), or to a carbon on the protein (C-glycoproteins). A glycoprotein may contain multiple glycans, may contain a combination of one or more monosaccharide glycans and one or more oligosaccharide glycans, or may contain a combination of N-linked glycans, O-linked glycans, and C-linked glycans. It is estimated that more than 50% of all proteins have some form of glycosylation and are therefore eligible to be glycoproteins.

[0060] The term "glycan" is used herein in its usual scientific sense and refers to a monosaccharide or oligosaccharide chain bound to a protein. Therefore, a glycan refers to the carbohydrate portion of a glycoprotein. A glycan is bound to a protein via a single C-1 carbon atom of a sugar, and may not involve additional substitutions (monosaccharide), or it may be further substituted with one or more of its hydroxyl groups (oligosaccharide). Naturally occurring glycans typically contain 1 to about 10 sugar units. However, if longer sugar chains are bound to a protein, these are also considered glycans. Glycans in glycoproteins may be monosaccharides. Glycans may also be oligosaccharides. Oligosaccharide chains in glycoproteins may be linear or branched. In oligosaccharides, the sugar directly bound to the protein is called the core sugar. In oligosaccharides, the sugar not directly bound to the protein but bound to at least two other sugars is called the internal sugar. In oligosaccharides, sugars that are not directly bound to a protein but are bound to a single other sugar, i.e., sugars that do not have additional sugar substituents on one or more of the other hydroxyl groups, are called terminal sugars. To avoid misunderstanding, oligosaccharides in glycoproteins can have multiple terminal sugars, but there is only one core sugar. Glycans can be O-linked glycans, N-linked glycans, or C-linked glycans. In delinked glycans, monosaccharide glycans or oligosaccharide glycans are bound to the carbon atom of an amino acid in a protein.

[0061] The term "glycosyltransferase" refers to a superfamily of enzymes involved in the synthesis of complex carbohydrates present on glycoproteins and glycolipids.

[0062] The term "N-acetylgalactosamine transferase" (GalNAc-T) refers to an N-acetyl-D-galactosamine transferase enzyme that catalyzes the addition of N-acetyl-D-galactosamine to proteins.

[0063] As used herein, the term "PEG unit" refers to the formula This refers to a polyethylene glycol subunit having TIFF2026082984000007.tif12128. In some embodiments, the PEG unit comprises a plurality of PEG subunits.

[0064] As used herein, the term "alkyl" refers to a saturated linear or branched hydrocarbon group having a specified number of carbon atoms. The term "C1-C6 alkyl" or "C 1~6 "Alkyl" refers to a methyl moiety containing 2 to 6 carbon atoms, or a linear or branched alkyl moiety.

[0065] Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, pentyl, and hexyl.

[0066] As used herein, the term "halo(alkyl)" refers to a saturated linear or branched hydrocarbon group having a specified number (n) of carbon atoms and one or more (up to 2n+1) halogen atoms. 1~4 Examples of alkyl groups include, but are not limited to, -CF3 (trifluoromethyl), -CCl3 (trichloromethyl), 1,1-difluoroethyl, 2,2,2-trifluoroethyl, and hexafluoroisopropyl.

[0067] As used herein, the term "alkenyl" refers to a linear or branched hydrocarbon group having a specified number of carbon atoms and at least one to three carbon-carbon double bonds. Examples include ethenyl and propenyl.

[0068] As used herein, the term "alkynyl" refers to a linear or branched hydrocarbon group having a specified number of carbon atoms and at least one to three carbon-carbon triple bonds. Examples include ethynyl and propynyl.

[0069] As used herein, the terms “alkoxy-” or “(alkyl)oxy-” refer to an “alkyl-oxy-” group containing an alkyl moiety having a specified number of carbon atoms bonded via an oxygen-linked atom. 1~4 Alkoxy- group or (C 1~4 The alkyl)oxy- group includes, without limitation, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, s-butoxy, and t-butoxy.

[0070] As used herein, the term "halo(alkyloxy)-" refers to a saturated, linear, or branched hydrocarbon group having a specified number (n) of carbon atoms bonded via oxygen-linked atoms and one or more (up to 2n+1) halogen atoms. An example of "halo(C)" is... 1~4 The alkoxy-" group includes, but is not limited to, -OCHF2 (difluoromethoxy), -OCF3 (trifluoromethoxy), -OCH2CF3 (trifluoroethoxy), and -OCH(CF3)2 (hexafluoroisopropoxy).

[0071] As used herein, the term "amino" refers to a substituent containing at least one nitrogen atom. Specifically, this includes -NH2 substituents, -NH(C) substituents, etc. 1~4 alkyl) substituent, alkylamino substituent, or (C 1~4 Alkyl)amino substituent or (C 1~4 Alkyl)(C 1~4 The term "amino" includes alkyl)amino substituents or dialkylamino substituents, amide substituents, carbamide substituents, urea substituents, and sulfamide substituents.

[0072] As used herein, the term "carbocyclic group or carbocyclic moiety" refers to a cyclic group or cyclic moiety whose ring members are carbon atoms, and the cyclic group or cyclic moiety may be saturated, partially unsaturated (non-aromatic), or fully unsaturated (aromatic).

[0073] As used herein, the term "cycloalkyl" refers to a non-aromatic saturated hydrocarbon ring group containing a specified number of carbon atoms within the ring. For example, the term "C 3~6 A "cycloalkyl" refers to a cyclic group having 3 to 6 ring carbon atoms. An example is "C 3~6 The "cycloalkyl" group includes cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0074] As used herein, the term "aryl" refers to an aromatic group, including "conjugated" or polycyclic systems having one or more aromatic rings that do not contain any heteroatoms within the ring structure. The term aryl includes both monovalent and divalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, and naphthyl. In some embodiments, the aryl is phenyl.

[0075] As used herein, the term “heterocyclic group or heterocyclic moiety” means a cyclic group or cyclic moiety having at least two different elemental atoms as ring members, which may be saturated, partially unsaturated (non-aromatic), or fully unsaturated (aromatic).

[0076] As used herein, the term "heteroatom" refers to a nitrogen atom, a sulfur atom, or an oxygen atom, for example, a nitrogen atom or an oxygen atom.

[0077] As used herein, the term "heterocycloalkyl" refers to a non-aromatic monocyclic or bicyclic group comprising 3 to 10 ring atoms and one or more (generally one or two) heteroatom ring members independently selected from oxygen, sulfur, and nitrogen. The bonding sites of the heterocycloalkyl group may be any preferred carbon or nitrogen atom.

[0078] As used herein, the term "heteroaryl" refers to an aromatic monocyclic or bicyclic group containing 5 to 10 ring atoms, including 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, where at least a portion of the group is aromatic. For example, the term encompasses bicyclic heterocyclic aryl groups containing either a phenyl ring fused to a heterocyclic portion or a heteroaryl ring fused to a carbocyclic portion. The bonding sites of the heteroaryl group may be any preferred carbon or nitrogen atoms.

[0079] As used herein, the terms "halogen" and "halo" refer to halogen radicals, such as fluoro substituents, chloro substituents, bromo substituents, or iodo substituents.

[0080] As used herein, the term "oxo" refers to the oxygen portion of a double bond, which, for example, forms a carbonyl moiety (C=O) when directly bonded to a carbon atom.

[0081] As used herein, the terms "hydroxy" or "hydroxyl" are intended to mean the radical -OH.

[0082] As used herein, the term "cyano" refers to the nitrile group, -C≡N.

[0083] As used herein, the term "may be substituted" indicates that a group (such as an alkyl group, cycloalkyl group, alkoxy group, heterocycloalkyl group, aryl group, or heteroaryl group), ring, or part may be unsubstituted, or may be substituted by one or more substituents. If a group can be selected from several alternative groups, the selected groups may be the same or different. Suitable substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamide, nitro, trifluoromethyl, cyano, azide, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties.

[0084] As used herein, the term "independently" means that when multiple substituents are selected from several possible substituents, those substituents may be the same or different.

[0085] As used herein, the term “pharmaceutically acceptable” means a compound, conjugate, material, composition, or dosage form suitable for use in contact with human and animal tissues, within the bounds of reasonable medical judgment, without excessive toxicity, irritation, or other problems or complications, and in proportion to a reasonable benefit-to-risk ratio.

[0086] As used herein, the terms “treating” or “treat” refer to the management and care of a patient to combat a disease, condition or disorder, and include the administration of the compounds disclosed herein or their pharmaceutically acceptable salts, polymorphs or solvates to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” may also include the treatment of cells in vitro or in animal models.

[0087] As used herein, the terms “preventing,” “prevent,” or “protecting against” mean reducing or eliminating the onset of symptoms or complications of such disease, condition, or disorder.

[0088] The term "subject" refers to an animal, preferably a mammal, most preferably a human, that was the subject of treatment, observation, or experimentation.

[0089] The term “therapeutic dose” refers to the amount of an active compound or medicinal product containing the conjugate of this disclosure that elicits a biological or medical response in a tissue system, animal or human, as sought by researchers, veterinarians, physicians or other clinicians, including relief or partial relief of symptoms of the disease, syndrome, condition or disorder being treated.

[0090] The therapeutic “effective dose” is intended to mean an amount of conjugate sufficient to effectively treat or prevent the condition of a patient in need of such treatment, as defined herein. A given amount of conjugate equivalent to such a dose is a specific conjugate (e.g., potency (pICso), effect (EC) of a specific conjugate). 50The duration of treatment and administration of a conjugate (the period between the dose and the timing of the dose, e.g., before / with / after meals) varies depending on factors such as the specific mammal requiring treatment (e.g., body weight), the specific conjugate and its properties (e.g., pharmacokinetic properties), the disease or disorder and its severity, and the specific composition and method used, but can still be determined by those skilled in the art.

[0091] The term “composition” refers to a product containing a specific component in a therapeutically effective amount, and any product that arises directly or indirectly from a specific combination of a specific component in a specific amount.

[0092] As used herein, the term “pharmaceutically acceptable excipient” means an excipient that is generally safe, non-toxic, and useful for preparing a pharmaceutical composition that is not biologically or otherwise undesirable, and includes excipients acceptable for veterinary and human pharmaceutical use. As used herein and in the claims, “pharmaceutically acceptable excipient” includes one and more such excipients.

[0093] In some embodiments, the conjugates of the present disclosure, or their enantiomers, diastereomers, solvates, or pharmaceutically acceptable salt forms thereof, may be useful in treating or improving diseases, syndromes, conditions, or disorders such as melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B.

[0094] As used herein, the terms “conjugate(s) of the disclosure” or “conjugate(s) of the present disclosure” mean any form, i.e., any tautomer form, any isomer form, any salt or non-salt form (e.g., as a free acid or free base form, or as a salt, in particular as a pharmaceutically acceptable salt thereof), and any physical form thereof (e.g., non-solid form (e.g., liquid or semi-solid form) and solid form (e.g., amorphous or crystalline form, certain polymorphic forms, solvate forms including hydrate forms (e.g., monohydrate, dihydrate and hemihydrate)), as well as mixtures of various forms, as defined herein.

[0095] Accordingly, conjugates of any salt or non-salt form and any physical form thereof, as well as mixtures of various forms, as disclosed herein are included in this disclosure. While such are included in this disclosure, it will be understood that conjugates of any salt or non-salt form and any physical form thereof, as disclosed herein, may have varying levels of activity, varying bioavailability, and varying handling properties for formulation purposes.

[0096] As used herein, expressions such as "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 the group consisting of A, B, and C," and "selected from A, B, and C" are used interchangeably and, unless otherwise specified, refer to any selection from the group consisting of A, B, and / or C, i.e., one or more A's, one or more B's, one or more C's, or any combination thereof.

[0097] Throughout this specification, when a composition is described as having, including, or comprising certain components, it is understood that the composition is also intended to be essentially composed of or consisting of the listed components. Similarly, when a method or process is described as having, including, or comprising certain process steps, the process is also intended to be essentially composed of or consisting of the listed processing steps. Furthermore, it should be understood that the order or sequence of steps for performing a particular operation is not important, as long as the invention remains operable. Moreover, two or more steps or operations may be performed simultaneously.

[0098] All percentages and ratios used herein are by weight unless otherwise specified. Other features and advantages of this disclosure are evident from various examples. The examples provided illustrate various components and methods useful for carrying out this disclosure. The examples do not limit the claimed disclosure. Based on this disclosure, a person skilled in the art can identify and use other components and methods useful for carrying out this disclosure.

[0099] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document were specifically and individually indicated to be incorporated herein by reference. The citation of publications and patent documents is not intended as an acknowledgment of relevant prior art and does not constitute any acknowledgment of their content or date. While the present invention has been described herein, those skilled in the art will recognize that the invention may be carried out in various embodiments, and the following descriptions and examples are illustrative and not intended to limit the scope of the appended claims.

[0100] When used herein in relation to two or more antibodies, the terms “compete with” or “cross-compete with” indicate that two or more antibodies compete for binding to B7-H4 in the assays described in Example 5 or 8, e.g., compete for B7-H4 binding. If an antibody competes with one or more other antibodies by 25% or more, the antibody “blocks” or “cross-blocks” the binding of one or more other antibodies to B7-H4, preferably determined using the assays of Examples 5 and 8, with 25% to 74% representing “partial blockage” and 75% to 400% representing “complete blockage.” For some pairs of antibodies, competition or blockage in the assays of Example 5 or 8 is observed only if one antibody is coated on the plate and the other is used to compete, and not the other way around. Unless otherwise defined or denied by context, when used herein, the terms “compete with,” “cross-compete with,” “block,” or “cross-block” are also intended to encompass such pairs of antibodies.

[0101] Antibody-drug conjugates and scaffolds In some aspects, this disclosure provides B7-H4 antibody-drug conjugates. In some embodiments, the B7-H4 antibody-drug conjugates are site-specific. In some embodiments, the B7-H4 antibody-drug conjugates are not site-specific. In some embodiments, the B7-H4 antibody-drug conjugates are biodegradable and biocompatible, as well as exhibiting high drug loading and strong binding to target antigens.

[0102] In some embodiments, the Disclosure provides a B7-H4 antibody-drug conjugate comprising a B7-H4 targeting moiety (e.g., an antibody) and one or more linker-drug moieties, wherein the targeting moiety is covalently bound to one or more linker-drug moieties.

[0103] In some embodiments, the B7-H4 targeting portion is an antibody, a cysteine-modified antibody, or a modified antibody.

[0104] In some embodiments, the B7-H4 targeting portion is a B7-H4 antibody, a cysteine-modified B7-H4 antibody, or a modified B7-H4 antibody.

[0105] In some embodiments, the B7-H4 targeting portion is a B7-H4 antibody.

[0106] In some embodiments, the B7-H4 targeting portion is a cysteine-modified B7-H4 antibody.

[0107] In some embodiments, the B7-H4 targeting portion is a modified B7-H4 antibody.

[0108] In some aspects, the present disclosure relates to a B7-H4 antibody-drug conjugate comprising a B7-H4 targeting moiety (e.g., an antibody) and one or more linker-drug moieties covalently bound to the targeting moiety, Each linker-drug portion, A polyfunctional linker that connects a targeting portion to one or more drug units (e.g., one or more therapeutic agents (D)) via a releaseable assembly unit for each drug unit, and connects hydrophilic groups to drug units of each linker-drug portion. Includes, The releaseable assembly unit can release a free drug in close proximity to the target site targeted by the targeting portion, and The present invention provides a B7-H4 antibody-drug conjugate in which a polyfunctional linker comprises a peptide moiety between a targeting moiety and a hydrophilic group, and the peptide moiety comprises at least two amino acids.

[0109] In some aspects, the present disclosure relates to a B7-H4 antibody-drug conjugate comprising a B7-H4 targeting moiety (e.g., an antibody) and one or more linker-drug moieties covalently bound to the targeting moiety, Each linker-drug portion, A polyfunctional linker that connects the B7-H4 targeting portion to one or more drug units (e.g., one or more therapeutic agents (D)) via a releaseable assembly unit for each drug unit, and connects the hydrophilic groups to the drug units of each linker-drug portion. including and The present invention provides a B7-H4 antibody-drug conjugate in which a releaseable assembly unit can release a free drug in proximity to a target site targeted by a targeting portion.

[0110] In some aspects, this disclosure provides a B7-H4 antibody-drug conjugate of formula (I'): TIFF2026082984000008.tif37131In formula, a2 is an integer between 1 and 3. a3 is an integer between 0 and 1. a4 is an integer between 1 and approximately 5. a5 is an integer between 1 and 3. d 13 These are integers from 1 to approximately 12. The antibody is a B7-H4 antibody, a cysteine-modified B7-H4 antibody, or a modified B7-H4 antibody. L P' M P This is the divalent linker portion that connects to the corresponding unvalent portion L. P This is a functional group W that can form a covalent bond with the reactive portion of the antibody. P Includes, M P This is a stretcher unit, L M is a bond, or a trivalent linker or tetravalent linker, L M If it is a bond, then a2 is 1 or L M If it is a trivalent linker, then a2 is 2 or L M If it is a tetravalent linker, then a2 is 3, L 3 If present, this is the carbonyl-containing portion. M A It contains a peptide moiety that includes at least two amino acids, T 1 It contains a hydrophilic group, T 1 and M A Between TIFF2026082984000009.tif8128 is T 1 and M A This indicates a direct or indirect connection with, Each of the D entities is independently a therapeutic agent having a molecular weight of approximately 5 kDa or less. L D Each of these entities independently controls D to M A It is a divalent linker portion that connects to and contains at least one cleavable bond, and as a result, when the bond is cleaved, D is released in its active form for its intended therapeutic effect.

[0111] In some embodiments, D is a cytotoxic drug moiety or a STING agonist drug moiety.

[0112] In some embodiments, D is the cytotoxic drug portion.

[0113] In some embodiments, D is the STING agonist drug portion.

[0114] In some embodiments, the antibody-drug conjugate is of formula (II') or (III'): TIFF2026082984000010.tif72128

[0115] In some aspects, the present disclosure provides a B7-H4 antibody scaffold comprising a B7-H4 targeting moiety (e.g., an antibody) and one or more linker moieties covalently bound to the B7-H4 targeting moiety.

[0116] In some embodiments, the present disclosure provides a B7-H4 antibody scaffold of any one of formulas (II) to (V): TIFF2026082984000011.tif110128 formula, a2 is an integer between 1 and 3. a3 is an integer between 0 and 1, if it exists. a4 is an integer between 1 and approximately 5. a5 is an integer between 1 and 3. d 13 These are integers from 1 to approximately 12. The antibody is a B7-H4 antibody, a cysteine-modified B7-H4 antibody, or a modified B7-H4 antibody. L P' M P This is the divalent linker portion that connects to the corresponding unvalent portion L. P This is a functional group W that can form a covalent bond with the functional group of an antibody. P Includes, M P This is a stretcher unit, L M If present, it is a bond, or a trivalent linker or tetravalent linker, L M If it is a bond, then a2 is 1 or L M If it is a trivalent linker, then a2 is 2 or L M If it is a tetravalent linker, then a2 is 3, L 3 If present, this is the carbonyl-containing portion. M A It contains a peptide moiety that includes at least two amino acids, T 1 It contains a hydrophilic group, T 1 and M A Between TIFF2026082984000012.tif9128 is T 1 and M A This indicates a direct or indirect connection with, W D Each of these entities, when present, is a functional group that can independently form a covalent bond with a functional group of a therapeutic agent ("D") having a molecular weight of approximately 5 kDa or less. L D Each of these beings is independent of W DOr D to M A This is the divalent linker portion that connects to L D It contains at least one cleavable bond, and as a result, when the bond is cleaved, D is released in an active form for its intended therapeutic effect.

[0117] The conjugates and scaffolds of this disclosure may include one or more of the following features, where applicable:

[0118] In some aspects, d 13 These are integers between 2 and 12, 2 and 10, 2 and 8, 2 and 6, 2 and 4, 1 and 2, 4 and 10, 4 and 8, 4 and 6, 6 and 12, 6 and 10, 6 and 8, 8 and 14, 8 and 12, or 8 and 10.

[0119] In some aspects, d 13 is an integer in the range of 1 to 2 (for example, d 13 (is 1 or 2). In some embodiments, d 13 is an integer in the range of 2 to 4 (for example, d 13 (is 2, 3 or 4). In some embodiments, d 13 is an integer in the range of 4 to 6 (for example, d 13 (is 4, 5, or 6). In some embodiments, d 13 is an integer in the range of 6 to 8 (for example, d 13 (is 6, 7, or 8). In some embodiments, d 13 is an integer in the range of 6 to 10 (for example, d 13 (is 6, 7, 8, 9 or 10). In some embodiments, d 13 It is 6. In some embodiments, d 13 It is 7.

[0120] In some aspects, d 13 8. In some embodiments, d 13 is 1 or 2. In some embodiments, d 13 is 1. In some embodiments, d 13 The answer is 2.

[0121] In some embodiments, L 3 does not exist.

[0122] In some embodiments, each L 3 , when present, is independently *-C 1~12 alkyl-C(O)-**, *-NH-C 1~12 alkyl-C(O)-**, or *-C 1~12 alkyl-C(O)-NH-C 1~12 alkyl-C(O)-**, where * represents, when present, a bond to another L 3 , or L M , and ** represents, when present, a bond to another L 3 , or M A .

[0123] In some embodiments, at least one L 3 is *-CH2CH2-C(O)-** or *-NH-CH2CH2-C(O)-**, where * represents, when present, a bond to another L 3 , or L M , and ** represents, when present, a bond to another L 3 , or M A .

[0124] In some embodiments, a3 is 2 or more, and at least one L 3 is *-C 1~12 alkyl-C(O)-** and at least one L 3 is *-NH-C 1~12 alkyl-C(O)-**.

[0125] In some embodiments, each L 3 is *-CH2CH2-C(O)-NH-CH2CH2-C(O)-** or *NH-CH2CH2-C(O)-CH2CH2-C(O)-**, where * represents a bond to L M , and ** represents a bond to M A .

[0126] In some embodiments, a4 is 1. In some embodiments, a4 is 2. In some embodiments, a4 is 3.

[0127] Variable L for conjugation with B7-H4 antibody or cysteine-modified B7-H4 antibody P and L P' In some embodiments, L P' This is the divalent linker portion. In some embodiments, L P' This involves using B7-H4 antibody or cysteine-modified B7-H4 antibody to modify the cysteine. P This is the divalent linker portion that connects to L. In some embodiments, L P' This involves the cysteine ​​of the B7-H4 antibody being M P This is the divalent linker portion that connects to L. In some embodiments, L P' This involves using a cysteine-modified B7-H4 antibody. P This is the divalent linker portion that connects to it.

[0128] In some embodiments, L P L is the corresponding monovalent part. In some embodiments, L P If the cysteine ​​of the B7-H4 antibody is not attached, or if the cysteine ​​of the cysteine-modified B7-H4 antibody is not attached, then L P' It is the corresponding unvalent part of L. In some embodiments, L P If the B7-H4 antibody is not bound to cysteine, L P' It is the corresponding unvalent part of L. In some embodiments, L P If the cysteine ​​in the cysteine-operated B7-H4 antibody is not attached, then L P' This is the corresponding single-valued part.

[0129] In some embodiments, each L P If not connected to the cysteine ​​of the B7-H4 antibody, or the cysteine ​​of the cysteine-modified B7-H4 antibody, then the terminal group W P Includes.

[0130] In some embodiments, L P The terminal group WP Includes each W P Independently, the following are: TIFF2026082984000013.tif130170In the formula, Ring B is a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring. R 1K is a leaving group, R 1A It is a sulfur protecting group; R 2J These are a hydrogen moiety, an aliphatic moiety, an aryl moiety, a heteroaliphatic moiety, or a carbocyclic moiety. R 3J is C 1~6 It is an alkyl group, and each of Z1, Z2, Z3, and Z7 is independently either a carbon atom or a nitrogen atom.

[0131] In some embodiments, each R 1K R is either a halo or RC(O)O-, where R is a hydrogen moiety, an aliphatic moiety, a heteroaliphatic moiety, a carbocyclic moiety, or a heterocycloalkyl moiety.

[0132] In some embodiments, each R 1A Independently, The file is TIFF2026082984000014.tif23134, where r is 1 or 2, and R s1 , R s2 and R s3 Each of these is independently a hydrogen moiety, an aliphatic moiety, a heteroaliphatic moiety, a carbocyclic moiety, or a heterocycloalkyl moiety.

[0133] In some embodiments, W P teeth It is TIFF2026082984000015.tif14128. In some aspects, W P teeth, The filename is TIFF2026082984000016.tif18128.

[0134] In some embodiments, W P but If it is TIFF2026082984000017.tif18128, then L P' teeth, Includes TIFF2026082984000018.tif17128.

[0135] Variable L for conjugation of modified B7-H4 antibody P and L P’ In some embodiments, L P’ L P Functional groups (e.g., W P It is formed by a reaction between the ) and the reactive portion of the modified antibody (for example, the modified GlcNAc portion of *-GlcNAc-S''-A'').

[0136] In some embodiments, L P’ L P Functional groups (e.g., W P It contains a triazolyl formed between the ) and the reactive portion of the modified antibody (for example, the modified GlcNAc portion of *-GlcNAc-S''-A'').

[0137] In some embodiments, each L P If not attached to a modified B7-H4 antibody, the terminal group W P Includes.

[0138] In some embodiments, at least one W P teeth The filename is TIFF2026082984000019.tif112153. During the ceremony, R 8j is hydrogen, halogen, C 1~24 Alkyl (e.g., C 1~6 Alkyl), C 6~24 Cycloalkyl, 6-24 member heterocycloalkyl, C 6~24 Aryl, 6-24 member heteroaryl, -(C 1~24 Alkyl)-(C 6~24 Cycloalkyl), -(C 1~24 Alkyl)-(6-24 member heterocycloalkyl),-(C 1~24 Alkyl)-(C6~24 (aryl), or -(C 1~24 It is an alkyl)-(6-24 member heteroaryl) and C 1~24 The alkyl group may be interrupted by one or more O, N, or S atoms, and C 1~24 Alkyl (e.g., C 1~6 Alkyl), C 6~24 Cycloalkyl, 6-24 member heterocycloalkyl, C 6~24 Aryl, 6-24 member heteroaryl, -(C 1~24 Alkyl)-(C 6~24 Cycloalkyl), -(C 1~24 Alkyl)-(6-24 member heterocycloalkyl),-(C 1~24 Alkyl)-(C 6~24 (aryl), or -(C 1~24 Alkyl)-(6-24 member heteroaryl) is one or more C1-C 12 Alkyl, C2~C 12 Alkenyl, C2~C 12 Alkinyl, C3~C 12 Cycloalkyl, -O(C1~C 12 Alkyl), -O(C2~C 12 Alkenyl), -O(C2~C 12 Alkinyl), -O(C3~C 12 They may be substituted with cycloalkyl, halogen, amino, oxo, or silyl, C1~C 12 Alkyl, C2~C 12 Alkenyl, C2~C 12 Alkinyl, C3~C 12 Cycloalkyl, -O(C1~C 12 Alkyl), -O(C2~C 12 Alkenyl), -O(C2~C 12 Alkinyl), -O(C3~C 12 The cycloalkyl group may be substituted, C1-C 12 Alkyl, C3~C 12 Cycloalkyl, -O(C1~C 12 Alkyl) or -O(C3~C 12The cycloalkyl group may be interrupted by one or more O, N, or S atoms. R 10j is hydrogen, halogen, C 1~24 Alkyl (e.g., C 1~6 Alkyl), C 6~24 Cycloalkyl, 6-24 member heterocycloalkyl, C 6~24 Aryl, 6-24 member heteroaryl, -(C 1~24 Alkyl)-(C 6~24 Cycloalkyl), -(C 1~24 Alkyl)-(6-24 member heterocycloalkyl),-(C 1~24 Alkyl)-(C 6~24 (aryl), or -(C 1~24 It is an alkyl)-(6-24 member heteroaryl) and C 1~24 Alkyl (e.g., C 1~6 Alkyl), C 6~24 Cycloalkyl, 6-24 member heterocycloalkyl, C 6~24 Aryl, 6-24 member heteroaryl, -(C 1~24 Alkyl)-(C 6~24 Cycloalkyl), -(C 1~24 Alkyl)-(6-24 member heterocycloalkyl),-(C 1~24 Alkyl)-(C 6~24 (aryl), or -(C 1~24 The alkyl)-(6-24 member heteroaryl) may be substituted, Each R 11j These are, independently, hydrogen and C 1~24 Alkyl (e.g., C 1~6 Alkyl), C 6~24 Cycloalkyl, 6-24 member heterocycloalkyl, C 6~24 Aryl, 6-24 member heteroaryl, -(C 1~24 Alkyl)-(C 6~24 Cycloalkyl), -(C 1~24 Alkyl)-(6-24 member heterocycloalkyl),-(C 1~24 Alkyl)-(C 6~24 (aryl), or -(C 1~24 It is an alkyl)-(6-24 member heteroaryl) and Each R 12j These are, independently, halogen, -OR 10j -NO2, -CN, -S(O)2R 10j , C 1~24 Alkyl (e.g., C 1~6 Alkyl), C 6~24 Cycloalkyl, 6-24 member heterocycloalkyl, C 6~24 Aryl, 6-24 member heteroaryl, -(C 1~24 Alkyl)-(C 6~24 Cycloalkyl), -(C 1~24 Alkyl)-(6-24 member heterocycloalkyl),-(C 1~24 Alkyl)-(C 6~24 (aryl), or -(C 1~24 It is an alkyl)-(6-24 member heteroaryl) and u2 is an integer in the range of 0 to 8.

[0139] In some embodiments, at least one W P teeth The filename is TIFF2026082984000020.tif32128.

[0140] In some embodiments, at least one W P teeth The filename is TIFF2026082984000021.tif34128.

[0141] In some embodiments, each R 11j is hydrogen. In some embodiments, u2 is 0. In some embodiments, R 8j It is hydrogen.

[0142] In some embodiments, at least one W P teeth The filename is TIFF2026082984000022.tif34128.

[0143] In some embodiments, at least one W P teeth The filename is TIFF2026082984000023.tif31128.

[0144] In some embodiments, at least one R 12j is an electron-withdrawing group, for example, a group whose Hammett substituent constant σ is positive. In some embodiments, suitable electron-withdrawing groups are known in the art. In some embodiments, at least one R 12j is a halogen (e.g., F or Cl), -OR 10j -NO2, -CN, -S(O)2R 7j , substitution C1~C 12 Alkyl or substituted C6-C 12 It is an aryl group, and at least one of its substituents is an electron-withdrawing group. In some embodiments, at least one R 12j Fluorinated C1~C 12 Alkyl (e.g., -CF3), fluorinated C5~C 12 Aryl (e.g., -C6F5) or haloalkylated C5~C 12 It is an aryl group (for example, -[3,5-(CF3)2(C6H3)]).

[0145] In some embodiments, at least one W P teeth The filename is TIFF2026082984000024.tif34128.

[0146] In some embodiments, at least one W P teeth The filename is TIFF2026082984000025.tif70139.

[0147] In some embodiments, at least one W P teeth The filename is TIFF2026082984000026.tif36128.

[0148] In some embodiments, each R 11j is hydrogen. In some embodiments, u2 is 0.

[0149] In some embodiments, at least one W P teeth The filename is TIFF2026082984000027.tif31128.

[0150] In some embodiments, each W P If present, independently, The filename is TIFF2026082984000028.tif31128.

[0151] In some embodiments, each W P teeth The filename is TIFF2026082984000029.tif29128.

[0152] In some embodiments, each L P’ When it is attached to a B7-H4 modified antibody, the binding group W P’ Includes.

[0153] In some embodiments, at least one W P’ teeth The filename is TIFF2026082984000030.tif44128.

[0154] In some embodiments, at least one W P’ teeth The filename is TIFF2026082984000031.tif45128.

[0155] In some embodiments, at least one W P’ teeth The filename is TIFF2026082984000032.tif45128.

[0156] In some embodiments, at least one W P’ teeth The filename is TIFF2026082984000033.tif45128.

[0157] In some embodiments, at least one W P’ teeth The filename is TIFF2026082984000034.tif44128.

[0158] Stretcher Unit M P In some embodiments, M P teeth The filename is TIFF2026082984000035.tif221138; In the formula, * represents L P' or L P This indicates a connection to L M or M A This shows a connection to, Each R 66 These are independently NH or O, Each R3 is independently -C(O)-NR5- or -NR5-C(O)-, Each R5 independently produces hydrogen and C 1~6 Alkyl, C 6~10 Ariel, C 3~8 Cycloalkyl, COOH, or COO-C 1~6 It is alkyl, R4 is bound or -NR5-(CR 20 R 21 )-C(O)-, Each R 20 and R 21 These are, independently, hydrogen and C 1~6 Alkyl, C 6~10 Aryl, hydroxylated C 6~10 Aryl, polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, hydroxylated C 3~8 Cycloalkyl, polyhydroxylated C 3~8 A cycloalkyl group, or a side chain of a natural or unnatural amino acid, Each R 7 These are independently -O- and -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)-, -(3-8 member heterocycloalkyl)-, -(5-8 member heteroaryl)-, -(C1-C 10 Alkyl)-(3-8 member heterocycloalkyl)-,-(C1-C 10 Alkyl)-(5-8 member heteroaryl)-,-(3-8 member heterocycloalkyl)-(C1-C 10 Alkyl)-,-(5-8 member heteroaryl)-(C1-C 10 Alkyl)-,-OC(O)-(CH2CH2O) r -(CH2)2-, -(CH2CH2O) r -, or -(CH2CH2O) r It is -(CH2)2-, Each b1 is an independent integer in the range of 0 to 6. Each e1 is an independent integer in the range of 0 to 8. Each f1 is an independent integer in the range of 1 to 6. Each f2 is an independent integer in the range of 1 to 12. Each g2 is an independent integer in the range of 1 to 4.

[0159] In some embodiments, b1 is 0. In some embodiments, b1 is 1.

[0160] In some embodiments, each f1 is independently 1 or 2. In some embodiments, f1 is 1. In some embodiments, f1 is 2.

[0161] In some embodiments, g2 is 1 or 2. In some embodiments, g2 is 1. In some embodiments, g2 is 2.

[0162] In some embodiments, f2 is an integer in the range of 4 to 6.

[0163] In some embodiments, R 7 is, -(C1~C 10Alkyl)-, -O-(C1~C8 alkyl)-, -(CH2CH2O) r -, -OC(O)-(CH2CH2O) r -(CH2)2-, or -(CH2CH2O) r It is -(CH2)2-.

[0164] In some embodiments, R 7 These are -O-, -NH, -N(CH3), -CH2-, -(CH2)2-, -(CH2)5-, -OC(O)-(CH2CH2O)6-(CH2)2-, -(CH2CH2O)-(CH2)2-, -(CH2CH2O)2-(CH2)2-, -(CH2CH2O)4-(CH2)2-, or -(CH2CH2O)6-(CH2)2-.

[0165] M P Regarding the manner, * is L P' or L P This indicates a connection to L M or M A It is understood that this indicates a connection to.

[0166] In some embodiments, M P teeth The filename is TIFF2026082984000036.tif163150.

[0167] In some embodiments, M P teeth The filename is TIFF2026082984000037.tif11128.

[0168] In some embodiments, M P teeth The filename is TIFF2026082984000038.tif13128.

[0169] In some embodiments, M P teeth The filename is TIFF2026082984000039.tif15130.

[0170] In some embodiments, M P teeth The filename is TIFF2026082984000040.tif17128.

[0171] In some embodiments, M P teeth The filename is TIFF2026082984000041.tif18128.

[0172] In some embodiments, M P teeth The filename is TIFF2026082984000042.tif21128.

[0173] In some embodiments, M P teeth The filename is TIFF2026082984000043.tif16128.

[0174] Variable L M In some embodiments, L M The linker is either a linker with two arms (e.g., a divalent linker or a linker with two arms) or a multi-arm linker (e.g., a trivalent or tetravalent linker or a linker with three or four arms), where each arm may be the same or different.

[0175] In some embodiments, L M The linkers are either linked (e.g., divalent linkers, or having two arms) or multi-arm linkers (e.g., tetravalent, or having four arms, or trivalent with three arms), where each arm may be the same or different.

[0176] As used herein, the term "arm" means (1) M if present. P (2) If it is attached to L 3 It is either bound to L 3 If it does not exist, then M A L is connected M It is understood that this refers to that part.

[0177] In some embodiments, LM This is a linkage (for example, a divalent linker, or one with two arms).

[0178] In some embodiments, L M The linker is a multi-arm linker (for example, trivalent or tetravalent, or having three or four arms), where each arm may be the same or different. In some embodiments, L M This is a multi-arm linker (for example, trivalent or tetravalent, or having three or four arms).

[0179] In some embodiments, L M This is a trivalent linker having three arms, each arm may be the same or different. In some embodiments, L M This is a trivalent linker having three arms, each of which may be the same. In some embodiments, L M This is a trivalent linker having three arms, each of which may be different.

[0180] In some embodiments, L M This is a tetravalent linker having four arms, each arm may be the same or different. In some embodiments, L M This is a tetravalent linker having four arms, each of which may be the same. In some embodiments, L M This is a tetravalent linker having four arms, each of which may be different.

[0181] In some embodiments, a2 is 2, L M teeth The filename is TIFF2026082984000044.tif98137. During the ceremony, TIFF2026082984000045.tif7128 is M if it exists. P Joining to, or M P If it does not exist, then L P Or L P' This shows a connection to, Y1 is L if it exists. 3 Binding to, or L 3 If it does not exist, then M A This shows a connection to, R2 and R'2 are, independently, hydrogen or substituted carbon. 1~6 Alkyl, possibly substituted C 2~6 Alkenyl, C may be substituted 2~6 Alkynyl, C may be substituted. 3~19 Branched alkyl, possibly substituted C 3~8 Cycloalkyl, may be substituted C 6~10 Aryl, possibly substituted heteroaryl, possibly substituted C 1~6 Heteroalkyl, C 1~6 Alkoxy, aryloxy, C 1~6 Heteroalkoxy, C 2~6 Alkanoyl, optionally substituted arylcarbonyl, C 2~6 Alkoxycarbonyl, C 2~6 Alkanoyloxy, arylcarbonyloxy, C may be substituted. 2~6 Alkanoyl, C may be substituted. 2~6 Alkanoyloxy, C may be substituted. 2~6 Substituting alkanoyloxy, -COOH, or -COO-C 1~6 It is alkyl, Each of c1, c2, c3, c4, c5, c7, and c8, if present, is an independent integer in the range of 0 to 10. Each of d1, d2, d3, d4, d5, and d7, if present, is an independent integer in the range of 0 to 10.

[0182] In some embodiments, a2 is 2, L M teeth The filename is TIFF2026082984000046.tif97134. During the ceremony, TIFF2026082984000047.tif9128 is M P This shows a connection to, Y1 is L if it exists. 3Binding to, or L 3 If it does not exist, then M A This shows a connection to, R2 and R'2 are, independently, hydrogen or substituted carbon. 1~6 Alkyl, possibly substituted C 2~6 Alkenyl, C may be substituted 2~6 Alkynyl, C may be substituted. 3~19 Branched alkyl, possibly substituted C 3~8 Cycloalkyl, may be substituted C 6~10 Aryl, possibly substituted heteroaryl, possibly substituted C 1~6 Heteroalkyl, C 1~6 Alkoxy, aryloxy, C 1~6 Heteroalkoxy, C 2~6 Alkanoyl, optionally substituted arylcarbonyl, C 2~6 Alkoxycarbonyl, C 2~6 Alkanoyloxy, arylcarbonyloxy, C may be substituted. 2~6 Alkanoyl, C may be substituted. 2~6 Alkanoyloxy, C may be substituted. 2~6 Substituting alkanoyloxy, -COOH, or -COO-C 1~6 It is alkyl, Each of c1, c2, c3, c4, c5, c7, and c8, if present, is an independent integer in the range of 0 to 10. Each of d1, d2, d3, d4, d5, and d7, if present, is an independent integer in the range of 0 to 10.

[0183] In some embodiments, a2 is 2, L M teeth The filename is TIFF2026082984000048.tif25128.

[0184] In some embodiments, a2 is 2, L M teeth The filename is TIFF2026082984000049.tif26128.

[0185] In some embodiments, c1, c2, c3, c4, c5, c7, and c8, if present, are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are independently 0 or 1, respectively. In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are independently 0, 1, or 2, respectively. In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are independently 0, respectively. In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are independently 1, respectively. In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are each independently 2.

[0186] In some embodiments, d1, d2, d3, d4, d5, and d7, if present, are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, d1, d2, d3, d4, d5, and d7 are independently 0 or 1. In some embodiments, d1, d2, d3, d4, d5, and d7 are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, d1, d2, d3, d4, d5, and d7 are independently 1, 2, 3, or 4. In some embodiments, d1, d2, d3, d4, d5, and d7 are independently 1. In some embodiments, d1, d2, d3, d4, d5, and d7 are independently 2. In some embodiments, d1, d2, d3, d4, d5, and d7 are each independently 3. In some embodiments, d1, d2, d3, d4, d5, and d7 are each independently 4.

[0187] In some embodiments, R2 and R'2 are, independently, hydrogen and C. 1~6 Alkyl, C 6~10 Ariel, C 3~8Cycloalkyl, -COOH, or -COO-C 1~6 It is alkyl. In some embodiments, R2 and R'2 are independently hydrogen or C 1~6 It is alkyl. In some embodiments, R2 and R'2 are each independently hydrogen. In some embodiments, R2 and R'2 are each independently C 1~6 It is alkyl.

[0188] In some embodiments, L M teeth The filename is TIFF2026082984000050.tif82154.

[0189] In some embodiments, a2 is 3, L M teeth The filename is TIFF2026082984000051.tif230170. During the ceremony, TIFF2026082984000052.tif8128 is M if it exists. P Joining to, or M P If it does not exist, then L P Or L P' This shows a connection to, Y1 is L if it exists. 3 Binding to, or L 3 If it does not exist, then M A This shows a connection to, R2 and R'2 are, independently, hydrogen or substituted carbon. 1~6 Alkyl, possibly substituted C 2~6 Alkenyl, C may be substituted 2~6 Alkynyl, C may be substituted. 3~19 Branched alkyl, possibly substituted C 3~8 Cycloalkyl, may be substituted C 6~10 Aryl, possibly substituted heteroaryl, possibly substituted C 1~6 Heteroalkyl, C 1~6 Alkoxy, aryloxy, C 1~6 Heteroalkoxy, C 2~6Alkanoyl, optionally substituted arylcarbonyl, C 2~6 Alkoxycarbonyl, C 2~6 Alkanoyloxy, arylcarbonyloxy, C may be substituted. 2~6 Alkanoyl, C may be substituted. 2~6 Alkanoyloxy, C may be substituted. 2~6 Substituting alkanoyloxy, -COOH, or -COO-C 1~6 It is alkyl, Each of c1, c2, c3, c4, c5, c6, c7, and c8 is an independent integer in the range of 0 to 10. Each of d1, d2, d3, d4, d5, d6, d7, and d8 is an independent integer in the range of 0 to 10. Each of e1, e2, e3, e4, e5, e6, e7, and e8 is an independent integer in the range of 0 to 10.

[0190] In some embodiments, a2 is 3, L M teeth TIFF2026082984000053.tif187155TIFF2026082984000054.tif33156, During the ceremony, TIFF2026082984000055.tif7128 is M P This shows a connection to, Y1 is L if it exists. 3 Binding to, or L 3 If it does not exist, then M A This shows a connection to, R2 and R'2 are, independently, hydrogen or substituted carbon. 1~6 Alkyl, possibly substituted C 2~6 Alkenyl, C may be substituted 2~6 Alkynyl, C may be substituted. 3~19 Branched alkyl, possibly substituted C 3~8 Cycloalkyl, may be substituted C 6~10 Aryl, possibly substituted heteroaryl, possibly substituted C 1~6 Heteroalkyl, C1~6 Alkoxy, aryloxy, C 1~6 Heteroalkoxy, C 2~6 Alkanoyl, optionally substituted arylcarbonyl, C 2~6 Alkoxycarbonyl, C 2~6 Alkanoyloxy, arylcarbonyloxy, C may be substituted. 2~6 Alkanoyl, C may be substituted. 2~6 Alkanoyloxy, C may be substituted. 2~6 Substituting alkanoyloxy, -COOH, or -COO-C 1~6 It is alkyl, Each of c1, c2, c3, c4, c5, c6, c7, and c8 is an independent integer in the range of 0 to 10. Each of d1, d2, d3, d4, d5, d6, d7, and d8 is an independent integer in the range of 0 to 10. Each of e1, e2, e3, e4, e5, e6, e7, and e8 is an independent integer in the range of 0 to 10.

[0191] In some embodiments, a2 is 3, L M teeth The filename is TIFF2026082984000056.tif27128.

[0192] In some embodiments, a2 is 3, L M teeth The filename is TIFF2026082984000057.tif27128.

[0193] In some embodiments, -L M -(L 3 ) a2 -teeth The filename is TIFF2026082984000058.tif46128.

[0194] In some embodiments where the amino acid unit has two binding sites (i.e., terminal drug units), one of the binding sites shown above is, for example, H, OH, or C. 1~3 It may be substituted with an unsubstituted alkyl group.

[0195] In some embodiments, L M It is a multi-arm linker, and stretcher unit M P If you are not yet connected to W M L M It is the end of W M Each of these entities independently forms a hydrogen atom, a protecting group, a leaving group, or a covalent bond with L M to M P It is a functional group that can be connected to [something].

[0196] In some embodiments, W M is an amine protecting group. In some embodiments, W M It is BOC.

[0197] In some embodiments, W M L is an amine protecting group, M teeth The filename is TIFF2026082984000059.tif25128.

[0198] In some embodiments, W M L is an amine protecting group, M teeth The filename is TIFF2026082984000060.tif23128.

[0199] In some embodiments, W M is BOC, L M teeth The filename is TIFF2026082984000061.tif24128.

[0200] In some embodiments, W M It contains an amine group. In some embodiments, W M is -C(O)-(CH2) w -NH2 is included in the formula, where w is an integer from 1 to 6. In some embodiments, W M It is -C(O)-CH2-NH2.

[0201] In some embodiments, WM It is -C(O)-CH2-NH2, and L M teeth The filename is TIFF2026082984000062.tif26128.

[0202] In some embodiments, W M It is -C(O)-CH2-NH2, and L M teeth The filename is TIFF2026082984000063.tif24128.

[0203] In some embodiments, W M H is H.

[0204] Variable L 3 In some embodiments, each L 3 It does not exist. In some embodiments, each L 3 This is the carbonyl-containing portion.

[0205] L 3 Regarding the aspect, * indicates another L if present. 3 , or L M This indicates a connection to **, and if present, another L 3 , or M A It is understood that this indicates a connection to.

[0206] In some embodiments, each L 3 If present, independently, *-C 1~12 Alkyl-C(O)-**, *-NH-C 1~12 It is alkyl-C(O)-** or *-C1~12alkyl-C(O)-NH-C1~12alkyl-C(O)-**.

[0207] In some embodiments, at least one L 3 is *-C 1~12 It is alkyl-C(O)-**.

[0208] In some embodiments, at least one L 3 It is *-CH2CH2-C(O)-**.

[0209] In some embodiments, L 3 It is *-CH2CH2-C(O)-**.

[0210] In some aspects, (L 3 ) a3 It is *-CH2CH2-C(O)-**.

[0211] In some embodiments, at least one L 3 is *-NH-C 1~12 It is alkyl-C(O)-**.

[0212] In some embodiments, at least one L 3 It is *-NH-CH2CH2-C(O)-**.

[0213] In some embodiments, L 3 It is *-NH-CH2CH2-C(O)-**.

[0214] In some aspects, (L 3 ) a3 It is *-NH-CH2CH2-C(O)-**.

[0215] In some embodiments, at least one L 3 is *-C 1~12 Alkyl-C(O)-NH-C 1~12 It is alkyl-C(O)-**.

[0216] In some embodiments, at least one L 3 The formula is *-CH2CH2-C(O)-NH-CH2CH2-C(O)-**.

[0217] In some embodiments, L 3 The formula is *-CH2CH2-C(O)-NH-CH2CH2-C(O)-**.

[0218] In some aspects, (L 3 ) a3The formula is *-CH2CH2-C(O)-NH-CH2CH2-C(O)-**.

[0219] In some embodiments, a3 is 2 or more, and at least one L 3 is *-C 1~12 It is alkyl-C(O)-** and has at least one L 3 is *-NH-C 1~12 It is alkyl-C(O)-**.

[0220] In some aspects, (L 3 ) a3 The formula is *-CH2CH2-C(O)-NH-CH2CH2-C(O)-**.

[0221] In some aspects, (L 3 ) a3 It is *NH-CH2CH2-C(O)-CH2CH2-C(O)-**.

[0222] Variable M A In some embodiments, M A L contains one or more drugs and one or more hydrophilic groups. P or L P’ This is a linker section that can be connected. In some embodiments, M A It comprises a peptide moiety of at least two amino acids. In some embodiments, a singular amino acid is referred to herein as "AA," and a plural amino acid is referred to herein as "AA's."

[0223] In some embodiments, the peptide portion is -L D-A portion that can form a covalent bond with the D unit, enabling the binding of multiple drugs. In some embodiments, the peptide portion 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), each of which is independently a native or non-native amino acid, an amino alcohol, an amino aldehyde, a diamine, a polyamine, or a combination thereof. In some embodiments, in order to have the required number of bonds, at least one of the AA units is -L D -It has a functionalized side chain that provides a bond for the D unit. In some embodiments, exemplary functionalized AA units (e.g., amino acids, amino alcohols, or amino aldehydes) include, for example, azide-functionalized AA units or alkyne-functionalized AA units (e.g., amino acids, amino alcohols, or amino aldehydes modified to have an azide group or an alkyne group). In some embodiments, the azide group or alkyne group is for bonding using click chemistry.

[0224] In some embodiments, the peptide portion has 2 to 12 AA units. In some embodiments, the peptide portion has 2 to 10 AA units. In some embodiments, the peptide portion has 2 to 6 AA units. In some embodiments, the peptide portion has 2, 3, 4, 5, or 6 AA units.

[0225] In some embodiments, the peptide portion has 2 AA units. In some embodiments, the peptide portion has 3 AA units. In some embodiments, the peptide portion has 4 AA units. In some embodiments, the peptide portion has 5 AA units. In some embodiments, the peptide portion has 6 AA units.

[0226] In some embodiments, the binding within the peptide moiety, or with other components of the conjugate, its intermediate, or scaffold, may be via, for example, amino, carboxy, or other functional groups. In some embodiments, each amino acid in the peptide moiety may independently be a D-isomer or L-isomer of a thiol-containing amino acid. In some embodiments, each amino acid in the peptide moiety may independently be a D-isomer of a thiol-containing amino acid. In some embodiments, each amino acid in the peptide moiety may independently be an L-isomer of a thiol-containing amino acid. In some embodiments, the thiol-containing amino acid may be, for example, cysteine, homocysteine, or penicillamine.

[0227] In some embodiments, each amino acid containing the peptide portion may independently be one of the following amino acids: alanine (including β-alanine), arginine, aspartic acid, asparagine, cysteine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine, penicillamine, aminoalkynic acid, aminoalkanediic acid, heterocyclocarboxylic acid, citrulline, statin, diaminoalkanoic acid, its stereoisomer, or an L-isomer or D-isomer of its derivative.

[0228] In some embodiments, each amino acid containing the peptide moiety 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.

[0229] In some embodiments, the peptide portion comprises a monopeptide, dipeptide, tripeptide, tetrapeptide, or pentapeptide. In some embodiments, the peptide portion comprises a pentapeptide.

[0230] In some embodiments, the peptide portion contains at least about 5 amino acids (e.g., 5, 6, 7, 8, 9, or 10 amino acids). In some embodiments, the peptide portion contains up to about 10 amino acids.

[0231] In some embodiments, the amino acids independently comprising the peptide moiety are glycine, serine, glutamic acid, lysine, aspartic acid, and cysteine.

[0232] In some embodiments, the peptide portion comprises at least four glycine molecules and at least one serine molecule, for example, (glycine)4 and serine, where the serine molecule is located at any position along the peptide chain, for example, (serine)-(glycine)4;(glycine)-(serine)-(glycine)3;(glycine)2-(serine)-(glycine)2;(glycine)3-(serine)-(glycine); or (glycine)4-(serine), etc.

[0233] In some embodiments, the peptide portion comprises (glycine)4-(serine) or (serine)-(glycine)4. In some embodiments, the peptide portion comprises (glycine)4-(serine). In some embodiments, the peptide portion comprises (serine)-(glycine)4.

[0234] In some embodiments, the peptide portion comprises at least four glycine molecules and at least one glutamic acid molecule, for example, (glycine)4 and glutamic acid, where the glutamic acid molecule is at any position along the peptide chain.

[0235] In some embodiments, the peptide portion comprises (glutamic acid)-(glycine)4 or (glycine)4-(glutamic acid).

[0236] In some embodiments, the peptide portion comprises (β-alanine)-(glycine)4-(serine), where the serine is at any position along the peptide chain.

[0237] In some embodiments, the peptide moiety comprises (glycine)4-(serine)-(glutamic acid), with serine at any position along the peptide chain. In some embodiments, the peptide moiety comprises (β-alanine)-(glycine)4-(serine)-(glutamic acid), with serine at any position along the peptide chain.

[0238] In some embodiments, the peptide portion is (glycine) 1~4 -(Serine) The peptide portion is transmitted via one of the glycine molecules, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound, and the peptide portion is via serine, if present, T 1 It is bound, and the peptide portion is transmitted via serine, if present, to L D It is connected.

[0239] In some embodiments, the peptide portion is (serine)-(glycine) 1~4 Includes, The peptide portion is transmitted via serine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound to the peptide portion, and if present, via glycine, T 1 It is bound, and the peptide portion is transmitted via serine, if present, to L D It is connected.

[0240] Regarding the form of the peptide portion, * indicates L if present. 3 to, or L 3 If it does not exist, then L M It is understood that this indicates a coupling to. In some aspects, ** is T if present. 1 to, or T 1 If not present, it indicates a bond to -OH. In some embodiments, *** is L if present. D to, or L DIf it is not present, it shows a bond to hydrogen.

[0241] In some embodiments, the peptide portion is Includes TIFF2026082984000064.tif17128.

[0242] In some embodiments, the peptide portion includes (glycine)-(serine), The peptide portion is transmitted via glycine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound, and the peptide portion is via serine, if present, T 1 It is bound, and the peptide portion is transmitted via serine, if present, to L D It is connected.

[0243] In some embodiments, the peptide portion includes (glycine)-(serine), The peptide portion is transmitted via serine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound to the peptide portion, and if present, via glycine, T 1 It is bound, and the peptide portion is transmitted via serine, if present, to L D It is connected.

[0244] In some embodiments, the peptide portion is Includes TIFF2026082984000065.tif18128.

[0245] In some embodiments, the peptide portion includes (glycine)4-(serine), The peptide portion is transmitted via one of the glycine molecules, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound, and the peptide portion is via serine, if present, T 1It is bound, and the peptide portion is transmitted via serine, if present, to L D It is connected.

[0246] In some embodiments, the peptide portion is Includes TIFF2026082984000066.tif18128.

[0247] In some embodiments, the peptide portion comprises (serine)-(glycine)4, The peptide portion is transmitted via serine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound to, and the peptide portion is transmitted via one of the glycines, if present, to T 1 It is bound, and the peptide portion is transmitted via serine, if present, to L D It is connected.

[0248] In some embodiments, the peptide portion is Includes TIFF2026082984000067.tif18128.

[0249] In some embodiments, the peptide portion is Includes TIFF2026082984000068.tif17128.

[0250] In some embodiments, the peptide portion is (β-alanine)-(glycine) 1~4 -(Serine) The peptide portion is transmitted via β-alanine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound, and the peptide portion is via serine, if present, T 1 It is bound, and the peptide portion is transmitted via serine, if present, to L D It is connected.

[0251] In some embodiments, the peptide portion is Includes TIFF2026082984000069.tif18128.

[0252] In some embodiments, the peptide portion comprises (β-alanine)-(glycine)4-(serine), The peptide portion is transmitted via β-alanine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound, and the peptide portion is via serine, if present, T 1 It is bound, and the peptide portion is transmitted via serine, if present, to L D It is connected.

[0253] In some embodiments, the peptide portion is Includes TIFF2026082984000070.tif17128.

[0254] In some embodiments, the peptide portion is (glycine) 1~4 -Contains glutamic acid, The peptide portion is transmitted via one of the glycine molecules, if present, through L 3 to, or L 3 If it does not exist, then L M It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0255] In some embodiments, the peptide portion is (glycine) 1~4 -Contains glutamic acid, The peptide portion, via glutamic acid, if present, is L 3 to, or L 3 If it does not exist, then L M It is bound to the peptide portion, and if present, via glycine, T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L DIt is connected.

[0256] In some embodiments, the peptide portion is Includes TIFF2026082984000071.tif23128.

[0257] In some embodiments, the peptide portion includes (glycine)-(glutamic acid), The peptide portion is transmitted via glycine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0258] In some embodiments, the peptide portion is Includes TIFF2026082984000072.tif21128.

[0259] In some embodiments, the peptide portion includes (glycine)4-(glutamic acid), The peptide portion is transmitted via one of the glycine molecules, if present, through L 3 to, or L 3 If it does not exist, then L M It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0260] In some embodiments, the peptide portion is Includes TIFF2026082984000073.tif23128.

[0261] In some embodiments, the peptide portion is (glutamic acid)-(glycine) 1~4 Includes, The peptide portion, via glutamic acid, if present, is L 3 to, or L 3 If it does not exist, then L M It is bound to, and the peptide portion is transmitted via one of the glycines, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0262] In some embodiments, the peptide portion is Includes TIFF2026082984000074.tif22128.

[0263] In some embodiments, the peptide portion is Includes TIFF2026082984000075.tif22128.

[0264] In some embodiments, the peptide portion comprises (glutamic acid)-(glycine)4, The peptide portion, via glutamic acid, if present, is L 3 to, or L 3 If it does not exist, then L M It is bound to, and the peptide portion is transmitted via one of the glycines, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0265] In some embodiments, the peptide portion is Includes TIFF2026082984000076.tif26128.

[0266] In some embodiments, the peptide portion includes (glutamic acid)-(glycine), The peptide portion, via glutamic acid, if present, is L 3 to, or L 3 If it does not exist, then L M It is bound to, and the peptide portion is transmitted via one of the glycines, if present, to T1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0267] In some embodiments, the peptide portion is Includes TIFF2026082984000077.tif26128.

[0268] In some embodiments, the peptide portion is (β-alanine)-(glycine) 1~4 -Contains glutamic acid, The peptide portion is transmitted via β-alanine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0269] In some embodiments, the peptide portion is Includes TIFF2026082984000078.tif20128.

[0270] In some embodiments, the peptide portion includes (β-alanine)-(glycine)4-(glutamic acid), The peptide portion is transmitted via β-alanine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0271] In some embodiments, the peptide portion is Includes TIFF2026082984000079.tif22128.

[0272] M A Regarding the aspect, * indicates L if present. 3 Binding to, or L 3 If it does not exist, then L M This indicates a connection to T 1 This indicates a bond to L D It is understood that this indicates a connection to.

[0273] In some embodiments, the peptide portion includes (β-alanine)-(glycine)-(glutamic acid), The peptide portion is transmitted via β-alanine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0274] In some embodiments, the peptide portion is Includes TIFF2026082984000080.tif29128.

[0275] Variable L D In some embodiments, each L D Independently, D to M A This is the divalent linker portion that connects to L. In some embodiments, each L D It contains at least one cleavable bond, and as a result, when the bond is cleaved, D is released in its active form for its intended therapeutic effect.

[0276] In some embodiments, L D It includes one severable bond. In some embodiments, L D This includes multiple severable parts or joints.

[0277] Each L D Before being connected to D, independently, the single-valent part L D'It is understood that this corresponds to the intended purpose.

[0278] In some embodiments, L D' It contains functional groups that can form cleavable bonds. Functional groups that can form cleavable bonds may include, for example, sulfhydryl groups for forming disulfide bonds, aldehyde groups, ketone groups or hydrazine groups for forming hydrazone bonds, hydroxylamine groups for forming oxime bonds, carboxyl groups or amino groups for forming peptide bonds, carboxyl groups or hydroxyl groups for forming ester bonds, and sugars for forming glycosidic bonds.

[0279] In some embodiments, each L D This includes a disulfide bond that can be cleaved by disulfide exchange, an acid-unstable bond that can be cleaved at an acidic pH, and / or a bond that can be cleaved by a hydrolase. In some embodiments, L D It contains a carbamate bond (i.e., -OC(O)-NR- where R is hydrogen or alkyl, etc.).

[0280] In some embodiments, L D The structure and arrangement of the cleavable bonds within may be such that the bonds are cleaved by the action of enzymes present at the target site. In some embodiments, the cleavable bonds may be cleavable by other mechanisms.

[0281] In some embodiments, L D The structure and arrangement of the cleavable bonds within may be such that the bonds are cleaved by the action of enzymes present at the target site. In some embodiments, the cleavable bonds may be cleavable by other mechanisms.

[0282] In some embodiments, the cleavable bond can be enzymatically cleaved by one or more enzymes, including tumor-associated proteases, to release a drug unit or D, and the conjugate or intermediate or scaffold of the present disclosure is protonated in vivo upon release to provide a drug unit or D.

[0283] In some embodiments, each L D Independently, The filename is TIFF2026082984000081.tif13128, and in the formula, L E If present, -NH-[(CH2CH2O) p -(CH2) 0~2 ] q -C(O)-, -NH-(C1~C6alkyl)-OC(O)-, or -NH-[(CH2CH2O) p -(CH2) 0~2 ] q -C(O)-NH-(C 1~ The formula is C6 alkyl)-OC(O)-, where p is an integer in the range of approximately 1 to approximately 20, and q is an integer in the range of approximately 1 to approximately 10. Each W is independently either a natural amino acid unit or a non-natural amino acid unit. w is an integer in the range of approximately 0 to approximately 12. *** is M A This shows a connection to, **** indicates a connection to D.

[0284] In some embodiments, each L D Independently, The filename is TIFF2026082984000082.tif11128.

[0285] In some embodiments, each L D Independently, The filename is TIFF2026082984000083.tif11128.

[0286] In some embodiments, each L D Independently, The filename is TIFF2026082984000084.tif11128.

[0287] In some embodiments, L E It includes at least one PEG unit.

[0288] In some embodiments, the PEG unit includes at least one subunit, at least two subunits, at least three subunits, at least four subunits, at least five subunits, or at least six subunits. In some embodiments, the PEG unit includes at least four subunits, at least three subunits, at least two subunits, or at least one subunit.

[0289] In some embodiments, the PEG unit includes at least one subunit.

[0290] In some embodiments, the PEG unit includes at least two subunits.

[0291] In some embodiments, p is an integer in the range of approximately 1 to approximately 15, approximately 1 to approximately 10, approximately 1 to approximately 9, approximately 1 to approximately 8, approximately 1 to approximately 7, approximately 1 to approximately 6, or approximately 1 to approximately 5.

[0292] In some embodiments, p is an integer in the range of approximately 1 to approximately 6. In some embodiments, p is an integer in the range of approximately 1 to approximately 4. In some embodiments, p is an integer in the range of approximately 1 to approximately 2.

[0293] In some embodiments, p is 2.

[0294] In some embodiments, q is an integer in the range of approximately 1 to approximately 15, approximately 1 to approximately 10, approximately 1 to approximately 9, approximately 1 to approximately 8, approximately 1 to approximately 7, approximately 1 to approximately 6, or approximately 1 to approximately 5.

[0295] In some embodiments, q is 1, 2, 3, 4, or 5. In some embodiments, q is 2.

[0296] In some embodiments, L E If present, -NH-(CH2CH2O) 1~4 It is -(CH2)2-C(O)-. In some embodiments, L E If present, it is -NH-(CH2CH2O)2-(CH2)2-C(O)-. In some embodiments, L E If present, -NH-(CH2CH2O)3-(CH2) 0~2 -C(O)-. In some embodiments, L E If present, it is -NH-(CH2CH2O)3-(CH2)-C(O)-. In some embodiments, L E If present, it is -NH-(CH2CH2O)3-(CH2)2-C(O)-. In some embodiments, L E If present, -NH-(CH2CH2O)-(CH2) 0~2 -C(O)-. In some embodiments, L E If present, it is -NH-CH2CH2O-C(O)-. In some embodiments, L E If present, it is -NH-(C1~C6 alkyl)-OC(O)-. In some embodiments, L E If present, it is -NH-CH2-CH(CH3)-OC(O)-. In some embodiments, L E If present, -NH-[(CH2CH2O) 1~4 It is -(CH2)2-C(O)-NH-(C1~C6 alkyl)-OC(O)-. In some embodiments, L E If present, it is -NH-CH2CH2O-(CH2)2-C(O)-NH-(CH2)2-OC(O)-.

[0297] In some embodiments, w is an integer in the range of about 1 to about 12 (e.g., 1 to 6, or 1 to 4, or 1 to 3, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

[0298] In some embodiments, w is 0, 1, 2, 3, 4, or 5. In some embodiments, w is 1, 2, 3, 4, or 5.

[0299] In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, w is 3.

[0300] In some embodiments, each W is independently a native amino acid or a non-native amino acid and / or a D isomer or an L isomer.

[0301] In some embodiments, each W is independently a natural or unnatural alpha-amino acid, beta-amino acid, or gamma-amino acid.

[0302] 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.

[0303] In some embodiments, W w It does not contain natural amino acids. In some embodiments, W w It does not contain unnatural amino acids.

[0304] In some embodiments, W w It contains natural amino acids bonded to non-natural amino acids.

[0305] In some embodiments, W w It contains natural amino acids bonded to the D-isomer of natural amino acids.

[0306] In some embodiments, W w These are dipeptides, such as -Val-Cit-, -Phe-Lys-, -Val-Ala-, or Glu-Ala.

[0307] In some embodiments, W wThese are monopeptide units, dipeptide units, tripeptide units, tetrapeptide units, pentapeptide units, hexapeptide units, heptapeptide units, octapeptide units, nonapeptide units, decapeptide units, undecapeptide units, or dodecapeptide units.

[0308] In some embodiments, W w This is a peptide (e.g., a peptide of 1 to 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.

[0309] In some embodiments, W w Each amino acid in the compound is independently selected from alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, selenocysteine, ornithine, penicillamine, aminoalkanoic acid, aminoalkynic acid, aminoalkanedioic acid, aminobenzoic acid, aminoheterocycloalkanoic acid, heterocyclocarboxylic acid, citrulline, statin, diaminoalkanoic acid and its derivatives.

[0310] In some embodiments, W w Each amino acid in the compound 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.

[0311] In some embodiments, W w Each amino acid in the mixture is independently selected from both proteinogenic and non-proteinogenic amino acids.

[0312] In some embodiments, W w Each amino acid in the compound is independently selected from the following amino acids: alanine, β-alanine, arginine, aspartic acid, asparagine, cysteine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine, penicillamine, aminoalkynic acid, aminoalkanediic acid, heterocyclocarboxylic acid, citrulline, statin, diaminoalkanoic acid, valine, and L-isomers or D-isomers of citrulline and its derivatives.

[0313] In some embodiments, W w Each amino acid in the compound 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.

[0314] In some embodiments, W w Each amino acid in the compound is independently selected from the following amino acids, namely, alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, citrulline, and the L-isomers of valine.

[0315] In some embodiments, W w Each amino acid in the compound is independently selected from the following amino acids, namely alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, citrulline, and the D-isomers of valine.

[0316] In some embodiments, W wThe amino acids in it are alanine, β-alanine, glycine, glutamine, glutamic acid, isoglutamic acid, isoaspartic acid, valinecitrulline, or aspartic acid.

[0317] In some embodiments, W w It contains β-alanine. In some embodiments, W w It contains (β-alanine)-(alanine). In some embodiments, W w It contains (β-alanine) and optionally glutamic acid, glutamine, isoglutamic acid, aspartic acid, isospartic acid, valine, (valine)-(alanine), (alanine)-(alanine), or (valine)-(citrulline).

[0318] In some embodiments, W w It contains (glutamic acid)-(alanine).

[0319] In some embodiments, W w It contains (β-alanine)-(glutamine).

[0320] In some embodiments, W w It contains (β-alanine)-(glutamine)-(alanine).

[0321] In some embodiments, W w It contains glutamic acid and optionally alanine, glycine, isoglutamic acid, aspartic acid, isoaspartic acid, valine, (valine)-(alanine), (alanine)-(alanine), or (valine)-(citrulline).

[0322] In some embodiments, W w It contains 2,3-diaminopropanoic acid. In some embodiments, W w It contains (R)-2,3-diaminopropanoic acid. In some embodiments, W w It contains glutamic acid. In some embodiments, W w It contains (glutamic acid)-(alanine). In some embodiments, W wIt contains (glutamic acid)-(glycine)-(alanine).

[0323] In some embodiments, W w This includes L-glutamic acid, D-glutamic acid, (L-glutamic acid)-(L-alanine), (L-glutamic acid)-(D-alanine), (D-glutamic acid)-(L-alanine), (D-glutamic acid)-(D-alanine), (L-glutamic acid)-(glycine)-(L-alanine), (D-glutamic acid)-(glycine)-(D-alanine), (L-glutamic acid)-(glycine)-(D-alanine), or (D-glutamic acid)-(glycine)-(L-alanine).

[0324] In some embodiments, W w It contains one or more amino acids in addition to a carbamate bond.

[0325] In some embodiments, L D (For example, W w ) is selective for enzymatic cleavage (e.g., by a specific enzyme). In some embodiments, the specific enzyme is a tumor-associated protease.

[0326] In some embodiments, L D (For example, W w ) contains a bond whose cleavage is catalyzed by cathepsins B, C, and D, or plasmin proteases.

[0327] In some embodiments, L D This includes a sugar cleavage site.

[0328] In some embodiments, L D It contains a sugar moiety (Su) attached to a self-immolative group via an oxygen glycosidic bond.

[0329] In some embodiments, the "self-sacrificing group" consists of three separated chemical moieties (i.e., a sugar moiety (via a glycosidic bond), a drug unit (directly or indirectly), and M AIf M exists A (Directly or indirectly), or M A If it does not exist, then A 1 It can be a trifunctional chemical moiety that can be covalently bonded together.

[0330] In some embodiments, the glycosidic bond can be cleaved at the target site to initiate a self-sacrificing reaction sequence that results in drug release.

[0331] In some embodiments, each L D If present, independently, the following applies: TIFF2026082984000085.tif93163TIFF2026082984000086.tif209163TIFF2026082984000087.tif217163In the formula, *** is M A The string indicates a connection to , and **** indicates a connection to D.

[0332] In some embodiments, each L D If present, independently, the following applies: TIFF2026082984000088.tif138163In formula, *** is M A This shows a connection to, **** indicates a connection to D.

[0333] In some embodiments, each L D If present, independently, the following applies: TIFF2026082984000089.tif16128

[0334] Therapeutic agent, drug unit, or variable D In some embodiments, the therapeutic agent is a cytotoxic drug portion. In some embodiments, the therapeutic agent is a STING agonist drug portion.

[0335] In some embodiments, the cytotoxic drug portion is a small molecule.

[0336] In some embodiments, the therapeutic agent has a molecular weight of approximately 5 kDa or less (for example, having a molecular weight of approximately 4 kDa or less, approximately 3 kDa or less, approximately 1.5 kDa or less, or approximately 1 kDa or less).

[0337] In some embodiments, the therapeutic agent has an IC of less than approximately 1 nM. 50 It has the following characteristics: In some embodiments, the cytotoxic drug portion or the STING agonist drug portion has an IC50 of less than 1 nM. 50 It has.

[0338] In some embodiments, the therapeutic agent has an IC of more than approximately 1 nM. 50 (For example, the cytotoxic drug portion or the STING agonist drug portion has an IC50 of about 1 to about 50 nM) 50 (Having) In some embodiments, the therapeutic agent has an IC of more than about 1 nM. 50 It has. In some embodiments, the therapeutic agent has an IC of greater than 1 nM. 50 (For example, the cytotoxic drug portion or the STING agonist drug portion has an IC50 of 1-50 nM) 50 (having). In some embodiments, the therapeutic agent has an IC of greater than 1 nM. 50 It has.

[0339] In some embodiments, ICs exceeding approximately 1 nM 50Therapeutic agents having a low potency (e.g., “low-potency drugs”) are not suitable for conjugation with antibodies using conjugation techniques recognized in the art. While we do not wish to be bound by theory, such therapeutic agents (i.e., cytotoxic drug moieties or STING agonist drug moieties) have insufficient potency for use in conventional targeted antibody-drug conjugates because it is not possible to conjugate a sufficient number of copies of the drug (i.e., more than 8) using techniques recognized in the art without degrading the pharmacokinetic and physicochemical properties of the conjugate. In some embodiments, the conjugation strategies described herein can be used to achieve sufficiently high loadings of these low-potency drugs, thereby resulting in a high loading of the therapeutic agent while maintaining desirable pharmacokinetic and physicochemical properties. In some embodiments, the present disclosure provides an antibody-drug conjugate comprising an antibody, a scaffold, and at least 8 therapeutic agents (i.e., cytotoxic drug moieties or STING agonist drug moieties), wherein the therapeutic agent has an IC50 greater than approximately 1 nM 50 This relates to antibody-drug conjugates having the following properties.

[0340] Cytotoxic drug portion (variable D) In some embodiments, the therapeutic agent is a cytotoxic drug moiety. In some embodiments, the cytotoxic drug moiety is (a) an auristatin compound, (b) a calicheamycin compound, (c) a duocalmycin compound, (d) SN38, (e) a pyrrolobenzodiazepine, (f) a vinca compound, (g) a tubulisin compound, (h) a non-natural camptothecin compound, (i) a meitansinoid compound, (j) a DNA conjugate, (k) a kinase inhibitor, (l) a MEK inhibitor, (m) a KSP inhibitor, (n) a topoisomerase inhibitor, (o) a DNA alkylating agent, (p) an RNA polymerase, (q) a PARP inhibitor, (r) a NAMPT inhibitor, (s) a topoisomerase inhibitor, (t) a protein synthesis inhibitor, (u) a DNA conjugate, (v) a DNA intercalation agent, or (w) a derivative of an immunomodulatory compound, as described in U.S. Patent No. 2018 / 0154018, the entirety of which is incorporated herein by reference.

[0341] In some embodiments, the cytotoxic drug portion is auristatin F-hydroxypropylamide-L-alanine.

[0342] In some embodiments, auristatin is a compound of formula (X): TIFF2026082984000090.tif21128 formula, R 31 and R 32 Each of them independently contains hydrogen or C 1~8 It is alkyl, R 31 and R 32 The maximum of these is H, R 33 is hydrogen, C 1~8 Alkyl, C 3~8 carbocycle, C 6~10 Ariel, C 1~8 Alkyl-C 6~10 Ariel, X 1 -(C 3~8 carbon ring), C 3~8 A complex algebra, or X 1 -(C 3~8 It is a complex algebra, R 34 is hydrogen, C1~8 Alkyl, C 3~8 carbocycle, C 6~10 Ariel, X 1 -C 6~10 Ariel, X 1 -(C 3~8 carbon ring), C 3~8 A complex algebra, or X 1 -(C 3~8 It is a complex algebra, R 35 is hydrogen or methyl, Or, R 34 and R 35 Together with the carbon atoms to which they are bonded, they form the formula -(CR 55 R 41 ) b - forms a carbon cyclic ring having, in the formula, R 55 and R 41 Each of them independently contains hydrogen or C 1~8 It is an alkyl group, and b is an integer between 3 and 7. R 36 is hydrogen or C 1~8 It is alkyl, R 37 is hydrogen, C 1~8 Alkyl, C 3~8 carbocycle, C 6~10 Ariel, -X 1 -C 6~10 Ariel, -X 1 -(C 3~8 carbon ring), C 3~8 A complex algebra, or -X 1 -(C 3~8 It is a complex algebra, Each R 38 These are independently hydrogen, OH, and C. 1~8 Alkyl, C 3~8 A carbon ring, or O-(C 1~8 It is alkyl, R 53 teeth, TIFF2026082984000091.tif19128 or R 54 And, R 39 is hydrogen, C 1~8 Alkyl, C 6~10 Ariel, -X 1 -C6~10 Ariel, C 3~8 carbocycle, C 3~8 The complex algebra, -X 1 -C 3~8 Complex algebra, -C 1~8 It is alkylene-NH2 or (CH2)2SCH3, each X 1 Independently, C 1~10 Alkylene or C 3~10 It is a cycloalkylene, R 44 is hydrogen or C 1~8 It is alkyl, R 45 X 3 -R 42 or NH-R 19 And, X 3 is either O or S, R 19 is hydrogen, OH, amino group, C 1~8 Alkylamino, or -[C(R 20 R 21 )] a -R 22 And, R 42 is an amino group, C 1~6 Alkylamino, or -[C(R 20 R 21 )] a -R 22 And, R 20 and R 21 Each of them independently contains hydrogen and C 1~6 Alkyl, C 6~10 Aryl, hydroxylated C 6~10 Aryl, polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, hydroxylated C 3~8 Cycloalkyl, polyhydroxylated C 3~8 A cycloalkyl group, or a side chain of a natural or unnatural amino acid, R 22 -OH, -NHR 23 -COOH, -R 82 -C(O)(CH2)c -C(H)(R 23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(O CH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 And, Each R 23 These are, independently, hydrogen and C 1~6 Alkyl, C 6~10 Ariel, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 It is alkyl, X 2 These are side chains of natural or unnatural amino acids. R 77 is hydrogen or X 2 And, NR 77 It forms nitrogen-containing cyclic compounds, R 82 -NR 23 or oxygen, R 54 -C(R 56 )2--C(R 56 )2-C 6~10 Aryl, -C(R 56 )2--C(R 56 )2-C 3~8 A heterogeneous ring, or C(R 56 )2--C(R 56 )2-C 3~8 It is a carbon ring, R 56 These are independently H, OH, and C. 1~8 Alkyl, C 3~8 Carbocycle, -OC 1~8 Alkyl, -OC(O)-R 29 , or -OR 23 -OC 1~6 It is alkyl-NH2, R 29 It is an amino group, a 5-12 member heterocycloalkyl group, -R 28 -C 1~6Alkyl-R 22 , R 28 -C 5~12 Heterocycloalkyl-C 1~6 Alkyl-R 22 ,-[C(R 20 R 21 )] a -R 22 , or -R 28 -C 1~6 Alkyl-C 6~12 Aryl-C 1~6 Alkyl-R 22 is or R 29 R is defined herein. 47 And, R 28 It does not exist, or NR 23 or oxygen, a is an integer between 1 and 6, c is an integer between 0 and 3, d is an integer between 1 and 3, and f is an integer between 1 and 12.

[0343] In some embodiments, in an auristatin compound of formula (X): R 39 is benzyl or TIFF2026082984000092.tif21128, R 44 It is hydrogen.

[0344] In some embodiments, auristatin is a compound of formula (Xa): The filename is TIFF2026082984000093.tif22128. During the ceremony, R 33 ~R 38 , and R 44 This is as defined herein, R 31 and R 32 One of them is hydrogen or C 1~8 It is alkyl, and the other is The filename is TIFF2026082984000094.tif26128. During the ceremony, R 83 is hydrogen or CH3, R 84 is C 1~6 alkyl or C 6~10 aryl, and each R 12 ’ is independently halogen, -C 1~8 alkyl, -O-C 1~8 alkyl, nitro, or cyano, and h is an integer from 0 to 4, and u is the integer 0 or 1, and R 53 is TIFF2026082984000095.tif21128 or R 54 and R 39 is hydrogen, C 1~8 alkyl, C 6~10 aryl, -X 1 -C 6~10 aryl, C 3~8 carbocyclic, C 3~8 heterocyclic, -X 1 -C 3~8 heterocyclic, -C 1~8 alkylene-NH2, or (CH2)2SCH3, and each X 1 is independently C 1~10 alkylene or C 3~10 cycloalkylene, and R 45 is X 3 -R 42 or NH-R 19 and X 3 is O or S, and R 19 is hydrogen, OH, an amino group, C 1~8 alkylamino, or -[C(R 20 R 21 )] a -R 22 and R 42 is hydrogen, an amino group, C 1~6 alkylamino, or -[C(R 20 R 21 )] a -R 22 and R 20 and R 21 Each of them independently contains hydrogen and C 1~6 Alkyl, C 6~10 Aryl, hydroxylated C 6~10 Aryl, polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, hydroxylated C 3~8 Cycloalkyl, polyhydroxylated C 3~8 A cycloalkyl group, or a side chain of a natural or unnatural amino acid, R 22 -OH, -NHR 23 -COOH, -R 82 -C(O)(CH2) c -C(H)(R 23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(O-CH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 And, Each R 23 These are, independently, hydrogen and C 1~6 Alkyl, C 6~10 Ariel, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 It is alkyl, X 2 These are side chains of natural or unnatural amino acids. R 77 is hydrogen or X 2 And, NR 77 It forms nitrogen-containing cyclic compounds, R 82 -NR 23 or oxygen, R 54 -C(R 56 )2--C(R 56 )2-C 6~10 Aryl, -C(R 56)2--C(R 56 )2-C 3~8 a complex ring, or -C(R 56 )2--C(R 56 )2-C 3~8 is a carbocyclic ring, R<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​The filename is TIFF2026082984000097.tif18128. R 83 It is either hydrogen or CH3.

[0346] In some embodiments, auristatin of formula (X) is a compound of formula (XI), formula (XII), or formula (XIII), Here, the compound of formula (XI) The filename is TIFF2026082984000098.tif23128. In the formula, R 31 is hydrogen or CH3, and R 42 is either -CH3 or one of the following structures: The filename is TIFF2026082984000099.tif191155. During the ceremony, a is an integer between 1 and 6, c is an integer between 0 and 3, and g is an integer between 2 and 6; Here, the compound of formula (XII) The filename is TIFF2026082984000100.tif24128. In the formula, R 31 is hydrogen or CH3, and R 40 is hydrogen, -OH, -NH2, or one of the following structures: TIFF2026082984000101.tif84153TIFF2026082984000102.tif147155, During the ceremony, a is an integer between 1 and 6, g is an integer between 2 and 6, and c is an integer between 0 and 3; Here, the compound of formula (XIII) The filename is TIFF2026082984000103.tif28134. During the ceremony, R 31 is hydrogen or CH3, R 29 It is an amino group, a 5-12 member heterocycloalkyl group, -R 28 -C 1~6 Alkyl-R 22 , R 28 -C 5~12Heterocycloalkyl-C 1~6 Alkyl-R 22 , -R 28 -[C(R 20 R 21 )] a -R 22 , or -R 28 -C 1~6 Alkyl-C 6~12 Aryl-C 1~6 Alkyl-R 22 is or R 29 R as defined herein 47 And, R 20 and R 21 Each of them independently contains hydrogen and C 1~6 Alkyl, C 6~10 Aryl, hydroxylated C 6~10 Aryl, polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, hydroxylated C 3~8 Cycloalkyl, polyhydroxylated C 3~8 A cycloalkyl group, or a side chain of a natural or unnatural amino acid, R 22 -OH, -NHR 23 -COOH, -R 82 -C(O)(CH2) c -C(H)(R 23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(O CH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 And, Each R 23 These are, independently, hydrogen and C 1~6 Alkyl, C 6~10 Ariel, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 It is alkyl, X 2These are side chains of natural or unnatural amino acids. R 77 is hydrogen or X 2 And, NR 77 It forms nitrogen-containing cyclic compounds, R 82 -NR 23 or oxygen, R 28 It does not exist, or NR 23 or oxygen, a is an integer between 1 and 6, c is an integer between 0 and 3, d is an integer between 1 and 3, and f is an integer between 1 and 12.

[0347] In some aspects of equation (XII), R 40 teeth The filename is TIFF2026082984000104.tif59160.

[0348] In some embodiments, the compound of formula (XII) is a compound of formula (XIIa), (XIIb), (XIIc), (XIId), (XIIe), (XIIf), (XIIg), or (XIIh): The files are TIFF2026082984000105.tif32128, TIFF2026082984000106.tif204100, and TIFF2026082984000107.tif201111.

[0349] In some embodiments of the compound of formula (XIII), R 29 -NH2, 5-membered heterocycloalkyl, -R 28 -C 1~6 Alkyl-R 22 , R 28 -C 5~12 Heterocycloalkyl-C 1~6 Alkyl-R 22 , or -R 28 -C 1~6 Alkyl-C 6~12 Aryl-C 1~6 Alkyl-R 22 is or R 29 R as defined herein 47 And, R 28 It does not exist, or NR 23 or oxygen, R 22 -OH, -NHR 23 -COOH, -R 82 -C(O)(CH2) c -C(H)(R 23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(O CH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 And, Each R 23 These are, independently, hydrogen and C 1~6 Alkyl, C 6~10 Ariel, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 It is alkyl, X 2 These are side chains of natural or unnatural amino acids. R 77 is hydrogen or X 2 And, NR 77 It forms nitrogen-containing cyclic compounds, R 82 -NR 23 or oxygen, c is an integer between 0 and 3, d is an integer between 1 and 3, and f is an integer between 1 and 12.

[0350] In some embodiments, R 29 teeth: TIFF2026082984000108.tif114157TIFF2026082984000109.tif156163, During the ceremony, a is an integer between 1 and 6, c is an integer between 0 and 3, and g is an integer between 2 and 6.

[0351] TIFF2026082984000110.tif23128 formula, R 42 H, -CH3 (m / z=760), The filename is TIFF2026082984000111.tif11150; In formula TIFF2026082984000112.tif26128, R40 is H, TIFF2026082984000113.tif152169 is; or TIFF2026082984000114.tif31139 In the formula, -C(O)-R 29 teeth, The filename is TIFF2026082984000115.tif41168.

[0352] In some embodiments, the cytotoxic drug portion (D) is as follows: TIFF2026082984000116.tif30128

[0353] STING agonist drug portion (variable D) In some embodiments, the STING agonist drug portion (D) is a compound of formula (A): TIFF2026082984000117.tif69128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: Y1, Y2, Z1, and Z2 are each independently O, S, C, or N. X1, X2, W1, and W2 are each independently either C or N. X3 and X4 can be independently S or NR f And, X5 is N or CR A2 And, X6 is N or CR A1 And, X7 is N or CR 4 And, R 3 and R 5 These are, independently, -CON(R d )(R f ), -CH2N(Rd )(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(R d )CO(R f ) and R 3 and R 5 The other is H, -COOH, or -CO2(R C ) and R c is C 1~4 It is alkyl, R A2 , R A1 and R 4 These are, independently, H, halogen, hydroxyl, amino, and amino(C) 1~4 Alkyl)-, may be substituted (C 1~6 Alkyl), or possibly substituted (C 1~6 It is alkyl)oxy-, and the substituted (C) is also alkyl. 1~6 Alkyl), or possibly substituted (C 1~6 C of alkyl)oxy 1~6 Alkyl is hydroxyl, C 1~4 Alkoxyl, -N(R e )(R f ), -CO2(R f ), -CON(R e )(R f ), and -COOH may each be independently selected from the group, and may be substituted with 1 to 4 substituents. Each R d These are independently H, hydroxyl, or C1~4 It is alkyl, R e H, (C 1~4 Alkyl), -CO(C 1~4 Alkyl), -OCO(C 1~4 Alkyl), and -CO2(C 1~4 Selected from alkyl, Each R f These are independently H, hydroxyl, or (C 1~4 It is alkyl, R 14 and R C2 These are, independently, non-existent, or C 1~4 It is alkyl, C 1~4 Alkyl is a halogen, -OR c , -NR c R d , -CO2R c ,-CONR c R d -SO2NR c R d , and -OCONR c R d They may be substituted by substituents selected from, R 16 and R C1 Each of these is independent, either nonexistent, or H or C 1~4 It is alkyl, R 15 , R 17 , R 18 or R 19 Each of these is independent, either nonexistent, or H or C 1~4 It is alkyl, C 1~4 Alkyl is a halogen, -OR c , -NR c R d , -CO2R c ,-CONR c R d -SO2NR c R d , and -OCONR c R d They may be substituted by substituents selected from, (i)R A2 and RA1 At least one of these exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L C If L exists C to, or L C If it does not exist, then A 1 (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L C If L exists C to, or L C If it does not exist, then A 1 It is connected to it directly or indirectly.

[0354] In some embodiments, the STING agonist drug portion (D) is a compound of formula (A'): TIFF2026082984000118.tif62128 or its prodrugs, solvates, pharmaceutically acceptable salts or tautomers, wherein the formula contains Y1, Y2, Z1, Z2, X1, X2, W1, W2, X3, X4, X5, X6, R 3 , R 5 , R c , R A1 , R A2 , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 and R 19 This is defined by equation (A).

[0355] In some embodiments, the STING agonist drug portion is a compound of formula (Aa): TIFF2026082984000119.tif76128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein in the formula, Y1, Y2, Z1, Z2, X1, X2, W1, W2, X3, X4, R 3 , R 5 , R c , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 and R 19 This is defined by equation (A), X5 is CR A2 And, R A2 (C) may be substituted with halogens, hydroxyls, or other elements. 1~6 Alkyl), substituted (C 1~6 Alkyl)oxy-, may be substituted (C 1~6 Alkyl)amino-, or possibly substituted (C 1~6 Alkyl)(C 1~4 It is alkyl)amino-, and the substituted (C) 1~6 Alkyl), or substituted (C 1~6 C of alkyl)oxy 1~6 Alkyl is hydroxyl, C 1~4 Alkoxyl, -N(R e )(R f ), -CO2(R f ), -CON(R e )(R f ), and -COOH may each be independently selected from the group, and may be substituted with 1 to 4 substituents. (i)R A2 R A2 L via the functional group D (ii)R C2 and R C1At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0356] In some embodiments, the STING agonist drug portion is a compound of formula (Ab): TIFF2026082984000120.tif76128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: Y1, Y2, Z1, Z2, X1, X2, W1, W2, X4, X5, X6, R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 and R 19 This is defined by equation (A), (i)R A2 and R A1 At least one of these exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0357] In some embodiments, the STING agonist drug portion is a compound of formula (Ac): TIFF2026082984000121.tif75128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: Y2, Z2, X2, W2, X3, X4, X5, X6, R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 and R 19 This is defined by equation (A), One of W1, X1, Y1, and Z1 is N, and the additional W1, X1, Y1, and Z1 are O, S, or C. (i)R A2 and R A1 At least one of these exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0358] In some embodiments, the STING agonist drug portion is a compound of formula (Ad): TIFF2026082984000122.tif76128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein in the formula, Y1, Y2, Z1, Z2, X1, X2, W1, W2, X3, R 3 , R 5 , R c , R d , R e , R f , R 14 , R A2 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 and R 19 This is defined by equation (A), X5 is CR A2 And, (i)R A2 R A2 L via the functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0359] In some embodiments, the STING agonist drug portion is a compound of formula (Ae): TIFF2026082984000123.tif76128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: Y1, Y2, Z1, Z2, X1, X2, X3, W1, W2, R A1 , R3, R 5 , R c , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 and R 19 This is defined by equation (A), X6 is CR A1 And, (i)R A1 R A1 L via the functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0360] In some embodiments, the STING agonist drug portion is a compound of formula (Af): TIFF2026082984000124.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: X2, X3, X4, X5, X6, W2, Y2, Z2R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 16 , R 17 , R 18 , R 19 , R C2 and R C1 This is defined by equation (A), (i)R A2 and R A1 At least one of these exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2and / or R C1 L via at least one functional group D It connects to the network.

[0361] In some embodiments, the STING agonist drug portion is a compound of formula (A-f1): TIFF2026082984000125.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein in the formula, X2, X3, X4, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R 16 , R A2 , R 17 , R 18 , R 19 , R C2 and R C1 This is defined by equation (A), X5 is CR A2 And, (i)R A2 R A2 L via the functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0362] In some embodiments, the STING agonist drug portion is a compound of formula (A-f2): TIFF2026082984000126.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: X2, X4, X5, X6, X7, W2, Y2, Z2R 3 , R 5 , R c , RA2 , R A1 , R 4 , R d , R e , R C1 , R C2 , R 16 , R 17 , R 18 , R 19 and R f This is defined by equation (A), (i)R A2 and R A1 At least one of these exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0363] In some embodiments, the STING agonist drug portion is a compound of formula (A-f3): TIFF2026082984000127.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R 16 , R A2 , R C2 , R 16 , R 17 , R 18 , R 19 and R C1 This is defined by equation (A), X5 is CR A2And, (i)R A2 R A2 L via the functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0364] In some embodiments, the STING agonist drug portion is a compound of formula (A-f4): TIFF2026082984000128.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R 16 , R A2 , R C2 , R 16 , R 17 , R 18 , R 19 and R C1 This is defined by equation (A), X5 is CR A2 And, (i)R A2 R A2 L via the functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0365] In some embodiments, the STING agonist drug portion is a compound of formula (A-f5): TIFF2026082984000129.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein in the formula, 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 This is defined by equation (A), X5 is CR A2 And, (i)R A2 R A2 L via the functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0366] In some embodiments, the STING agonist drug portion is a compound of formula (A-f6): TIFF2026082984000130.tif76128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: Y1, Y2, Z1, Z2, X1, X2, W1, W2, X4, X5, X6, R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 14, R C2 , R 16 , R C1 , R 15 , R 17 , R 18 and R 19 This is defined by equation (A), (i)R A2 and R A1 At least one of these exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0367] In some embodiments, the STING agonist drug portion is a compound of formula (A-f7): TIFF2026082984000131.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein in the formula, X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R 16 , R A2 , R C2 , R 16 , R 17 , R 18 , R 19 and R C1 This is defined by equation (A), X5 is CR A2 And, (i)R A2 R A2 L via the functional group D(ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0368] In some embodiments, the STING agonist drug portion is a compound of formula (Ag): TIFF2026082984000132.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, in which, X3, X4, X5, X6, X7, R 3 , R 5 , R c , R A2 , R A1 , R 4 , R C2 , R 17 , R 18 , R 19 , R d , R e , R f , R 16 and R C1 This is defined by equation (A), Y2 and Z2 are independently O, S, C, or N. X2 and W2 are independently either C or N. (i)R A2 and R A1 At least one of these exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or RC1 L via at least one functional group D It connects to the network.

[0369] In some embodiments, the STING agonist drug portion is a compound of formula (A-g1): TIFF2026082984000133.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein in the formula, X2, W2, Y2, Z2, X3, X4, X5, R 3 , R 5 , R c , R d , R e , R f , R A2 , R C2 , R 17 , R 18 , R 19 , R 16 and R C1 This is defined by equation (A), and (i)R A2 R A2 L via the functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0370] In some embodiments, the STING agonist drug portion is a compound of formula (A-g2): TIFF2026082984000134.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein in the formula, X2, X4, X5, X6, X7, W2, Y2, Z2, R 3 , R 5 , R c , R A2 , R A1 , R 4 , R C2 , R17 , R 18 , R 19 , R d , R e , R f , R 16 and R C1 This is defined by equation (A), (i)R A2 and R A1 At least one of these exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0371] In some embodiments, the STING agonist drug portion is a compound of formula (A-g3): TIFF2026082984000135.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R A2 , R C2 , R 17 , R 18 , R 19 , R 16 and R C1 This is defined by equation (A), X5 is CR A2 And, (i)R A2 R A2 L via the functional group D (ii)R C2 and R C1At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D Connected to Optionally, R A2 R A2 L via the functional group D It connects to the network.

[0372] In some embodiments, the STING agonist drug portion is a compound of formula (A-g4): TIFF2026082984000136.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: X2, X4, W2, Y2, Z2R 3 , R 5 , R c , R d , R e , R f , R A2 , R C2 , R 17 , R 18 , R 19 , R 16 and R C1 This is defined by equation (A), X5 is CR A2 And, (i)R A2 R A2 L via the functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0373] In some embodiments, the STING agonist drug portion is a compound of formula (A-g5): TIFF2026082984000137.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, in which, X2, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R A2 , R C2 , R 17 , R 18 , R 19 , R 16 and R C1 This is defined by equation (A), X5 is CR A2 And, (i)R A2 R A2 L via the functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0374] In some embodiments, the STING agonist drug portion is a compound of formula (A-g6): TIFF2026082984000138.tif70128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein in the formula, X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R A2 , R C2 , R 17 , R 18 , R 19 , R 16 and R C1 This is defined by equation (A), X5 is CR A2 And, (i)R A2 R A2 L via the functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D Connected to Optionally, R A2 R A2 L via the functional group D It connects to the network.

[0375] In some embodiments, the STING agonist drug portion is a compound of formula (Ah): TIFF2026082984000139.tif78128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: X1, W1, Y1, Z1, X3, X4, X5, X6, R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 14 , R 15 , R 18 , R 19 , R 16 and R C1 This is defined by equation (A), and (i)R A2 and R A1 At least one of these exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0376] In some embodiments, the STING agonist drug portion is a compound of formula (A-h1): TIFF2026082984000140.tif78128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: 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 This is defined by equation (A), (i)R A2 and R A1 At least one of these exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0377] In some embodiments, the STING agonist drug portion is a compound of formula (A-h2): TIFF2026082984000141.tif80128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: X1, X3, W1, Y1, Z1, R 3 , R 5 , R c , R d , R e , R f , R A2 , R 14 , R 15 , R 18 , R 19 , R 16 and R C1 This is defined by equation (A), X5 is CR A2 And, (i)R A2 and R A1 At least one of these exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0378] In some embodiments, the STING agonist drug portion (D) is a compound of formula (A), and the compound is of formula (Ai): TIFF2026082984000142.tif63128 or its prodrug, solvate, pharmaceutically acceptable salt or tautomer, wherein the formula is: Y1, Y2, Z1, Z2, X1, X2, X3, X6, X7, W1, W2, R A1 , R A2 , R 4 , R c , R d, R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 and R 19 This is defined by equation (A), (i)R A2 and R A1 At least one of these exists, R A2 and R A1 At least one of them is R A2 and / or R A1 L via at least one functional group D (ii)R C2 and R C1 At least one of these exists, R C2 and R C1 At least one of them is R C2 and / or R C1 L via at least one functional group D It connects to the network.

[0379] In some embodiments, each STING agonist drug portion (D) is independently: TIFF2026082984000143.tif203131TIFF2026082984000144.tif215129TIFF2026082984000145.ti f218128TIFF2026082984000146.tif214126TIFF2026082984000147.tif217128TIFF202608298400 0148.tif212128TIFF2026082984000149.tif217131TIFF2026082984000150.tif208125TIFF20260 82984000151.tif198126TIFF2026082984000152.tif201126TIFF2026082984000153.tif95129In the formula, R 2 It does not exist, or is -O-, or -NR4 - and R 4 is H or C 1~3 It is alkyl, TIFF2026082984000154.tif4128 is L D This indicates a connection to [the specified location].

[0380] In some embodiments, each STING agonist drug portion (D) is independently: TIFF2026082984000155.tif52128TIFF2026082984000156.tif216130TIFF2026082984000157.tif168130TIFF2026082984000158.tif103128In the formula, R 2 It does not exist, or is -O-, or -NR 4 - and R 4 is H or C 1~3 It is alkyl, TIFF2026082984000159.tif4128 is L D This indicates a connection to [the specified location].

[0381] In some embodiments, each STING agonist drug portion (D) is independently: TIFF2026082984000160.tif52128TIFF2026082984000161.tif108135In the formula, R 2 It does not exist, or is -O-, or -NR 4 - and R 4 is H or C 1~3 It is alkyl, TIFF2026082984000162.tif4128 is L D This indicates a connection to [the specified location].

[0382] In some embodiments, each STING agonist drug portion (D) is independently: TIFF2026082984000163.tif50128TIFF2026082984000164.tif98138In the formula, R 2 It does not exist, or is -O-, or -NR 4 - and R 4 is H or C 1~3 It is alkyl, TIFF2026082984000165.tif4128 is L D This indicates a connection to [the specified location].

[0383] In some embodiments, each STING agonist drug portion (D) is independently: TIFF2026082984000166.tif106138In formula, R 2 It does not exist, or is -O-, or -NR 4 - and R 4 is H or C 1~3 It is alkyl, TIFF2026082984000167.tif4128 is L D This indicates a connection to [the specified location].

[0384] Hydrophilic group (variable T 1 ) In some embodiments, the hydrophilic groups contained in the conjugates or scaffolds of the present disclosure are water-soluble and substantially non-antigenic polymers. Examples of hydrophilic groups include, but are not limited to, polyalcohols, polyethers, polyanions, polycations, polyphosphates, polyamines, polysaccharides, polyhydroxy compounds, polylysine and its derivatives. In some embodiments, one end of the hydrophilic group is connected by an inseparable bond or via a cleavable bond. A Linker (for example, M AThe hydrophilic group may be functionalized to be covalently bonded to an amino acid in the linker. In some embodiments, the functionalization may be via, for example, an amine, thiol, NHS ester, maleimide, alkyne, azide, carbonyl, or other functional group. In some embodiments, the other end (terminus) (or terminus) of the hydrophilic group is free and unbonded. In some embodiments, "unbonded" means that the hydrophilic group is not bonded to another part, such as D or a drug unit, or other components of the conjugate or scaffold of this disclosure. In some embodiments, the free and unbonded end of the hydrophilic group may include a methoxy, carboxylic acid, alcohol, or other suitable functional group. In some embodiments, the methoxy, carboxylic acid, alcohol, or other suitable functional group acts as a cap for the terminus or terminus of the hydrophilic group.

[0385] In some embodiments, a cleavable bond refers to a bond that is substantially insensitive to cleavage while circulating in plasma but sensitive to cleavage in the intracellular or intratumoral environment. In some embodiments, an uncleavable bond is one 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 bonds of hydrophilic groups are mediated by amide bonds, ether bonds, ester bonds, hydrazone bonds, oxime bonds, disulfide bonds, peptide bonds, or triazole bonds. In some embodiments, M A (For example, M) A The attachment of hydrophilic groups to amino acids in the linker is via amide bonds.

[0386] In some embodiments of the Conjugate or Scaffold of the Disclosure, which comprises multiple hydrophilic groups, the multiple hydrophilic groups may be the same or different chemical moieties (e.g., hydrophilic groups with different molecular weights, number of subunits, or chemical structures). In some embodiments, the multiple hydrophilic groups may be M at a single binding site or at different sites. A It can be linked to a linker.

[0387] In some embodiments, the addition of a hydrophilic group can have two potential effects on the pharmacokinetics of the resulting conjugate. In some embodiments, the desired effect is a decrease in clearance (and consequently, an increase in exposure) resulting from a reduction in nonspecific interactions induced by the exposed hydrophobic element of the drug or drug-linker. In some embodiments, the undesirable effect is a decrease in volume and distribution rate, which may result from an increase in the molecular weight of the conjugate. In some embodiments, increasing the molecular weight of the hydrophilic group may increase the hydrodynamic radius of the conjugate and decrease its diffusivity, thereby reducing the conjugate's ability to penetrate tumors. Due to these two competing pharmacokinetic effects, it may be desirable to use a hydrophilic group that is large enough to reduce conjugate clearance and thus increase plasma exposure, but not large enough to significantly reduce its diffusivity, thereby reducing the conjugate's ability to reach the intended target cell population.

[0388] In some embodiments, the hydrophilic group includes, but is not limited to, sugar alcohols (polyalcohols, polyhydric alcohols, alditols or glycitols, also known as inositol, glycerol, erythritol, treitol, arabitol, xylitol, ribitol, galactitol, mannitol, sorbitol, etc.) or derivatives thereof (e.g., aminopolyalcohols), carbohydrates (e.g., sugars), polyvinyl alcohols, carbohydrate polymers (e.g., dextran), hydroxypropyl methacrylamide (HPMA), polyalkylene oxides and / or copolymers thereof.

[0389] In some aspects, T 1 It includes a portion that incorporates multiple hydroxyl ("-OH") groups, such as monosaccharides, oligosaccharides, and polysaccharides.

[0390] In some aspects, T 1 is multiple -(CR 58 It contains an OH)- group, and in the formula, R 58 is -H, or C 1~8 It is alkyl.

[0391] In some aspects, T 1 is -OH or The file is TIFF2026082984000168.tif6128, and in the formula, n1 is an integer between 0 and approximately 6. Each R 58 These are independently -H, or C 1~8 It is alkyl, R 60 is a bond, C 1~6 Alkyl linker, or -CHR 59 - and in the formula, R 59 is -H, C 1~8 Alkyl, cycloalkyl, or arylalkyl, R 61 CH2OR 62 COOR 62 ,-(CH2) n2 COOR 62 , or heterocycloalkyl substituted with one or more hydroxyls, R 62 is -H, or C 1~8 It is alkyl, n² is an integer between 1 and approximately 5.

[0392] In some aspects, T 1 It is -OH.

[0393] In some aspects, T 1 teeth The filename is TIFF2026082984000169.tif6128.

[0394] In some embodiments, R 58 is -H, and R 60 is a combination or C 1~6 It is an alkyl linker, where n1 is an integer from 1 to approximately 6, and R 61 It is either CH2OH or COOH.

[0395] In some embodiments, R 58 is -H, and R 60 ha-CHR 59 - and n1 is 0, R 61 This is a heterocycloalkyl group, such as a monosaccharide, that is substituted with one or more hydroxyls.

[0396] In some aspects, T 1 This includes glucosylamines, diamines, or triamines.

[0397] In some aspects, T 1 This includes one or more of the following fragments or their stereoisomers: TIFF2026082984000170.tif179169In formula, R 59 is -H, C 1~8 Alkyl, cycloalkyl, or arylalkyl, n1 is an integer between 1 and approximately 6. n² is an integer between 1 and approximately 5. n3 is an integer between approximately 1 and approximately 3.

[0398] It is understood that in this specification, all stereochemical forms of hydrophilic groups are intended. For example, in the above formula, the hydrophilic group may be derived from ribose, xylose, glucose, mannose, galactose, or other sugars, and may retain the stereochemical configuration of the pendant hydroxyl group and alkyl group present on those molecules.

[0399] It is understood that various deoxy compounds are also intended in the aforementioned formula. For example, where applicable, one or more of the following characteristics are intended for the hydrophilic group:

[0400] In some embodiments, n3 is 2 or 3.

[0401] In some embodiments, n1 is 1, 2, or 3.

[0402] In some embodiments, n2 is 1.

[0403] In some embodiments, R 59 It is hydrogen.

[0404] In some aspects, T 1 teeth It is TIFF2026082984000171.tif16128. In some aspects, T 1 teeth The filename is TIFF2026082984000172.tif24128.

[0405] In some aspects, T 1 teeth The filename is TIFF2026082984000173.tif24128.

[0406] In some aspects, T 1 teeth TIFF2026082984000174.tif15128, and in the formula, n4 is an integer between 1 and approximately 25. Each R 63 These are independently -H, or C 1~8 It is alkyl, R 64 is a combination or C 1~8 It is an alkyl linker, R 65 is -H, C 1~8 Alkyl, -(CH2) n2 COOR 62 , or -(CH2) n2 COR66 And, R 62 is H, or C 1~8 It is alkyl, R 66 H, The filename is TIFF2026082984000175.tif49143. n² is an integer between 1 and approximately 5.

[0407] In some aspects, T 1 teeth, The filename is TIFF2026082984000176.tif19128. In the formula, R 67 The following is true: (1) OH In formula TIFF2026082984000177.tif66149, n4 is an integer between approximately 2 and 20, approximately 4 and 16, approximately 6 and 12, and approximately 8 and 12.

[0408] In some aspects, T 1 teeth The filename is TIFF2026082984000178.tif12128.

[0409] In some embodiments, n4 is an integer between approximately 2 and approximately 20, approximately 4 and approximately 16, approximately 6 and approximately 12, and approximately 8 and approximately 12.

[0410] In some embodiments, n4 is 6, 7, 8, 9, 10, 11, or 12.

[0411] In some embodiments, n4 is 8 or 12.

[0412] In some aspects, T 1 teeth, The file is TIFF2026082984000179.tif50128, where n4 is an integer between approximately 2 and 24, approximately 4 and 16, approximately 6 and 12, and approximately 8 and 12.

[0413] In some embodiments, n4 is 6, 7, 8, 9, 10, 11, or 12.

[0414] In some embodiments, n4 is 8. In some embodiments, n4 is 12.

[0415] In some aspects, T 1 teeth The filename is TIFF2026082984000180.tif26128, and in the formula, n4 is 8.

[0416] In some aspects, T 1 This includes polyethers, such as polyalkylene glycols (PAOs). PAOs include, but are not limited to, polymers of lower alkylene oxides, particularly polymers of ethylene oxides, such as propylene oxide, polypropylene glycol, polyethylene glycol (PEG), polyoxyethylene-derived polyols, copolymers thereof, and block copolymers thereof.

[0417] In some embodiments, polyalkylene glycol is polyethylene glycol (PEG), including, but not limited to, polydisperse PEG, monodisperse PEG, and individual PEGs. Polydisperse PEG is a heterogeneous mixture of size and molecular weight, while monodisperse PEG is typically purified from a heterogeneous mixture and therefore provides a single chain length and molecular weight. In some embodiments, the PEG unit is individual PEG, providing a single molecule with a defined specific chain length. In some embodiments, polyethylene glycol is mPEG.

[0418] In some aspects, T 1A PEG unit comprises one or more PEG chains. The PEG chains can be linked to each other in, for example, linear, branched, or star-shaped configurations. In addition to containing repeating PEG subunits, the PEG unit may also contain non-PEG material (for example, to facilitate the coupling of multiple PEG chains to each other or to facilitate coupling to amino acids). Non-PEG material refers to atoms in the PEG chain that are not part of the repeating -CH2CH2O- subunit. In some embodiments, the PEG chain may include two monomeric PEG chains linked to each other via non-PEG elements. In some embodiments, the PEG unit may include two linear PEG chains linked to a central core bonded to an amino acid (i.e., the PEG unit itself is branched).

[0419] The PEG unit is M via a reactive group. A Linker (for example, M A The linker may be covalently bonded to the amino acids. The reactive group is one to which the activated PEG molecule can be bound (e.g., a free amino group or a carboxyl group). In some embodiments, the N-terminal amino acid and lysine (K) have a free amino group, and the C-terminal amino acid residue has a free carboxyl group. Sulfhydryl groups (e.g., as found in cysteine ​​residues) can also be used as reactive groups for binding PEG.

[0420] In some embodiments, the PEG unit is made by using methoxylated PEG ("mPEG") having various reactive moieties including succinimidyl succinate (SS), succinimidyl carbonate (SC), mPEG-imidate, para-nitrophenyl carbonate (NPC), succinimidyl propionate (SPA), and cyanuryl chloride, without limiting M A Linker (for example, M AIt can be bound to amino acids in the linker. Examples of mPEG include, but are not limited to, mPEG-succinimidyl succinate (mPEG-SS), mPEG2-succinimidyl succinate (mPEG2-SS), mPEG-succinimidyl carbonate (mPEG-SC), mPEG2-succinimidyl carbonate (mPEG2-SC), mPEG-imidate, mPEG-para-nitrophenyl carbonate (mPEG-NPC), mPEG-imidate, and mPEG2-para-nitrophenyl Examples include carbonate (mPEG2-NPC), mPEG-succinimidylpropionate (mPEG-SPA), mPEG-N-hydroxysuccinimide (mPEG-NHS), mPEG-N-hydroxysuccinimide (mPEG2-NHS), mPEG-cyanurate chloride, mPEG2-cyanurate chloride, mPEG2-lycinol-NPC, and mPEG2-Lys-NHS. A wide variety of PEG species can be used, and substantially any suitable reactive PEG reagent can be used. In some embodiments, the reactive PEG reagent is a polyfunctional linker or M A Linker (for example, M AWhen it binds to an amino acid in the linker, it forms a carbamate bond or an amide bond. Reactive PEG reagents include, but are not limited to, mPEG2-N-hydroxysuccinimide (mPEG2-NHS), difunctional PEG propionaldehyde (mPEG2-ALD), and multi-arm PEG (multi-Arm PEG). PEG), maleimide-containing PEG (mPEG(MAL)2, mPEG2(MAL)), mPEG-NH2, mPEG-succinimidylpropionate (mPEG-SPA), succinimide of mPEG-butanoic acid (mPEG-SBA), mPEG-thioester, mPEG-double ester, mPEG-BTC, mPEG-ButyrALD, mPEG-acetaldehyde diethyl acetal (mPEG-ACET), heterofunctional PEG (e.g., NH2-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-vinyl sulfone (NHS-PEG-VS), or NHS-PEG-MAL), PEG acrylate (ACRL-PEG Multi-armed PEGs in the SUNBRITE® series, including SUNBRITE-activated PEGs (without limiting them), such as carboxyl-PEG, p-NP-PEG, Tresyl-PEG, aldehyde-PEG, acetal-PEG, amino-PEG, thiol-PEG, maleimide-PEG, hydroxyl-PEG-amine, amino-PEG-COOK hydroxyl-PEG-aldehyde, carboxylic acid anhydride-type PEG, functionalized PEG-phospholipids, and other similar and / or preferred reactive PEGs.

[0421] In some embodiments, the PEG unit includes at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, 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 includes about 72 or fewer subunits.

[0422] In some embodiments, the PEG unit includes at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, 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.

[0423] In some embodiments, the PEG unit includes at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, 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.

[0424] In some embodiments, the PEG unit includes at least six subunits, at least seven subunits, at least eight subunits, at least nine subunits, at least ten subunits, at least eleven subunits, or at least twelve subunits.

[0425] In some embodiments, the PEG unit includes at least eight subunits, at least nine subunits, at least ten subunits, at least eleven subunits, or at least twelve subunits.

[0426] In some embodiments, the PEG unit includes at least six subunits, at least seven subunits, or at least eight subunits.

[0427] In some embodiments, linear PEG units are The filename is TIFF2026082984000181.tif33145. During the ceremony, TIFF2026082984000182.tif12128 is M A (For example, M) A It shows the binding site (to the amino acid in the linker), Y 71 This is a PEG binding unit, Y 72 This is a PEG capping unit, Y 73 This is a PEG coupling unit (i.e., for coupling multiple PEG subunit strands together), d9 is an integer between 2 and 72. each d 10 These are independent integers from 1 to 72. d 11 It is an integer between 2 and 5.

[0428] In some embodiments, d9 is an integer between 2 and 24. In some embodiments, d9 is an integer between 4 and 24. In some embodiments, d9 is an integer between 6 and 24, 8 and 24, 10 and 24, or 12 and 24.

[0429] In some embodiments, there are at least six PEG subunits in a PEG unit. In some embodiments, there are at least eight PEG subunits in a PEG unit. In some embodiments, there are at least ten PEG subunits in a PEG unit. In some embodiments, there are at least twelve PEG subunits in a PEG unit.

[0430] In some embodiments, d9 is 8 or about 8, 12 or about 12, 24 or about 24.

[0431] In some embodiments, each Y 72 -C 1~10 Alkyl, -C 2~10 Alkyl-CO2H, -C 2~10 alkyl-OH, -C 2~10 Alkyl-NH2, -C 2~10 Alkyl-NH(C 1~3 Alkyl), or C 2~10 Alkyl-N(C 1~3 It is alkyl(2).

[0432] In some embodiments, Y 72 is -C 1~10 Alkyl, -C 2~10 Alkyl-CO2H, -C 2~10 alkyl-OH, or -C 2~10 It is alkyl-NH2.

[0433] In some embodiments, the PEG coupling unit is a non-PEG material that is part of the PEG unit and acts to connect two or more chains of repeating CH2CH2O- subunits. In some embodiments, the PEG coupling unit Y 73 is -C 2~10 Alkyl-C(O)-NH-, -C2~10 Alkyl-NH-C(O)-, -C 2~10 Alkyl-NH-,-C 2~10 Alkyl-C(O)-, -C 2~10 Alkyl-O-, or -C 2~10 It is alkyl-S-.

[0434] In some embodiments, each Y 73 -C 1~10 Alkyl-C(O)-NH-, -C 1~10 Alkyl-NH-C(O)-, -C 2~10 Alkyl-NH-,-C 2~10 Alkyl-O-, -C 1~10 Alkyl-S- or -C 1~10 It is alkyl-NH-.

[0435] In some embodiments, the PEG-binding unit is part of the PEG unit, and the PEG unit is M A Linker (for example, M AIt acts to bind to the amino acid in the linker. In some embodiments, the amino acid has a functional group that forms a bond with the PEG unit. In some embodiments, the functional groups for binding the PEG unit to the amino acid include sulfhydryl groups for forming a disulfide bond or thioether bond, aldehyde groups, ketone groups or hydrazine groups for forming a hydrazone bond, hydroxylamines for forming an oxime bond, carboxyl groups or amino groups for forming a peptide bond, carboxyl groups or hydroxyl groups for forming an ester bond, sulfonic acids for forming a sulfonamide bond, alcohols for forming a carbamate bond, and amines for forming a sulfonamide bond or carbamate bond or amide bond. In some embodiments, the PEG unit can be bound to the amino acid via, for example, a disulfide bond, thioether bond, hydrazone bond, oxime bond, peptide bond, ester bond, sulfonamide bond, carbamate bond or amide bond. In some embodiments, the reaction for binding the PEG unit may be a cyclization-addition, addition, addition / elimination or substitution reaction, or a combination thereof, where applicable.

[0436] Examples of linear PEG units include: TIFF2026082984000183.tif59138 was cited. During the ceremony, TIFF2026082984000184.tif12128 is a polyfunctional linker or M A (For example, M) A It indicates the binding site (to an amino acid in the linker), and each d9 is independently an integer between 4 and 24, 6 and 24, 8 and 24, 10 and 24, 12 and 24, 14 and 24, or 16 and 24.

[0437] In some embodiments, d9 is about 8, about 12, or about 24.

[0438] In some embodiments, d9 is approximately 8.

[0439] In some embodiments, the PEG unit is approximately 300 Da to approximately 5 kDa, approximately 300 Da to approximately 4 kDa, approximately 300 Da to approximately 3 kDa, approximately 300 Da to approximately 2 kDa, or approximately 300 Da to approximately 1 kDa. In some embodiments, the PEG unit has at least 6 subunits or at least 8, 10, or 12 subunits. In some embodiments, the PEG unit has at least 6 subunits, or at least 8, 10, or 12 subunits but no more than 24 subunits.

[0440] In some embodiments, the preferred polyethylene glycol may have free hydroxyl groups at each end of the polymer molecule, or one hydroxyl group etherified by a lower alkyl group, such as a methyl group. In some embodiments, the preferred polyethylene glycol is a derivative of polyethylene glycol having an esterifiable carboxyl group. In some embodiments, polyethylene glycol is typically marketed under the trade name PEG as a mixture of polymers characterized by some average molecular weight. In some embodiments, polyethylene glycol having an average molecular weight of about 300 to about 5000. In some embodiments, polyethylene glycol having an average molecular weight of about 600 to about 1000.

[0441] In some embodiments, examples of hydrophilic groups suitable for the conjugates, scaffolds, and methods disclosed herein can be found, for example, in U.S. Patent No. 8,367,065, Column 13, U.S. Patent No. 8,524,696, Column 6, International Publication No. 2015 / 057699, and International Publication No. 2014 / 062697, the entire contents of which are incorporated herein by reference.

[0442] antibody In some embodiments, a glycoprotein containing a core-N-acetylglucosamine substituent (core-GlcNAc moiety) is an antibody containing a core-N-acetylglucosamine substituent (core-GlcNAc moiety). In some embodiments, the glycoprotein is a monoclonal antibody (mAb) IgA antibody, IgD antibody, IgE antibody, IgG antibody, or IgM antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, if the antibody is a whole antibody, the antibody contains one or more (e.g., one) core-GlcNAc moieties on each heavy chain, and the core-GlcNAc moieties may be fucosylated. In some embodiments, the whole antibody contains two or more (e.g., two) fucosylated core-GlcNAc moieties. In some embodiments, if the antibody is a single-chain antibody or antibody fragment, e.g., a Fab fragment or an Fc fragment, the antibody contains one or more fucosylated core-GlcNAc moieties. In some embodiments, within an antibody containing a core-GlcNAc moiety, the core-GlcNAc moiety may be located anywhere on the antibody, provided that the substituent does not interfere with the antibody's antigen-binding site. In some embodiments, the core-GlcNAc moiety is located at the antibody's natural N-glycosylation site. In some embodiments, the antibody contains, or is engineered to contain, at least one chemically reactive group or chemically reactive amino acid moiety or chemically reactive side chain.

[0443] In some embodiments, antibodies can direct conjugates to specific tissues, cells, or intracellular locations. In some embodiments, antibodies can direct conjugates in a culture, in a whole organism, or both. In some embodiments, antibodies contain ligands present on the cell surface of target cells to which the antibody binds with effective specificity, affinity, and avidity. In some embodiments, antibodies direct conjugates to tissues other than the liver. In some embodiments, antibodies direct conjugates to specific tissues, such as the liver, kidney, lung, or pancreas. In some embodiments, antibodies direct conjugates to target cells (e.g., cancer cells), receptors expressed on cells (e.g., cancer cells), matrix tissue, or cancer-related proteins (e.g., tumor antigens). In some embodiments, cells including tumor vascular systems may be targeted. In some embodiments, antibodies can direct conjugates to specific types of cells, for example, specifically targeting hepatocytes in the liver rather than Kupffer cells. In some embodiments, the antibody can deliver the conjugate to reticular endothelial or lymphoid cells, or to professional phagocytic cells such as macrophages or eosinophils. In some embodiments, the conjugate itself is an effective delivery system that does not require specific targeting.

[0444] In some embodiments, antibodies can guide conjugates to a location within the cell (e.g., the nucleus, cytoplasm, or endosomes). In some embodiments, antibodies enhance cell binding to receptors, or cytoplasmic transport to the nucleus, and nuclear entry or release from endosomes or other intracellular vesicles.

[0445] In some embodiments, the conjugate comprises the B7-H4 antibody or modified B7-H4 antibody of the Disclosure.

[0446] B7-H4 antibody This disclosure provides isolated antibodies that bind to B7-H4, a type I transmembrane protein found on the surface of antigen-presenting cells (APCs), for example. B7-H4 antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising heavy and / or light chain CDRs as described herein.

[0447] In some embodiments, the B7-H4 antibody of this disclosure has the amino acid sequence: It specifically binds to the epitope on the full-length human B7-H4 protein, including TIFF2026082984000185.tif33160.

[0448] In some embodiments, the B7-H4 antibody is a human antibody. In some embodiments, the B7-H4 antibody modulates B7-H4 activity. In some embodiments, the antibody is an antibody that induces an ADCC response in the subject to which the antibody is administered. In some embodiments, the B7-H4 antibody does not inhibit the T-cell suppressive activity of B7-H4. The B7-H4 antibody of this disclosure may be, for example, a full-length antibody. Alternatively, the antibody may be an antibody fragment, such as a Fab fragment, a Fab' fragment, or a Fab'2 fragment, or a single-chain antibody (e.g., scFv). In some embodiments, the antibody is an IgG1 antibody.

[0449] In some embodiments, the B7-H4 antibody includes a heavy chain variable region and a light chain variable region. In some embodiments, the B7-H4 antibody includes at least one heavy chain comprising a heavy chain variable region and at least a portion of the heavy chain constant region, and at least one light chain comprising a light chain variable region and at least a portion of the light chain constant region. In some embodiments, the B7-H4 antibody includes two heavy chains, each comprising a heavy chain variable region and at least a portion of the heavy chain constant region, and two light chains, each comprising a light chain variable region and at least a portion of the light chain constant region.

[0450] In some embodiments, the human B7-H4 antibody comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is an isotype selected from IgA, IgG (e.g., IgG1, IgG2, IgG3, or IgG4, and IgD). In some embodiments, the human light chain constant region is an isotype selected from kappa (κ) and lambda (λ). In some embodiments, the human antibody described herein comprises a human IgG constant region. In some embodiments, the human antibody described herein comprises a human IgG4 heavy chain constant region. In some embodiments, the human antibody described herein comprises a human IgG4 constant region and a human κ light chain.

[0451] In some embodiments, when effector function is desired, a human B7-H4 antibody containing a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, when effector function is not desired, a human B7-H4 antibody containing a human IgG4 heavy chain constant region or a human IgG2 heavy chain constant region is selected.

[0452] In some embodiments, the antibodies of this disclosure are characterized by specific functional features or properties of the antibody. For example, the antibody binds specifically to human B7-H4. Typically, the antibodies of this disclosure have high affinity for B7-H4, for example, 1 × 10⁻⁶ -7 K below M D It binds via [method]. The anti-B7-H4 antibodies of this disclosure typically exhibit one or more of the following characteristics: (a) 1 × 10 -7 K below M D Binds to human B7-H4; and / or (b) Binds to human CHO cells transfected with B7-H4 (e.g., human B7-H4).

[0453] In some embodiments, the antibody is 5 × 10 -8 K below M D It binds to human B7-H4, or 2 × 10 -8 K below M DIt binds to human B7-H4, or 5 × 10 -9 K below M D It binds to human B7-H4, or 4 × 10 -9 K below M D It binds to human B7-H4, or 3 × 10 -9 K below M D It binds to human B7-H4, or 2 × 10 -9 K below M D It binds to human B7-H4, or 1 × 10⁻⁶ -9 K below M D It then binds to human B7-H4.

[0454] Standard assays for evaluating the binding ability of antibodies to B7-H4 are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry. Antibody binding kinetics (e.g., binding affinity) can also be evaluated by standard assays known in the art, such as ELISA, Scatchard, and Biacore® system analysis.

[0455] Potential therapeutic mAbs must not only bind to their targets but also be free from "developability issues," such as low stability or high levels of aggregation. The inventors describe five metrics considered relevant to low developability: the total length of the complementarity-determining region (CDR), the degree and size of surface hydrophobicity, the positive and negative charges within the CDR, and guideline values ​​for the asymmetry of the net heavy-chain and light-chain surface charges. Guideline cutoffs for each property are derived from values ​​observed in the CST, and a flagging system is proposed to identify unsuitable candidates.

[0456] The exemplary B7-H4 antibodies in this disclosure include B7-H4_2F9 (disclosed in U.S. Patent No. 8,609,816, the entirety of which is incorporated herein by reference, and also referred to herein as the “B7-H4_2F9 parent antibody,” the “2F9 parent antibody,” or the “parent antibody”).

[0457] Potential therapeutic mAbs must not only bind to their targets, but also be free from "developability issues," such as low stability, high levels of aggregation, low levels of expression, low solubility, covalent integrity, conformational and colloidal instability, high polyspecificity, and high immunogenicity.

[0458] The B7-H4_2F9 parent antibody sequence was analyzed for metrics thought to be related to low development potential, such as the total length of the complementarity-determining region (CDR), the degree and size of surface hydrophobicity, the positive and negative charges within the CDR, the asymmetry of the net heavy-chain and light-chain surface charges, and post-translational modifications (PTMs). This development potential analysis of the B7-H4_2F9 parent antibody sequence revealed three potential sequence liabilities. In particular, the B7-H4_2F9 parent antibody sequence contained unpaired cysteine ​​residues, aspartate isomerization sequences, and methionine oxidation sites. Each of these three sequence liabilities could potentially lead to developments in areas such as antibody stability, potency, and uniformity, and could introduce complex processes into downstream development.

[0459] To address the potential sequence liability of the B7-H4_2F9 parent antibody, 20 variants of B7-H4_2F9 were designed and generated to address these potential developmental concerns. These variant antibodies include B7-H4_2F9V1, B7-H4_2F9V2, B7-H4_2F9V3, B7-H4_2F9V4, B7-H4_2F9V5, B7-H4_2F9V6, B7-H4_2F9V7, B7-H4_2F9V8, B7-H4_2F9V9, B7-H4_2F9V10, B The study included 7-H4_2F9V11, B7-H4_2F9V12, B7-H4_2F9V13, B7-H4_2F9V14, B7-H4_2F9V15, B7-H4_2F9V16, B7-H4_2F9V17, B7-H4_2F9V18, B7-H4_2F9V19, and B7-H4_2F9V20. Each of the 20 mutants was characterized for B7-H4 binding, polyspecificity, and other properties.

[0460] The nucleic acid sequence and amino acid sequence of the monoclonal B7-H4 antibody disclosed herein are shown below. In the amino acid sequence shown below, the complementarity-determining regions (CDRs) of the heavy and light chains are underlined. The amino acids that comprise the complementarity-determining regions (CDRs) shown below are defined according to the IMGT numbering system (IMGT®, international ImMunoGeneTics information system®, available online at http: / / www.imgt.org / ).

[0461] B7-H4 2F9 Variable Area All 20 B7-H4 2F9 parental mutants (i.e., B7-H4_2F9V1 to B7-H4_2F9V20) share a common light chain variable region. (B7-H4_2F9 V in this specification) L (called)

[0462] In some embodiments, the entire antibody of the present disclosure includes or comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:50.

[0463] B7-H4_2F9_V L V L The chain (SEQ ID NO: 50) contains or consists of the following amino acid sequence: TIFF2026082984000186.tif11160

[0464] The parent VH chain (SEQ ID NO:1) of B7-H4_2F9 contains or consists of the following amino acid sequence: TIFF2026082984000187.tif18160

[0465] The VH chain (SEQ ID NO: 5) of B7-H4_2F9V1 contains or consists of the following amino acid sequence: TIFF2026082984000188.tif19161

[0466] The VH chain (SEQ ID NO:8) of B7-H4_2F9V2 contains or consists of the following amino acid sequence: TIFF2026082984000189.tif19161

[0467] The VH chain (SEQ ID NO:11) of B7-H4_2F9V3 contains or consists of the following amino acid sequence: TIFF2026082984000190.tif19160

[0468] The VH chain (SEQ ID NO: 14) of B7-H4_2F9V4 contains or consists of the following amino acid sequence: TIFF2026082984000191.tif18160

[0469] The VH chain (SEQ ID NO: 17) of B7-H4_2F9V5 contains or consists of the following amino acid sequence: TIFF2026082984000192.tif18159

[0470] The VH chain of B7-H4_2F9V6 (SEQ ID NO:20) contains or consists of the following amino acid sequence: TIFF2026082984000193.tif18161

[0471] The VH chain (SEQ ID NO:22) of B7-H4_2F9V7 contains or consists of the following amino acid sequence: TIFF2026082984000194.tif19161

[0472] The B7-H4_2F9V7 variable heavy chain is also referred to herein as the XMT-1603 variable heavy chain.

[0473] The VH chain (SEQ ID NO:24) of B7-H4_2F9V8 contains or consists of the following amino acid sequence: TIFF2026082984000195.tif18160

[0474] The VH chain (SEQ ID NO:26) of B7-H4_2F9V9 contains or consists of the following amino acid sequence: TIFF2026082984000196.tif19160

[0475] The VH chain (SEQ ID NO:28) of B7-H4_2F9V10 contains or consists of the following amino acid sequence: TIFF2026082984000197.tif18159

[0476] The VH chain (SEQ ID NO:30) of B7-H4_2F9V11 contains or consists of the following amino acid sequence: TIFF2026082984000198.tif18161

[0477] The VH chain (SEQ ID NO:32) of B7-H4_2F9V12 contains or consists of the following amino acid sequence: TIFF2026082984000199.tif18161

[0478] The VH chain (SEQ ID NO:34) of B7-H4_2F9V13 contains or consists of the following amino acid sequence: TIFF2026082984000200.tif19160

[0479] The VH chain (SEQ ID NO:36) of B7-H4_2F9V14 contains or consists of the following amino acid sequence: TIFF2026082984000201.tif18160

[0480] The VH chain (SEQ ID NO:38) of B7-H4_2F9V15 contains or consists of the following amino acid sequence: TIFF2026082984000202.tif19159

[0481] The VH chain (SEQ ID NO: 40) of B7-H4_2F9V16 contains or consists of the following amino acid sequence: TIFF2026082984000203.tif18161

[0482] The VH chain (SEQ ID NO: 42) of B7-H4_2F9V17 contains or consists of the following amino acid sequence: TIFF2026082984000204.tif18161

[0483] The VH chain of B7-H4_2F9V18 (SEQ ID NO: 44) (also referred to herein as XMT-1604 VH) contains or consists of the following amino acid sequence: TIFF2026082984000205.tif18161

[0484] The B7-H4_2F9V18 variable heavy chain is also referred to herein as the XMT-1604 variable heavy chain.

[0485] The VH chain (SEQ ID NO: 46) of B7-H4_2F9V19 contains or consists of the following amino acid sequence: TIFF2026082984000206.tif19161

[0486] The VH chain (SEQ ID NO: 48) of B7-H4_2F9V20 contains or consists of the following amino acid sequence: TIFF2026082984000207.tif18161

[0487] CDR Table I below summarizes the CDRs of the B7-H4 antibody in this disclosure.

[0488] (Table I) Amino acid sequences of the complementarity-determining regions of the heavy and light chains. TIFF2026082984000208.tif78165TIFF2026082984000209.tif166165

[0489] It is well known in the art that the CDR3 domain, independently of the CDR1 and / or CDR2 domains alone, can determine the binding specificity of B7-H4 antibodies to congener antigens, and that multiple antibodies with the same binding specificity can be predictably generated based on a common CDR3 sequence.

[0490] In some embodiments, the antibody comprises one or more heavy and / or light chain CDR3 domains derived from a non-human antibody, such as a mouse antibody or a rat antibody, and the monoclonal antibody can specifically bind to B7-H4. In some embodiments, such an antibody of the present invention comprising one or more heavy and / or light chain CDR3 domains derived from a non-human antibody (a) can compete for binding with the corresponding parental non-human antibody, (b) retains functional characteristics similar to the corresponding parental non-human antibody, (c) binds to the same epitope as the corresponding parental non-human antibody, and / or (d) has a binding affinity similar to the corresponding parental non-human antibody.

[0491] In some embodiments, a monoclonal antibody comprising one or more heavy and / or light chain CDR3 domains derived from a first human antibody, such as a human antibody obtained from a non-human animal, the first human antibody can specifically bind to B7-H4, and the CDR3 domains derived from the first human antibody replace the CDR3 domain of a human antibody that lacks binding specificity to B7-H4, thereby generating a second human antibody that can specifically bind to B7-H4. In some embodiments, an antibody comprising one or more heavy and / or light chain CDR3 domains derived from the first human antibody (a) can compete for binding with a corresponding first parental human antibody, (b) retains functional characteristics similar to the corresponding first parental human antibody, (c) binds to the same epitope as the corresponding first parental human antibody, and / or (d) has similar binding affinity to the corresponding first parental human antibody.

[0492] B7-H4 2F9 Steady-state region The B7-H4 2F9 parental antibody and the 20 variants of this disclosure have the following amino acid sequences: It has a light chain constant region that includes or consists of TIFF2026082984000210.tif11160.

[0493] The B7-H4 2F9 light chain steady-state region (SEQ ID NO: 51) is also referred to as B7-H4 2F9 LC in this specification.

[0494] In some embodiments, the antibody of the present disclosure comprises a light chain comprising or consisting of a light chain variable region amino acid sequence and a light chain constant region amino acid sequence. The antibody light chain of the present disclosure (variable region and constant region) comprising or consisting of the amino acid sequence of SEQ ID NO: 52.

[0495] The B7-H4 2F9 parental antibody and the 20 variants of this disclosure have the following amino acid sequences: It has an IgG1 heavy chain constant region containing or consisting of TIFF2026082984000211.tif40161. The B7-H4 2F9 IgG1 heavy chain constant region (SEQ ID NO:6) is also referred to herein as B7-H4 2F9 HC.

[0496] In some embodiments, the IgG1 heavy chain constant region comprising or consisting of SEQ ID NO:6 further comprises one or more amino acids at its N-terminus or C-terminus. In some embodiments, the IgG1 heavy chain constant region comprises C-terminal lysine.

[0497] In some embodiments, the antibodies of this disclosure include a heavy chain comprising or consisting of a heavy chain variable region amino acid sequence and a heavy chain constant region amino acid sequence. The antibody heavy chains (variable region and constant region) of this disclosure are described in Table II and in the sequence listings filed together with this specification.

[0498] In some embodiments, the antibody of this disclosure has the amino acid sequence: It may contain or consist of an IgG double-chain constant region containing TIFF2026082984000212.tif40160.

[0499] In some embodiments, the antibody of this disclosure has the amino acid sequence: It may contain or consist of an IgG quadruple chain constant region containing TIFF2026082984000213.tif40160.

[0500] (Table II) Antibody heavy chain amino acid sequence (heavy chain variable region + IgG1 heavy chain constant region) TIFF2026082984000214.tif95138

[0501] Specific manner In yet another embodiment, the B7-H4 antibody of the Disclosure comprises a heavy-chain variable region and a light-chain variable region having amino acid sequences homologous to the amino acid sequences of preferred antibodies described herein, and the antibody retains the desired functional properties of the anti-B7-H4 antibody of the Disclosure. For example, the Disclosure provides an isolated monoclonal antibody or its antigen-binding moiety comprising a heavy-chain variable region and a light-chain variable region, (a) The heavy chain variable region includes an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NO: 44, 22, 30, 40, and 42. (b) The light chain variable region contains an amino acid sequence that is at least 80% homologous to the amino acid sequence of SEQ ID NO:50. (c) Antibodies should have a K content of 1 × 10⁻⁷ M or less. D It binds to human B7-H4, (d) The antibody binds to human CHO cells transfected with B7-H4.

[0502] In various embodiments, the antibody may be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0503] In other embodiments, the VH and / or VL amino acid sequences may be 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the sequences described above. B7-H4 antibodies having VH and VL regions with high homology (i.e., 80% or more) to the VH and VL regions of the sequences described above can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of nucleic acid molecules encoding the amino acid sequences described in SEQ ID NO: 44, 22, 30, 40, and 42, followed by testing the encoded altered antibody for retained function (i.e., the function described in (c) and (d) above) using the functional assays described herein.

[0504] In a preferred embodiment, the heavy chain variable region CDR2 sequence includes an amino acid sequence selected from the group consisting of amino acid sequences with SEQ ID NO: 44, 22, 30, 40, and 42 and their conserved modifications, and the light chain variable region CDR2 sequence includes an amino acid sequence with SEQ ID NO: 54 and its conserved modifications. In another preferred embodiment, the heavy chain variable region CDR1 sequence includes an amino acid sequence selected from the group consisting of amino acid sequences with SEQ ID NO: 44, 22, 30, 40, and 42 and their conserved modifications, and the light chain variable region CDR1 sequence includes an amino acid sequence with SEQ ID NO: 50 and its conserved modifications.

[0505] In various embodiments, the antibody may be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0506] In some embodiments, the antibody binds to the same epitope on human B7-H4 as any of the B7-H4 monoclonal antibodies of the Disclosure (i.e., an antibody having the ability to cross-compete with any of the monoclonal antibodies of the Disclosure for binding to B7-H4).

[0507] Accordingly, another aspect of the present disclosure relates to an isolated monoclonal antibody or its antigen-binding moiety comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences, each containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 22, 30, 40, and 42, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, each containing an amino acid sequence of SEQ ID NOs: 53, 54, and 55, respectively.

[0508] Accordingly, in another embodiment, the present disclosure provides an isolated anti-B7-H4 monoclonal antibody or its antigen-binding moiety comprising a heavy chain variable region comprising (a) a VH CDR1 region comprising an amino acid sequence comprising SEQ ID NO:2, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO:2; (b) a VH CDR2 region comprising an amino acid sequence comprising SEQ ID NO:3, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO:3; and (c) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:16, 10, or 4, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO:16, 10, or 4, or a VH CDR3 region.

[0509] In some embodiments, the antibodies disclosed herein include a heavy chain having an amino acid sequence identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of a sequence selected from the group consisting of SEQ ID NO: 45, 23, 31, 41, or 43, and a light chain having an amino acid sequence identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of a sequence selected from the group consisting of SEQ ID NO: 52.

[0510] In some embodiments, the antibodies disclosed herein are (i) heavy chain amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 45, and light chain amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 52, and (ii) heavy chain amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 23, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to the amino acid sequence of SEQ ID NO: 52 (iii) Light chain amino acid sequences that are 98%, 99% or more identical, (iii) At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to the amino acid sequence of SEQ ID NO:31, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to the heavy chain amino acid sequence of SEQ ID NO:52, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to the light chain amino acid sequence of SEQ ID NO:41, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to the heavy chain amino acid sequence of SEQ ID NO:50, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to the light chain amino acid sequence of SEQ ID NO:41, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to the heavy chain amino acid sequence of SEQ ID NO:50 This includes combinations of heavy chain amino acid sequences and light chain amino acid sequences selected from the group consisting of (v) SEQ ID NO:43 amino acid sequences that are 98%, 99% or more identical to light chain amino acid sequences, and (v) SEQ ID NO:43 amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to heavy chain amino acid sequences, and SEQ ID NO:52 amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to light chain amino acid sequences.

[0511] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:45, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:52.

[0512] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:23, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:52.

[0513] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:31, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:52.

[0514] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:41, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:52.

[0515] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:43, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQ ID NO:52.

[0516] In some embodiments, the antibodies disclosed herein include a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 45, 23, 31, 41, or 43, and a light chain having an amino acid sequence of SEQ ID NO: 52.

[0517] In some embodiments, the antibodies disclosed herein include combinations of heavy-chain amino acid sequences and light-chain amino acid sequences selected from the group consisting of (i) a heavy-chain amino acid sequence of SEQ ID NO: 45 and a light-chain amino acid sequence of SEQ ID NO: 52, (ii) a heavy-chain amino acid sequence of SEQ ID NO: 23 and a light-chain amino acid sequence of SEQ ID NO: 52, (iii) a heavy-chain amino acid sequence of SEQ ID NO: 31 and a light-chain amino acid sequence of SEQ ID NO: 52, (iv) a heavy-chain amino acid sequence of SEQ ID NO: 41 and a light-chain amino acid sequence of SEQ ID NO: 52, and (v) a heavy-chain amino acid sequence of SEQ ID NO: 43 and a light-chain amino acid sequence of SEQ ID NO: 52.

[0518] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence of SEQ ID NO:45 and a light chain amino acid sequence of SEQ ID NO:52.

[0519] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence of SEQ ID NO:23 and a light chain amino acid sequence of SEQ ID NO:52.

[0520] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence of SEQ ID NO:31 and a light chain amino acid sequence of SEQ ID NO:52.

[0521] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence of SEQ ID NO:41 and a light chain amino acid sequence of SEQ ID NO:52.

[0522] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence of SEQ ID NO:43 and a light chain amino acid sequence of SEQ ID NO:52.

[0523] The antibodies disclosed herein include a heavy chain variable region having an amino acid sequence identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of a sequence selected from the group consisting of SEQ ID NO: 44, 22, 30, 40, or 42, and a light chain variable region having an amino acid sequence identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of a sequence consisting of SEQ ID NO: 50.

[0524] In some embodiments, the three heavy chain CDRs of the antibody disclosed herein include: Heavy chain complementarity determination region 1 (CDRH1) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences to the sequence containing SEQ ID NO:2; Heavy chain complementarity determination region 2 (CDRH2) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences to the sequence containing SEQ ID NO:3; and Heavy chain complementarity determination region 3 (CDRH3) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences to a sequence selected from the group consisting of SEQ ID NO:16, 10, or 4; and SEQ ID NO: Contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical heavy chain amino acid sequences to amino acid sequences of NO: 45, 23, 31, 41, or 43.

[0525] The three light chain CDRs of the antibodies disclosed herein include a light chain complementarity determination region 1 (CDRL1) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical amino acid sequences to the sequence containing SEQ ID NO: 53; a light chain complementarity determination region 2 (CDRL2) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical amino acid sequences to the sequence containing SEQ ID NO: 54; and a light chain complementarity determination region 3 (CDRL3) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical amino acid sequences to the sequence containing SEQ ID NO: 55.

[0526] The antibody contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical amino acid sequences to the sequence containing SEQ ID NO:2; CDRH2 contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical amino acid sequences to the sequence containing SEQ ID NO:3; CDRH3 contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical amino acid sequences to a sequence selected from the group consisting of SEQ ID NO:16, 10, or 4; and a sequence selected from the group consisting of SEQ ID NO:53 contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical amino acid sequences to a sequence containing SEQ ID NO:2. CDRL1 containing 98%, 99%, or more identical amino acid sequences; CDRL2 containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical amino acid sequences to the sequence of SEQ ID NO: 54; CDRL3 containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical amino acid sequences to the sequence of SEQ ID NO: 55a; and combinations of heavy chain CDR sequences and light chain CDR sequences containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical heavy chain amino acid sequences to the amino acid sequences of SEQ ID NO: 45, 23, 31, 41, or 43.

[0527] The three heavy chain CDRs of the antibodies disclosed herein include CDRH1 containing an amino acid sequence selected from the group including SEQ ID NO:2, CDRH2 containing an amino acid sequence including SEQ ID NO:3, CDRH3 containing an amino acid sequence selected from the group consisting of SEQ ID NO:10 or 16, and a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:45 or 23.

[0528] The three light chain CDRs of the antibody disclosed herein include CDRL1 having the amino acid sequence of SEQ ID NO: 53, CDRL2 having the amino acid sequence of SEQ ID NO: 54, and CDRL3 having the amino acid sequence of SEQ ID NO: 55. The antibodies disclosed herein include a combination of a heavy chain CDR sequence and a light chain CDR sequence, including the CDHR1 sequence containing a selected amino acid sequence including SEQ ID NO:2, the CDRH2 sequence containing an amino acid sequence containing SEQ ID NO:3, the CDRH3 sequence containing an amino acid sequence containing SEQ ID NO:10 or 16, the CDRL1 sequence having the amino acid sequence of SEQ ID NO:53, the CDRL2 sequence having the amino acid sequence of SEQ ID NO:54, and the CDRL3 sequence having the amino acid sequence of SEQ ID NO:55, as well as the heavy chain CDR sequence having the heavy chain CDR sequence of SEQ ID NO:45 or 23. The antibodies disclosed herein include (i) the CDRH1 amino acid sequence of SEQ ID NO:2, the CDRH2 amino acid sequence of SEQ ID NO:3, the CDRH3 amino acid sequence of SEQ ID NO:16, the CDRL1 amino acid sequence of SEQ ID NO:53, the CDRL2 amino acid sequence of SEQ ID NO:54, the CDRL3 amino acid sequence of SEQ ID NO:55, and the heavy chain CDR sequence of SEQ ID NO:45, and (ii) the SEQ ID (iii) CDRH1 amino acid sequence of NO:2, CDRH2 amino acid sequence of SEQ ID NO:3, CDRH3 amino acid sequence of SEQ ID NO:10, CDRL1 amino acid sequence of SEQ ID NO:53, CDRL2 amino acid sequence of SEQ ID NO:54, CDRL3 amino acid sequence of SEQ ID NO:55, and heavy chain amino acid sequence of SEQ ID NO:23, (iii) CDRH1 amino acid sequence of SEQ ID NO:2, CDRH2 amino acid sequence of SEQ ID NO:3, CDRH3 amino acid sequence of SEQ ID NO:4, CDRL1 amino acid sequence of SEQ ID NO:53, CDRL2 amino acid sequence of SEQ ID NO:54, CDRL3 amino acid sequence of SEQ ID NO:55, and heavy chain amino acid sequence of SEQ ID NO:31, (iv) CDRH1 amino acid sequence of SEQ ID NO:2, CDRH2 amino acid sequence of SEQ ID NO:3, SEQ IDThis includes combinations of a heavy chain complementarity-determining region amino acid sequence and a light chain complementarity-determining region amino acid sequence selected from the group consisting of the CDRH3 amino acid sequence of NO:4, the CDRL1 amino acid sequence of SEQ ID NO:53, the CDRL2 amino acid sequence of SEQ ID NO:54, the CDRL3 amino acid sequence of SEQ ID NO:55, and the heavy chain amino acid sequence of SEQ ID NO:41, as well as (v) the CDRH1 amino acid sequence of SEQ ID NO:2, the CDRH2 amino acid sequence of SEQ ID NO:3, the CDRH3 amino acid sequence of SEQ ID NO:10, the CDRL1 amino acid sequence of SEQ ID NO:53, the CDRL2 amino acid sequence of SEQ ID NO:54, the CDRL3 amino acid sequence of SEQ ID NO:55, and the heavy chain amino acid sequence of SEQ ID NO:43.

[0529] In some embodiments, the antibodies disclosed herein include the CDRH1 amino acid sequence of SEQ ID NO:2, the CDRH2 amino acid sequence of SEQ ID NO:3, the CDRH3 amino acid sequence of SEQ ID NO:16, the CDRL1 amino acid sequence of SEQ ID NO:53, the CDRL2 amino acid sequence of SEQ ID NO:54, the CDRL3 amino acid sequence of SEQ ID NO:55, and the heavy chain amino acid sequence of SEQ ID NO:45.

[0530] In some embodiments, the antibodies disclosed herein include the CDRH1 amino acid sequence of SEQ ID NO:2, the CDRH2 amino acid sequence of SEQ ID NO:3, the CDRH3 amino acid sequence of SEQ ID NO:10, the CDRL1 amino acid sequence of SEQ ID NO:53, the CDRL2 amino acid sequence of SEQ ID NO:54, the CDRL3 amino acid sequence of SEQ ID NO:55, and the heavy chain amino acid sequence of SEQ ID NO:23.

[0531] In some embodiments, the antibodies disclosed herein include the CDRH1 amino acid sequence of SEQ ID NO:2, the CDRH2 amino acid sequence of SEQ ID NO:3, the CDRH3 amino acid sequence of SEQ ID NO:4, the CDRL1 amino acid sequence of SEQ ID NO:53, the CDRL2 amino acid sequence of SEQ ID NO:54, and the CDRL3 amino acid sequence of SEQ ID NO:55.

[0532] In some embodiments, the antibodies disclosed herein include the CDRH1 amino acid sequence of SEQ ID NO:2, the CDRH2 amino acid sequence of SEQ ID NO:3, the CDRH3 amino acid sequence of SEQ ID NO:4, the CDRL1 amino acid sequence of SEQ ID NO:53, the CDRL2 amino acid sequence of SEQ ID NO:54, and the CDRL3 amino acid sequence of SEQ ID NO:55.

[0533] In some embodiments, the antibodies disclosed herein include the CDRH1 amino acid sequence of SEQ ID NO:2, the CDRH2 amino acid sequence of SEQ ID NO:3, the CDRH3 amino acid sequence of SEQ ID NO:10, the CDRL1 amino acid sequence of SEQ ID NO:53, the CDRL2 amino acid sequence of SEQ ID NO:54, and the CDRL3 amino acid sequence of SEQ ID NO:55.

[0534] Preferred antibodies disclosed herein include, for example, B7-H4-2F9V7 (also known herein as XMT 1603 antibody) and B7-H4-2F9V18 (also known herein as XMT 1604 antibody). These antibodies exhibit specificity for human B7-H4 and have been shown to inhibit the functional activity of B7-H4 in vitro.

[0535] B7-H4 antibody generation B7-H4 antibodies are produced, for example, by methods described in the examples provided herein. Alternatively, or in addition, various procedures known in the art may be used to produce monoclonal antibodies against B7-H4 or its derivatives, fragments, analog homologs, or orthologues. (For example, see Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Kozbor, et al., 1983 Immunol Today 4:72, each of which is incorporated herein in its entirety by reference); Cole, et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96; Cole, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030; Cole, et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0536] The monoclonal antibodies disclosed herein include fully human antibodies or humanized antibodies. These antibodies are suitable for administration to humans without inducing a human immune response to the administered immunoglobulin. Humanized B7-H4 antibodies or fully human B7-H4 antibodies are developed, for example, using phage display methods that use antibodies containing only human sequences. Such methods are well known in the art, for example, in International Publication No. 92 / 01047 and U.S. Patent No. 6,521,404, which are incorporated herein by reference.

[0537] Antibodies are purified by well-known techniques, such as affinity chromatography, which primarily provides the IgG fraction of the immunoserum. Subsequently, or alternatively, the specific antigen or epitope that is the target of the desired immunoglobulin may be immobilized on a column, and the immuno-specific antibody may be purified by immunoaffinity chromatography. The purification of immunoglobulins is described, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28, incorporated herein by reference).

[0538] The present invention also includes F v Anti-B7-H4 fragment, F ab Anti-B7-H4 fragment, F ab' Anti-B7-H4 fragment and F (ab')2 The present invention comprises an anti-B7-H4 fragment or anti-B7-H4 fragment, a single-chain anti-B7-H4 antibody, a multispecific antibody having at least one arm that binds to B7-H4, and a heteroconjugate anti-B7-H4 antibody.

[0539] A bispecific antibody is an antibody that has binding specificity to at least two different antigens. In this case, one binding specificity is for B7-H4. The second binding target is any other antigen, including cell surface proteins, receptors, or receptor subunits.

[0540] Methods for producing bispecific antibodies are known in the art. Conventionally, the recombinant generation of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).

[0541] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments are described in the literature.

[0542] Antibodies with more than two binding titers are intended. For example, triplicate antibodies can be prepared. Tutt et al., J.Immunol. 147:60 (1991).

[0543] An exemplary bispecific antibody can bind to two different epitopes, at least one of which is derived from a protein antigen disclosed herein. Alternatively, an anti-antigenic arm of an immunoglobulin molecule may be combined with an arm that binds to a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or to an IgG Fc receptor (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), in order to concentrate cellular defense mechanisms on cells expressing a specific antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells expressing a specific antigen. These antibodies have an antigen-binding arm and an arm that binds to a cytotoxic agent or radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds to a protein antigen described herein and further binds to tissue factor (TF).

[0544] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies have been proposed, for example, to target immune system cells against undesirable cells (see U.S. Patent No. 4,676,980) and to treat HIV infection (see International Publication Nos. 91 / 00360, International Publication Nos. 92 / 200373, EP03089). Antibodies are thought to be able to be prepared in vitro using known methods in synthetic protein chemistry, including synthetic protein chemistry with crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolates and methyl-4-mercaptobutylimidate, as disclosed, for example, in U.S. Patent No. 4,676,980.

[0545] For example, it may be desirable to modify the antibodies disclosed herein with respect to effector function in order to enhance the efficacy of antibodies in treating diseases and disorders associated with abnormal B7-H4 expression and / or activity. For instance, a cysteine ​​residue can be introduced into the Fc region, thereby forming an interchain disulfide bond in this region. The homodimeric antibodies thus produced may have improved internalization ability and / or increased complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, antibodies can be manipulated to have a double Fc region, thereby enabling enhanced complement lysis and ADCC ability. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0546] Cysteine-modified B7-H4 antibody In some embodiments, the cysteine-modified B7-H4 antibody directs a conjugate containing a peptide linker to a specific tissue, cell, or intracellular location. In some embodiments, the cysteine-modified B7-H4 antibody contains modified cysteine.

[0547] In some embodiments, a B7-H4 cysteine-modified antibody or B7-H4 cysteine-modified antibody fragment is a B7-H4 antibody or B7-H4 antibody fragment in which one or more amino acids of the corresponding parental antibody or parental antibody fragment (e.g., the corresponding wild-type B7-H4 antibody or wild-type B7-H4 antibody fragment) are substituted with cysteine ​​(e.g., modified cysteine). In some embodiments, the parental B7-H4 antibody or parental B7-H4 antibody fragment is one described herein.

[0548] In some embodiments, a B7-H4 antibody is manipulated to form a cysteine-modified antibody. In some embodiments, the cysteine-modified B7-H4 antibody or cysteine-modified B7-H4 antibody fragment retains the antigen-binding ability of its corresponding wild-type B7-H4 antibody or wild-type B7-H4 antibody fragment. In some embodiments, the cysteine-modified antibody or cysteine-modified antibody fragment can bind to one or more antigens relating to its corresponding wild-type B7-H4 antibody or wild-type B7-H4 antibody fragment.

[0549] In some embodiments, the engineered cysteine ​​is not part of an intrachain disulfide unit or an interchain disulfide unit. In some embodiments, the engineered cysteine ​​contains a free thiol group that is reactive with an electrophilic functional group. In some embodiments, the engineered cysteine ​​(e.g., its free thiol group) on the surface of a B7-H4 antibody or B7-H4 antibody fragment may enable conjugation between the B7-H4 antibody or B7-H4 antibody fragment and a linker-drug moiety containing a thiol-reactive group (e.g., maleimide or haloacetyl).

[0550] It is understood that by substituting one or more non-cysteine ​​amino acids in a B7-H4 antibody or B7-H4 antibody fragment with cysteine, one or more manipulated cysteines can be created as sites available for conjugation. In some embodiments, by substituting non-cysteine ​​amino acids in a B7-H4 antibody or B7-H4 antibody fragment with cysteine, the reactive thiol group is positioned as an accessible site in the antibody or antibody fragment and can be used to conjugate the antibody or antibody fragment to other parts (e.g., a drug portion or a linker-drug portion) and create the conjugate of the present disclosure. In some embodiments, the V205 (Kabat numbering or EU numbering) amino acid of the light chain or the A118 or S442 (EU numbering) amino acid of the heavy chain of the parent B7-H4 antibody or parent B7-H4 antibody fragment is substituted with cysteine. In some embodiments, the cysteine-manipulated antibody can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0551] In some embodiments, the cysteine-modified B7-H4 antibody is conjugated to the linker-drug moiety by forming a covalent bond via the sulfhydryl group of the modified cysteine ​​and the functional group of the linker-drug moiety.

[0552] Modified B7-H4 antibody In some embodiments, the B7-H4 antibody is a modified B7-H4 antibody.

[0553] In some embodiments of the modified B7-H4 antibody, * indicates direct or indirect binding to the remainder of the modified B7-H4 antibody. In some embodiments, S'' is a sugar or derivatized sugar. In some embodiments, A'' is a functional group that can form a covalent bond with the functional group of the linker-drug moiety.

[0554] In some embodiments, the modified B7-H4 antibody before conjugation contains a sugar-derivative moiety of *-S''-A''.

[0555] In some embodiments, the modified B7-H4 antibody contains an asparagine group (e.g., N297; in EU numbering) within region 290-305. In some embodiments, the sugar-derivative moiety is directly or indirectly conjugated to this asparagine group (e.g., N297).

[0556] In some embodiments, the pre-conjugation modified B7-H4 antibody contains a modified GlcNAc moiety, *-GlcNAc-S''-A'', where GlcNAc is N-acetylglucosamine.

[0557] In some embodiments, the modified GlcNAc moiety is connected to the remainder of the modified B7-H4 antibody via the C1 position of the GlcNAc. In some embodiments, the modified GlcNAc moiety further comprises fucose.

[0558] In some embodiments, the modified GlcNAc moiety is directly or indirectly bonded to the asparagine group (for example, to N297).

[0559] In some embodiments, the modified B7-H4 antibody is conjugated to the linker-drug moiety via a covalent bond formed between A'' and the functional group of the linker-drug moiety.

[0560] In some embodiments, the modified B7-H4 antibody of this disclosure is (a) A step of contacting a glycoprotein (e.g., B7-H4 antibody glycan) containing a B7-H4 antibody and a core-GlcNAc moiety with an endoglycosidase to form an intermediate antibody containing an antibody and a terminal GlcNAc moiety, wherein optionally the terminal GlcNAc moiety further contains fucose, and (b) A modified B7-H4 antibody is obtained by a process comprising contacting an intermediate antibody with a compound having the structure P''-S''-A'' in the presence of a glycosyltransferase, thereby forming a modified B7-H4 antibody comprising the antibody and a modified GlcNAc moiety, *-GlcNAc-S''-A'', wherein optionally the modified GlcNAc moiety is bound to the remainder of the modified B7-H4 antibody via the C1 position of GlcNAc, GlcNAc is N-acetylglucosamine, S'' is a sugar or a derivatized sugar, A'' is azid, keto, or alkinyl, P'' is uridine diphosphate (UDP), guanosine diphosphate (GDP), or cytidine diphosphate (CDP).

[0561] In some embodiments, steps (a) and (b) are performed sequentially. In some embodiments, steps (a) and (b) are performed simultaneously.

[0562] In some embodiments, the antibody glycan comprises a mixture of glycoforms G0, G1, G2, G0F, GIF, G2F, and M5 (for example, the glycoforms shown in Figure 1).

[0563] In some embodiments, the antibody is a monoclonal antibody (mAb).

[0564] In some embodiments, the antibody is an IgA antibody, an IgD antibody, an IgE antibody, an IgG antibody, or an IgM antibody.

[0565] In some embodiments, the antibody is an IgG antibody, such as an IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody.

[0566] In some embodiments, the antibody is a full-length antibody, and the antibody glycan comprises one or more core-GlcNAc moieties.

[0567] In some embodiments, the antibody is a full-length antibody, and the antibody glycan comprises one or more core-GlcNAc moieties attached to each heavy chain of the antibody.

[0568] In some embodiments, the core-GlcNAc portion further includes fucose.

[0569] In some embodiments, the antibody is a full-length antibody, and the antibody glycan comprises two or more core-GlcNAc moieties attached to the full-length antibody.

[0570] In some embodiments, the antibody is a full-length antibody, and the antibody glycan contains two core-GlcNAc moieties attached to the full-length antibody.

[0571] In some embodiments, at least one of two or more core-GlcNAc portions further includes fucose.

[0572] In some embodiments, each of two or more core-GlcNAc portions further includes fucose.

[0573] In some embodiments, the antibody is a single-chain antibody or a B7-H4 antibody fragment (e.g., a Fab fragment or an Fc fragment), and the antibody glycan comprises one or more core-GlcNAc moieties (optionally further comprising fucose) attached to the antibody.

[0574] In some embodiments, the core-GlcNAc portion is attached to a site on the antibody, and the core-GlcNAc portion does not substantially interfere with the antigen-binding site of the antibody.

[0575] In some embodiments, the core-GlcNAc moiety is attached to the Fc fragment of the antibody. In some embodiments, the core-GlcNAc moiety is attached to the CH domain. In some embodiments, the core-GlcNAc moiety is attached to the Fab or Fc fragment of the antibody. In some embodiments, the core-GlcNAc moiety is attached to the antibody via an N-glycosidic bond to the amide nitrogen atom of the side chain of the asparagine amino acid of the antibody. In some embodiments, the core-GlcNAc moiety is attached to the native N-glycosylation site of the antibody.

[0576] In some embodiments, the antibody is an IgG antibody, and the core-GlcNAc moiety is connected to the natural N-glycosylation site of IgG.

[0577] In some embodiments, the antibody is an IgG antibody, and the core-GlcNAc moiety is connected to a native N-glycosylation site of IgG (e.g., the N297 N-glycosylation site of IgG). In some embodiments, the N297 N-glycosylation site is located in a conserved Fc region of the heavy chain of the IgG antibody in asparagine within region 290-305 (e.g., at N297).

[0578] In some embodiments, the intermediate antibody is of formula (XXII): The filename is TIFF2026082984000215.tif21128. During the ceremony, Ab is the B7-H4 antibody, GlcNAc is N-acetylglucosamine, Fuc is fucose, u3 is 0 or 1, and u4 is an integer in the range of 1 to 16.

[0579] In some embodiments, u4 is an integer in the range of 1 to 10. In some embodiments, u4 is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, u4 is 1, 2, 3, 4, 5, or 6. In some embodiments, u4 is 1, 2, 3, or 4. In some embodiments, u4 is 2 or 4. In some embodiments, u4 is 1 or 2. In some embodiments, u4 is 1. In some embodiments, u4 is 2.

[0580] In some embodiments, the antibody contains one core-GlcNAc moiety (e.g., u4 is 1). In some embodiments, the antibody contains two core-GlcNAc moieties (e.g., u4 is 2).

[0581] In some embodiments, the modified B7-H4 antibody is obtained by the process outlined in Scheme 1. As shown below, contact of an intermediate antibody of formula (XXIII) containing one terminal GlcNAc moiety with a compound having the structure P''-S''-A'' in the presence of glycosyltransferase yields a modified B7-H4 antibody containing one modified GlcNAc moiety (e.g., a modified B7-H4 antibody of formula (XXIIIa)).

[0582] In some embodiments, a modified B7-H4 antibody is obtained by contacting an intermediate antibody of formula (XXIV) containing two terminal GlcNAc moieties with a compound having a P''-S''-A'' structure in the presence of a glycosyltransferase, providing a modified B7-H4 antibody containing two modified GlcNAc moieties (e.g., a modified B7-H4 antibody of formula (XXIVa)).

[0583] In formula TIFF2026082984000216.tif51144, u3, Ab, S'', A'', and P'' are as defined herein.

[0584] In some embodiments, the antibody glycan modified by the process according to the present disclosure comprises a glycan, the glycan comprising a core-GlcNAc moiety, i.e., a GlcNAc moiety located at the non-reducing end of the glycan. In some embodiments, the glycan comprises one or more sugar moieties and may be linear or branched.

[0585] In some embodiments, reaction with endoglycosidase may form an intermediate antibody containing a terminal GlcNAc moiety (e.g., an intermediate antibody of formula (XXIII) or (XXIV)).

[0586] In some embodiments, step (a) of the process (deglycosylation or trimming) is as shown in Figure 2, where a mixture of antibody glycoforms G2F, GIF, G0F, G2, G1, G0, and M5 (see, for example, Figure 1) and optionally additional glycoforms (e.g., triple-stranded glycans) is converted into an intermediate antibody containing a terminal GlcNAc moiety which may contain fucose (e.g., u3 is 0 or 1).

[0587] In some embodiments, the endoglycosidase is endoglycosidase Endo S, Endo SH, Endo S2, Endo S49, Endo F1, Endo F2, Endo F3, or a combination thereof.

[0588] In some embodiments, the endoglycosidase is Endo S, Endo SH, Endo S2, Endo S49, or a combination thereof.

[0589] In some embodiments, the endoglycosidase is Endo S, Endo SH, or a combination thereof. In some embodiments, the endoglycosidase is Endo SH.

[0590] In some embodiments, step (b) of the process (formation of modified B7-H4 antibody) is as shown in Figure 3, where the intermediate antibody comprises a monoclonal antibody (mAb) and terminal GlcNAc moieties on each heavy chain of the monoclonal antibody (mAb) (which may contain fucose (e.g., u3 is 0 or 1)). In some embodiments, in step (b), the terminal GlcNAc moieties are converted to modified GlcNAc moieties. In some embodiments, this conversion may be carried out by the reaction of the terminal GlcNAc moieties with a P''-S''-A'' compound in the presence of glycosyltransferase.

[0591] In some embodiments, the P''-S''-A'' compound is GalNAz-UDP (e.g., 4-AzGalNAc-UDP). In some embodiments, the terminal GlcNAc portion is *-GlcNAc-GalNAz or *-GlcNAc(Fuc)-GalNAz, where * represents binding to the remainder of the modified B7-H4 antibody.

[0592] In some embodiments, the deglycosylation / trimming step and the step of forming the modified B7-H4 antibody are carried out sequentially.

[0593] In some embodiments, the deglycosylation / trimming step and the step of forming the modified B7-H4 antibody are performed simultaneously.

[0594] In some embodiments, the process for preparing the modified B7-H4 antibody is carried out in a suitable buffer, such as buffered saline (e.g., phosphate-buffered saline, Tris-buffered saline), citrate, HEPES, Tris, and glycine. In some embodiments, the buffer is phosphate-buffered saline (PBS) or Tris-buffered saline. In some embodiments, the buffer is phosphate-buffered saline (PBS).

[0595] In some embodiments, the process is carried out at a temperature in the range of approximately 4 to approximately 50°C. In some embodiments, the process is carried out at a temperature in the range of approximately 10 to approximately 45°C. In some embodiments, the process is carried out at a temperature in the range of approximately 20 to approximately 40°C. In some embodiments, the process is carried out at a temperature in the range of approximately 30 to approximately 37°C. In some embodiments, the process is carried out at a temperature of approximately 30°C. In some embodiments, the process is carried out at a temperature of 30°C.

[0596] In some embodiments, the process is carried out at a pH value in the range of about 5 to about 9 (e.g., about 5.5 to about 8.5, about 6 to about 8, or about 7 to about 8). In some embodiments, the process is carried out at a pH value of about 7.4.

[0597] In some embodiments, the process for preparing the modified B7-H4 antibody is as shown in Figure 4.

[0598] In some embodiments, the process for preparing a modified B7-H4 antibody is: (a) A step of contacting a glycoprotein (e.g., B7-H4 antibody glycan) containing a B7-H4 antibody and a core-GlcNAc moiety connected to site N297 of the antibody with endoglycosidase Endo SH, thereby forming an intermediate antibody containing the terminal GlcNAc moiety, and (b) A step comprising contacting an intermediate antibody with 4-AzGalNAc-UDP in the presence of the β-(1,4)-GalNAcT enzyme, thereby forming a modified B7-H4 antibody containing a modified GlcNAc moiety, Processes (a) and (b) are performed simultaneously.

[0599] In some embodiments, the endoglycosidase is Endo SH, which is a fusion of two endoglycosidases, Endo S and Endo H, linked by a glycy-rich spacer containing an internal 6xHis tag, resulting in a total molecular weight of 139 kDa.

[0600] In some embodiments, the β-(1,4)-GalNAcT enzyme contains an N-terminal 6xHis tag and has a total molecular weight of 45.7 kDa. In some embodiments, the β-(1,4)-GalNAcT enzyme containing an N-terminal 6xHis tag is derived from the nettle moth (Trichoplusia ni).

[0601] In some embodiments, the process is carried out in PBS buffer at a pH of approximately 7.4 and a temperature of approximately 30°C.

[0602] Endoglycosidase Endoglycosidases are enzymes that can cleave internal glycosidic bonds within a glycan structure, thereby reconstructing or trimming the glycan structure. For example, endoglycosidases can be used for the easy homogenization of heterogeneous glycan populations when cleaved at predictable sites within conserved glycan regions. One class of endoglycosidases includes endo-β-N-acetylglucosaminidase (EC 3.2.1.96, commonly known as Endo S or ENGase), a class of hydrolytic enzymes that remove N-glycans from glycoproteins by hydrolyzing the β-1,4-glycosidic bonds of the N,N'-diacetylchitobiose core, leaving a single-core N-linked GlcNAc residue, as described in Wong et al. Chem. Rev. 2011, 111, 4259 (whose entirety is incorporated herein by reference). Endo-β-N-acetylglucosaminidases are widely found in nature, along with common chemioenzyme variants including Endo D, which is specific to pausimannose; Endo A and Endo H, which are specific to high-mannose; the Endo F subtype, which extends from high-mannose to double-stranded complexes; and Endo M, which can cleave most N-glycan structures (high-mannose / complex / hybrid types) except for fucosylated glycans. The hydrolytic activity of high-mannose oligosaccharides is significantly higher than that of complex and hybrid oligosaccharides. In some embodiments, these ENGases exhibit specificity for distal N-glycan structures but not for proteins presenting distal N-glycan structures, and are useful for cleaving most N-linked glycans from glycoproteins under natural conditions.

[0603] In some embodiments, endoglycosidases F1, F2, and F3 are suitable for deglycosylating native proteins. The binding specificity of Endo F1, F2, and F3 suggests a general strategy for protein deglycosylation that can remove any class of N-linked oligosaccharides without denaturing the protein. In some embodiments, double-stranded and triple-stranded structures can be immediately removed by endoglycosidases F2 and F3, respectively. In some embodiments, oligomannoses and hybrid structures can be removed by Endo F1.

[0604] Endo S is an endoglycosidase secreted by Streptococcus pyogenes and belongs to the glycoside hydrolase family 18, as disclosed by Collin et al. (EMBO J., 2001, 20, 3046), whose entire work is incorporated herein by reference. In contrast to the above ENGase, Endo S has even clearer specificity, being specific to cleave only the conserved N-glycan within the Fc domain of human IgG (other substrates have not been identified to date), suggesting that the protein-protein interaction between the enzyme and IgG gives rise to this specificity.

[0605] Endo S49, also known as Endo S2, is a homolog of Endo S, isolated from Streptococcus pyogenes NZ131, and is described in its entirety in International Publication No. 2013 / 037824, which is incorporated herein by reference. Endo S49 has specific endoglycosidase activity for native IgG and cleaves a wider variety of Fc glycans than Endo S.

[0606] Endo SH is a fusion of two endoglycosidases, Endo S and Endo H, linked by a glycy-rich spacer. Endo SH specifically cleaves the N-linked glycan between two N-acetylglucosamine (GluNAc) moieties within the core region of the glycan chain.

[0607] In some embodiments, the endoglycosidase for deglycosylating antibodies is Endo S, Endo SH, Endo S2, Endo S49, Endo F1, Endo F2, Endo F3, Endo H, Endo M, Endo A, or a combination thereof. In some embodiments, the endoglycosidase for deglycosylating antibodies is Endo S, Endo SH, Endo S2, Endo S49, Endo F1, Endo F2, Endo F3, Endo H, or a combination thereof. In some embodiments, the endoglycosidase is Endo S, Endo SH, Endo S2, or Endo S49.

[0608] In some embodiments, when the glycan being trimmed is a complex bifurcated structure, the endoglycosidase is Endo S, Endo SH, Endo S2, Endo S49, Endo F1, Endo F2, Endo F3, or a combination thereof.

[0609] In some embodiments, when the glycoprotein is a B7-H4 antibody and the trimmed oligosaccharide is a complex bifurcated structure present at the IgG-conserved N-glycosylation site at N297, the endoglycosidase is Endo S, Endo SH, Endo S2, Endo S49, Endo F1, Endo F2, Endo F3, or a combination thereof. In some embodiments, the endoglycosidase is Endo S, Endo SH, Endo S2, Endo S49, or a combination thereof.

[0610] In some embodiments, when the glycoprotein is a B7-H4 antibody and the glycan to be trimmed is a complex bifurcated structure and is not present at the IgG-conserved N-glycosylation site at N297, the endoglycosidase is Endo F1, Endo F2, Endo F3, or a combination thereof.

[0611] In some embodiments, when the glycan being trimmed is high in mannose, the endoglycosidase is Endo H, Endo M, Endo A, Endo F1, or a combination thereof.

[0612] In some embodiments, when the glycoprotein is a B7-H4 antibody, and the trimmed oligosaccharide is high in mannose, in addition to having a complex bifurcated structure, and is present at the IgG-conserved N-glycosylation site at N297, the endoglycosidase is Endo S, Endo SH, Endo S2, Endo S49, or a combination thereof. In some embodiments, the endoglycosidase is Endo S or Endo SH. In some embodiments, the endoglycosidase is Endo SH.

[0613] In some embodiments, the endoglycosidase enzymes as defined herein include a sequence encoding a tag for facilitating purification. In some embodiments, the tag includes, but is not limited to, a FLAG tag, a poly(His)-tag, an HA-tag, a Myc-tag, a SUMO-tag, a GST-tag, an MBP-tag, or a CBP-tag. In some embodiments, the tag is a 6xHis tag. In some embodiments, the tag is covalently bound to the endoglycoside enzyme at the C-terminus or as an internal residue. In some embodiments, the tag is covalently bound to the endoglycoside enzyme at the N-terminus.

[0614] In some embodiments, Endo SH is a fusion of two endoglycosidases, Endo S and Endo H, linked by a glycy-rich spacer containing an internal 6xHis tag, resulting in a total molecular weight of 139 kDa.

[0615] Glycosyltransferase The process for forming a modified B7-H4 antibody includes treating a deglycosylated / trimmed antibody having a fucosylated or otherwise deglycosylated terminal N-acetylglucosamine (Gal-NAc) moiety with a compound of formula S''(A'')-P'' in the presence of glycosyltransferase to form a modified B7-H4 antibody having a GlcNAc-S''(A'') substituent bound to the antibody at C1 of the GalNAc moiety via a β-1,4-O-glycosidic bond.

[0616] In some embodiments, glycosyltransferases are β-1,4-galactosyltransferase (4Gal-T), β-(1,4)-acetylgalactosaminyltransferase (β-(1,4)-GalNAcT or GalNAcT), or variants thereof.

[0617] β-(1,4)-acetylgalactosaminyltransferase (β-(1,4)-GalNAcT or β-(1,4)-GalNAcT) has been identified in numerous organisms, including humans, the nematode Caenorhabditis elegans (Kawar et al, J. Biol. Chem. 2002, 277, 34924, whole text incorporated herein by reference), Drosophila melanogaster (Hoskins et al., Science 2007, 316, 1625, whole text incorporated herein by reference), and the nettle moth (Vadaie et al, J. Biol. Chem. 2004, 279, 33501, whole text incorporated herein by reference).

[0618] β-(1,4)-N-acetylgalactosaminyltransferase (β-(1,4)-GalNAcT) is known in the art. In some embodiments, β-(1,4)-GalNAcT is an enzyme that catalyzes the transfer of N-acetylgalactosamine (GalNAc) from uridine diphosphate-GalNAc (UDP-GalNAc, also called GalNAc-UDP) to the terminal GlcNAc portion of a glycoprotein glycan, where C1 of the GalNAc portion is bound to an antibody via a β-1,4-O-glycosidic bond. In some embodiments, the terminal GlcNAc portion is fucosylated.

[0619] In some embodiments, the β-(1,4)-GalNAcT enzyme used in the process of the present invention is an invertebrate β-(1,4)-GalNAcT enzyme or derived therefrom, for example, a β-(1,4)-GalNAcT from an invertebrate species or derived therefrom. The β-(1,4)-GalNAcT enzyme may be or be derived from any invertebrate β-(1,4)-GalNAcT enzyme known to those skilled in the art. In some embodiments, the β-(1,4)-GalNAcT enzyme may be a β-(1,4)-GalNAcT enzyme from the phylum Nematoda, for example, a class such as Chromadorea or Seernentea, or from the phylum Arthropoda, for example, a class such as Insecta, or derived therefrom. In some aspects, the β-(1,4)-GalNAcT enzyme is found in *Elegans* nematodes, *Caenorhabditis remanei*, *Caenorhabditis briggsae*, *Ascaris suum*, *Urtica dioica*, *Drosophila melanogaster*, *Wuchereria bancrofti*, *Loa loa*, *Cerapachys biroi*, *Zootermopsis nevadensis*, *Camponotus floridanus*, *Crassostrea gigas*, or *Danaus* The β-(1,4)-GalNAcT enzyme is derived from or is derived from *Plexippus*, (e.g., *Plexippus elegans*, *Ranunculus porcini*, *Galium saccharina*, or *Drosophila melanogaster*. In some embodiments, the β-(1,4)-GalNAcT enzyme is derived from or is derived from *Plexippus*, *Ranunculus porcini*, or *Galium saccharina*. In other embodiments, the β-(1,4)-GalNAcT enzyme is derived from or is derived from *Galium saccharina*.

[0620] The term “derived from” includes, for example, cleavage enzymes, mutant enzymes, enzymes with tags to facilitate purification, or combinations thereof. Therefore, “derived from” means having an amino acid sequence modified by substituting, inserting, deleting, or adding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 or more) amino acids from the naturally occurring β-(1,4)-GalNAcT enzyme. A β-(1,4)-GalNAcT enzyme derived from a β-(1,4)-GalNAcT enzyme is also referred to herein as an inducible β-(1,4)-GalNAcT enzyme, a modified β-(1,4)-GalNAcT enzyme, or a β-(1,4)-GalNAcT mutant enzyme.

[0621] In some embodiments, the inducible β-(1,4)-GalNAcT enzyme is modified by adding or deleting additional N-terminal or C-terminal amino acids or chemical moieties to increase stability, solubility, activity, and / or ease of purification.

[0622] In some embodiments, the β-(1,4)-GalNAcT enzyme is modified by deleting the N-terminal cytoplasmic domain and transmembrane domain, and is called a cleavage enzyme.

[0623] A β-(1,4)-GalNAcT enzyme in which one or more amino acids are substituted, added, or deleted is also referred to herein as a mutant β-(1,4)-GalNAcT enzyme or an inducible β-(1,4)-GalNAcT enzyme. In some embodiments, a β-(1,4)-GalNAcT enzyme is modified by deleting the N-terminal cytoplasmic domain and the transmembrane domain, and is mutated by substituting one or more amino acids. Substitution of one or more amino acids is also referred to herein as a mutation. An enzyme containing one or more substituted amino acids is also referred to as a mutant enzyme.

[0624] In some embodiments, if the glycosyltransferase is a β-(1,4)-GalNAcT enzyme or a cleavage-type β-(1,4)-GalNAcT enzyme, the enzyme further comprises one or more mutations. In some embodiments, these mutations include, but are not limited to, substitutions of isoleucine (I, also known as He) at position 257 by leucine (Leu, also known as L), methionine (Met, also known as M), or alanine (Ala, also known as A). In some embodiments, substitutions of methionine (Met, also known as M) at position 312 by histidine (His, also known as H). The amino acid position numbering herein is based on the amino acid position numbering in the wild-type β-(1,4)-GalNAcT enzyme. If the β-(1,4)-GalNAcT enzyme is, for example, a cleavage-type enzyme, the numbers used herein to indicate the amino acid substitution positions correspond to the numbering of amino acid positions in the corresponding wild-type β-(1,4)-GalNAcT enzyme.

[0625] In some embodiments, glycosyltransferase is a β(1,4)-GalT enzyme containing a mutant catalytic domain.

[0626] The catalytic domain may have an amino acid sequence similar to that found in the wild-type enzyme, or it may have an amino acid sequence different from the wild-type sequence. A catalytic domain having an amino acid sequence different from the wild-type sequence is referred to herein as a mutant catalytic domain. In some embodiments, the mutation may include a single amino acid change (e.g., a point mutation), multiple amino acid changes (e.g., 1 to 10, or 1 to 6, or 1, 2, 3, or 4, or 1 or 2 amino acids), or the deletion or insertion of one or more amino acids (e.g., 1 to 10, or 1 to 6, or 1, 2, 3, or 4, or 1 or 2 amino acids). In some embodiments, the mutant catalytic domain may be present in a full-length enzyme, such as β(1,4)-galactosyltransferase or α(1,3)-N-galactosyltransferase, but may also be present in polypeptide fragments or recombinant polypeptides containing the mutant catalytic domain optionally bound to additional amino acids.

[0627] β(1,4)-galactosyltransferase I is referred to herein as GalT. Such mutant GalT catalytic domains are disclosed, for example, in International Publication No. 2004 / 063344, which is incorporated herein in its entirety by reference. International Publication No. 2004 / 063344 also discloses Tyr-289 variants of GalT and methods for their preparation. These variants are referred to as Y289L, Y289N, or Y289I.

[0628] In some embodiments, the GalT mutation-catalyzing domain is Y289L, Y289N, Y289I, Y284L, or R228K. In some embodiments, the GalT mutation-catalyzing domain is Y289L.

[0629] In some embodiments, GalT Y289F, GalT Y289M, GalT Y289V, GalT Y289G, GalT Y289I, GalT Y289A, GalT Y289N, and GalT Y289L variants may be generated via site-directed mutagenesis processes described, for example, in International Publication No. 2004063344, Qasba et al, Prot.Expr.Pur.2003,30,219, and Qasba et al, J.Biol.Chem.2002,277,20833 (all of which are incorporated herein by reference in their entirety). In GalT Y289F, the tyrosine amino acid (Y) at position 289 is substituted with the amino acid phenylalanine (F); in GalT Y289M, the tyrosine is substituted with the amino acid methionine (M); in GalT Y289V, it is substituted with the amino acid valine (V); in GalT Y289G, it is substituted with the amino acid glycine (G); in GalT Y289I, it is substituted with the amino acid isoleucine (I); and in Y289A, it is substituted with the amino acid analine (A).

[0630] In some embodiments, the β-(1,4)-GalNAcT enzyme includes a sequence encoding a tag for facilitating purification. In some embodiments, the tag includes, but is not limited to, a FLAG tag, a poly(His)-tag, an HA-tag, a Myc-tag, a SUMO-tag, a GST-tag, an MBP-tag, or a CBP-tag. In other embodiments, the tag is a 6xHis tag. In some embodiments, the tag is covalently bound to the β-(1,4)-GalNAcT enzyme at its C-terminus. In some embodiments, the tag is covalently bound to the β-(1,4)-GalNAcT enzyme at its N-terminus.

[0631] In some embodiments, the β-(1,4)-GalNAcT enzyme contains an N-terminal 6xHis tag and has a total molecular weight of 45.7 kDa. In some embodiments, the β-(1,4)-GalNAcT enzyme containing an N-terminal 6xHis tag is derived from nettle moth.

[0632] P''-S''-A'' molecule In some embodiments, the P''-S''-A'' molecule for use in the process of preparing the modified B7-H4 antibody of this disclosure may be any sugar derivative nucleotide that is a substrate for a suitable galactosyltransferase catalyst.

[0633] In some embodiments, S''-A'' is a sugar derivative moiety, in the formula, S'' is a sugar or a derivatized sugar, and A'' is a functional group that can form a covalent bond with the functional group of the linker-drug moiety.

[0634] In some embodiments, A'' is an azid moiety, a keto moiety, or an alkynyl moiety. In some embodiments, A'' is an azid moiety or a keto moiety. In some embodiments, A'' is an azid moiety. In some embodiments, A'' is -N 3 In some aspects, A'' is the keto part.

[0635] In some embodiments, A'' is -[C(R 8k )2] x2 C(O)R 9k And in the formula, R 9k C is methyl or may be substituted. 2~24 It is alkyl, Each R 8k These are independently hydrogen, halogen, or R 9k And, x² is an integer in the range of 0 to 24.

[0636] In some embodiments, x2 is an integer in the range of 0 to 10. In some embodiments, x2 is 0, 1, 2, 3, 4, 5, or 6.

[0637] In some embodiments, each R 8k It is hydrogen.

[0638] In some embodiments, A'' is an alkynyl moiety. In some embodiments, A'' is a terminal alkynyl moiety, a cycloalkynyl moiety, or a heterocycloalkynyl moiety. In some embodiments, A'' is a terminal alkynyl moiety. In some embodiments, A'' is a cycloalkynyl moiety. In some embodiments, A'' is a heterocycloalkynyl moiety.

[0639] In some aspects, A'' is -[C(R 8k )2] x2 -C≡CR 8k It is a base, and in the formula, R 8k and x2 are as defined herein. In some embodiments, A'' is -[CH2] x2 -C ≡ CH

[0640] In some embodiments, S''-A'' is derived from a sugar or a derivatized sugar, such as an amino sugar or other derivatized sugar. In some embodiments, examples of sugars and derivatized sugars include, but are not limited to, galactose (Gal), mannose (Man), glucose (Glc), glucuronic acid (Gcu), and fucose (Fuc). An amino sugar is understood to be a sugar in which a hydroxyl (OH) group is substituted with an amine group. Examples of amino sugars include, but are not limited to, N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). Examples of sugars derivatized by other means include, but are not limited to, glucuronic acid (Gcu) and N-acetylneuraminic acid (sialic acid).

[0641] In some embodiments, S''-A'' is derived from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc), or N-acetylneuraminic acid (sialic acid). In some embodiments, S''-A'' is derived from GlcNAc, Glc, Gal, or GalNAc. In some embodiments, S''-A'' is derived from GlcNAc. In some embodiments, S''-A'' is derived from Glc. In some embodiments, S''-A'' is derived from Gal or GalNAc. In some embodiments, S''-A'' is derived from Gal. In some embodiments, S''-A'' is derived from GalNAc.

[0642] In some embodiments, the functional group A'' can be bonded to S'' in various ways.

[0643] In some embodiments, A'' is directly bonded to the C2, C3, C4, or C6 carbon atoms of the sugar or derivatized sugar S'' (for example, instead of the hydroxyl at the corresponding position).

[0644] In some embodiments, S'' is fucose or derivatized fucose lacking any hydroxyl C6 position. In some embodiments, if A'' is bonded to the C6 position of fucose or derivatized fucose, A'' is directly bonded to the carbon atom at the C6 position.

[0645] In some embodiments, A'' is an azide moiety, and A'' is bonded to the C2, C4, or C6 position of the sugar or derivatized sugar of S''.

[0646] In some embodiments, A'' is an azide moiety, and A'' is directly bonded to the C2, C3, C4, or C6 carbon atom of the sugar or derivatized sugar S'' (e.g., instead of the hydroxyl at the corresponding position). In some embodiments, S''-A'' is 6-azidofucose (6-AzFuc). In some embodiments, A'' is an azide moiety, and A'' is bonded to the N-acetyl moiety of the amino sugar or derivatized amino sugar (e.g., by substituting the acetyl moiety with the azidoacetyl moiety). In some embodiments, S''-A'' is 2-azidoacetamidogalactose (GalNAz), 6-azido-6-deoxygalactose (6-AzGal), 6-azido-6-deoxy-2-acetamidogalactose (6-AzGalNAc), 4-azido-4-deoxy-2-acetamidogalactose (4-AzGalNAc), 6-azido-6-deoxy-2-azidoacetamidogalactose (6-A S''-A'' is GalNAz, 4-AzGalNAc, GlcNAz, 6-AzGlc, 6-AzGlcNAc, 4-AzGlcNAc, or 6-AzGlcNAz. In some embodiments, S''-A'' is GalNAz, 4-AzGalNAc, GlcNAz, or 6-AzGlcNAz.

[0647] In some embodiments, P''-S''-A'' is a compound or salt of formula (XXIVb), (XXXIVc), or (XXIVd).

[0648] In some embodiments, A'' keto and A'' are directly bonded to the carbon atom at the C2 position of the sugar or derivatized sugar S'' (for example, instead of the hydroxyl at the corresponding position).

[0649] In some embodiments, A'' is bonded to the nitrogen atom of an amino sugar or derivatized amino sugar, such as a C2-derivative amino sugar. In some embodiments, the derivatized amino sugar is -NC(O)-R 9k The part includes, in the formula, R 9k C is methyl or may be substituted. 2~24 It is an alkyl group (for example, ethyl).

[0650] In some embodiments, R 9k It is ethyl.

[0651] In some embodiments, S''-A'' is 2-deoxy-(2-oxopropyl)-galactose (2-keto-Gal), 2-N-propionyl-galactosamine (2-N-propionylGal-NAc), 2-N-(4-oxopentanoyl)-galactosamine (2-N-Lev-Gal), or 2-N-butyryl-galactosamine (2-N-butyryl-GalNAc). In some embodiments, S''-A'' is 2-ketoGalNAc or 2-N-propionyl-GalNAc.

[0652] In some embodiments, P''-S''-A'' is a compound of formula (XXIVe) or (XXIVf) or a salt thereof.

[0653] In some embodiments, A'' is a terminal alkynyl, cycloalkynyl, or heterocycloalkynyl. In some embodiments, A'' is bonded to the C2-derivative amino sugar of S''.

[0654] In some embodiments, S''-A'' is 2-(buta-3-iodine amide)-2-deoxy-galactose.

[0655] In some embodiments, P''-S''-A'' is a compound of formula (XXIVg) or a salt thereof. In some embodiments, P''-S''-A'' is a compound of formula (XXIVd) or a salt thereof.

[0656] In some embodiments, the P''-S''-A'' compounds can be synthesized according to various methods known in the art. In some embodiments, the compounds are synthesized by attaching a nucleoside monophosphate P'' or nucleoside diphosphate P'' to a sugar derivative S''-A'', as disclosed, for example, in Wang et al. (Chem.Eur.J.16:13343-13345 (2010)), Piller et al. (ACS Chem.Biol.7:753 (2012)), Piller et al. (Bioorg.Med.Chem.Lett.15:5459-5462 (2005)), and PCT Publication International Publication No. 2009 / 102820, each of which is incorporated herein by reference in its entirety.

[0657] In some embodiments, P'' is a nucleoside monophosphate or a nucleoside diphosphate. In some embodiments, P'' is uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP), or cytidine monophosphate (CMP). In some embodiments, P'' is uridine diphosphate (UDP).

[0658] In some embodiments, P''-S''-A'' is a compound of formula (XXIVb), (XXIVc), (XXIVd), (XXIVe), (XXIVf), or (XXIVg): TIFF2026082984000217.tif87128 or a salt thereof, where R 9k is C 2~24 It is an alkyl group.

[0659] In some embodiments, P''-S''-A'' is GalNAz-UDP (e.g., formula (XXIVb)), 6-AzGal-UDP (e.g., formula (XXIVc)), 6-AzGalNAc-UDP (e.g., formula (XXIVd)), 4-AzGalNAz-UDP, 6-AzGalNAz-UDP, 6-AzGlc-UDP, 6-AzGlcNAz-UDP, 2-ketoGal-UDP (e.g., formula (XXIVe)), 2-N-propionylGalNAc-UDP (e.g., formula (XXIVf), where R 9k (is ethyl), or 2-(buta-3-iodamide)-2-deoxy-galactose-UDP (e.g., formula (XXIVg)).

[0660] In some embodiments, P''-S''-A'' is GalNAz-UDP or 4-AzGalNAc-UDP. In some embodiments, P''-S''-A'' is a compound of formula (XXIVb) or (XXIVd). The synthesis of GalNAz-UDP (e.g., formula (XXIVb)) and 6-AzGalNAc-UDP (e.g., formula (XXIVd)) is disclosed in Piller et al. (Bioorg. Med. Chem. Lett. 15:5459-5462 (2005)) and Wang et al. (Chem. Eur. J. 16:13343-13345 (2010)), each of which is incorporated herein by reference in its entirety.

[0661] In some embodiments, P''-S''-A'' is 4-AzGalNAc-UDP. In some embodiments, P''-S''-A'' is the compound of formula (XXIVd) or a salt thereof. The synthesis of 2-ketoGal-UDP(XXIVe) is disclosed in Qasba et al. (J.Am.Chem.Soc.125:16162(2003)) and its supplementary information, which are incorporated herein by reference in their entirety.

[0662] The synthesis of 2-(buta-3-ino acid amide)-2-deoxy-galactose-UDP is disclosed in PCT Publication International 2009 / 102820, which is incorporated herein by reference in its entirety.

[0663] variable d 13 In some aspects, d 13 It is an integer in the range of approximately 2 to 14, approximately 2 to 12, approximately 2 to 10, approximately 2 to 8, approximately 2 to 6, approximately 2 to 4, approximately 4 to 10, approximately 4 to 8, approximately 4 to 6, approximately 6 to 14, approximately 6 to 12, approximately 6 to 10, approximately 6 to 8, approximately 8 to 14, approximately 8 to 12, or approximately 8 to 10.

[0664] In some aspects, d 13 It is an integer in the range of approximately 2 to approximately 8.

[0665] In some aspects, d 13 is 2, 4, 6, or 8. In some embodiments, d 13 is 2, 6, or 8. In some embodiments, d 13 8. In some embodiments, d 13 It is 6. In some embodiments, d 13 The answer is 2.

[0666] B7-H4 antibody-drug conjugate In some embodiments, the conjugate of the present disclosure comprises one or more entities of D, where D is a cytotoxic drug portion or a STING agonist drug portion, and the one or more entities of D may be the same or different.

[0667] In some embodiments, the presence of one or more B7-H4 antibodies or B7-H4 modified antibodies is bound to the linker-drug moiety, and the presence of one or more B7-H4 antibodies or B7-H4 modified antibodies may be the same or different. In some embodiments, one or more linker-drug moieties containing one or more D are bound to one B7-H4 antibody or B7-H4 modified antibody.

[0668] In some embodiments, the B7-H4 antibody is either a B7-H4 antibody or a cysteine-modified B7-H4 antibody.

[0669] In some embodiments, the targeting ligands, linkers, and drug fragments or prodrug fragments described herein may be incorporated into the conjugates or scaffolds of this disclosure, for example, according to the techniques and methods disclosed herein. Therapeutic conjugates and targeting conjugates of this disclosure, as well as methods for generating them, are described below as non-limiting examples.

[0670] In some embodiments, the total number of sulfide bonds (or total number of binding sites) formed between the linker-drug moiety and the B7-H4 antibody or cysteine-modified B7-H4 antibody is 10 or less (e.g., 8, 6, 4, or 2).

[0671] In some embodiments, the total number of sulfide bonds (or total number of binding sites) formed between the linker-drug moiety and the B7-H4 antibody or cysteine-modified B7-H4 antibody is 8 or less.

[0672] In some embodiments, the total number of sulfide bonds (or binding sites) formed between the linker-drug moiety and the B7-H4 antibody or cysteine-modified B7-H4 antibody is 8. In some embodiments, the total number of sulfide bonds (or binding sites) formed between the linker-drug moiety and the B7-H4 antibody or cysteine-modified B7-H4 antibody is 6. In some embodiments, the total number of sulfide bonds (or binding sites) formed between the linker-drug moiety and the B7-H4 antibody or cysteine-modified B7-H4 antibody is 5. In some embodiments, the total number of sulfide bonds (or binding sites) formed between the linker-drug moiety and the B7-H4 antibody or cysteine-modified B7-H4 antibody is 4. In some embodiments, the total number of sulfide bonds (or binding sites) formed between the linker-drug moiety and the B7-H4 antibody or cysteine-modified B7-H4 antibody is 3. In some embodiments, the total number of sulfide bonds (or total number of bond sites) formed between the linker-drug moiety and the B7-H4 antibody or cysteine-modified B7-H4 antibody is 2.

[0673] In some embodiments, the linker-drug ratio to B7-H4 antibody is approximately 1:1 to approximately 8:1. In some embodiments, the linker-drug ratio to B7-H4 antibody or cysteine-modified B7-H4 antibody is approximately 1:1 to approximately 6:1. In some embodiments, the linker-drug ratio to B7-H4 antibody is approximately 1:1 to approximately 4:1. In some embodiments, the linker-drug ratio to B7-H4 antibody or cysteine-modified B7-H4 antibody is approximately 2:1 to approximately 2:1.

[0674] In some embodiments, the linker-drug ratio to the B7-H4 antibody or cysteine-modified B7-H4 antibody is approximately 6:1 to approximately 8:1.

[0675] In some embodiments, the linker-drug ratio to the B7-H4 antibody or cysteine-modified B7-H4 antibody is approximately 8:1.

[0676] In some embodiments, the linker-drug ratio to the B7-H4 antibody is approximately 6:1.

[0677] In some embodiments, the disclosure also comprises at least two portions of a cysteine-operated B7-H4 antibody or a linker-drug portion, wherein each portion can be conjugated to a thiol group of the B7-H4 antibody to form a protein-linker-drug conjugate.

[0678] In some embodiments, one or more thiol groups of the B7-H4 antibody or cysteine-modified B7-H4 antibody are generated by reducing a protein. These one or more thiol groups of the B7-H4 antibody or cysteine-modified B7-H4 antibody can then react with one or more linker-drug moieties that can conjugate the thiol groups from the B7-H4 antibody or cysteine-modified B7-H4 antibody with the linker-drug moiety. In some embodiments, at least two moieties attached to the B7-H4 antibody or cysteine-modified B7-H4 antibody are maleimide groups. In these embodiments, D is a cytotoxic drug moiety or a STING agonist drug moiety.

[0679] In some embodiments, the antibody may be activated for conjugation with a linker-drug moiety by treatment with a reducing agent such as DTT (Clealand's Reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride). In some embodiments, a full-length monoclonal antibody may be reduced with excess TCEP to reduce the disulfide bonds (e.g., between cysteine ​​present in the corresponding parent antibody or cysteine-manipulated antibody) to obtain a reduced antibody. The newly introduced unpaired cysteine ​​may remain available for reaction with the linker-drug moiety to form the antibody conjugate of this disclosure. In some embodiments, conjugation is performed by adding excess linker-drug moiety to form an antibody-drug conjugate, and the conjugation mixture is purified to remove excess linker-drug intermediates and other impurities.

[0680] In some embodiments, the ratio of the B7-H4 antibody or cysteine-modified B7-H4 / linker-drug portion is approximately 1:1 to 1:8, approximately 1:1 to 1:6, approximately 1:1 to 1:5, approximately 1:1 to 1:4, approximately 1:1 to 1:3, or approximately 1:1 to 1:2.

[0681] The conjugates disclosed herein can be purified by extensive diafiltration (i.e., removal of any starting material). If necessary, additional purification by size exclusion chromatography can be performed to remove aggregated conjugates. Generally, the purified conjugate typically contains less than 5% (e.g., less than 2% w / w) of aggregated conjugate as determined by SEC; less than 0.5% (e.g., less than 0.1% w / w) of free (unconjugated) drug as determined by RP-HPLC; less than 1% of drug-carrying peptide-containing scaffold as determined by SEC; and less than 2% (e.g., less than 1% w / w) of unconjugated B7-H4 antibody as determined by HIC-HPLC.

[0682] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 conjugated to the STING agonist drug moiety is selected from the conjugates listed in Tables A1 and A2.

[0683] (Table A1) TIFF2026082984000218.tif176158TIFF2026082984000219.tif218158TIFF2026082984000220.tif217158TIFF2026082984000221.tif199158TIFF2026082984000222.tif212158TIFF2026082984000223.tif103158In the formula, d 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R C1 , R C2 , R 4 X3, X4, X6, X7, X1, W1, Y1, Z1, X2, W2, Y2, and Z2 are as defined herein, and ANTIBODY is B7-H4 antibody or cysteine-modified B7-H4 antibody.

[0684] In some embodiments, in the case of the conjugate in Table A1, d 13 The integer is between 6 and 8.

[0685] In some embodiments, in the case of the conjugate in Table A1, d 13 It is 8. In some embodiments, in the case of the conjugate in Table A1, d 13 It is 7. In some embodiments, in the case of the conjugate in Table A1, d 13 It is 6.

[0686] In some embodiments, in the case of the conjugate in Table A1, d 13 It is 8.

[0687] (Table A2) TIFF2026082984000224.tif167158TIFF2026082984000225.tif168158TIFF2026082984000226.tif164158 TIFF2026082984000227.tif154158TIFF2026082984000228.tif199158TIFF2026082984000229.tif172158T IFF2026082984000230.tif204158TIFF2026082984000231.tif169158TIFF2026082984000232.tif198158T IFF2026082984000233.tif204158TIFF2026082984000234.tif204158TIFF2026082984000235.tif212158TI FF2026082984000236.tif214158TIFF2026082984000237.tif209158TIFF2026082984000238.tif209158TI FF2026082984000239.tif184158TIFF2026082984000240.tif187158TIFF2026082984000241.tif185158TIF F2026082984000242.tif209158TIFF2026082984000243.tif189158TIFF2026082984000244.tif201158TIFF2026082984000245.tif199158TIFF2026082984000246.tif182158TIFF2026082984000247.tif178158 In the formula, d 13 The term is as defined herein, and the antibody is a B7-H4 antibody or a cysteine-modified B7-H4 antibody.

[0688] In some embodiments, in the case of the conjugate in Table A2, d 13 The integer is between 6 and 8.

[0689] In some embodiments, in the case of the conjugate in Table A2, d 13 It is 8. In some embodiments, in the case of the conjugate in Table A2, d13 It is 7. In some embodiments, in the case of the conjugate in Table A2, d 13 It is 6.

[0690] In some embodiments, in the case of the conjugate in Table A2, d 13 It is 8.

[0691] In some aspects, the STING agonist drug conjugate is as follows: TIFF2026082984000248.tif172137TIFF2026082984000249.tif105134In formula, d 13 The antibody is 8, and the antibody is a B7-H4 antibody or a cysteine-modified B7-H4 antibody. The B7-H4 antibody contains variable heavy chain complementarity determination region 1 (CDRH1) with amino acid sequence GFIVSRNY (SEQ ID NO:2), variable heavy chain complementarity determination region 2 (CDRH2) with amino acid sequence IYGSGRT (SEQ ID NO:3), and amino acid sequence TIFF2026082984000250.tif4128 or amino acid sequence Variable heavy chain complementarity determination region 3 (CDRH3) containing TIFF2026082984000251.tif4128, variable light chain complementarity determination region 1 (CDRL1) containing amino acid sequence QSVSSSY (SEQ ID NO: 53), variable light chain complementarity determination region 2 (CDRL2) containing amino acid sequence GAS (SEQ ID NO: 54), amino acid sequence Includes variable light chain complementarity determination region 3 (CDRL3), which includes TIFF2026082984000252.tif4128.

[0692] In some aspects, the STING agonist drug conjugate is as follows: TIFF2026082984000253.tif170148, d 13 The antibody is 8, and the antibody is a B7-H4 antibody or a cysteine-modified B7-H4 antibody. The B7-H4 antibody contains variable heavy chain complementarity determination region 1 (CDRH1) with amino acid sequence GFIVSRNY (SEQ ID NO:2), variable heavy chain complementarity determination region 2 (CDRH2) with amino acid sequence IYGSGRT (SEQ ID NO:3), and amino acid sequence TIFF2026082984000254.tif4128 or amino acid sequence Variable heavy chain complementarity determination region 3 (CDRH3) containing TIFF2026082984000255.tif4128, variable light chain complementarity determination region 1 (CDRL1) containing amino acid sequence QSVSSSY (SEQ ID NO: 53), variable light chain complementarity determination region 2 (CDRL2) containing amino acid sequence GAS (SEQ ID NO: 54), amino acid sequence Includes variable light chain complementarity determination region 3 (CDRL3), which includes TIFF2026082984000256.tif4128.

[0693] In some aspects, the STING agonist drug conjugate is as follows: TIFF2026082984000257.tif56128 formula, d 13 8 is ANTIBODY is B7-H4 antibody, or d 13 The antibody is 2, and the antibody is a cysteine-modified B7-H4 antibody. The B7-H4 antibody contains variable heavy chain complementarity determination region 1 (CDRH1) with amino acid sequence GFIVSRNY (SEQ ID NO:2), variable heavy chain complementarity determination region 2 (CDRH2) with amino acid sequence IYGSGRT (SEQ ID NO:3), and amino acid sequence TIFF2026082984000258.tif4128 or amino acid sequence Variable heavy chain complementarity determination region 3 (CDRH3) containing TIFF2026082984000259.tif4128, variable light chain complementarity determination region 1 (CDRL1) containing amino acid sequence QSVSSSY (SEQ ID NO: 53), variable light chain complementarity determination region 2 (CDRL2) containing amino acid sequence GAS (SEQ ID NO: 54), amino acid sequence Includes variable light chain complementarity determination region 3 (CDRL3), which contains TIFF2026082984000260.tif4128.

[0694] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 conjugated to the cytotoxic drug moiety is selected from the conjugates listed in Table B1.

[0695] (Table B1) TIFF2026082984000261.tif210142TIFF2026082984000262.tif221142TIFF2026082984000263.tif135142TIFF2026082984000264.tif135142T IFF2026082984000265.tif203142TIFF2026082984000266.tif215142TIFF2026082984000267.tif173142TIFF2026082984000268.tif174142TI FF2026082984000269.tif180142TIFF2026082984000270.tif211142TIFF2026082984000271.tif209142TIFF2026082984000272.tif186142TIF In the formula, d 13 The term is as defined herein, and the antibody is a B7-H4 antibody or a cysteine-modified B7-H4 antibody.

[0696] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 antibody conjugated to the cytotoxic drug moiety is a conjugate of formula (XXX): TIFF2026082984000277.tif31142In formula, each R A The following is true: TIFF2026082984000278.tif143150d 13 It is 2, 4, 6, or 8.

[0697] In other embodiments, the B7-H4 antibody or cysteine-modified B7-H4 antibody conjugated to the cytotoxic drug moiety is a conjugate of formula (XXX). In the formula, each R A The following is true: TIFF2026082984000279.tif203126TIFF2026082984000280.tif220132TIFF202608298400 0281.tif204143TIFF2026082984000282.tif191150TIFF2026082984000283.tif79128In the formula, d 13 It is 2, 4, 6, or 8.

[0698] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 antibody conjugated to the cytotoxic drug moiety is a conjugate of formula (XXX), and each R A The following is true: TIFF2026082984000284.tif86128

[0699] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 antibody conjugated to the cytotoxic drug moiety is a conjugate of formula (XXXI-1), (XXXI-2), (XXXI-3), or (XXXI-4): TIFF2026082984000285.tif155160

[0700] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 antibody conjugated to the cytotoxic drug moiety is a conjugate of formula (XXXII): TIFF2026082984000286.tif80136

[0701] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 antibody conjugated to the cytotoxic drug moiety is a conjugate of formula (XXXII): In the TIFF2026082984000287.tif33128 formula, each R B The following is true: TIFF2026082984000288.tif196126

[0702] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 antibody conjugated to the cytotoxic drug moiety is a conjugate of formula (XXXI-1), (XXXI-2), (XXXI-3), (XXXI-4), or (XXXII), where the variable -L D -D is as follows: TIFF2026082984000289.tif143164

[0703] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 antibody conjugated to the cytotoxic drug moiety is a conjugate of formula (XXXI-1), (XXXI-2), (XXXI-3), (XXXI-4), or (XXXII), where the variable -L D -D is as follows: TIFF2026082984000290.tif70128

[0704] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 antibody conjugated to the cytotoxic drug moiety is a conjugate of formula (XXXXIII-3): TIFF2026082984000291.tif22142, each R A The following is true: TIFF2026082984000292.tif105128

[0705] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 antibody conjugated to the cytotoxic drug moiety is a conjugate of formula (XXXXIII-8): TIFF2026082984000293.tif102156

[0706] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 antibody conjugated to the cytotoxic drug moiety is a conjugate of formula (XXXIII-5): TIFF2026082984000294.tif135151, d 13 This is as defined herein.

[0707] In some embodiments, the B7-H4 antibody or cysteine-modified B7-H4 antibody conjugated to the cytotoxic drug moiety is a conjugate of formula (XXXIII-8): TIFF2026082984000295.tif102158 formula, d 13 This is as defined herein.

[0708] Modified B7-H4 antibody drug conjugate In some embodiments, the modified B7-H4 antibody-drug conjugate of the disclosed herein comprises a modified B7-H4 antibody that can form a covalent bond with a functional group (e.g., W) in the modified GlcNAc moiety within the modified B7-H4 antibody, the functional group A'' of *-GlcNAc-S''-A''. P It can be obtained by reacting a linker containing the drug portion with the drug portion.

[0709] In some embodiments, W P This includes alkynyls, such as cycloalkynyls, heterocycloalkynyls, or terminal alkynyls.

[0710] In some embodiments, the functional group A'' of the modified B7-H4 antibody is azide, keto, or alkynyl. In some embodiments, the functional group A'' of the modified B7-H4 antibody is azide. In some embodiments, the azide functional group A'' of the modified B7-H4 antibody is W of the linker-drug moiety. PIt reacts with an alkynyl group (e.g., cycloalkynyl, heterocycloalkynyl, or terminal alkynyl) to form a triazole moiety (e.g., via cycloaddition). Cycloaddition reactions between azide groups and alkynyl groups are well known in the art as "click chemistry".

[0711] In some embodiments, the linker-drug portion W P The molecule contains a terminal alkynyl, and the cycloaddition reaction can be carried out in the presence of a catalyst (e.g., a Cu(I) catalyst).

[0712] In some embodiments, the linker-drug portion W P This includes cycloalkynyl or heterocycloalkynyl (e.g., strained cycloalkynyl or strained heterocycloalkynyl).

[0713] In some embodiments, the linker-drug portion W P The catalyst comprises strained cycloalkynyls or strained heterocycloalkynyls, and the cycloaddition reaction may occur in or without the catalyst. In some embodiments, the cycloaddition reaction may occur spontaneously by a reaction known in the art as "metal-free click chemistry," called strain-enhanced azide-alkyne cycloaddition (SPAAC). In some embodiments, the strained cycloalkynyls or strained heterocycloalkynyls are as described herein.

[0714] In some embodiments, during conjugation, the functional group A'' of the modified B7-H4 antibody and the W of the linker-drug moiety are combined. P This forms the triazole portion.

[0715] In some embodiments, during conjugation, the functional group A'' of the modified B7-H4 antibody and the W of the linker-drug moiety are combined. P This is expressed by equation (XXXV): Forms the triazole portion of TIFF2026082984000296.tif25128, In the formula, * represents direct or indirect binding to the remainder of the modified B7-H4 antibody, and ** represents M if present. p , or L M Or M A This indicates a connection to [the specified location].

[0716] In some embodiments, when the azide-modified B7-H4 antibody of the present disclosure is reacted with a linker-drug moiety containing an alkynyl group to form an antibody-drug conjugate via a cycloaddition reaction, the triazole moiety formed within the antibody-drug conjugate may be resistant to hydrolysis and / or other degradation pathways.

[0717] In some embodiments, when an aldehyde-modified B7-H4 antibody or a ketone-modified B7-H4 antibody of the present disclosure is reacted with a linker-drug moiety containing hydroxylamine or hydrazine, the resulting oxime moiety or hydrazone moiety in the modified B7-H4 antibody-drug conjugate may be relatively inactive under neutral conditions.

[0718] In some embodiments, the modified B7-H4 antibody-drug conjugates of the present disclosure may have high stability.

[0719] In some embodiments, the modified B7-H4 antibodies and modified B7-H4 antibody-drug conjugates of the present disclosure can be synthesized by practical synthetic routes, since the process for introducing functional group A'' (e.g., azide, keto, or alkynyl) into the antibody is straightforward and generally applicable.

[0720] In some embodiments, the site-specific B7-H4 antibody-drug conjugate of the present disclosure is obtained by a process comprising the step of reacting a modified B7-H4 antibody with a linker-drug moiety. The linker-drug portion contains cycloalkynyl or heterocycloalkynyl. The modified B7-H4 antibody, prior to conjugation, consists of the B7-H4 antibody and a modified GlcNAc moiety of *-GlcNAc-S''-A'' conjugated to the B7-H4 antibody via the C1 position of GlcNAc, where GlcNAc is N-acetylglucosamine, S'' is a sugar or derivatized sugar, and A'' is an azide.

[0721] In some embodiments, A'' is a cycloalkynyl or heterocycloalkynyl. In some embodiments, A'' is a cycloalkynyl. In some embodiments, A'' is a heterocycloalkynyl.

[0722] In some embodiments, A'' is a strained cycloalkynyl or a strained heterocycloalkynyl. In some embodiments, A'' is a strained cycloalkynyl. In some embodiments, A'' is a strained heterocycloalkynyl.

[0723] In some embodiments, the site-specific B7-H4 antibody-drug conjugate of the present disclosure is (a) An intermediate antibody of formula (XXII): TIFF2026082984000297.tif22128 formula, Ab is the B7-H4 antibody, GlcNAc is N-acetylglucosamine, Fuc is fucose, u3 is 0 or 1, and d 13 However, the intermediate antibody is an integer in the range of 1 to 12. The compound P''-S''-A'', in which, Compound P''-S''-A'' is a compound in which S'' is a sugar or a derivatized sugar, A'' is an azide, and P is uridine diphosphate (UDP), guanosine diphosphate (GDP), or cytidine diphosphate (CDP). The process involves contacting the modified GlcNAc moiety in the presence of galactosyltransferase to form a modified B7-H4 antibody containing the modified GlcNAc moiety, *-GlcNAc-S''-A'' (optionally, the modified GlcNAc moiety is bound to the remainder of the modified antibody via the C1 position of GlcNAc), and (b) A step of reacting a modified B7-H4 antibody with a linker-drug moiety containing a strained cycloalkynyl or strained heterocycloalkynyl to form an antibody-drug conjugate. It is obtained by a process that includes this.

[0724] In some embodiments, the process for preparing a site-specific B7-H4 antibody-drug conjugate is as shown in Figure 5.

[0725] In some embodiments, a modified antibody containing an azide at each amino acid N297 of a B7-H4 antibody is conjugated by metal-free click chemistry with a linker-drug moiety containing a strained cycloalkynyl or strained heterocycloalkynyl to form the site-specific antibody-drug conjugate of the present disclosure.

[0726] In some embodiments, if the modified B7-H4 antibody contains at least one azide moiety and the linker-drug moiety contains a strained cycloalkynyl, the presence of a copper catalyst is not required for the cycloaddition reaction between the azide in the modified antibody and the strained cycloalkynyl or strained heterocycloalkynyl in the linker-drug moiety. In some embodiments, the cycloaddition reaction proceeds in the absence of a copper catalyst, thereby mitigating some of the potential drawbacks of using a copper catalyst in the process.

[0727] In some embodiments, Cu(I) catalysts are generally required for the cyclization and addition of the azide moiety to the terminal alkyne moiety of an antibody. In some embodiments, extensive optimization and fine-tuning of conditions may be necessary to find the optimal parameters for efficient conversion. Nevertheless, even under such conditions, the co-formation of reactive oxygen species is not always completely avoided, which can induce oxidative damage to the antibody / protein (e.g., oxidation of methionine, histidine, cysteine, or disulfide bonds). Other protocols use Cu(I) sources such as CuBr to label fixed cells and synthesize glycoproteins. In these cases, the instability of Cu(I) in air requires an extremely large excess of Cu (e.g., more than 4 mm) and ligands for efficient reaction, which can increase the risk of residual metals after purification, in addition to damage or precipitation of the antibody / protein. Therefore, conjugation of azide-containing antibodies with terminal alkynes in the presence of copper catalysts can result in extensive byproduct formation due to undesirable amino acid oxidation.

[0728] In some embodiments, a modified B7-H4 antibody containing an azide (for example, at each amino acid N297 of the antibody) is conjugated with a linker-drug moiety containing a strained cycloalkynyl or strained heterocycloalkynyl (for example, by metal-free click chemistry).

[0729] In some embodiments, during conjugation, the azide portion of the modified B7-H4 antibody and the strained cycloalkynyl or strained heterocycloalkynyl of the linker-drug portion are combined using formula (XXXV): Forms the triazole portion of TIFF2026082984000298.tif25128, In the formula, * represents direct or indirect binding to the remainder of the modified antibody, and ** represents M if present. p , or L M Or M A This shows a connection to

[0730] In some embodiments, the B7-H4 antibody-drug conjugate of the present disclosure comprises the presence of one or more D, each D independently being a therapeutic agent (e.g., a cytotoxic drug portion), and the presence of one or more D may be the same or different.

[0731] In some embodiments, one or more specific sites of a B7-H4 antibody are bound to a linker-drug moiety, and the linker-drug moieties bound to one or more specific sites may be the same or different. In some embodiments, one or more linker-drug moieties containing one or more D (i.e., cytotoxic drug moieties) are bound to a single B7-H4 antibody.

[0732] In some embodiments, D is a cytotoxic drug moiety, and the cytotoxic drug moiety is (a) an auristatin compound, (b) a calicheamycin compound, (c) a duocalmycin compound, (d) SN38, (e) a pyrrolobenzodiazepine, (f) a vinca compound, (g) a tubulisin compound, (h) a non-natural camptothecin compound, (i) a meitansinoid compound, (j) a DNA binding agent, (k) a kinase inhibitor, (l) a MEK inhibitor, (m) a KSP inhibitor, (n) a topoisomerase inhibitor, (o) a DNA alkylating agent, (p) an RNA polymerase, (q) a PARP inhibitor, (r) a NAMPT inhibitor, (s) a topoisomerase inhibitor, (t) a protein synthesis inhibitor, (u) a DNA binding agent, (v) a DNA intercalation agent, or (w) an immunomodulatory compound.

[0733] In some embodiments, D, the cytotoxic drug portion, is (a) an auristatin compound, (b) a calicheamycin compound, (c) a duocalmycin compound, (d) a camptothecin compound, (e) a pyrrolobenzodiazepine compound, (f) a vinca compound or an analogue thereof.

[0734] In some embodiments, the auristatin compound is auristatin, drastatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F, AF-HPA, MMAF-HPA, or phenylenediamine (AFP).

[0735] In some embodiments, duocalmycin or its analogues are duocalmycin A, duocalmycin B1, duocalmycin B2, duocalmycin C1, duocalmycin C2, duocalmycin D, duocalmycin SA, CC-1065, adzelesin, biceresin, or calzelsin.

[0736] In some embodiments, the camptothecin compound is camptothecin, CPT-11 (irinotecan), SN-38, or topotecan.

[0737] In some embodiments, the pyrrolobenzodiazepine compound is a pyrrolobenzodiazepine monomer, a symmetric pyrrolobenzodiazepine dimer, or an asymmetric pyrrolobenzodiazepine dimer.

[0738] In some embodiments, the modified B7-H4 antibody is modified with amino acid N297.

[0739] In some embodiments, the total number of specific bonds (or binding sites) formed between the linker-drug moiety and the modified B7-H4 antibody is 12 or less. In some embodiments, the total number of specific bonds (or binding sites) formed between the linker-drug moiety and the modified B7-H4 antibody is 10 or less. In some embodiments, the total number of specific bonds (or binding sites) formed between the linker-drug moiety and the modified B7-H4 antibody is 8 or less. In some embodiments, the total number of specific bonds (or binding sites) formed between the linker-drug moiety and the modified B7-H4 antibody is 6 or less. In some embodiments, the total number of specific bonds (or binding sites) formed between the linker-drug moiety and the modified B7-H4 antibody is 4 or less. In some embodiments, the total number of specific bonds (or binding sites) formed between the linker-drug moiety and the modified B7-H4 antibody is 2 or less.

[0740] In some embodiments, the total number of specific bindings (or binding sites) formed between the linker-drug moiety and the modified B7-H4 antibody is 2.

[0741] In some embodiments, the modified B7-H4 antibodies, linkers, or therapeutic agents described herein may be incorporated into the conjugates or scaffolds of this disclosure according to various techniques and methods known in the art. The conjugates of this disclosure and methods for producing them are described herein (e.g., by non-limiting embodiments and examples).

[0742] In some embodiments, the total number of bonds (or binding sites) formed between the linker-drug moiety and the modified B7-H4 antibody is 12 or less.

[0743] In some embodiments, the linker-drug moiety ratio to the modified B7-H4 antibody is greater than 1:1 and less than or equal to 12:1. In some embodiments, the linker-drug moiety ratio to the modified B7-H4 antibody is approximately 12:1, approximately 11:1, approximately 10:1, approximately 9:1, approximately 8:1, approximately 7:1, approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, approximately 2:1, or approximately 1:1. In some embodiments, the linker-drug moiety ratio to the modified B7-H4 antibody is between 2:1 and 10:1. In some embodiments, the linker-drug moiety ratio to the modified B7-H4 antibody is approximately 10:1, approximately 9:1, approximately 8:1, approximately 7:1, approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, or approximately 2:1. In some embodiments, the linker-drug moiety ratio to the modified B7-H4 antibody is approximately 2:1 to approximately 4:1. In some embodiments, the linker-drug moiety ratio to the modified B7-H4 antibody is approximately 4:1, approximately 3:1, or approximately 2:1. In some embodiments, the linker-drug moiety ratio to the modified B7-H4 antibody is approximately 2:1, or 1:1.

[0744] In some embodiments, a2 is 3, the linker-drug moiety to modified B7-H4 antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified B7-H4 antibody is approximately 8:1, approximately 7:1, approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, approximately 2:1, or approximately 1:1. In some embodiments, a2 is 3, the linker-drug moiety to modified B7-H4 antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified B7-H4 antibody is approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, approximately 2:1, or approximately 1:1. In some embodiments, a2 is 3, the linker-drug moiety to modified B7-H4 antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified B7-H4 antibody is approximately 6:1, approximately 5:1, approximately 4:1, or approximately 3:1. In some embodiments, a2 is 3, the linker-drug ratio to the modified B7-H4 antibody is 1:1, and the ratio of therapeutic agent (D) to the modified B7-H4 antibody is approximately 3:1, approximately 2:1, or approximately 1:1.

[0745] In some embodiments, a2 is 3, the linker-drug moiety to modified B7-H4 antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified B7-H4 antibody is approximately 8:1. In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified B7-H4 antibody is approximately 6:1. In some embodiments, a2 is 3, the linker-drug moiety to modified B7-H4 antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified B7-H4 antibody is approximately 5:1. In some embodiments, a2 is 3, the linker-drug moiety to modified B7-H4 antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified B7-H4 antibody is approximately 4:1. In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified B7-H4 antibody is approximately 3:1. In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified B7-H4 antibody is approximately 2:1. In some embodiments, a2 is 3, the linker-drug moiety to modified B7-H4 antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified B7-H4 antibody is approximately 1:1.

[0746] In some embodiments, the ratio of the linker-drug moiety to the modified B7-H4 antibody is approximately 2:1.

[0747] In some embodiments, the antibody contains an asparagine group at region 290-305 (e.g., at N297) attached to the sugar-derivative moiety containing functional group A'', and the modified B7-H4 antibody is conjugated to the linker-drug moiety by a covalent bond formed between A'' and the functional group of the linker-drug moiety.

[0748] In some embodiments, the linker-drug moiety comprises at least two functional groups, each of which can form a covalent bond with the functional group A'' of the sugar-derivative moiety of the modified B7-H4 antibody (for example, to amino acid N297 of the antibody) to form an antibody-drug conjugate.

[0749] In some embodiments, the ratio of the modified B7-H4 antibody to the linker-drug moiety is approximately 1:1 to approximately 1:2.

[0750] In some embodiments, the modified B7-H4 antibody-drug conjugates and scaffolds of the present disclosure may be purified by extensive diafiltration (e.g., to remove any starting material). If necessary, additional purification by size exclusion chromatography may be performed to remove aggregated conjugates. In some embodiments, the purified conjugate or scaffold may contain less than 5% w / w (e.g., less than 2% w / w) of aggregated conjugate as determined by SEC; less than 0.5% w / w (e.g., less than 0.1% w / w) of free (unconjugated) drug as determined by RP-HPLC; less than 1% w / w of drug-carrying peptide-containing scaffold as determined by SEC; and / or less than 2% w / w (e.g., less than 1% w / w) of unconjugated antibody as determined by HIC-HPLC.

[0751] In some embodiments, the modified B7-H4 antibody-drug conjugate for the cytotoxic drug moiety is selected from the conjugates listed in Table B2.

[0752] (Table B2) TIFF2026082984000299.tif206142TIFF2026082984000300.tif211142TIFF2026082984000301.tif190142 TIFF2026082984000302.tif202142TIFF2026082984000303.tif186142TIFF2026082984000304.tif208142T IFF2026082984000305.tif139142TIFF2026082984000306.tif139142TIFF2026082984000307.tif139142TI FF2026082984000308.tif196142TIFF2026082984000309.tif189142TIFF2026082984000310.tif216142In the formula, Antibody is a modified B7-H4 antibody, ■ is GlcNAc, △ is Fuc, □ is GalNAc, and d 13 This is as defined herein.

[0753] Unless otherwise specified, the symbol ■ is understood to refer to GlcNAc in this disclosure. Unless otherwise specified, the symbol △ is understood to refer to fucose in this disclosure. Unless otherwise specified, the symbol □ is understood to refer to GalNAc in this disclosure.

[0754] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXIV): TIFF2026082984000311.tif32161In formula, Each R A The following is: TIFF2026082984000312.tif99128d 13 The molecule is 2, and one or more linker-drug moieties are bound to the asparagine group at N297 of the antibody. In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXIV), where each R A The following is: TIFF2026082984000313.tif113128TIFF2026082984000314.tif210126TIFF2026082984000 315.tif215132TIFF2026082984000316.tif201137TIFF2026082984000317.tif171150, d 13 The modified B7-H4 antibody contains one or more asparagine groups at N297, which is connected to the remainder of the conjugate.

[0755] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXIV), where each R A The following applies: TIFF2026082984000318.tif106128

[0756] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXIV), where each R A The following applies: TIFF2026082984000319.tif85128

[0757] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXIV), where each R A The following applies: TIFF2026082984000320.tif97128

[0758] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXIV), where each R A The following applies: TIFF2026082984000321.tif90128

[0759] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXIV), where each R A The following applies: TIFF2026082984000322.tif90128

[0760] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXIV), where each R A The following applies: TIFF2026082984000323.tif88137

[0761] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXIV), where each R A The following applies: TIFF2026082984000324.tif87137

[0762] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXIV), where each R A The following is true: TIFF2026082984000325.tif98135

[0763] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXIV), where each R A The following applies: TIFF2026082984000326.tif98135

[0764] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXIV-1), (XXXIV-2), (XXXIV-3), or (XXXIV-4): TIFF2026082984000327.tif138161

[0765] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXV): TIFF2026082984000328.tif33148, each R B The following is true: TIFF2026082984000329.tif152154

[0766] In some embodiments, the modified B7-H4 antibody-drug conjugate is of the formula (XXXIV-1), (XXXIV-2), (XXXIV-3), (XXXIV-4), or (XXXV), where -L D -The D part is as follows: TIFF2026082984000330.tif141164

[0767] In some embodiments, the modified B7-H4 antibody-drug conjugate is of the formula (XXXIV-1), (XXXIV-2), (XXXIV-3), (XXXIV-4), or (XXXV), where -L D -The D part is as follows: TIFF2026082984000331.tif74128

[0768] In some embodiments, the modified B7-H4 antibody-drug conjugate is of formula (XXXVI): TIFF2026082984000332.tif23148, each R A The following applies: TIFF2026082984000333.tif90128TIFF2026082984000334.tif179128TIFF2026082984000335.tif167144

[0769] In some embodiments, the modified B7-H4 antibody-drug conjugate is a conjugate of formula (XXXVI), where each R A The following is: The TIFF2026082984000336.tif94128 modified B7-H4 antibody contains one or more asparagine groups at N297, which is attached to the remainder of the conjugate.

[0770] In some embodiments, the modified B7-H4 antibody-drug conjugate is a conjugate of formula (XXXVII): TIFF2026082984000337.tif110137In formula, d 13 It is 2, ANTIBODY contains variable heavy chain complementarity determination region 1 (CDRH1) containing the amino acid sequence GFIVSRNY (SEQ ID NO:2), variable heavy chain complementarity determination region 2 (CDRH2) containing the amino acid sequence IYGSGRT (SEQ ID NO:3), and amino acid sequence TIFF2026082984000338.tif4128 or amino acid sequence Variable heavy chain complementarity determination region 3 (CDRH3) containing TIFF2026082984000339.tif4128, variable light chain complementarity determination region 1 (CDRL1) containing amino acid sequence QSVSSSY (SEQ ID NO: 53), variable light chain complementarity determination region 2 (CDRL2) containing amino acid sequence GAS (SEQ ID NO: 54), amino acid sequence This is a B7-H4 antibody containing variable light chain complementarity determining region 3 (CDRL3), which includes TIFF2026082984000340.tif4128. The linker-drug portion is bound to the asparagine group at N297 of the B7-H4 antibody; ■ represents GlcNAc, △ represents Fuc, and □ represents GalNAc.

[0771] In some embodiments, the modified B7-H4 antibody-drug conjugate is a conjugate of formula (XXXVIII): TIFF2026082984000341.tif87160 formula, d 13 is an integer 2, ANTIBODY contains variable heavy chain complementarity determination region 1 (CDRH1) containing the amino acid sequence GFIVSRNY (SEQ ID NO:2), variable heavy chain complementarity determination region 2 (CDRH2) containing the amino acid sequence IYGSGRT (SEQ ID NO:3), and amino acid sequence TIFF2026082984000342.tif4128 or amino acid sequence Variable heavy chain complementarity determination region 3 (CDRH3) containing TIFF2026082984000343.tif4128, variable light chain complementarity determination region 1 (CDRL1) containing amino acid sequence QSVSSSY (SEQ ID NO: 53), variable light chain complementarity determination region 2 (CDRL2) containing amino acid sequence GAS (SEQ ID NO: 54), amino acid sequence This is a B7-H4 antibody containing variable light chain complementarity determining region 3 (CDRL3), which includes TIFF2026082984000344.tif4128. The linker-drug portion is bound to the asparagine group at N297 of the antibody; ■ represents GlcNAc, △ represents Fuc, and □ represents GalNAc.

[0772] How to use In some embodiments, the Disclosure provides a method for treating or preventing a disease or disorder in a subject, comprising the step of administering a therapeutically effective dose of the conjugate disclosed herein to a subject in need of such treatment or prevention.

[0773] In some embodiments, the Disclosure provides a method for treating a disease or disorder in a subject, comprising the step of administering a therapeutically effective dose of the conjugate disclosed herein to a subject in need.

[0774] In some embodiments, the Disclosure provides a method for treating or preventing a disease or disorder of a subject, comprising the step of administering a conjugate disclosed herein to a subject in need of such treatment or prevention.

[0775] In some embodiments, the Disclosure provides a method for treating a disease or disorder of a subject, comprising the step of administering a conjugate disclosed herein to a subject in need of treatment.

[0776] In some embodiments, the Disclosure relates to a method for treating a target cancer, comprising the step of administering an effective amount of the conjugate disclosed herein to a target in need. In some embodiments, the Disclosure relates to a method for treating a target B7-H4 positive cancer, comprising the step of administering an effective amount of the conjugate disclosed herein to a target in need.

[0777] In some aspects, the disclosure provides conjugates disclosed herein for use in treating or preventing a disease or disorder of which there is a need.

[0778] In some aspects, the disclosure provides conjugates disclosed herein for use in treating a disease or disorder of a subject where such treatment is necessary.

[0779] In some embodiments, the Disclosure provides the use of the conjugates disclosed herein for treating cancers of a subject that require such treatment. In some embodiments, the Disclosure provides the use of the conjugates disclosed herein for treating B7-H4 positive-expressing cancers of a subject that require such treatment.

[0780] In some aspects, the disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for treating a target disease or disorder for which such use is necessary.

[0781] In some aspects, the disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for treating or preventing a disease or disorder of which there is a need.

[0782] In some aspects, the disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for treating cancers of a subject where such treatment is necessary. In some aspects, the disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for treating B7-H4 positive-expressing cancers of a subject where such treatment is necessary.

[0783] In some aspects, the disclosure provides the use of a conjugate to treat or prevent a disease or disorder in which such treatment is necessary.

[0784] In some aspects, the disclosure provides the use of a conjugate to treat a disease or disorder in which such treatment is necessary.

[0785] In some embodiments, the Disclosure provides the use of a conjugate for treating a target cancer, comprising the step of administering an effective amount of the conjugate disclosed herein to a target in need. In some embodiments, the Disclosure provides the use of a conjugate for treating a target B7-H4 positive-expressing cancer, comprising the step of administering an effective amount of the conjugate disclosed herein to a target in need.

[0786] In some embodiments, the Disclosure provides a method for treating or preventing a disease or disorder in a subject, comprising the step of administering an efficient amount of at least one conjugate of the Disclosure to a subject in need, wherein the conjugate releases one or more therapeutic agents upon biodegradation.

[0787] In some embodiments, the Disclosure provides a method for treating a disease or disorder in a subject, comprising the step of administering an efficient amount of at least one conjugate of the Disclosure to the subject in need, wherein the conjugate releases one or more therapeutic agents upon biodegradation.

[0788] In some aspects, the disease is cancer.

[0789] In some embodiments, the present disclosure provides a method comprising the step of administering a therapeutically effective dose of the B7-H4 antibody-drug conjugate disclosed herein.

[0790] In some embodiments, the present disclosure provides a method comprising the step of administering a B7-H4 antibody-drug conjugate disclosed herein.

[0791] In some embodiments, the present disclosure provides a method for inhibiting the proliferation of B7-H4-positive cells, comprising the step of exposing cells to a B7-H4 antibody-drug conjugate under conditions that allow for the binding of the B7-H4 antibody-drug conjugate to B7-H4 on the surface of the cells, followed by internalization, thereby inhibiting cell proliferation. In certain embodiments, the method is in vitro or in vivo. In further embodiments, the cells are mammary cells, ovarian cells, or endometrial cells.

[0792] Inhibition of cell proliferation in vitro can be assayed using the CellTiter-Glo® Luminescent Cell Viability Assay, commercially available from Promega (Madison, Wis.). This assay determines the number of viable cells in culture based on the quantification of ATP present, an indicator of metabolically active cells. See Crouch et al. (1993) J.Immunol.Meth. 160:81-88, U.S. Patent No. 6,602,677. The assay can be performed in a 96 or 384-well format, making it suitable for automated high-t...

Claims

1. Variable heavy chain complementarity determination region 1 (CDRH1) containing amino acid sequence GFIVSRNY (SEQ ID NO:2), Variable heavy chain complementarity determination region 2 (CDRH2) containing amino acid sequence IYGSGRT (SEQ ID NO:3), amino acid sequence or amino acid sequence Variable heavy chain complementarity determination region 3 (CDRH3) containing the amino acid sequence QSVSSSY (SEQ ID NO: 53), variable light chain complementarity determination region 1 (CDRL1) containing the amino acid sequence GAS (SEQ ID NO: 54), variable light chain complementarity determination region 2 (CDRL2) containing the amino acid sequence Variable light chain complementarity determination region 3 (CDRL3) including An isolated antibody that specifically binds to B7-H4, containing [specific component].

2. The isolated antibody according to claim 1, comprising a heavy chain variable sequence containing the amino acid sequence of SEQ ID NO:44 and a light chain variable sequence containing the amino acid sequence of SEQ ID NO:

50.

3. The isolated antibody according to claim 1, comprising a heavy chain containing the amino acid sequence of SEQ ID NO:45 and a light chain containing the amino acid sequence of SEQ ID NO:

52.

4. An isolated antibody according to any one of the claims, comprising a heavy chain variable sequence containing the amino acid sequence of SEQ ID NO:22 and a light chain variable sequence containing the amino acid sequence of SEQ ID NO:

50.

5. An isolated antibody according to any one of the claims, comprising a heavy chain containing the amino acid sequence of SEQ ID NO:23 and a light chain containing the amino acid sequence of SEQ ID NO:

52.

6. An isolated antibody according to any one of the above claims, which is a monoclonal antibody.

7. An isolated antibody according to any one of the claims, which is a rabbit monoclonal antibody, a mouse monoclonal antibody, a chimeric monoclonal antibody, a humanized monoclonal antibody, or a fully human monoclonal antibody.

8. An isolated antibody according to any one of the claims, which is an IgG isotype.

9. An isolated antibody according to any one of the claims, wherein the IgG1 isotype.

10. Variable heavy chain complementarity determination region 1 (CDRH1) containing amino acid sequence GFIVSRNY (SEQ ID NO:2), Variable heavy chain complementarity determination region 2 (CDRH2) containing amino acid sequence IYGSGRT (SEQ ID NO:3), amino acid sequence Variable heavy chain complementarity determination region 3 (CDRH3) containing the amino acid sequence QSVSSSY (SEQ ID NO: 53), variable light chain complementarity determination region 1 (CDRL1) containing the amino acid sequence GAS (SEQ ID NO: 54), variable light chain complementarity determination region 2 (CDRL2) containing the amino acid sequence Variable light chain complementarity determination region 3 (CDRL3) including An isolated antibody according to any one of the claims, which competes with an isolated antibody containing for specific binding to human B7-H4.

11. An isolated antibody according to any one of the claims, which competes for specific binding to human B7-H4 with an isolated antibody comprising a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO:44 and a light chain variable sequence comprising the amino acid sequence of SEQ ID NO:50, or an isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:45 and a light chain comprising the amino acid sequence of SEQ ID NO:

52.

12. A B7-H4 antibody-drug conjugate comprising the isolated antibody according to any one of the above claims.

13. It comprises one or more linker-drug moieties covalently bonded to the targeting portion, Each linker-drug portion, A polyfunctional linker that connects a targeting portion to one or more drug units (e.g., one or more therapeutic agents (D)) via a releaseable assembly unit for each drug unit, and connects hydrophilic groups to drug units of each linker-drug portion. Includes, The releaseable assembly unit can release a free drug in close proximity to the target site targeted by the targeting portion, and The polyfunctional linker includes a peptide moiety between the targeting moiety and the hydrophilic group, and the peptide moiety contains at least two amino acids. The conjugate according to claim 12.

14. A conjugate selected from either the conjugates in Table A1 or Table A2.

15. A conjugate selected from either the conjugates in Table B1 or Table B2.

16. The conjugate according to claim 12, which is of formula (XXXVII): During the ceremony, d 13 It is 2, ANTIBODY consists of variable heavy chain complementarity determination region 1 (CDRH1) containing the amino acid sequence GFIVSRNY (SEQ ID NO:2), variable heavy chain complementarity determination region 2 (CDRH2) containing the amino acid sequence IYGSGRT (SEQ ID NO:3), and amino acid sequence or amino acid sequence Variable heavy chain complementarity determination region 3 (CDRH3) containing the amino acid sequence QSVSSSY (SEQ ID NO: 53), variable light chain complementarity determination region 1 (CDRL1) containing the amino acid sequence GAS (SEQ ID NO: 54), variable light chain complementarity determination region 2 (CDRL2) containing the amino acid sequence It is a B7-H4 modified antibody containing variable light chain complementarity determining region 3 (CDRL3), The linker-drug portion is bound to the asparagine group at N297 of the B7-H4 antibody, and ■ represents GlcNAc, △ represents Fuc, and □ represents GalNAc.

17. The conjugate according to claim 12, which is of formula (XXXVIII): During the ceremony, d 13 is an integer 2, ANTIBODY consists of variable heavy chain complementarity determination region 1 (CDRH1) containing the amino acid sequence GFIVSRNY (SEQ ID NO:2), variable heavy chain complementarity determination region 2 (CDRH2) containing the amino acid sequence IYGSGRT (SEQ ID NO:3), and amino acid sequence or amino acid sequence Variable heavy chain complementarity determination region 3 (CDRH3) containing the amino acid sequence QSVSSSY (SEQ ID NO: 53), variable light chain complementarity determination region 1 (CDRL1) containing the amino acid sequence GAS (SEQ ID NO: 54), variable light chain complementarity determination region 2 (CDRL2) containing the amino acid sequence This is a B7-H4 antibody containing variable light chain complementarity determining region 3 (CDRL3), The linker-drug portion is bound to the asparagine group at N297 of the antibody, ■ represents GlcNAc, △ represents Fuc, and □ represents GalNAc.

18. The conjugate is one of the following: In the formula, d 13 It is 8, The antibody is a B7-H4 antibody or a cysteine-modified B7-H4 antibody. The B7-H4 antibody contains variable heavy chain complementarity determination region 1 (CDRH1) with amino acid sequence GFIVSRNY (SEQ ID NO:2), variable heavy chain complementarity determination region 2 (CDRH2) with amino acid sequence IYGSGRT (SEQ ID NO:3), and amino acid sequence or amino acid sequence Variable heavy chain complementarity determination region 3 (CDRH3) containing the amino acid sequence QSVSSSY (SEQ ID NO: 53), variable light chain complementarity determination region 1 (CDRL1) containing the amino acid sequence GAS (SEQ ID NO: 54), variable light chain complementarity determination region 2 (CDRL2) containing the amino acid sequence It includes variable light chain complementarity determination region 3 (CDRL3).

19. A method for treating or preventing a disease or disorder of a subject, comprising the step of administering a conjugate according to any one of claims 12 to 18 to a subject in need of such treatment or prevention.

20. A conjugate according to any one of claims 12 to 18, for use in treating or preventing a disease or disorder in which such treatment is necessary.

21. Use of the conjugate according to any one of claims 12 to 18 in the manufacture of a pharmaceutical product for the treatment of a disease or disorder for which such treatment is necessary.

22. Use of the conjugate according to any one of claims 12 to 18 for the treatment or prevention of a disease or disorder in which such treatment is necessary.

23. The method, conjugate, or use according to any one of claims 19 to 22, wherein the conjugate releases one or more therapeutic agents upon biodegradation.

24. The method, conjugate, or use according to any one of claims 19 to 23, wherein the disease or disorder is cancer.

25. The method, conjugate, or use according to claim 24, wherein the cancer is a B7-H4 positive cancer.

26. The method, conjugate, or use according to claim 25, wherein the B7-H4 positive cancer is bile duct carcinoma, breast cancer, endometrial cancer, ovarian cancer, non-small cell lung cancer, small cell lung cancer, uterine cancer, thyroid cancer, kidney cancer, head and neck cancer, gastric cancer, melanoma, bile duct carcinoma, cholangial carcinoma, pancreatic cancer, colon cancer, or bladder cancer.

27. The method, conjugate, or use according to any one of claims 19 to 26, wherein the subject is a human.

28. The method according to any one of claims 19 to 27, further comprising administering a therapeutic agent to the subject.

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