Folate receptor targeting chimera and application thereof

Abstract

The invention relates to folate receptor targeting chimeras (FRTACs), further relates to folate-PEG conjugates, further relates to pharmaceutical compositions containing the folate receptor targeting chimeras, and further relates to application of the folate receptor targeting chimeras in preparation of drugs for treating and / or preventing diseases.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to folic acid receptor-targeting chimeras (FRTACs), folic acid-PEG conjugates, pharmaceutical compositions comprising folic acid receptor-targeting chimeras, and the use of these folic acid receptor-targeting chimeras in the preparation of medicaments for the treatment and / or prevention of diseases. Background Technology

[0002] Over the past two decades, targeted protein degradation (TPD) technology has emerged as a promising therapeutic strategy in chemical biology and drug discovery, offering advantages over traditional occupation-driven inhibitors. TPD technologies can not only specifically recognize and degrade proteins with drug-resistant mutations and those difficult to drug, but also catalyze and persistently knock down protein levels. Generally, TPD technologies include traditional protein hydrolysis-targeting chimeras (ROTACs), tag-based degradation systems (dTAGs), lysosomal-targeting chimeras (LYTACs), and related technologies. Due to their intracellular mechanism utilizing the ubiquitin-proteasome system (UPS), PROTACs are limited to degrading intracellular target proteins (POIs) and require cell-permeable degradation products. To overcome this challenge, LYTACs have been successfully developed to utilize the lysosomal pathway to degrade target proteins. By coupling target-binding fragments (antibodies or small molecules) to ligands binding to lysosomal transport receptors (LTRs) on the cell surface, LYTACs promote lysosomal degradation of related plasma membrane or secretory proteins.

[0003] While LYTACs offer novel strategies for disease treatment, the types of LYTACs currently being developed remain limited, with only a few LTRs investigated, such as the mannose-6-phosphate / IGF-II receptor (M6P / IGFIIR), the liver-specific desialyl glycoprotein receptor (ASGPR), and integrins. Other cell surface receptors, including transferrin receptors and folate receptors, also possess the ability to deliver drugs intracellularly. However, the potential of these receptors as suitable LTRs to mediate membrane protein degradation has not yet been explored. The folate receptor (FRs) family includes FRα, FRβ, FRγ, and FRδ, with FRα being a promising target for anticancer drug development because it is known to be overexpressed in many solid tumors, including ovarian cancer, triple-negative breast cancer (TNBC), endometrial cancer, and lung cancer, while its expression is limited in non-malignant tissues. Furthermore, FRα exhibits a high binding affinity for folate compared to other folate receptors, FRβ, FRγ, and FRδ, making it a dominant receptor for folate binding. Therefore, FRα-targeting strategies have been used for diagnostic and therapeutic purposes. The FRα-targeting antibody-drug conjugate (ADC) mirvetuximab soravtansine-gyynx (Elahere) has been approved for the treatment of FRα-expressing cancers, and several other FRα-targeting drugs are currently undergoing phase II / III clinical trials. As a clinically validated target, there is an urgent need to develop novel LYTACs that can bind to FRα to provide more options for treating cancer. Summary of the Invention

[0004] The inventors have developed a folic acid receptor-targeting chimeric conjugate (FRTACs) that links folic acid and an antibody via polyethylene glycol (PEG). These FRTACs can be selectively internalized by FRα-overexpressing cancer cells, subsequently transporting membrane point-of-injection (POI) to lysosomes for degradation. The FRTACs then release FRα and cycle back to the plasma membrane. These FRTACs can target tumor sites and utilize FRα as a lysosomal transport receptor (LTR) for lysosomal degradation, exhibiting unexpectedly high sub-nanomolar activity. Importantly, these FRTACs demonstrate significant target degradation efficacy in vivo, showing stronger antitumor activity than conventional antibody therapies.

[0005] Traditional folic acid conjugates [Nat Rev Clin Oncol. 2020; 17(6):349-359.] or folic acid antibody-drug conjugates [Drugs 83,265-273(2023)] are low conjugation ratio forms, with a folic acid antibody-drug conjugate ratio (FAR) of only 1-6 and an affinity for the folic acid receptor at only nM levels. For example Figure 15 , Figure 16 and Figure 17 As shown, FRTAC with a low FAR value exhibits degradation activity.

[0006] The inventors unexpectedly discovered that by using polyethylene glycol (PEG), for example PEG with an average molecular weight of 200-6000, preferably PEG with an average molecular weight of 800-1500, to conjugate folic acid and antibodies, folic acid receptor-targeting chimeras (FRTACs) with a high folic acid-antibody conjugation ratio can be obtained. The folic acid receptor-targeting chimeras (FRTACs) provided by this invention have a folic acid-antibody conjugation ratio greater than 8, preferably 10-40, more preferably 12-38, and even more preferably 15-35, and their affinity for folic acid can reach 10. -5 nM, thereby leveraging lysosomal targeted degradation of target proteins. The FRTACs provided by this invention can not only target tumor sites, but also utilize FRα as an LTR for lysosomal targeted degradation of membrane proteins, achieving unexpected sub-nanomolar degradation activity. They exhibit significant target degradation efficacy in vivo, demonstrating stronger anti-tumor activity than traditional antibody therapies.

[0007] On the one hand, the present invention relates to compounds of formula I or pharmaceutically acceptable salts, hydrates or solvates thereof.

[0008]

[0009] Among them, structural fragments That is, -(C2H4O) n - is PEG with an average molecular weight of 200 to 6000;

[0010] T represents the folic acid antibody conjugate ratio (FAR), which is a number between 8 and 40;

[0011] R represents a polypeptide, small molecule compound, or antibody targeting a target protein, or its antigen-binding fragment.

[0012] In some implementations, the structural fragment in Equation I It is PEG with an average molecular weight of 300-6000.

[0013] In some implementations, the structural fragment in Equation I PEG with average molecular weights of approximately 400, 600, 800, 1000, 1450, 1500, 3350, 4000, 5000, and 6000.

[0014] In some implementations, the structural fragment in Equation I It is PEG with an average molecular weight of 800-1500.

[0015] In some implementations, the structural fragment in Equation I It is PEG with an average molecular weight of approximately 1000.

[0016] In some implementations, T in Equation I is a number between 10 and 40.

[0017] In some implementations, T in Equation I is a number between 12 and 38.

[0018] In some implementations, T in Equation I is a number between 15 and 35.

[0019] In some implementations, T in Formula I is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, or about 35.

[0020] In some implementations, R in Formula I is an antibody targeting the target protein or its antigen-binding fragment.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof targeting the target protein described in Formula I is selected from monoclonal antibodies, mouse antibodies, rabbit antibodies, humanized antibodies, fully human antibodies, chimeric antibodies (e.g., human-mouse chimeric antibodies), bispecific antibodies, multispecific antibodies, single-chain antibodies, dAb, complementarity-determining region fragments, Fv, single-chain Fv (scFv), Fd, Fab, Fab', and F(ab')2.

[0022] In some embodiments, R in Formula I is an anti-EGFR antibody (e.g., cetuximab), an anti-PD-L1 antibody (e.g., Atezolizumab), an EGFR-targeting peptide (e.g., GE11), an anti-HER2 antibody (e.g., Herceptin), a CPCR-associated antibody, an anti-Trop2 antibody (e.g., Sacituzumab), an anti-FGFR antibody, an anti-VEGFR antibody, an anti-CTLA4 antibody, an anti-IL-5Rα antibody, or an antigen-binding fragment thereof (e.g., single-chain antibodies, dAbs, complementarity-determining region fragments, Fvs, single-chain Fv(scFv), Fd, Fab, Fab', and F(ab')2).

[0023] In some embodiments, R in Formula I is cetuximab, Atezolizumab, Herceptin, Sacituzumab or an antigen-binding fragment thereof (e.g., single-chain antibodies, dAbs, complementarity-determining region fragments, Fv, single-chain Fv (scFv), Fd, Fab, Fab' and F(ab')2).

[0024] In some implementations, R in Formula I is cetuximab, Atezolizumab, Herceptin, or Sacituzumab.

[0025] On the other hand, the present invention also relates to compounds represented by Formula II or pharmaceutically acceptable salts, hydrates or solvates thereof.

[0026]

[0027] Structural segments That is, -(C2H4O) n - represents PEG with an average molecular weight of 200 to 6000.

[0028] In some implementations, the structural fragment in Formula II It is PEG with an average molecular weight of 300-6000.

[0029] In some implementations, the structural fragment in Formula II PEG with average molecular weights of approximately 400, 600, 800, 1000, 1450, 1500, 3350, 4000, 5000, and 6000.

[0030] In some implementations, the structural fragment in Formula II It is PEG with an average molecular weight of 800-1500.

[0031] In some implementations, the structural fragment in Formula II It is PEG with an average molecular weight of approximately 1000.

[0032] On the other hand, the present invention also relates to the use of the compound of Formula II or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of antibody-drug conjugates, particularly in the preparation of the compound of Formula I or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0033] On the other hand, the present invention also relates to a method for preparing a compound of formula I or a pharmaceutically acceptable salt, hydrate or solvate thereof, comprising:

[0034] (1) Prepare the compound of formula II or its pharmaceutically acceptable salt, hydrate or solvate;

[0035] (2) React the compound of Formula II or its pharmaceutically acceptable salt, hydrate or solvate with a polypeptide, small molecule compound or antibody or its antigen-binding fragment containing a free amino group to target a protein.

[0036] In some implementations, step (1) includes:

[0037]

[0038] i) React tBuCOO-PEG-NH2 with folic acid (1.0 eq.) to obtain tBuCOO-PEG-FA;

[0039] ii) Remove the protecting group tert-butyl from tBuCOO-PEG-FA to obtain the compound shown in Formula III;

[0040] iii) The compound shown in Formula III reacts with NHS to obtain the compound shown in Formula II.

[0041] On the other hand, the present invention also relates to compositions comprising a compound of formula I or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a compound of formula II or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0042] On the other hand, the present invention also relates to pharmaceutical compositions comprising a compound of formula I or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0043] On the other hand, the present invention also relates to the use of the compound of Formula I or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a medicament for the targeted degradation of proteins. In some embodiments, the protein is EGFR, PD-L1, HER2, Trop2, CPCR, FGFR, VEGFR, CTLA4 or IL-5Rα protein.

[0044] On the other hand, the present invention also relates to the use of the compound of Formula I or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a medicament, wherein the medicament is used to inhibit tumor cell activity or to treat tumors.

[0045] On the other hand, the present invention also relates to compounds of Formula I or pharmaceutically acceptable salts, hydrates or solvates thereof for targeted degradation of proteins, inhibition of tumor cell activity or for the treatment of tumors.

[0046] On the other hand, the present invention also relates to methods for treating tumors or inhibiting tumor cell activity, including administering to a subject in need an effective amount of the compound of formula I or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0047] In some embodiments, the tumor cells are cancer cells expressing FRα. In some embodiments, the tumor is a tumor expressing FRα.

[0048] In some embodiments, the tumor cells are breast cancer cells, oral cancer cells, liver cancer cells, gastric cancer cells, pancreatic cancer cells, colorectal cancer cells, bladder cancer cells, or prostate cancer cells. In some embodiments, the tumor cells are ovarian cancer cells, triple-negative breast cancer (TNBC) cells, endometrial cancer cells, mesothelioma cells, or lung cancer cells.

[0049] In some embodiments, the tumor is ovarian cancer, triple-negative breast cancer (TNBC), endometrial cancer, mesothelioma, or lung cancer. In some embodiments, the tumor is breast cancer, oral cancer, liver cancer, stomach cancer, pancreatic cancer, colorectal cancer, bladder cancer, or prostate cancer.

[0050] As used herein, the term "polyethylene glycol (PEG)" has a meaning known in the art, with an average molecular weight generally between 200 and 20,000. PEG with different average molecular weights can be synthesized as needed. Those skilled in the art can modify PEG with different average molecular weights in various ways as needed. For example, after modification, the compound represented by Formula II of the present invention can be obtained. The compound represented by Formula II can be further conjugated with an antibody to obtain the compound represented by Formula I of the present invention.

[0051] In formula I or formula II of the present invention, structural fragments That is, -(C2H4O) n - represents polyethylene glycol (PEG) with an average molecular weight of 200 to 6000, where n is approximately 4 to 150.

[0052] As used herein, the term “about” is understood to mean within + / -10%, + / -9%, + / -8%, + / -7%, + / -6%, + / -5%, + / -4%, + / -3%, + / -2%, + / -1%, + / -0.5%, + / -0.4%, + / -0.3%, + / -0.2%, and + / -0.1% of the stated value. Unless otherwise apparent from the context, all numerical values ​​provided herein are modified by the term “about”.

[0053] For example, PEG with an average molecular weight of approximately 200 has a molecular weight between 190 and 210 and an n value between 3.5 and 4.5, which equals approximately 4; PEG with an average molecular weight of approximately 300 has a molecular weight between 285 and 315 and an n value between 5.5 and 6.5, which equals approximately 6; PEG with an average molecular weight of approximately 400 has a molecular weight between 380 and 420 and an n value between 8 and 9, which equals approximately 8.7; PEG with an average molecular weight of approximately 600 has a molecular weight between 570 and 630 and an n value between 12 and 14, which equals approximately 13.2; and PEG with an average molecular weight of approximately 800 has a molecular weight between 760 and 840 and an n value between 17 and 19, which equals approximately... Approximately 18; PEG with an average molecular weight of approximately 1000, wherein the molecular weight is between 900 and 1100 (preferably between 950 and 1050), and the n value is between 20 and 25, which equals approximately 22.3; PEG with an average molecular weight of approximately 1500, wherein the molecular weight is between 1350 and 1650 (preferably between 1425 and 1575), and the n value is between 30 and 37, which equals approximately 34; PEG with an average molecular weight of approximately 4000, wherein the molecular weight is between 3600 and 4400, and the n value is between 80 and 100, which equals approximately 90; PEG with an average molecular weight of approximately 6000, wherein the molecular weight is between 5400 and 6600, and the n value is between 122 and 150, which equals approximately 136.

[0054] As used herein, the term "peptide" refers to a chain containing at least two consecutively linked amino acid residues, with no upper limit on the chain length. One or more amino acid residues in a polypeptide may contain modifications, such as, but not limited to, optional substituted polysarcosine residue modifications, optional substituted glycosylated polyethylene glycol modifications, glycosylation modifications, phosphorylation modifications, or disulfide bond modifications.

[0055] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. The term "antibody" is not limited to any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0056] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefrance et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0057] The CDR contained in the antibody or antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment of the present invention is preferably determined by the Kabat, Chothia, or IMGT numbering system.

[0058] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide that contains a fragment of the full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0059] As used herein, the term “Fd fragment” refers to an antibody fragment consisting of the VH and CH1 domains; the term “Fv fragment” refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody; the term “dAb fragment” refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546 (1989)); the term “Fab fragment” refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; the term “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region; and the term “Fab' fragment” refers to one of two Fab' fragments formed by breaking the disulfide bond on the hinge region of the F(ab')2 fragment through reduction of the F(ab')2 fragment.

[0060] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), where the VL and VH domains pair to form a monovalent molecule by enabling them to generate linkers as single polypeptide chains (see, for example, Bird et al., Science 242:423 426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879 5883 (1988)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other connectors that may be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0061] In some cases, the antigen-binding fragment of an antibody is a biantibody, i.e., a bivalent antibody, which is a small antibody fragment with two antigen-binding sites. The fragment contains a heavy chain variable domain (VH) and a light chain variable domain (VL) linked thereto in the same polypeptide chain (VH-VL or VL-VH), wherein the VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with a complementary domain on another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993), and Poljak RJ et al., Structure 2:11211123 (1994)).

[0062] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be screened for specificity in the same manner as those used for intact antibodies.

[0063] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0064] As used herein, the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are usually obtained using hybridoma techniques first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see USP 4,816,567).

[0065] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain is derived from a portion of an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).

[0066] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, please refer to, for example, Jones et al., Nature, 321:522 525 (1986); Reichmann et al., Nature, 332:323 329 (1988); Presta, Curr. Op. Struct. Biol., 2:593 596 (1992); and Clark, Immunol. Today 21:397 402 (2000).

[0067] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity to at least two different epitopes on the same antigen or different antigens. A multispecific antibody can be a full-length antibody or a fragment of said antibody. The term "bispecific antibody" refers to an antibody that has binding specificity to two different antigens or to two different epitopes of a single antigen.

[0068] As used herein, the term "human antibody" or "fully human antibody" includes antibodies having variable and constant regions (if present) derived from human immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, the term "human antibody" does not include antibodies whose CDR sequences derived from another mammalian species lineage (e.g., mice) have been grafted onto human framework sequences (i.e., humanized antibodies). Fully human antibodies or human antibodies may be derived from transgenic mice carrying human antibody genes or from human cells.

[0069] As used herein, the terms "specific binding," "specificity," or "specific to..." refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10... -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (KD) binds to the antigen.

[0070] As used herein, the terms “monoclonal antibody” and “monoclonal antibody” have the same meaning and are used interchangeably; the terms “polyclonal antibody” and “polyclonal antibody” have the same meaning and are used interchangeably; and the terms “peptide” and “protein” have the same meaning and are used interchangeably.

[0071] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological efficacy and properties of a compound and is biologically or otherwise suitable for use in pharmaceuticals. In many cases, the compounds disclosed herein are capable of forming acid and / or base salts in the presence of amino and / or carboxyl or similar groups. Pharmaceutically acceptable acid addition salts can consist of inorganic and organic acids. Inorganic acids that can be derivatized to form salts include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids that can be derivatized to form salts include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Pharmaceutically acceptable base addition salts can consist of inorganic and organic bases. Inorganic bases that can be derived to form salts include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum; particularly preferred are ammonium, potassium, sodium, calcium, and magnesium salts. Organic bases that can be derived to form salts include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc., specifically, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Many such salts are known in the art, such as those described in WO87 / 05297 by Johnston et al., published on September 11, 1987 (which is incorporated herein by reference in its entirety).

[0072] In cases where the compound name used in this article differs from the chemical structural formula, the chemical structural formula shall prevail.

[0073] The pharmaceutical compositions described herein comprise a compound of Formula I or a pharmaceutically acceptable salt, hydrate, or solvate thereof, together with a conventionally pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions may be administered via, for example, oral or parenteral routes, such as intravenous, intraperitoneal, intramuscular, or subcutaneous injection.

[0074] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutically acceptable carriers and / or excipients include, but are not limited to: pH adjusters, surfactants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives, and stabilizers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and their analogues. Agents for delaying absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art for stabilizing the desired activity of the active ingredient in a pharmaceutical product, including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0075] As used in this article, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic effect, such as an amount that reduces symptoms associated with the disease to be treated.

[0076] It should also be noted that the dosage and method of administration of the compounds of this invention depend on many factors, including the patient's age, weight, sex, natural health condition, nutritional status, the activity intensity of the compound, the time of administration, the metabolic rate, the severity of the illness, and the subjective judgment of the treating physician. The preferred dosage is between 0.01 and 1000 mg / kg body weight / day.

[0077] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0078] The inventors unexpectedly discovered that by using polyethylene glycol (PEG), such as PEG with an average molecular weight of 200 to 6000, to conjugate folic acid and antibodies, folic acid receptor-targeting chimeras (FRTACs) with a high folic acid-antibody conjugation ratio can be obtained.

[0079] The folic acid receptor-targeting chimeric conjugates (FRTACs) provided by this invention have a folic acid antibody conjugation ratio greater than 8, preferably 10-40, more preferably 12-38, and even more preferably 15-35, and their affinity for the folic acid receptor (FRα) can reach 10. -5 nM.

[0080] The FRTACs provided by this invention have a simple structure and are easy to prepare, and can be used as targeted protein degraders. The FRTACs provided by this invention can not only target tumor sites, but also utilize FRα as an LTR for lysosomal targeted degradation of membrane proteins, achieving unexpected sub-nanomolar degradation activity. They exhibit significant target degradation efficacy in vivo, demonstrating stronger anti-tumor activity than traditional antibody therapies.

[0081] Abbreviations / Tags

[0082] TPD (targeted protein degradation): Targeted protein degradation

[0083] ROTACs (proteolysis targeting chimeras): proteolysis-targeting chimeras

[0084] LYTACs (lysosome-targeting chimeras): Lysosome-targeting chimeras

[0085] POI (proteins of interest): target protein

[0086] M6P / IGFIIR (mannose 6-phosphate / IGF-II receptor): mannose 6-phosphate / IGF-II receptor

[0087] LTR (lysosome-trafficking receptors): Lysosomal transport receptors

[0088] UPS (ubiquitin proteasome system): The ubiquitin-proteasome system

[0089] dTAG (tag-based degradation systems): Tag degradation systems

[0090] ASGPR (asialoglycoprotein receptor): Asialic acid glycoprotein receptor

[0091] FRα (folate receptor α): Folate α receptor

[0092] TNBC (triple-negative breast cancer):

[0093] ADC (antibody-drug conjugate): Targeted antibody-drug conjugate

[0094] FRTACs (folate receptor-targeting chimeras): Folate receptor-targeting chimeras

[0095] mAb (Monoclonal Antibody): Monoclonal antibody

[0096] Ctx (Cetuximab): Cetuximab

[0097] Atz (Atezolizumab): Atezolizumab

[0098] Ttz (Trastuzumab): Trastuzumab

[0099] Stz (Sacituzumab): Trop2 monoclonal antibody

[0100] Fr-Ctx (folate-Cetuximab): Folic acid-cetuximab conjugate

[0101] Fr-Atz (folate-Atezolizumab): Folic acid-atezolizumab conjugate

[0102] FAR (folate-antibody ratio): Folate-antibody conjugation ratio, the number of folate molecules conjugated to each antibody.

[0103] HIC (hydrophobic interaction chromatography): A chromatography technique involving hydrophobic interactions. Attached Figure Description

[0104] Figure 1 and Figure 2 The mass spectra and NMR spectra of FA-PEG1K-NHS are shown respectively;

[0105] Figure 3 and Figure 4The UV spectrum and HIC chromatogram of FR-Ctx (FAR 15) are shown respectively;

[0106] Figure 5 The ultraviolet spectrum of FR-Ctx(FAR 2) is shown;

[0107] Figure 6 The ultraviolet spectrum of FR-Ctx (FAR 30) is shown;

[0108] Figure 7 and Figure 8 The UV spectrum and HIC chromatogram of FR-Atz (FAR 15) are shown respectively;

[0109] Figure 9 The ultraviolet spectrum of FR-Atz (FAR 2) is shown;

[0110] Figure 10 and Figure 11 The UV spectrum and HIC chromatogram of FR-Ttz (FAR 15) are shown.

[0111] Figure 12 and Figure 13 The UV spectrum and HIC chromatogram of FR-Stz (FAR 20) are shown.

[0112] Figure 14 The ultraviolet spectrum of FR-Stz (FAR 2) is shown;

[0113] Figure 15 The affinity of FR-Ctx for FRα with different coupling ratios (FAR 2, FAR 15, FAR 30) was shown. The results showed that at high coupling ratios (FAR greater than 15), the affinity of FR-Ctx for FRα increased exponentially;

[0114] Figures 16 to 18 The degradation effects of FR-Ctx (FAR 2 and FAR 15), FR-Atz (FAR 2 and FAR 15), and FR-Stx (FAR 2 and FAR 20) with different coupling ratios on target proteins (EGFR, PD-L1, and Trop2) were compared. The results showed that FRTAC with a high coupling ratio had a good degradation effect on the target proteins.

[0115] Figure 19 The study demonstrated the degradation of EGFR on the surface of SKOV3 cells by different concentrations of FR-Ctx (FAR 15) using flow cytometry. The results showed that after administration of different concentrations of FR-Ctx (FAR 15) for 48 hours, the degradation of EGFR on the surface of SKOV3 cells was concentration-dependent.

[0116] Figure 20 The study demonstrated the degradation of PD-L1 on the surface of H292 cells by different concentrations of FR-Atz (FAR 15) using flow cytometry. The results showed that after administration of different concentrations of FR-Atz (FAR 15) for 48 hours, PD-L1 on the surface of H292 cells exhibited concentration-dependent degradation.

[0117] Figure 21 The study showed the degradation of EGFR on the surface of SKOV3 cells after treatment with FR-Ctx (FAR 15) for different time periods using flow cytometry. The results showed that EGFR on the cell surface of SKOV3 cells was degraded in a time-dependent manner after treatment with 10 nM FR-Ctx (FAR 15).

[0118] Figure 22 The study showed the degradation of PD-L1 on the surface of H292 cells after treatment with FR-Atz (FAR 15) for different time periods using flow cytometry. The results showed that PD-L1 on the surface of H292 cells was degraded in a time-dependent manner after treatment with 10 nM FR-Atz (FAR 15) for different time periods.

[0119] Figure 23 and 24 The study demonstrated the degradation of cell membrane proteins by different concentrations of FR-Ctx (FAR 15) using Western blotting. The results showed that the degradation of EGFR on the surface of SKOV3 and H292 cells by FR-Ctx (FAR 15) was concentration-dependent.

[0120] Figure 25 and 26 The study showed that Western blotting was used to analyze the degradation of cell membrane proteins by different concentrations of FR-Atz (FAR 15). The results showed that the degradation of PD-L1 on the surface of H292 cells and HCC827 cells by FR-Atz (FAR 15) was concentration-dependent.

[0121] Figure 27 The study showed that Western blotting was used to analyze the degradation of EGFR on the surface of H292 cells by FR-Ctx(FAR 15) at different treatment times. The results showed that the degradation of EGFR in H292 cells mediated by FR-Ctx(FAR 15) was time-dependent.

[0122] Figure 28The study showed that Western blotting was used to analyze the degradation of PD-L1 on the surface of HCC827 cells by FR-Atz (FAR 15) at different treatment times. The results showed that the degradation of PD-L1 in HCC827 cells mediated by FR-Atz (FAR 15) was time-dependent.

[0123] Figure 29 and 30 The results of proteomic analysis of FR-Ctx(FAR 15) degradation of EGFR on the surface of H292 cells are presented. The results show that FR-Ctx(FAR 15) can significantly reduce EGFR levels, while Ctx does not affect EGFR levels.

[0124] Figure 31 and 32 The results of proteomic analysis of FR-Atz (FAR 15) degradation of PD-L1 on the surface of H292 cells were presented. The results showed that 10 nM FR-Atz (FAR 15) reduced PD-L1 levels in H292 cells, but Atezolizumab did not change PD-L1 levels.

[0125] Figure 33 and 34 Proteomic analysis of the effects of FR-Atz (FAR 15) on FRα levels in H292 cells showed that neither FR-Atz (FAR 15) nor Atezolizumab treatment altered FRα levels.

[0126] Figure 35 The distribution and accumulation time of Dylight 680-FR-Atz in mice were shown;

[0127] Figure 36 The distribution of Dylight 680-FR-Atz in various tissues of mice was shown, and the results showed that the fluorescence intensity in tumor tissues was significantly higher than that in normal mouse tissues.

[0128] Figure 37 The tumor growth curves in mice are shown, demonstrating that FR-Atz (FAR 15) inhibits the growth of RM-1 tumors;

[0129] Figure 38 The changes in tumor weight in mice after administration were shown in different drug groups;

[0130] Figure 39 The changes in mouse body weight after administration were shown in different drug groups;

[0131] Figure 40The results show the changes in PD-L1 levels in tumor tissues after 48 hours of different drug administrations, analyzed using Western blotting.

[0132] Figure 41 The results show the expression of PD-L1 in tumor tissues after immunohistochemical staining analysis 48 hours after different drug administrations;

[0133] Figure 42 The effects of different drug administrations on major normal organs (heart, liver, kidney, and spleen) in mice were shown 48 hours after treatment.

[0134] Figure 43 The degradation of PD-L1 by FR-Atz (FAR 15) after HCC827 cells were pretreated with the lysosomal inhibitor bafimycin A1 and the proteasome inhibitor MG132 was shown.

[0135] Figure 44 The degradation of EGFR by FR-Ctx(FAR 15) after SKOV3 cells were pretreated with the lysosomal inhibitor bafimycin A1 and the proteasome inhibitor MG132 was shown.

[0136] Figure 45 The results showed that FR-Atz (FAR 15) effectively degraded PD-L1 in FRα-positive H292 cells, but had no effect on PD-L1 degradation in FRα-negative H460 cells, proving that the degradation of PD-L1 by FR-Atz (FAR 15) is FRα-dependent.

[0137] Figure 46 The results showed that FR-Ctx (FAR 15) effectively degraded EGFR in FRα-positive SKOV3 cells, but had no effect on EGFR degradation in FRα-negative Hep3B cells, demonstrating that the degradation of EGFR by FR-Ctx (FAR 15) is FRα-dependent.

[0138] Figure 47 The results showed that after FRα knockout, the degradation of EGFR by FR-Ctx(FRA15) was significantly eliminated, while EGFR could still be degraded in control cells, proving that the degradation of EGFR by FR-Ctx(FRA15) is FRα-dependent.

[0139] Figure 48 The results showed that FR-Ctx(FRA15) could effectively degrade EGFR after upregulation of FRα, proving that the degradation of EGFR by FR-Ctx(FRA15) is FRα-dependent;

[0140] Figure 49The results showed that FR-Atz (FRA15) could effectively degrade PD-L1 after upregulation of FRα, proving that the degradation of PD-L1 by FR-Ctx (FRA15) is FRα-dependent. Detailed Implementation

[0141] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments.

[0142] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in the art. Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available, and the room temperature described in the embodiments of this invention is 25-30°C.

[0143] Example 1: Synthesis of intermediate FA-PEG1K-NHS

[0144]

[0145] Weigh 500 mg of tBuCOO-PEG1K-NH2 (purchased from Xi'an Ruixi Biotechnology Co., Ltd., where PEG1K represents polyethylene glycol with an average molecular weight of 1000 (g / mol), and in the structural formula, PEG1K is represented as -(CH2CH2O). n - (n = 21-24) was dissolved in 3 mL of DMSO, and folic acid (1.0 eq.), EDC (2.0 eq.) and DMAP (0.1 eq.) were added and dissolved completely. The mixture was reacted at 40 °C for 6 h, and then poured into a large amount of ice-cold ether to precipitate. The product was collected by filtration and vacuum drying to obtain tBuCOO-PEG1K-FA.

[0146]

[0147] Weigh 500 mg of tBuCOO-PEG1K-FA and dissolve it in 6 mL of dichloromethane. Add 2 mL of trifluoroacetic acid under ice bath and stir for 1 h. Wash the reaction solution with water 3 times, dry with anhydrous sodium sulfate, concentrate the solution by rotary evaporation under reduced pressure, pour it into a large amount of ice-cold ether to precipitate, filter to collect the product, and dry under vacuum to obtain FA-PEG1K-COOH.

[0148]

[0149] Weigh 500 mg of COOH-PEG1K-folic acid and dissolve it in 6 mL of dichloromethane. Add EDC (2.0 eq.) and NHS (N-hydroxysuccinimide, 2.0 eq.) until completely dissolved. React at 40 °C for 3 h. Concentrate the reaction solution by rotary evaporation under reduced pressure, precipitate with a large amount of ice-cold diethyl ether, filter and collect the product, and dry under vacuum to obtain FA-PEG1K-NHS. Mass spectra are shown below. Figure 1 See MRI Figure 2 .

[0150] Example 2: Synthesis of FR-Ctx (FAR 15), a product of FA-PEG1K-NHS conjugated with EGFR antibody cetuximab.

[0151]

[0152] 5.0 mg / mL of cetuximab (Ctx) (Erbitux, Merck, Germany) dissolved in PBS was reacted with 50 eq of FA-PEG1K-NHS (4.5 mg / mL PBS, pH 7.4) and incubated overnight at room temperature. FA-PEG1K-NHS formed an amide bond with the free amino group in the cetuximab antibody, releasing N-hydroxysuccinimide (NHS). The solution was then purified by ultrafiltration (5500 rpm, 15 min × 4) in an Amicon centrifuge filter (30 kDa) to remove unlinked FA-PEG1K-NHS, yielding FRTAC represented by the formula FA-PEG1K-MAB, where MAB represents the cetuximab antibody.

[0153] The FAR value is calculated after ultraviolet spectroscopy testing. The calculation formula is as follows:

[0154] A 370nm =ε (FA-PEG1K-NHS,370nm) ×C (FA-PEG1K-NHS) (1)

[0155] A 280nm =ε (FA-PEG1K-NHS , 280nm) ×C (FA-PEG1K-NHS) +ε (mAb,280nm) ×C (mAb) (2)

[0156] FAR = C (FA-PEG1K-NHS) / C(mAb) (3)

[0157] in:

[0158] ε (FA-PEG1K-NHS,370nm) The molar extinction coefficient of FA-PEG1K-NHS at a wavelength of 370 nm is represented.

[0159] ε (FA-PEG1K-NHS,280nm)The molar extinction coefficient of FA-PEG1K-NHS at a wavelength of 280 nm is represented.

[0160] ε (mAb,280nm) The molar extinction coefficient of the monoclonal antibody at a wavelength of 280 nm;

[0161] A 370nm This represents the absorption intensity of FRTAC at a wavelength of 370 nm;

[0162] A 280nm This represents the absorption intensity of FRTAC at a wavelength of 280 nm;

[0163] C (FA-PEG1K-NHS) This represents the concentration of FA-PEG1K-NHS contained in FRTAC;

[0164] C (mAb) This represents the concentration of monoclonal antibodies contained in FRTAC.

[0165] Calculations showed that each antibody was conjugated to approximately 15 folic acid molecules, and the final product was designated FR-Ctx (FAR 15). Its UV absorption spectrum is shown below. Figure 3 As shown, its hydrophobic interaction chromatography (HIC) is as follows: Figure 4 As shown.

[0166] Example 3: Synthesis of FR-Ctx (FAR 2), a product of FA-PEG1K-NHS conjugated with EGFR antibody cetuximab.

[0167] 5.0 mg / mL of Ctx dissolved in PBS was reacted with 5 eq of FA-PEG1K-NHS (4.5 mg / mL PBS, pH 7.4) and incubated overnight at room temperature. The solution was then purified by ultrafiltration (5500 rpm, 15 min × 4) in an Amicon centrifuge filter (30 kDa) to remove unconjugated FA-PEG1K-NHS, yielding FRTAC (FA-PEG1K-MAB), where MAB represents the antibody cetuximab. UV spectroscopy analysis showed that each antibody conjugated approximately two folic acid molecules. The final product was designated FR-Ctx (FAR 2), and its UV absorption spectrum is shown below. Figure 5 As shown.

[0168] Example 4: Synthesis of FR-Ctx (FAR 30), a product of FA-PEG1K-NHS conjugated with EGFR antibody cetuximab.

[0169] 5.0 mg / mL of Ctx dissolved in PBS was reacted with 100 eq of FA-PEG1K-NHS (4.5 mg / mL PBS, pH 7.4) and incubated overnight at room temperature. The solution was then purified by ultrafiltration (5500 rpm, 15 min × 4) in an Amicon centrifuge filter (30 kDa) to remove unlinked FA-PEG1K-NHS, yielding FRTAC (FA-PEG1K-MAB), where MAB represents the antibody cetuximab. UV spectroscopy analysis showed that each antibody contained approximately 30 folic acid molecules. The final product was designated FR-Ctx (FAR 30), and its UV absorption spectrum is shown below. Figure 6 As shown.

[0170] Example 5: Synthesis of FR-Atz (FAR 15), a product of FA-PEG1K-NHS conjugated with the PD-L1 antibody Atezolizumab.

[0171] Atezolizumab (Atz) (Tecentriq, Roche Diagnostics, Germany) dissolved in 5.0 mg / mL PBS was reacted with 50 eq of folic acid-PEG1K-NHS (4.5 mg / mL PBS, pH 7.4) and incubated overnight at room temperature. The solution was then purified by ultrafiltration (5500 rpm, 15 min × 4) in an Amicon centrifuge filter (30 kDa) to remove unconjugated FA-PEG1K-NHS, yielding FRTAC (FA-PEG1K-MAB), where MAB represents the antibody Atezolizumab. UV spectroscopy analysis showed that each antibody conjugated approximately 15 folic acid molecules, and the final product was designated FR-Atz (FAR 15). Its UV absorption spectrum is shown below. Figure 7 As shown, its hydrophobic interaction chromatography (HIC) is as follows: Figure 8 As shown.

[0172] Example 6: Synthesis of FR-Atz (FAR 2), a product of FA-PEG1K-NHS conjugated with the PD-L1 antibody Atezolizumab

[0173] 5.0 mg / mL of Atz dissolved in PBS was reacted with 5 eq of folic acid-PEG1K-NHS (4.5 mg / mL PBS, pH 7.4) and incubated overnight at room temperature. The solution was then purified by ultrafiltration (5500 rpm, 15 min × 4) in an Amicon centrifuge filter (30 kDa) to remove unconjugated FA-PEG1K-NHS, yielding FRTAC (FA-PEG1K-MAB), where MAB represents the antibody Atezolizumab. UV spectroscopy analysis showed that each antibody conjugated approximately two folic acid molecules, and the final product was designated FR-Atz (FAR 2). Its UV absorption spectrum is shown below. Figure 9 As shown.

[0174] Example 7: Synthesis of FR-Ttz (FAR 15), a product of FA-PEG1K-NHS conjugated with the HER2 antibody Trastuzumab.

[0175] 5.0 mg / mL Trastuzumab (Ttz) (HY-P9907, MedChemExpressFunctional) dissolved in PBS was reacted with 50 eq of FA-PEG1K-NHS (4.5 mg / mL PBS, pH 7.4) and incubated overnight at room temperature. The solution was then purified by ultrafiltration (5500 rpm, 15 min × 4) in an Amicon centrifuge filter (30 kDa) to remove unconjugated FA-PEG1K-NHS, yielding FRTAC (FA-PEG1K-MAB), where MAB represents the antibody Trastuzumab. UV spectroscopy analysis showed that each antibody conjugated approximately 15 folic acid molecules, and the final product was designated FR-Ttz (FAR 15). Its UV absorption spectrum is shown below. Figure 10 As shown, its hydrophobic interaction chromatography (HIC) is as follows: Figure 11 As shown.

[0176] Example 8: Synthesis of FR-Stz (FAR 20), a product of FA-PEG1K-NHS conjugated with Trop2 antibody Sacituzumab

[0177] 5.0 mg / mL of Sacituzumab (Stz) (HY-P99045 MedChemExpressFunctional) dissolved in PBS was reacted with 50 eq of FA-PEG1K-NHS (4.5 mg / mL PBS, pH 7.4) and incubated overnight at room temperature. The solution was then purified by ultrafiltration (5500 rpm, 15 min × 4) in an Amicon centrifuge filter (30 kDa) to remove unconjugated FA-PEG1K-NHS, yielding FRTAC (FA-PEG1K-MAB), where MAB represents the antibody Sacituzumab. UV spectroscopy analysis showed that each antibody conjugated approximately 20 folic acid molecules, and the final product was designated FR-Stz (FAR 20). Its UV absorption spectrum is shown below. Figure 12 As shown, its hydrophobic interaction chromatography (HIC) is as follows: Figure 13 As shown.

[0178] Example 9: Synthesis of FR-Stz (FAR 2), a product of FA-PEG1K-NHS conjugated with Trop2 antibody Sacituzumab

[0179] 5.0 mg / mL of Stz dissolved in PBS was reacted with 5 eq of FA-PEG1K-NHS (4.5 mg / mL PBS, pH 7.4) and incubated overnight at room temperature. The solution was then purified by ultrafiltration (5500 rpm, 15 min × 4) in an Amicon centrifuge filter (30 kDa) to remove unconjugated FA-PEG1K-NHS, yielding FRTAC (FA-PEG1K-MAB), where MAB represents the antibody Sacituzumab. UV spectroscopy analysis showed that each antibody conjugated approximately two folic acid molecules. The final product was denoted as FR-Stz (FAR 2), and its UV absorption spectrum is shown below. Figure 14 As shown.

[0180] Example 10: Synthesis of Dylight 680-FR-Atz

[0181] 5.0 mg / mL Atz dissolved in PBS was reacted with 7 eq of Dylight 680-NHS (Thermo, #46418) and incubated overnight at room temperature. The solution was then purified by ultrafiltration (5500 rpm, 15 min × 4) in an Amicon centrifuge filter (30 kDa) to remove unlinked Dylight 680-NHS, yielding Dylight 680-Atz. Dylight 680-Atz (4.2 mg / mL PBS, pH 7.4) was reacted with 50 eq of FA-PEG1K-NHS (4.5 mg / mL PBS, pH 7.4) and incubated overnight at room temperature. The solution was then purified by ultrafiltration (5500 rpm, 15 min × 4) in an Amicon centrifuge filter (30 kDa) to remove unlinked FA-PEG1K-NHS, yielding Dylight 680-FR-Atz. Calculations based on ultraviolet spectroscopy tests show that each antibody is conjugated to approximately 2 fluorescent molecules and approximately 15 folic acid molecules.

[0182] Example 11: Determination of the affinity of FR-Ctx (FAR 2, FAR 15 and FRA30) for folic acid receptors

[0183] The binding affinity of FR-Ctx to FRα was determined using a SPR-based Biacore 8K instrument (Cytiva, Washington, MA, USA). FRα protein was immobilized on a CM5 sensor chip at 25°C using the standard amine coupling method with flow buffer (PBS-P), achieving an immobilization level of approximately 9000 RU. Different concentrations of FR-Ctx in PBS-P solutions (FAR 2, FAR 15, and FRA 30) were sequentially injected into the channels to assess binding affinity. Additional washing with 5 mM NaOH was performed to remove any remaining sample from the channels. The dataset was fitted to a 1:1 Langmuir binding model using Biacore 8K evaluation software to obtain the equilibrium dissociation constant KD of the compounds. SPR results are shown below. Figure 15 As shown, the affinities of FR-Ctx (FAR 2), FR-Ctx (FAR15), and FR-Ctx (FAR 30) for FRα are 5.75 × 10⁻⁶, respectively. -1 nM, 1.79×10 -5 nM, 6.80×10 -5 The results showed that when the antibody was conjugated with 15 to 30 folic acid molecules, the affinity for FRα was exponentially increased compared with FRTAC and small folic acid molecules with low conjugation ratios. This indicates that FRTAC conjugated with 15 to 30 folic acid molecules has significantly higher activity than FRTAC with low conjugation ratios.

[0184] Example 12: Evaluation of the degradation effect of FR-Ctx (FAR 2 and FAR 15) on EGFR protein in H292 tumor cells (after 24 hours of incubation)

[0185] EGFR antibodies Cetuximab (50 nM), FR-Ctx(FAR2) (10 nM), FR-Ctx(FAR 2) (50 nM), FR-Ctx(FAR 15) (10 nM), and FR-Ctx(FAR 15) (50 nM) prepared in cell culture medium were added to H292 cells (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) cultured in RPMI 1640 medium (Gibco, USA). After incubation for 24 hours, the cells were washed three times with PBS, and cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 10 mM β-glycerophosphate, 5 mM EGTA, 1 mM sodium acid pyrophosphate, 5 mM sodium fluoride, 1 mM Na3VO4, 0.5% Triton X-100, and 1 mM MTT) was added. The cells were then incubated on ice for 30 minutes with a protease inhibitor (Sigma-Aldrich). Centrifuge at 21000g for 15 minutes at 4℃, collect the supernatant, and determine the protein concentration using the BCA method. Separate the protein by SDS-PAGE and electrotransfer to a PVDF membrane. Add primary antibodies EGF Receptor Rabbit mAb (Cell Signaling Technology, Cat#4267) and GAPDHabbit mAb (Cell Signaling Technology, Cat#5174), incubate overnight at 4℃, wash three times with PBS, then add secondary antibody HRP-conjugated and incubate at room temperature for 1 hour, wash three times with PBS. Image using a Tanon 5200 system (Tanon, Shanghai, China). Quantify the intensity using ImageJ software (version v.1.53e). Results are as follows. Figure 16 As shown, EGFR protein was expressed in both the control group and the Ctx-treated group. In the FR-Ctx (FAR 15) treatment group, EGFR protein was degraded by more than 89% compared to the control group. In the FR-Ctx (FAR 2) treatment group, EGFR protein was degraded by only 25-49% compared to the control group. These results indicate that FR-Ctx at FAR 15 has a significant degradative effect on EGFR protein in H292 cells.

[0186] Example 13: Evaluation of the degradation effect of FR-Atz (FAR 2 and FAR 15) on PD-L1 protein in HCC827 tumor cells (after 24 hours of incubation)

[0187] PD-L1 antibody Atezolizumab (50 nM), FR-Atz (FAR 2) 10 nM, FR-Atz (FAR 2) 50 nM, FR-Atz (FAR 15) 10 nM, and FR-Atz (FAR 15) 50 nM, prepared in cell culture medium, were added to HCC827 cells (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) cultured in RPMI 1640 medium (Gibco, USA). After incubation for 24 hours, protein was collected according to the experimental procedure in Example 12, and the expression of PD-L1 (PD-L1 Rabbit mAb, Cell Signaling Technology, Cat#13684) was detected by Western blot. The results are as follows. Figure 17 As shown. The results showed that the Atz treatment group could degrade 32% of PD-L1 protein; the FR-Atz (FAR 15) treatment group,

[0188] Compared to the control group, PD-L1 protein was degraded by 72-84%; in the FR-Atz (FAR 2) treatment group, PD-L1 protein was degraded by 44-61% compared to the control group. The results indicate that FR-Atz at FAR 15 has a significant degradation effect on PD-L1 protein in HCC827 cells in a dose-dependent manner.

[0189] Example 14: Evaluation of the degradation effect of FR-Stz (FAR 2 and FAR 20) on Trop2 protein in SKOV3 tumor cells (after 24 hours of incubation)

[0190] Trop2 antibody Sacituzumab (50 nM), FR-Stz (FAR2) 10 nM, FR-Stz (FAR 2) 50 nM, FR-Stz (FAR 20) 10 nM, and FR-Stz (FAR 20) 50 nM, prepared in cell culture medium, were added to SKOV3 cells (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) cultured in DMEM medium (Gibco, USA). After incubation for 24 hours, protein was collected according to the experimental procedure in Example 12. The expression of Trop2 (Trop2 Rabbit mAb, Cell Signaling Technology, Cat#47866) was detected by Western blot (the internal control Tubulin used was β-Tubulin Rabbit mAb, Cell Signaling Technology, Cat#2128). The results are as follows. Figure 18As shown in the figure. The results showed that the Stz treatment group could degrade 28% of Trop2 protein; the FR-Stz (FAR 20) treatment group, compared with the control group, degraded Trop2 protein by 67-76%; and the FR-Stz (FAR 2) treatment group, compared with the control group, degraded Trop2 protein by 37-59%. The results indicate that FR-Stz at FAR 20 has a significant degradation effect on Trop2 protein in SKOV3 cells.

[0191] Example 15: Flow cytometry analysis of the effects of different concentrations of FRTAC on cell membrane protein degradation

[0192] One day prior to the experiment, SKOV3 cells were seeded in 24-well plates (approximately 100,000 cells per well). Cells were incubated for 48 h in 250 μL of complete growth medium containing different concentrations of FR-Ctx (FAR 15) or 50 nM cetuximab. Cells were washed three times with FACS buffer (PBS + 0.5% BSA + 5 mM EDTA), and incubated on ice for 30 min after adding the primary antibody EGFR Monoclonal Antibody (ThermoFisher, Cat#MA5-13319). Cells were then washed three times with FACS buffer and incubated with the fluorescently labeled secondary antibody Goat anti-Mouse IgG, IgM (H+L), and Alexa Fluor. TM After incubation at 488 (Thermo Fisher, Cat#A10680) on ice for 30 min, data were acquired by flow cytometry using guavaSoft 3.1 on a Guava easyCyte (Merck Luminex, Austin, Texas, USA) instrument. Analysis was performed using FlowJo software (v10.8.0). Figure 19 As shown, the results indicate that EGFR on the surface of SKOV3 cells in the FR-Ctx treatment group exhibits concentration-dependent degradation. FR-Ctx (FDR15) can still effectively degrade more than 50% of EGFR even at concentrations as low as 1 nM.

[0193] Using the same experimental method, flow cytometry was used to analyze the degradation of PD-L1 (Extracellular Domain Specific) Rabbit mAb (Alexa) on the cell surface of H292 cells after treatment with different concentrations of Atz or FR-Atz (FAR 15) for 48 hours. 647 Conjugate, Cell Signaling Technology, Cat#41726), results as follows Figure 20As shown, at a FR-Atz concentration of 0.1 nM, approximately 50% of cell surface PD-L1 can be degraded.

[0194] Example 16: Flow cytometry analysis of the effect of FRTAC treatment at different times on cell membrane protein degradation

[0195] One day prior to the experiment, SKOV3 cells were seeded in 24-well plates (approximately 100,000 cells per well). Cells were incubated for varying durations in 250 μL of complete growth medium containing 10 nM FR-Ctx (FAR 15) or 50 nM cetuximab. Following the experimental procedures in Example 15, flow cytometry was performed using guavaSoft 3.1 on a Guava easyCyte (Merck Luminex, Austin, Texas, USA) cell analyzer for data acquisition. Analysis was performed using FlowJo software (v10.8.0). Figure 21 As shown, the results indicate that treatment of SKOV3 cells with 10 nM FR-Ctx resulted in time-dependent degradation of surface EGFR, with degradation exceeding 50% at 6 h and the degradation effect lasting up to 72 h.

[0196] Using the same method, flow cytometry was used to analyze the time-dependent degradation of PD-L1 (Extracellular Domain-Specific) Rabbit mAb (Alexa) on the cell surface of H292 cells after treatment with 50 nM Atz or 10 nM FR-Atz (FAR 15) for different time periods. 647 Conjugate, Cell Signaling Technology, Cat#41726), results as follows Figure 22 As shown, degradation exceeds 50% within 6 hours, and its degradation effect can last up to 72 hours.

[0197] Example 17: Western blotting analysis of the degradation of cell membrane proteins by FRTAC at different concentrations

[0198] SKOV3 or H292 cells were incubated with complete growth medium containing different concentrations of FR-Ctx (FAR 15) or 50 nM cetuximab for a certain period of time. Proteins were collected according to the experimental procedures in Example 12, and EGFR expression was detected by Western blot. The results are as follows... Figure 23 As shown, FR-Ctx(FAR 15) treatment significantly reduced the expression level of EGFR in the SKOV3 cell membrane in a concentration-dependent manner. Figure 24As shown, FR-Ctx(FAR 15) treatment significantly reduced the expression level of EGFR on the surface of H292 cells in a concentration-dependent manner.

[0199] Using the same method, H292 and HCC827 cells (PD-L1 Rabbit mAb, Cell Signaling Technology, Cat#13684) were treated with different concentrations of FR-Atz (FAR 15). Proteins were collected according to the experimental procedures in Example 12, and PD-L1 expression was detected by Western blot. The results showed that even at concentrations as low as 0.1 nM, FR-Atz (FAR 15) could significantly reduce PD-L1 expression in H292 cells (PD-L1 Rabbit mAb, Cell Signaling Technology, Cat#13684). Figure 25 ) and HCC827 cells ( Figure 26 PD-L1 expression on the surface.

[0200] Example 18: Western blotting analysis of the effect of FRTAC treatment at different times on cell membrane protein degradation

[0201] H292 cells were incubated for different durations with complete growth medium containing 10 nM FR-Ctx (FAR 15) or 50 nM cetuximab. Proteins were collected following the experimental procedures in Example 12, and EGFR expression was detected by Western blot. Results are as follows... Figure 27 As shown, the degradation of EGFR on the surface of H292 cells mediated by FR-Ctx(FAR 15) is time-dependent, with the maximum degradation rate (Dmax) of 10 nM FR-Ctx(FAR 15) being 80% after 48 hours of incubation with the cells.

[0202] Using the same method, HCC827 cells (PD-L1Rabbit mAb, CellSignaling Technology, Cat#13684) were treated with 10 nM FR-Atz (FAR 15), and proteins were collected according to the experimental procedures in Example 12. EGFR expression was detected by Western blot. The results are as follows: Figure 28 As shown, FR-Atz (FAR 15) mediates the time-dependent degradation of PD-L1 on the surface of HCC827 cells.

[0203] Example 19: Proteomics analysis of the effect of FRTAC on cell membrane protein degradation

[0204] Quantitative mass spectrometry was used to investigate protein changes in cells treated with FRTACs. After 48 hours of treatment in H292 cells with either 10 nM FR-Ctx (FAR 15) or 50 nM cetuximab, which targets and degrades EGFR, proteomic analysis of whole-cell lysates showed that FR-Ctx (FAR 15) significantly reduced EGFR levels. Figure 29 Ctx does not affect EGFR levels. Figure 30 Consistently, whole-cell lysate proteomics analysis also showed that 10 nM FR-Atz (FAR 15) reduced PD-L1 levels in H292 cells. Figure 31 However, Atezolizumab did not change PD-L1 levels. Figure 32 In addition, FR-Atz(FAR 15) ( Figure 33 ) and Atezolizumab ( Figure 34 The treatments did not change the FRα level.

[0205] Example 20: In vivo imaging study of small animals

[0206] Six- to eight-week-old female BALB / c nude mice (purchased from Beijing Hanfei Biotechnology Co., Ltd.). All mice were placed in a specific pathogen-free environment, and all procedures were performed in accordance with the guidelines approved by the Animal Ethics Committee of the Institute of Pharmaceutical Biotechnology, Chinese Academy of Medical Sciences. For in vivo imaging studies in small animals, H292 cells (1×10⁻⁶) were used. 6 Cells were subcutaneously inoculated into the right axilla of BALB / c nude mice. When the average tumor volume reached approximately 300-400 mm², the cells were inoculated. 3 Nude mice were intravenously injected with 20 mg / kg of Dylight 680-FR-Atz prepared in Example 10. Images of Dylight 680-FR-Atz fluorescence at different time points were captured using an IVIS SPECTRUM mouse in vivo imaging system (PerkinElmer, Shelton, CT, USA). Figure 35 As shown, FR-Atz effectively accumulated at the tumor site 5 hours after intravenous injection. The fluorescence intensity at the tumor site persisted for 5 days, decreasing on days 7 and 9. Furthermore, the fluorescence intensity in tumor tissue was significantly higher than in normal mouse tissues, such as the heart, liver, spleen, or other organs. Figure 36 ).

[0207] Example 21: In vivo pharmacodynamic study of FRTAC

[0208] To conduct in vivo tumor suppression studies of FRTAC, mouse RM-1 cells (1×10⁻⁶) were used. 6Cells (from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were subcutaneously injected into the right side of C57BL / 6J mice (Beijing Huafukang Biotechnology Co., Ltd., China). Seven days later, FR-Atz (FAR 15) or Atz (5 mg / kg) was injected intraperitoneally at designated time points. Tumor growth and body weight changes were monitored and recorded every 3 days. Tumor volume was calculated as π / 6 × tumor length × (tumor width). 2 After the experiment, mice were euthanized, tumor tissue was isolated, and flow cytometry was used to detect tumor-infiltrating immune cells, while Western blotting was used to detect PD-L1 abundance. The isolated organs (heart, liver, spleen, and kidney) were fixed in 4% paraformaldehyde, stained with paclitaxel and eosin (H&E), and examined using an AxioVert A1 microscope (Carl Zeiss, Oberkochen, Germany).

[0209] Tumor growth curves showed that FR-Atz (FAR 15) inhibited the growth of RM-1 tumors. Figure 37 This observation was validated by reducing tumor weight using FR-Atz. Figure 38 The body weight of the mice did not change significantly. Figure 39 Western blotting results showed that, compared with Atz treatment, 5 mg / kg FR-Atz (FAR 15) treatment for 48 hours effectively reduced PD-L1 levels in tumor tissue. Figure 40 Immunohistochemical staining further confirmed that, compared with Atz treatment, FR-Atz (FAR 15) significantly reduced the levels of PD-L1 and Ki67 (proliferation markers) in tumor tissue. Figure 41 This demonstrates the effectiveness of FRTAC-mediated target degradation in vivo. No adverse effects were observed on major normal organs (heart, liver, kidney, and spleen). Figure 42 This verified the biosafety of FRTAC.

[0210] Example 22: Validation of FRTAC-dependent FRα-triggered target protein degradation

[0211] 1. Verify that FRTAC degrades target proteins in lysosomes.

[0212] HCC827 cells (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were pretreated for 1 hour with either 100 nM lysosomal acidification inhibitor bafimycin A1 (MCE, HY-100558) or 10 μM proteasome inhibitor MG132 (MCE, HY-13259). The blank control group used normal RPMI 1640 medium (Gibco, USA). PD-L1 antibodies Atezolizumab (50 nM) and FR-Atz (FAR 15) (50 nM) prepared in cell culture medium were added to HCC827 cells. After 24 hours of incubation, proteins were collected according to the experimental procedures in Example 12, and PD-L1 expression (PD-L1 Rabbit mAb, CellSignaling Technology, Cat#13684) was detected by Western blot. The results showed that bafimycin A1 pretreatment eliminated FRTAC-mediated PD-L1 degradation, while MG132 had no effect. Figure 43 Similarly, validation was performed in SKOV3 tumor cells using FR-Ctx (FAR 15), and the results showed that bafimycin A1 pretreatment eliminated FRTAC-mediated EGFR degradation, while MG132 had no effect. Figure 44 This confirms that the FRTAC provided by this invention mediates the degradation of target proteins via a lysosome-dependent pathway.

[0213] 2. Verify that FRTAC-dependent FRα triggers target protein degradation

[0214] To determine that the degradation of surface proteins by FRTAC depends on FRα, H292 cells, which highly express both FRα and PD-L1, and H460 cells, which did not express or did not express FRα but highly expressed PD-L1, were selected for verification. First, 10 nM of FR-Atz(FAR 15), 1 nM of FR-Atz(FAR 15), and 0.1 nM of FR-Atz(FAR 15) were added to H292 and H460 cells (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) cultured in RPMI 1640 medium (Gibco, USA) and incubated for 24 hours. Proteins were collected according to the experimental procedures in Example 12. PD-L1 expression (PD-L1 Rabbit mAb, Cell Signaling Technology, Cat#13684) was detected by Western blot, and FRα expression was detected by FOLR2 mAb (Themro, MA5-37763). The results are as follows: Figure 45As shown in the figure. The results showed that FR-Atz (FAR 15) treatment effectively degraded PD-L1 in H292 cells, but had no effect on PD-L1 degradation in H460 cells, proving that the degradation of PD-L1 by FR-Atz is FRα-dependent. The same method was used to validate this on SKOV3 (high expression of both EGFR and FRα) and Hep3B (undetectable or absent FRα, but high expression of EGFR) cells, and the results are shown in the figure. Figure 46 As shown in the figure. The results showed that FR-Ctx(FAR 15) treatment effectively degraded EGFR in SKOV3 cells, but had no effect on EGFR degradation in Hep3B cells. This demonstrates that the degradation of EGFR by FR-Ctx(FAR 15) is FRα-dependent.

[0215] 3. Knockout verification confirms that FRTAC is FRα-dependent in triggering target protein degradation.

[0216] First, 100 nM FRα-targeting siRNA and control siRNA (GenePharm, Shanghai) were transfected into cells at H292 via Vigofect (Vigorous Biotechnology, Beijing). After 48 hours, Western blot analysis showed FRα knockout. Then, 10 nM FR-Ctx (FRA15) was added to the cells, and after 24 hours of incubation, protein was collected following the experimental procedures in Example 12. EGFR expression was detected by Western blot, and the results are as follows: Figure 47 As shown, after FRα knockout, the degradation of EGFR by FR-Ctx (FRA15) was significantly eliminated, while EGFR could still be degraded in control cells, proving that the degradation of EGFR by FR-Ctx (FRA15) is FRα-dependent. Furthermore, FRα expression was upregulated in Hep3B and H460 cells transfected with pCMV-Myc-FRα plasmid (Sinochem, Beijing). After adding 10 nM FR-Ctx (FRA15) and 10 nM FR-Atz (FRA15) to the cells and incubating for 24 hours, proteins were collected according to the experimental procedures in Example 12. The expression of EGFR and PD-L1 was detected by Western blot. The results showed that upregulating FRα expression could promote EGFR degradation mediated by FRTAC. Figure 48 ) or PD-L1 ( Figure 49 The degradation of FRTAC provided by this invention demonstrates that the protein degradation effect is FRα-dependent.

[0217] The above experiments verify that the FRTACs provided by this invention utilize FRα as an LTR to perform lysosomal targeted degradation of membrane proteins.

[0218] The FRTACs provided by this invention can not only target tumor sites, but also utilize FRα as an LTR to perform lysosomal targeted degradation of membrane proteins, achieving unexpected sub-nanomolar degradation activity. They exhibit significant target degradation efficacy in vivo and show stronger anti-tumor activity than traditional antibody therapy.

[0219] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt, hydrate or solvate thereof. in, Structural fragments That is, -(C2H4O) n - represents PEG with an average molecular weight of 200-6000; T is a number between 8 and 40; R represents a polypeptide, small molecule compound, or antibody targeting a target protein, or its antigen-binding fragment.

2. The compound of claim 1 or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the structural fragment PEG represents an average molecular weight of 300–6000; Preferably, structural fragments PEGs representing average molecular weights of approximately 400, 600, 800, 1000, 1450, 1500, 3350, 4000, 5000, and 6000; Preferably, structural fragments This represents PEG with an average molecular weight of 800–1500.

3. The compound of claim 1 or 2 or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein T is a number between 10 and 40; Preferably, T is a number between 12 and 38; Preferably, T is a number between 15 and 35; Preferably, T is about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34 or about 35.

4. The compound of any one of claims 1-3 or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R is an antibody targeting a target protein or an antigen-binding fragment thereof; Preferably, the antibody targeting the target protein or its antigen-binding fragment is selected from monoclonal antibodies, mouse antibodies, rabbit antibodies, humanized antibodies, fully human antibodies, chimeric antibodies (e.g., human-mouse chimeric antibodies), bispecific antibodies, multispecific antibodies, single-chain antibodies, dAb, complementarity-determining region fragments, Fv, single-chain Fv (scFv), Fd, Fab, Fab', and F(ab')2; Preferably, R is an anti-EGFR antibody (e.g., cetuximab), an anti-PD-L1 antibody (e.g., Atezolizumab), an EGFR-targeting peptide (e.g., GE11), an anti-HER2 antibody (e.g., Herceptin), a CPCR-related antibody, an anti-Trop2 antibody (e.g., Sacituzumab), an anti-FGFR antibody, an anti-VEGFR antibody, an anti-CTLA4 antibody, an anti-IL-5Rα antibody, or an antigen-binding fragment thereof (e.g., single-chain antibodies, dAbs, complementarity-determining region fragments, Fvs, single-chain Fvs (scFvs), Fd, Fabs, Fab's, and F(ab')2s of the above antibodies).

5. The compound represented by Formula II or its pharmaceutically acceptable salt, hydrate, or solvate. Structural segments That is, -(C2H4O) n - represents PEG with an average molecular weight of 200-6000; Preferably, structural fragments PEG represents an average molecular weight of 300–6000; Preferably, structural fragments PEGs representing average molecular weights of approximately 400, 600, 800, 1000, 1450, 1500, 3350, 4000, 5000, and 6000; Preferably, structural fragments This represents PEG with an average molecular weight of 800–1500.

6. The use of the compound of claim 5 or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of antibody-drug conjugates, preferably in the preparation of the compound of any one of claims 1-4 or a pharmaceutically acceptable salt, hydrate or solvate thereof.

7. A method for preparing the compound of any one of claims 1-4 or a pharmaceutically acceptable salt, hydrate, or solvate thereof, comprising: (1) Prepare the compound of claim 5 or a pharmaceutically acceptable salt, hydrate or solvate thereof; (2) Reacting the compound of claim 5 or a pharmaceutically acceptable salt, hydrate or solvate thereof with a polypeptide, small molecule compound or antibody or an antigen-binding fragment thereof containing a free amino group. Preferably, step (1) includes: i) React tBuCOO-PEG-NH2 with folic acid (1.0 eq.) to obtain tBuCOO-PEG-FA; ii) Remove the protecting group tert-butyl from tBuCOO-PEG-FA to obtain the compound shown in Formula III; iii) The compound shown in Formula III reacts with NHS to obtain the compound shown in Formula II.

8. A composition comprising the compound of any one of claims 1-4 or a pharmaceutically acceptable salt, hydrate or solvate thereof, or the compound of claim 5 or a pharmaceutically acceptable salt, hydrate or solvate thereof.

9. A pharmaceutical composition comprising the compound of any one of claims 1-4 or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a pharmaceutically acceptable carrier and / or excipient.

10. The use of the compound of any one of claims 1-4, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in the preparation of a medicament for the targeted degradation of proteins. Preferably, the protein is EGFR, PD-L1, HER2, Trop2, CPCR, FGFR, VEGFR, CTLA4, or IL-5Rα protein.

11. The use of the compound of any one of claims 1-4 or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a medicament, wherein the medicament is used to inhibit tumor cell activity or to treat tumors; Preferably, the tumor cells are cancer cells expressing FRα; Preferably, the tumor is a tumor expressing FRα; Preferably, the tumor cells are breast cancer cells, oral cancer cells, liver cancer cells, gastric cancer cells, pancreatic cancer cells, colorectal cancer cells, bladder cancer cells, or prostate cancer cells; or the tumor cells are ovarian cancer cells, triple-negative breast cancer (TNBC) cells, endometrial cancer cells, mesothelioma cells, or lung cancer cells. Preferably, the tumor is ovarian cancer, triple-negative breast cancer (TNBC), endometrial cancer, mesothelioma, or lung cancer; or the tumor is breast cancer, oral cancer, liver cancer, stomach cancer, pancreatic cancer, colorectal cancer, bladder cancer, or prostate cancer.

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