Compound and conjugate thereof

Abstract

The present disclosure relates to a new camptothecin derivative and a conjugate comprising same. The present disclosure further relates to use of the conjugate in combating tumors.

Description

Compounds and their conjugates

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to Chinese patent applications CN202410504376.X, filed on April 25, 2024, and CN202410696822.1, filed on May 31, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of biomedicine, and more specifically, to a novel topoisomerase inhibitor and a conjugate containing the compound. Background Technology

[0004] Antibody-drug conjugates (ADCs) bridge antibodies and cytotoxic molecules via linkers, simultaneously exerting the targeting ability of antibodies and the cell-killing effects of cytotoxic molecules on tumor cells, thus expanding the therapeutic window for tumors. Among the cytotoxic molecules used in ADCs, a class of topoisomerase I inhibitors, such as camptothecin, have attracted considerable attention; for example, some ADC drugs have already been approved for marketing. and The camptothecin derivatives Dxd and SN38 were used as toxin molecules, respectively.

[0005] The linkers used in ADC drugs are mainly divided into two categories: cleavable and non-cleavable. Among them, cleavable linkers release toxin molecules at the target cell under specific conditions. Common types include acid-cleavable linkers, linkers that can be cleaved under reducing conditions, and enzyme-mediated cleavage linkers. A typical example of such enzymes is cathepsin B.

[0006] For enzyme-mediated catalytic inhibitor (ADC) drugs, the successful release and activity of the toxin molecule depends on various factors, such as the choice of linker, the linkage mode with the toxin molecule, and the steric hindrance of the toxin molecule itself. Nakada et al. discussed a series of ADC molecules of eczematidine derivatives in Bioorg Med Chem Lett. 2016 Mar 15; 26(6):1542-1545. Some of these ADCs showed decreased antitumor activity, which was speculated to be due to steric hindrance of the drug portion. The degrading enzymes in tumor cells may not be able to work effectively, indicating that the amide position between the drug and the cleavable peptide linker is very important for the affinity of the degrading enzyme. A similar situation also appeared in the report by Lyski et al. Mol Cancer Ther; 20(2) February 2021. When the camptothecin derivative CPT1 was linked to the dipeptide linker, the ADC could not be effectively cleaved and release the toxin. When it was linked to the tripeptide, it cleaved to obtain the toxin with residual amino acids, resulting in a decrease in cell activity.

[0007] The structure of compound CPT24 is shown below. Its racemic form was first reported in patent document CA2087898, and its antitumor activity has been reported in other documents such as Wadkins et al. CANCER RESEARCH 64, 6679–6683, September 15, 2004; Westover et al. Molecular Cancer (2015) 14:92. Summary of the Invention

[0008] In a first aspect, this disclosure provides a coupling with CPT24 as the load, having the structure shown in Formula I:

[0009] In this design, LU is the ligand unit, SP1 is the first spacer unit, L1 is the dipeptide linker, SP2 is the second spacer unit, L2 is the linker connected to LU, and n = 1–20. The conjugates disclosed herein exhibit good antitumor activity and a strong bystander killing effect.

[0010] The conjugates disclosed herein also exhibit improved hydrophilic properties, which help reduce their clearance rate and immunogenicity in vivo, resulting in better in vivo activity. Furthermore, metabolite identification indicates that the conjugates of this disclosure can rapidly release the effective payload CPT24 within tumor cells.

[0011] Secondly, this disclosure provides a pharmaceutical composition comprising the novel conjugate described herein, and a pharmaceutically acceptable carrier.

[0012] Thirdly, this disclosure also provides a method for treating and / or preventing cancer, including administering the conjugate described herein to a subject in need, and further provides the use of the conjugate in the preparation of a medicament for treating and / or preventing cancer.

[0013] Fourthly, this disclosure also provides a novel linker-load combination, such as the compound shown in Formula V, and its salts and solvates:

[0014] SP1, L1, and SP2 are defined as described above, and L2' is a connector unit used to connect with the ligand unit. Attached Figure Description

[0015] Figure 1 shows the fabrication process of ADC1.

[0016] Figure 2 shows the hydrophobicity detection results for each ADC.

[0017] Figure 3 shows the bystander killing effect of ADC1 and ADC4 on 293T-GFP cells.

[0018] Figure 4 shows the bystander killing ability of each ADC on 293T-Luc2 cells.

[0019] Figure 5 shows the tumor-suppressing effect of ADC5 on the human gastric cancer NCI-N87 nude mouse xenograft model.

[0020] Figure 6 shows the killing effect of ADC molecules on A431 tumor cells.

[0021] Figure 7 shows the load release of ADC1 after incubation in serum of different species. Detailed Implementation

[0022] Terminology Definition

[0023] Unless otherwise indicated or defined, all terms used herein have their ordinary meaning as will be understood by those skilled in the art. References include, for example, standard manuals such as Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), Volumes 1–3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York (1990); and Roitt et al., “Immunology” (2nd edition), Gower Medical Publishing, London, New York (1989), and general prior art cited herein; furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as will be understood by those skilled in the art. Also refer to, for example, standard manuals, the aforementioned general prior art, and other references cited therein.

[0024] Unless otherwise stated, the interchangeable terms “antibody” or “immunoglobulin”, whether referring herein to heavy-chain antibodies or conventional four-chain antibodies, are used generally to include full-length antibodies, their individual chains, and all their portions, domains, or fragments (including, but not limited to, antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively). Furthermore, the term “sequence” as used herein (e.g., in the terms “immunoglobulin sequence,” “antibody sequence,” or “protein sequence,” etc.) should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding said sequence, unless a more specific interpretation is required herein.

[0025] The terms "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules that have the molecular structure of natural immunoglobulins. In the case of a conventional four-chain IgG antibody, a full-length antibody comprises two heavy chains (H) and two light chains (L) linked together by disulfide bonds. The term "antigen-binding fragment" of an antibody is a molecule distinct from a full-length antibody, containing a portion of the full-length antibody but capable of binding antigens to the full-length antibody or competing with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), and nanobodies.

[0026] As used herein, the term "multispecific antibody" refers to an antibody having at least two antigen-binding regions, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. The term "bispecific antibody" refers to an antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to one antigen or epitope and the second antigen-binding region binds to another antigen or another epitope.

[0027] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any or a combination of many known antibody CDR assignment systems, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (on the World Wide Web imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)).

[0028] In this invention, unless otherwise specified, the Kabat definition scheme is used to determine the CDR region:

[0029] The term "epitope" or "antigenic epitope" generally refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also known in the art as an "antigenic determinant." An epitope or antigenic determinant typically consists of chemically active surface groups of a molecule, such as amino acids or carbohydrate or sugar side chains, and usually has specific three-dimensional structural features and specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation, which can be "linear" or "conformal." See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996). In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In a conformational epitope, points of interaction are separated by protein amino acid residues.

[0030] The term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein can bind. Specificity can be determined based on the affinity and / or cohesion of the antigen-binding protein. Affinity, expressed as the dissociation equilibrium constant (KD) between the antigen and the antigen-binding protein, is a measure of the strength of binding between the epitope and the antigen-binding site on the antigen-binding protein: the smaller the KD value, the stronger the binding between the epitope and the antigen-binding protein (or, affinity can also be expressed as the association constant (KA), which is 1 / KD). As those skilled in the art will understand, affinity can be determined in known ways depending on the specific antigen of interest. Affinity is a measure of the strength of binding between an antigen-binding protein (e.g., an immunoglobulin, antibody, a single variable domain of an immunoglobulin, or a polypeptide containing such a domain) and the associated antigen. Affinity relates to both the affinity between the antigen and the antigen-binding site on the antigen-binding protein and the number of associated binding sites present on the antigen-binding protein.

[0031] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, the immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, the amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In this document, the term "Fc region" does not include the heavy chain variable region VH and light chain variable region VL of immunoglobulins, nor the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region. In some embodiments, the Fc region of the present invention is derived from IgG1, IgG2, IgG3, or IgG4.

[0032] The term "conjugate," also known as a conjugate, generally refers to a binding protein or polypeptide (e.g., an antibody or its antigen-binding fragment) linked to one or more chemical drugs, such as antibody-drug conjugates (ADCs). The chemical drug can be any therapeutic agent and / or cytotoxic agent. The antibody-drug conjugate can have any number of drugs conjugated to the antibody from 1 to 20, for example, it can include 2, 4, 6, or 8 drug-loaded species. In some embodiments, the conjugate is an antibody-drug conjugate (ADC).

[0033] The term "N-glycan" refers to a glycan chain attached to asparagine (Asn) of a protein's Asn-X-Ser / Thr sequence (Ser is serine, Thr is threonine, and X is any amino acid except proline).

[0034] The term "G0 glycoform" refers to a glycosemyform, such as Fc glycosylated sugars, that does not contain terminal galactose residues or a core fucose residue. in It is N-acetylglucosamine. It is mannose. The term "GOF glycotype" refers to a glycotype in which the structure of sugar molecules, such as Fc glycosylated sugar molecules, does not contain terminal galactose residues but contains core fucose.

[0035] As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms.

[0036] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon group, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 10 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0037] The term "cycloalkylene" refers to a residue derived from the same carbon atom or two different carbon atoms removed from the parent ring of a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon group.

[0038] The term "heterocyclic alkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon group containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, but excluding the ring portions of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon.

[0039] The term "heterocyclic alkyl" refers to a residue derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent ring of a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon group, wherein the cyclic hydrocarbon group comprises 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, but excluding the ring portions of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon.

[0040] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein the definitions of alkyl or cycloalkyl are as described above.

[0041] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, with phenyl being more preferred. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring. The aryl group may be substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, amino, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxy, heterocyclic alkoxy, cycloalkylthio, and heterocyclic alkylthio.

[0042] The term "triaryl" refers to residues derived from removing two hydrogen atoms from two different carbon atoms of a parent aromatic ring of a 6- to 14-membered all-carbon monocyclic or fused polycyclic ring with a conjugated π-electron system.

[0043] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered, more preferably 5- or 6-membered, such as furanyl, thiophene, pyridyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, amino, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0044] The term "hybrid aryl" refers to a residue derived from the removal of two hydrogen atoms from two different carbon atoms of the parent aromatic ring of a heteroaromatic polycyclic aromatic ring containing 1 to 4 heteroatoms and 5 to 14 ring atoms.

[0045] The term "optional" or "optionally" means that the event or condition described below either occurs or does not occur, and the description includes instances where the event or condition occurs as well as instances where the event or condition does not occur. For example, when a group or structure is "optionally substituted," the group or structure may or may not be substituted.

[0046] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the compounds or conjugates of the present invention, and that such salt is not biologically or otherwise undesirable. The compounds and conjugates of the present invention can exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the compounds or conjugates of the present invention with an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the compounds or couplings of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by reacting with an organic base containing an N group.

[0047] The term "glycosidic bond" refers to the chemical bond that connects the sugar and the other hydroxyl-containing compound (such as an alcohol, phenol, or another sugar) to form an acetal derivative through dehydration condensation of the hydroxyl group on the hemiacetal structure of a sugar.

[0048] The term "solvent" refers to an association formed by one or more solvent molecules with the ADC antibody-drug conjugate of this invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0049] The term "drug-antibody ratio" or "DAR" refers to the ratio of the drug portion to the ligand portion conjugated herein. In some embodiments described herein, the DAR may be determined by n in Formula I, for example, the DAR may be 1 to 16, such as 2-16, 2-10, 2-8, 2-6, or 3-4, such as 2, 3, 4, 5, or 6. The DAR may also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the drug portion to the ligand portion conjugated herein to the product, as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA, electrophoresis, and / or HPLC), this DAR is referred to herein as the average DAR or the measured DAR. In some embodiments, the average DAR value of the conjugates of the present invention is 1 to 16, for example 2-16, 2-8, 2-6, 3-4, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, The range is defined as 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0, with two of these values ​​as endpoints. It should be understood that when referring to the average DAR value, the couplings of this invention refer to a population or mixture of coupling molecules containing coupling molecules having the same and / or different DAR values.

[0050] Where there is no contradiction in the context, "pharmaceutical acceptable" and "medicinal" are used interchangeably in this article.

[0051] As used herein, the term “and / or” refers to any one of the options or two or more of the options.

[0052] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps.

[0053] The term "administration" generally refers to a method of giving a subject (e.g., a patient) a dose of a compound, conjugate, or pharmaceutical composition. Administration can be performed by any suitable means, including parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration (if necessary for local treatment). Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0054] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. Unless otherwise specified, all values ​​mentioned in this application are considered to be modified by "about". In case of doubt, or if the error range for a particular value or parameter is not generally understood in the art, "about" means ±5% of that value or parameter.

[0055] "Conservative substitution" refers to the replacement of a polypeptide sequence without substantially altering its intended functional activity. For example, conservative substitution often results in an amino acid being replaced by a chemically similar amino acid. Eight exemplary groups of amino acids containing mutually conserved substitutions are listed below: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0056] The term "effective amount" refers to such an amount or dose of the compound, conjugate, or composition or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. Depending on the intended effect, it may include "therapeutic effective amount" and "preventive effective amount".

[0057] "Therapeutic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. A therapeutic effective amount is also a amount in which any toxic or harmful effects of the compounds, conjugates, or compositions or combinations of the present invention are less than the beneficial therapeutic effects. "Prophylactic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired preventative outcome. Generally, because the prophylactic dose is used in the subject before or at an earlier stage of the disease, the prophylactic effective amount will be less than the therapeutic effective amount.

[0058] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0059] The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. Cancer can be in its early, middle, or late stages, or it can be metastatic.

[0060] The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. “Tumor” encompasses solid tumors and hematologic malignancies as well as metastatic lesions. The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusive when used in this article.

[0061] The terms “pharmaceutical excipients” and “pharmaceuticalally acceptable carriers” refer to diluents, adjuvants (e.g., Freund’s adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are administered together with the active substance.

[0062] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0063] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0064] Invention Details

[0065] Coupled

[0066] In a first aspect, this disclosure provides a coupling having the structure shown in Formula I:

[0067] Wherein, LU is the ligand unit, SP1 is the first spacer unit, L1 is the dipeptide linker, SP2 is the second spacer unit, L2 is the linker connected to LU, and n = 1 to 20.

[0068] In some implementations, SP1 is absent, or SP1 is selected from:

[0069] (The side connected to L1 is labeled L1, and the side connected to the CPT24 hydroxyl group is labeled O).

[0070] R1 is independently selected from hydrogen, C 1-6 Alkyl, hydroxyl, amino, halogen, nitro, cyano d is an integer from 1 to 20, and e is an integer from 1 to 20; R2 and R3 are each independently selected from hydrogen and C. 1-6 alkyl;

[0071] The alkyl group may optionally be substituted with hydroxyl, amino, halogen, nitro, and cyano groups.

[0072] In some implementations, SP1 is

[0073] In some implementations, SP1 is

[0074] In some implementations, SP1 is selected from: Where d is an integer from 1 to 10, and e is an integer from 1 to 10.

[0075] In other implementations, SP1 is

[0076] In some implementations, SP1 is selected from

[0077] In some embodiments, the dipeptide linker L1 can be cleaved by cathepsin; preferably, the cathepsin can be selected from: cathepsin A, B, C, D, E, F, G, H, K, L1, L2, O, S, W and Z; more preferably, the cathepsin is cathepsin B.

[0078] In some implementations, L1 is (The side connected to SP1 is labeled SP1, and the side connected to SP2 is labeled SP2). AA1 and AA2 are each an amino acid residue, and AA1 is more hydrophobic than AA2.

[0079] Those skilled in the art should know that the differences in hydrophobicity of different amino acids or amino acid residues are mainly caused by their side chain groups. The strength of hydrophobicity of amino acid side chain groups is well known in the art. For example, see the description in Monera et al. J Pept Sci. 1995 Sep-Oct; 1(5):319-29.

[0080] In some implementations, AA1 is selected from Val, Phe, Leu, Ile, and Trp, and AA2 is selected from Ala, Lys, Cit, Gly, Arg, Gln, Asn, Ser, His, Glu, Thr, and Asp.

[0081] In other embodiments, AA1 is selected from Val, Phe and Leu, and AA2 is selected from Ala, Lys, Cit, Gly and Arg; preferably, AA1 is Val and AA2 is selected from Ala, Lys, Cit and Gly; even more preferably, AA1 is Val and AA2 is selected from Ala, Lys and Cit.

[0082] In some implementations, L1 is selected from the following dipeptide residues:

[0083] SP2 -Val-Ala- SP1 , SP2 -Val-Lys- SP1 , SP2 -Val-Cit- SP1 , SP2 -Val-Gly- SP1 , SP2 -Val-Arg- SP1 , SP2 -Val-Glu- SP1 , SP2 -Val-Asp- SP1 , SP2 -Phe-Lys- SP1 , SP2 -Phe-Cit- SP1 , SP2 -Phe-Ala- SP1 , SP2 -Phe-Arg- SP1 , SP2 -Leu-Ala- SP1 , SP2 -Leu-Lys- SP1 , SP2 -Leu-Cit- SP1 , SP2 -Ile-Ala- SP1 , SP2 -Ile-Lys- SP1 , SP2 -Ile-Cit- SP1 and SP2 -Trp-Cit- SP1 .

[0084] Preferably, L1 is selected from the following dipeptide residues:

[0085] SP2 -Val-Ala- SP1 , SP2 -Val-Lys- SP1 , SP2 -Val-Cit- SP1 , SP2 -Val-Gly- SP1 , SP2 -Val-Arg- SP1 , SP2 -Phe-Lys- SP1, SP2 -Phe-Cit- SP1 , SP2 -Phe-Ala- SP1 , SP2 -Leu-Ala- SP1 , SP2 -Leu-Lys- SP1 and SP2 -Leu-Cit- SP1 .

[0086] More preferably, L1 is selected from the following dipeptide residues: SP2 -Val-Ala- SP1 , SP2 -Val-Lys- SP1 , SP2 -Val-Cit- SP1 and SP2 -Phe-Lys- SP1 For example, L1 is SP2 -Val-Ala- SP1 .

[0087] In some embodiments, this disclosure provides the coupling having the structure shown in Formula II:

[0088] AA'2 is selected from Ala, Lys, Cit, Gly, Arg, Gln, Asn, Ser, His, Glu, Thr and Asp, preferably from Ala, Lys, Cit, Gly, Arg and Glu, more preferably from Ala, Lys and Cit, and even more preferably from Ala.

[0089] In some embodiments, the coupling described in this disclosure is provided, wherein the structure of SP2 is as shown in Formula III:

[0090] (The side connected to L1 is labeled L1, and the side connected to L2 is labeled L2).

[0091] Where a1 = 0 or 1, a2 = integers from 0 to 6, b1 = 0 or 1, b2 = integers from 0 to 16, b3 = integers from 0 to 16, c = integers from 0 to 6, and at least one of b2 and b3 is 0.

[0092] In some implementations, a1 = 1 in the structure of SP2; in other implementations, a1 = 0.

[0093] In some implementations, a2 = 0, 1, 2, 3, 4, 5, or 6.

[0094] In some implementations, b1 = 0 or 1.

[0095] In some implementations, b2 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0096] In some implementations, b3 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0097] In some implementations, c = 0, 1, 2, 3, 4, 5, or 6.

[0098] The above options a1, a2, b1, b2, b3 and c can be combined in any way, provided that at least one of b2 and b3 is 0.

[0099] In some specific implementations, the structure of SP2 is selected from the following group:

[0100] (1) a1 = 0, a2 = 2, 3, 4, 5 or 6, b1 = 0, b2 = 0, b3 = 0, c = 0;

[0101] (2) a1 = 0, a2 = 0, b1 = 0, b2 = 0, b3 = 0, c = 2, 3, 4, 5 or 6;

[0102] (3) a1 = 1, a2 = 2, 3, 4, 5 or 6, b1 = 1, b2 = 2, 3, 4, 5, 6, 7 or 8, b3 = 0, c = 0;

[0103] (4) a1 = 0, a2 = 2, 3, 4, 5 or 6, b1 = 1, b2 = 2, 3, 4, 5, 6, 7 or 8, b3 = 0, c = 0;

[0104] (5) a1 = 1, a2 = 0, b1 = 0, b2 = 0, b3 = 2, 3, 4, 5, 6, 7 or 8, c = 2, 3, 4, 5 or 6; and

[0105] (6) a1 = 0, a2 = 0, b1 = 1, b2 = 2, 3, 4, 5, 6, 7 or 8, b3 = 0, c = 0.

[0106] In some implementations, the structure of SP2 is specifically selected from the following group:

[0107] (1.1) a1=0, a2=2, b1=0, b2=0, b3=0, c=0;

[0108] (1.2) a1=0, a2=3, b1=0, b2=0, b3=0, c=0;

[0109] (1.3)a1=0, a2=4, b1=0, b2=0, b3=0, c=0;

[0110] (1.4)a1=0, a2=5, b1=0, b2=0, b3=0, c=0;

[0111] (1.5)a1=0, a2=6, b1=0, b2=0, b3=0, c=0;

[0112] (2.1)a1=0, a2=0, b1=0, b2=0, b3=0, c=2;

[0113] (2.2)a1=0, a2=0, b1=0, b2=0, b3=0, c=3;

[0114] (2.3)a1=0, a2=0, b1=0, b2=0, b3=0, c=4;

[0115] (2.4)a1=0, a2=0, b1=0, b2=0, b3=0, c=5;

[0116] (2.5)a1=0, a2=0, b1=0, b2=0, b3=0, c=6;

[0117] (3.1)a1=1, a2=2, b1=1, b2=2, b3=0, c=0;

[0118] (3.2)a1=1, a2=2, b1=1, b2=3, b3=0, c=0;

[0119] (3.3)a1=1, a2=2, b1=1, b2=4, b3=0, c=0;

[0120] (3.4)a1=1, a2=2, b1=1, b2=5, b3=0, c=0;

[0121] (3.5)a1=1, a2=2, b1=1, b2=6, b3=0, c=0;

[0122] (3.6)a1=1, a2=2, b1=1, b2=7, b3=0, c=0;

[0123] (3.7)a1=1, a2=2, b1=1, b2=8, b3=0, c=0;

[0124] (3.8)a1=1, a2=3, b1=1, b2=2, b3=0, c=0;

[0125] (3.9)a1=1, a2=3, b1=1, b2=3, b3=0, c=0;

[0126] (3.10)a1=1, a2=3, b1=1, b2=4, b3=0, c=0;

[0127] (3.11)a1=1, a2=3, b1=1, b2=5, b3=0, c=0;

[0128] (3.12)a1=1, a2=3, b1=1, b2=6, b3=0, c=0;

[0129] (3.13)a1=1, a2=3, b1=1, b2=7, b3=0, c=0;

[0130] (3.14)a1=1, a2=3, b1=1, b2=8, b3=0, c=0;

[0131] (3.15)a1=1, a2=4, b1=1, b2=2, b3=0, c=0;

[0132] (3.16)a1=1, a2=4, b1=1, b2=3, b3=0, c=0;

[0133] (3.17)a1=1, a2=4, b1=1, b2=4, b3=0, c=0;

[0134] (3.18)a1=1, a2=4, b1=1, b2=5, b3=0, c=0;

[0135] (3.19)a1=1, a2=4, b1=1, b2=6, b3=0, c=0;

[0136] (3.20)a1=1, a2=4, b1=1, b2=7, b3=0, c=0;

[0137] (3.21)a1=1, a2=4, b1=1, b2=8, b3=0, c=0;

[0138] (4.1)a1=0, a2=2, b1=1, b2=2, b3=0, c=0;

[0139] (4.2)a1=0, a2=2, b1=1, b2=3, b3=0, c=0;

[0140] (4.3)a1=0, a2=2, b1=1, b2=4, b3=0, c=0;

[0141] (4.4)a1=0, a2=2, b1=1, b2=5, b3=0, c=0;

[0142] (4.5)a1=0, a2=2, b1=1, b2=6, b3=0, c=0;

[0143] (4.6)a1=0, a2=2, b1=1, b2=7, b3=0, c=0;

[0144] (4.7)a1=0, a2=2, b1=1, b2=8, b3=0, c=0;

[0145] (4.8)a1=0, a2=3, b1=1, b2=2, b3=0, c=0;

[0146] (4.9)a1=0, a2=3, b1=1, b2=3, b3=0, c=0;

[0147] (4.10)a1=0, a2=3, b1=1, b2=4, b3=0, c=0;

[0148] (4.11)a1=0, a2=3, b1=1, b2=5, b3=0, c=0;

[0149] (4.12)a1=0, a2=3, b1=1, b2=6, b3=0, c=0;

[0150] (4.13)a1=0, a2=3, b1=1, b2=7, b3=0, c=0;

[0151] (4.14)a1=0, a2=3, b1=1, b2=8, b3=0, c=0;

[0152] (4.15)a1=0, a2=4, b1=1, b2=2, b3=0, c=0;

[0153] (4.16)a1=0, a2=4, b1=1, b2=3, b3=0, c=0;

[0154] (4.17)a1=0, a2=4, b1=1, b2=4, b3=0, c=0;

[0155] (4.18)a1=0, a2=4, b1=1, b2=5, b3=0, c=0;

[0156] (4.19)a1=0, a2=4, b1=1, b2=6, b3=0, c=0;

[0157] (4.20)a1=0, a2=4, b1=1, b2=7, b3=0, c=0;

[0158] (4.21) a1=0, a2=4, b1=1, b2=8, b3=0, c=0;

[0159] (5.1) a1=1, a2=0, b1=0, b2=0, b3=2, c=2;

[0160] (5.2) a1=1, a2=0, b1=0, b2=0, b3=3, c=2;

[0161] (5.3) a1=1, a2=0, b1=0, b2=0, b3=4, c=2;

[0162] (5.4) a1=1, a2=0, b1=0, b2=0, b3=5, c=2;

[0163] (5.5) a1=1, a2=0, b1=0, b2=0, b3=6, c=2;

[0164] (5.6) a1=1, a2=0, b1=0, b2=0, b3=7, c=2;

[0165] (5.7) a1=1, a2=0, b1=0, b2=0, b3=8, c=2;

[0166] (6.1) a1=0, a2=0, b1=1, b2=2, b3=0, c=0;

[0167] (6.2) a1=0, a2=0, b1=1, b2=3, b3=0, c=0;

[0168] (6.3) a1=0, a2=0, b1=1, b2=4, b3=0, c=0;

[0169] (6.4) a1=0, a2=0, b1=1, b2=5, b3=0, c=0;

[0170] (6.5) a1=0, a2=0, b1=1, b2=6, b3=0, c=0;

[0171] (6.6) a1 = 0, a2 = 0, b1 = 1, b2 = 7, b3 = 0, c = 0; and

[0172] (6.7) a1=0, a2=0, b1=1, b2=8, b3=0, c=0;

[0173] In some embodiments, the coupling described in this invention is provided, wherein L2 is selected from:

[0174] (The side connected to SP2 is labeled SP2, and the side connected to LU is labeled LU), where Ar represents C. 6-10 Aryl groups, which are optionally coated with halogens, C 1-6 Alkyl substitution; R4 is selected from hydrogen, halogen, and C. 1-6 Alkyl group; Z is selected from straight bond, C 2-6 Ethyne group, C 2-6 imidene group, C 6-10 aryl, 5-10 heteroaryl, amide, sulfonamide, imino, and CF2; m is an integer from 1 to 6.

[0175] In some implementations, L2 is selected from

[0176] In some embodiments, in the couplings described herein, -L2-SP2-L1-SP1- is selected from the following structures:

[0177]

[0178] (The side connected to the ligand unit is marked as LU, and the side connected to the CPT24 hydroxyl group is marked as O); where k is an integer from 1 to 20, for example, k = 2, 4, 6, 8, 10, 12, 14 or 16.

[0179] In some embodiments, the couplings of this disclosure have a structure selected from the following:

[0180] Where k is as defined above, n = 1 to 20; preferably, n = 1 to 15, for example n = 1 to 10, n = 1 to 8, n = 2 to 6 or n = 2 to 5.

[0181] In this disclosure, the ligand unit LU refers to any kind of substance capable of specifically binding to a target molecule, such as an antibody or its antigen-binding fragment, a polypeptide, a small molecule, an aptamer, etc.

[0182] In some embodiments, the ligand unit may include a polypeptide, such as a cyclic polypeptide.

[0183] In some embodiments, the ligand unit may comprise an antibody or an antigen-binding fragment thereof; in other embodiments, the ligand unit of this disclosure may be a cell-binding agent that specifically targets cells.

[0184] In some implementations, the ligand unit comprises an antibody or an antigen-binding fragment thereof.

[0185] In some embodiments, the ligand unit comprises an antibody or an antigen-binding fragment thereof, which specifically binds to an antigen.

[0186] In some embodiments, the antibody or its antigen-binding fragment has an N-glycan chain.

[0187] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a bispecific antibody or a multispecific antibody.

[0188] In some embodiments, the antigen is a tumor-associated antigen (TAA) or a receptor. In some embodiments, the antigen is selected from one or more of the following:

[0189] 4-1BB, 4-1BBL, A33, Adenosine A2a receptor, Akt, ALK, androgen receptor, Ang-1, Ang-2, Annexin A3, Aurora A, Aurora B, B7-H3, B7-H4, Bcl-2, Bcr-Abl, BRAF, BTK, BTLA, BTN2A1, CA-125, CAIX, CCR4, CD105 / endoglin, CD109, CD123, CD155, CD16, CD160, CD19, CD20, CD200, CD200R, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD4 0L, CD47, CD48, CD52, CD70, CD79b, CD80, CD86, CD96, CDK4, CDK6, CDK9, CEA, CEACAM1, ChK1, ChK2, c-KIT, c-Met, CO X2, CSF-1R, CSF2, CTLA-4, CXCR2, CXCR4, DDR2, DLL3, DLL4, DNAM-1, DR5, EGFR, EpCAM, EPHA3, EphB4, ERK1, ERK2 / p38 MAPK, FAK, FAP, FGF-2, FGFR1, FGFR2, FGFR3, FGFR4, Flt-3, Gal-9, GITR, GITRL, Glypican-3, HDAC1, HDAC2, HDAC3 , HDAC4, HDAC5, HDAC7, HDAC8, HDAC9, HER2, HER3, HER4 / ERBB4, HGF, HHLA2, HIF-1α, HSP27, HSP90, HVEM, ICOS, ICOS Ligand, IDO, IGF1R, IL-13, IL-6, Integrin, JAK1, JAK2, JAK3, KRAS, LAG-3, LIGHT, MDM2, MEK1, MEK2, MMP-1, MMP-10, MMP-11, MMP-13, MMP-2, MMP-7, MMP-9, mTOR, Mucin1. Myc, NaPi2b, NF-κB, NKG2A, NRAS, NTRK1, NTRK2, NTRK3, OX40, OX40L, p53, PAF, PARP1, PARP2, PD1, PDGFR-α, PDGFR-β, PD-L2, PI3Kα, PI3Kβ, PI3Kγ, PI3Kδ, PIM 1. PIM3, PSMA, PTEN, RAF-1, RANKL, RET, S100A4, SIRPα, SLAMF7, SMO, Src, STAT3, STEAP-1, SYK, TDO, TGFβ, Tie-2, TIGIT, TIM-3, TLR8, TMIGD2, TNF-α, Toll-like receptor 3. TRAIL, TRAILR1, TROP-2, VEGF, VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3 and VISTA.

[0190] In some embodiments, the ligand unit comprises an antibody or antigen-binding fragment thereof that specifically binds to HER3 and / or EGFR. In some embodiments, the ligand unit comprises a bispecific antibody or fragment thereof that binds to HER3 and EGFR. As an example, the antibody may be Duligotuzumab (MEHD7945A), whose amino acid sequence is referenced from WHO Drug Information, Vol. 28, No. 3, 2014 Recommended INN: List 72, as follows:

[0191] In some embodiments, the ligand unit comprises an antibody or antigen-binding fragment thereof that binds to HER3 and / or EGFR, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region.

[0192] In some embodiments, the heavy chain variable region comprises CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 5, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 6, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 8, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 9, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 10.

[0193] In some embodiments, the heavy chain variable region includes CDR-H1 shown in SEQ ID NO: 5, CDR-H2 shown in SEQ ID NO: 6, and CDR-H3 shown in SEQ ID NO: 7, and the light chain variable region includes CDR-L1 shown in SEQ ID NO: 8, CDR-L2 shown in SEQ ID NO: 9, and CDR-L3 shown in SEQ ID NO: 10.

[0194] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises (1) SEQ ID NO: 1; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 1, preferably conserved amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, or consisting thereof. In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0195] In some embodiments, the light chain variable region of the antibody or its antigen-binding fragment comprises (1) the amino acid sequence shown in SEQ ID NO: 2; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2; or (3) an amino acid sequence comprising one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2, preferably conserved amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, or consisting thereof. In some embodiments, the light chain variable region of the antibody or its antigen-binding fragment comprises or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0196] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2.

[0197] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region. In some embodiments, the heavy chain constant region comprises an immunoglobulin Fc region.

[0198] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises or is composed of a heavy chain variable region and a heavy chain constant region, and the light chain comprises or is composed of a light chain variable region and a light chain constant region. In some embodiments, the heavy chain constant region comprises an immunoglobulin Fc region.

[0199] In some embodiments, the antibody or antigen-binding fragment thereof described in this disclosure further comprises an immunoglobulin Fc region. Preferably, the immunoglobulin Fc region is a human immunoglobulin Fc region, such as the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4.

[0200] In some embodiments, the immunoglobulin Fc region can be a variant of the human immunoglobulin Fc region, such as a variant of the human IgG1 Fc region.

[0201] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises (1) the amino acid sequence shown in SEQ ID NO: 3; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 3; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 3, preferably conserved amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, or composed thereof. In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises or is composed of the amino acid sequence shown in SEQ ID NO: 3.

[0202] In some embodiments, the light chain of the antibody or its antigen-binding fragment comprises (1) the amino acid sequence shown in SEQ ID NO: 4; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 4; or (3) an amino acid sequence comprising one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 4, preferably conserved amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, or consisting thereof. In some embodiments, the light chain of the antibody or its antigen-binding fragment comprises or consists of the amino acid sequence shown in SEQ ID NO: 4.

[0203] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:3, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the antibody or its antigen-binding fragment comprises one heavy chain and one light chain. In some embodiments, the antibody comprises two heavy chains and two light chains, for example, two identical heavy chains and two identical light chains.

[0204] In some embodiments, the antibody comprises the heavy chain shown in SEQ ID NO: 3, and optionally, the light chain shown in SEQ ID NO: 4.

[0205] In other embodiments, the ligand unit comprises an antibody or antigen-binding fragment thereof that binds to HER2. In some embodiments, the antibody or antigen-binding fragment thereof may bind to different epitopes of HER2, for example, it may specifically bind to extracellular domain II and extracellular domain IV of human HER2. As an example, the bispecific antibody is derived from WO2016110267, and its amino acid sequence is as follows:

[0206] In some embodiments, the HER2-binding antibody or its antigen-binding fragment comprises a first heavy chain, a second heavy chain, and a common light chain, wherein the first heavy chain includes a variable region of the first heavy chain, the second heavy chain includes a variable region of the second heavy chain, and the common light chain includes a variable region of the common light chain.

[0207] In some embodiments, the HER2-binding antibody or its antigen-binding fragment comprises a first heavy chain, a second heavy chain, and a common light chain, wherein the variable region of the first heavy chain comprises CDR-H1-1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H1-2 containing the amino acid sequence shown in SEQ ID NO: 18, and CDR-H1-3 containing the amino acid sequence shown in SEQ ID NO: 19; the variable region of the second heavy chain comprises CDR-H2-1 containing the amino acid sequence shown in SEQ ID NO: 20, CDR-H2-2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-H2-3 containing the amino acid sequence shown in SEQ ID NO: 22; and the variable region of the common light chain comprises CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 23, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 24, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 25.

[0208] In some embodiments, the HER2-binding antibody or its antigen-binding fragment comprises a first heavy chain, a second heavy chain, and a common light chain, wherein the variable region of the first heavy chain comprises CDR-H1-1 shown in SEQ ID NO: 17, CDR-H1-2 shown in SEQ ID NO: 18, and CDR-H1-3 shown in SEQ ID NO: 19; the variable region of the second heavy chain comprises CDR-H2-1 shown in SEQ ID NO: 20, CDR-H2-2 shown in SEQ ID: 21, and CDR-H2-3 shown in SEQ ID NO: 22; and the variable region of the common light chain comprises CDR-L1 shown in SEQ ID NO: 23, CDR-L2 shown in SEQ ID NO: 24, and CDR-L3 shown in SEQ ID NO: 25.

[0209] In some embodiments, the variable region of the first heavy chain comprises (1) the amino acid sequence shown in SEQ ID NO: 11; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 11, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, or consisting thereof. In some embodiments, the variable region of the first heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 11.

[0210] In some embodiments, the variable region of the second heavy chain comprises (1) the amino acid sequence shown in SEQ ID NO: 12; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 12; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 12, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, or consisting thereof. In some embodiments, the variable region of the second heavy chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 12.

[0211] In some embodiments, the variable region of the common light chain comprises (1) the amino acid sequence shown in SEQ ID NO: 13; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 13; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 11, preferably conserved amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, or consisting thereof. In some embodiments, the variable region of the common light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 13.

[0212] In some embodiments, the antibody comprises a first heavy chain, a second heavy chain, and two common light chains, wherein the first heavy chain includes a first heavy chain variable region, the second heavy chain includes a second heavy chain variable region, and the common light chains each include a light chain variable region.

[0213] The first heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO: 11;

[0214] The second heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO: 12; and

[0215] The light chain variable region contains or consists of the amino acid sequence shown in SEQ ID NO: 13.

[0216] In some embodiments, the first heavy chain comprises (1) the amino acid sequence shown in SEQ ID NO: 14; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 14; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 14, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, or consisting thereof. In some embodiments, the first heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 14.

[0217] In some embodiments, the second heavy chain comprises (1) the amino acid sequence shown in SEQ ID NO: 15; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 15; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 15, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, or consisting thereof. In some embodiments, the variable region of the second heavy chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 15.

[0218] In some embodiments, the common light chain comprises (1) the amino acid sequence shown in SEQ ID NO: 16; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 16; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 16, preferably conserved amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions, or consisting thereof. In some embodiments, the variable region of the common light chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 16.

[0219] In other embodiments, the antibody comprises a first heavy chain, a second heavy chain, and two common light chains, wherein

[0220] The first heavy chain contains or is composed of the amino acid sequence shown in SEQ ID NO: 14;

[0221] The second chain contains or is composed of the amino acid sequence shown in SEQ ID NO: 15; and

[0222] The common light chain contains or consists of the amino acid sequence shown in SEQ ID NO: 16.

[0223] In some embodiments, in the conjugates of this disclosure, the L2 is linked to the ligand unit via an oligosaccharide, such as an N-glycan. Preferably, the oligosaccharide is derived from a natural glycan of an antibody or its antigen-binding fragment.

[0224] In some embodiments, the oligosaccharide is derived from the N-glycan chain of an antibody or its antigen-binding fragment.

[0225] In some embodiments, the oligosaccharide is composed of 2 to 15 monosaccharides; preferably, the oligosaccharide is composed of 2 to 10 monosaccharides.

[0226] In some embodiments, the oligosaccharide has the structure shown in formula IV-a or formula IV-b:

[0227] Wherein, LU* is a ligand unit as defined above; preferably, LU* is an antibody or its antigen-binding fragment as defined above, for example, it can be the portion remaining after removing the N-glycan chain from the ligand unit or antibody or its antigen-binding fragment.

[0228] GlcNAc is N-acetylglucosamine, Fuc is fuc, Man is mannose, f is 0 or 1, and j is 1 to 20;

[0229] Gal* is a modified galactose selected from the following structures:

[0230] The oligosaccharide is connected to LU* via a core GlcNAc. The core GlcNAc mentioned in this disclosure refers to the leftmost GlcNAc in the structure shown in formula IV-a or IV-b.

[0231] In some implementations, the modified galactose is linked to GlcNAc via a β-1,4-glycosidic bond.

[0232] In some embodiments, the oligosaccharide is linked to the Fc fragment of LU*; preferably to the CH2 domain of the Fc fragment; more preferably to the Asn297 of the Fc fragment (according to the EU index number of Kabat).

[0233] In some specific embodiments, the coupling described in this application has the structure shown in Formula VII:

[0234] Wherein, LU* is a ligand unit as defined above; preferably, LU* is an antibody or its antigen-binding fragment as defined above, for example, it can be the portion remaining after removing the N-glycan chain from the ligand unit or the antibody or its antigen-binding fragment;

[0235] GlcNAc is N-acetylglucosamine, Fuc is fuc, Man is mannose, f is 0 or 1, and j is 1 to 20;

[0236] Gal* is a modified galactose selected from the following structures:

[0237] The core GlcNAc is connected to LU*;

[0238] LP is a structure selected from one of the following (a) to (f):

[0239] (a) and / or

[0240] (b) and / or

[0241] (c)

[0242] (d) and / or

[0243] (e) and / or

[0244] (f) Where k is an integer from 1 to 20, for example, k = 2, 4, 6, 8, 10, 12, 14 or 16.

[0245] In some implementation schemes, j = 1 to 10, for example j = 1 to 8, j = 1.2 to 6, j = 1.5 to 3, j = 1.5 to 2.5.

[0246] In some embodiments, the overall DAR value of the coupling is 1 to 8, for example 1 to 6, for example 2-6, for example 2, 3, 4, 5 or 6. In some embodiments, the overall DAR value of the coupling is 1-8, for example 1 to 6, for example 2-6, for example about 2, about 3, about 4, about 5 or about 6.

[0247] In some embodiments, the average DAR value of the conjugates described in this disclosure is 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 3-6, for example, about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0.

[0248] In some implementations, the modified galactose is linked to GlcNAc via a β-1,4-glycosidic bond.

[0249] In some implementations, the core GlcNAc is connected to the Fc segment of LU*; preferably to the CH2 domain of the Fc segment; more preferably to the Asn297 of the Fc segment (according to the EU index number of Kabat).

[0250] As previously stated, the oligosaccharides in the conjugates disclosed herein can be derived from the natural sugar chains of the antibody. Specifically, the preparation method of the oligosaccharide precursor includes the following steps: reacting an antibody with an N-glycan glycoform mainly of G0F / G0 with UDP-GalNAz or its salt, or with other UDP-GalNAc azide derivatives, in the presence of a catalyst to obtain the aforementioned oligosaccharide precursor.

[0251] The UDP-GalNAz has the following structure:

[0252] In some embodiments, the antibody or antigen-binding fragment thereof in the conjugates suitable for use in this invention is a G0F / G0 glycoform antibody or antigen-binding fragment thereof.

[0253] Methods for obtaining G0F / G0 glycoform antibodies are well known in the art. For example, antibodies expressed by eukaryotic cells (e.g., CHO cells) are post-translational modified to convert the glycan into the G0F / G0 form by galactosidase treatment, which removes any terminal galactose residues and leaves terminal N-acetylglucosamine residues.

[0254] In other embodiments, the starting glycan of the G0F / G0 glycoform described herein can also be obtained by expression and purification using a cell line with the B4GALT1 gene knocked out. One example of knocking out the B4GALT1 gene in a cell line is through homologous recombination technology. Other examples of knocking out the B4GALT1 gene include the use of zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), as described in Nature Biotechnology, volume 33, pages 842-844 (2015). Int. J. Mol. Sci. 2015, 16(10), 23849-23866, etc.

[0255] In some embodiments, the aforementioned catalyst is a galactosyltransferase or a functional variant or fragment thereof.

[0256] In some embodiments, the catalyst is β-1,4-galactosyltransferase or a functional variant or fragment thereof.

[0257] In some embodiments, the catalyst is bovine β-1,4-galactosyltransferase, human β-1,4-galactosyltransferase, or a functional variant or fragment thereof (e.g., an N-terminal truncated fragment, such as one truncated by 62 amino acids at the N-terminus).

[0258] In some embodiments, the fragment of the β-1,4-galactosyltransferase comprises or consists of the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:26.

[0259] In some embodiments, the catalyst is human β-(1,4)-GalT1 with the Y285L mutation, for example, comprising or consisting of the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:27.

[0260] In some embodiments, the catalyst is bovine β-(1,4)-GalT1 with the Y289L mutation, for example, comprising or consisting of the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:27.

[0261] In some embodiments, the catalyst is β-1,4-acetylgalactosyltransferase disclosed in patent application WO2016170186 (which is incorporated herein by reference in its entirety).

[0262] In some embodiments, the catalyst comprises any of the sequences shown in SEQ ID NO: 26-28.

[0263] Composition

[0264] In a second aspect, this disclosure provides a composition, for example, preferably, a pharmaceutical composition, comprising one or a combination of conjugates of this disclosure formulated together with a pharmaceutically acceptable carrier.

[0265] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the conjugate of this disclosure, may be encapsulated in a material to protect the conjugate from acids and other natural conditions that could inactivate it.

[0266] The amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation varies depending on the target population and the specific route of administration. Generally, the amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation is the amount of the composition that produces the therapeutic effect. Typically, this amount, expressed as 100%, ranges from about 0.01% to about 99% of the active ingredient, for example, from about 0.1% to about 70%, or from about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0267] The actual dose level of the active ingredient in the pharmaceutical compositions disclosed herein may be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response in a particular patient, composition, and route of administration, without toxicity to the patient. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the specific conjugate of this disclosure or its salt applied, the route of administration, the time of administration, the excretion rate of the specific conjugate applied, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific conjugate applied, the age, sex, weight, condition, general health status, and medical history of the patient being treated, and similar factors known in the medical field.

[0268] The compositions of this disclosure can be administered via one or more routes of administration using one or more methods known in the art. Those skilled in the art will understand that the route and / or manner of administration varies depending on the desired outcome. Preferred routes of administration for the conjugates of this disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteric and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.

[0269] In some embodiments, this disclosure provides a composition comprising the conjugate described herein, and optionally a pharmaceutically acceptable carrier. Preferably, the DAR value of the composition, for example, an average DAR value of 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 3-6, 6-10, such as 1.0-8.0, 2.0-6.0, 3.0-5.0, 3.5-4.0, such as approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0.

[0270] This disclosure also provides pharmaceutical combinations or combination products or kits comprising conjugates of this disclosure or pharmaceutical compositions thereof, and one or more other therapeutic agents, such as any therapeutic agent effective for treating and / or preventing tumors or cancers, such as cytotoxic agents, chemotherapeutic agents, small molecule compounds, angiogenic inhibitors, other immunotherapeutic agents such as other antibodies, or other conjugates, etc.

[0271] When this disclosure refers to "drug combination" or "combination product," it includes, but is not limited to, a pillbox or a pharmaceutical composition. Drug combination encompasses both non-fixed and fixed combinations. The term "non-fixed combination" refers to the simultaneous, unspecified, or sequential administration of active ingredients in separate entities at the same or different time intervals. The term "fixed combination" means that two or more active agents are administered simultaneously to a patient in the form of a single entity. When referring to "drug combination" or "combination product," it also covers situations where two active ingredients are present in different formulations but are administered in combination.

[0272] Treatment methods and uses

[0273] Thirdly, this disclosure also provides a method for treating and / or preventing tumors or cancers, such as solid tumors or non-solid tumors, comprising administering the conjugate of this disclosure or a pharmaceutical composition thereof to a patient in need. This application also provides a method for treating and / or preventing tumors or cancers, such as advanced or metastatic solid malignancies, comprising administering the conjugate of this disclosure or a pharmaceutical composition thereof to a patient in need.

[0274] In some embodiments, the conjugates or pharmaceutical compositions thereof disclosed herein may also be administered in combination with one or more therapies. Therapies may be any treatment or prevention effective in tumors or cancers, such as treatments (e.g., radiation therapy or surgery), or other therapeutic agents such as cytotoxic agents, chemotherapeutic agents, small molecule compounds, angiogenic inhibitors, other immunotherapeutic agents such as other antibodies, or other conjugates.

[0275] The descriptions of "administering a combination of drugs" or "combining drugs" in this disclosure include both cases of simultaneous administration of multiple drugs and cases of sequential or separate administration of multiple drugs. When administered sequentially, the interval between administrations of the multiple drugs shall not exceed 24 hours, for example, not exceeding 18 hours, not exceeding 15 hours, not exceeding 12 hours, not exceeding 10 hours, not exceeding 8 hours, not exceeding 5 hours, not exceeding 3 hours, not exceeding 2 hours, not exceeding 1 hour, or not exceeding 0.5 hours.

[0276] On the other hand, this disclosure also relates to the use of the conjugate in the preparation of a medicament for the treatment and / or prevention of tumors. In some embodiments, the tumor includes solid tumors and / or non-solid tumors.

[0277] In some embodiments, the tumor is cancer. In some embodiments, the cancer is in the early, middle, or late stage, or it is metastatic.

[0278] In some embodiments, when the ligand unit of the conjugate contains an antibody or antigen-binding fragment thereof that specifically binds to a tumor-associated antigen (TAA), the tumor or cancer is a TAA-positive tumor or cancer.

[0279] In some implementations, the TAA is selected from EGFR, HER3, or HER2.

[0280] In some embodiments, the TAA-positive tumor or cancer refers to an individual suffering from the tumor or cancer that contains abnormally expressed TAA or abnormal TAA activity. In some embodiments, the individual has elevated levels of TAA-encoding nucleic acid or elevated levels of TAA protein expression or activity compared to a control, for example, the control being the level of TAA-encoding nucleic acid or the protein expression or activity of TAA in a healthy individual or healthy tissue or cells. In some embodiments, the individual's biological sample (e.g., tumor cells or tumor tissue) contains (e.g., elevated levels, such as nucleic acid or protein levels or activity) TAA (e.g., compared to a biological sample from a healthy subject (e.g., corresponding tissue or cells in a healthy subject), or compared to TAA in adjacent healthy tissue or cells of the subject).

[0281] In some implementations, the tumor or cancer is selected from skin cancer, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancies, head and neck cancer, glioma, stomach cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, endometrial cancer, and osteosarcoma, etc.

[0282] compound

[0283] Fourthly, this disclosure also relates to compounds of formula V, and their salts and solvates:

[0284] In this context, SP1 is a first spacer unit as defined in the first aspect, L1 is a dipeptide linker as defined in the first aspect, SP2 is a second spacer unit as defined in the first aspect, and L2' is a linker unit used to connect with the ligand unit.

[0285] In some embodiments, the dipeptide linker can be cleaved by cathepsins; preferably, the cathepsins are selected from cathepsins A, B, C, D, E, F, G, H, K, L1, L2, O, S, W and Z.

[0286] In some embodiments, the compound of formula V has the structure shown in formula VI:

[0287] Wherein, AA'2 is as defined in the first aspect. For example, AA'2 may be selected from Ala, Lys, Cit, Gly, Arg, Gln, Asn, Ser, His, Glu, Thr and Asp, preferably from Ala, Lys, Cit, Gly, Arg and Glu, more preferably from Ala, Lys and Cit, and even more preferably from Ala.

[0288] In some embodiments, in compounds of formula V and formula VI, L2' is selected from:

[0289] Where Ar represents C 6-10 Aryl groups, which are optionally coated with halogens, C 1-6 Alkyl substitution; R4 is selected from hydrogen, halogen, and C. 1-6 Alkyl group; Z is selected from straight bond, C 2-6 Ethyne group, C 2-6 imidene group, C 6-10 aryl, 5-10 heteroaryl, amide, sulfonamide, imino, and CF2; m is an integer from 1 to 6; X is a halogen.

[0290] In some embodiments, the compound of formula V has a structure selected from 1)-8):

[0291] 1)

[0292] 2)

[0293] 3)

[0294] 4)

[0295] 5)

[0296] 6)

[0297] 7)

[0298] 8) Where k is an integer from 1 to 20, for example, k = 2, 4, 6, 8, 10, 12, 14 or 16.

[0299] On the other hand, this disclosure also relates to compounds represented by formula VIII, and their salts and solvates:

[0300] Wherein, R5 is selected from hydrogen or a protecting group; preferably, the protecting group is selected from fluoreneoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), p-methoxybenzyl (PMB), benzyl (Bn), triphenylmethyl (Trt), p-toluenesulfonyl (Tos), phthaloyl (Pht), and allyloxycarbonyl (Alloc).

[0301] In some embodiments, the structure of the compound of formula VIII is as shown in formula VIII-a or VIII-b:

[0302] It should be understood in the art that, when referring to the compounds or conjugates mentioned above, references may also include compounds substituted with deuterium or tritium. “Deuterated” means that hydrogen atoms in the molecule are replaced by deuterium, for example, one or more hydrogen atoms, such as 1 to 10 hydrogen atoms, are replaced by deuterium or tritium.

[0303] Example

[0304] 1. Preparation of compounds

[0305] abbreviation

[0306] The following abbreviations have the following meanings: ACN represents acetonitrile; BF3Et2O represents boron trifluoride ether; CHO cells represent Chinese hamster ovary cells; DAR represents drug:antibody ratio; DIEA represents diisopropylethylamine; DMF represents dimethylformamide; DMSO represents dimethyl sulfoxide; ESI-MS represents electrospray ionization mass spectrometry; HATU represents 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; LCMS represents liquid chromatography-mass spectrometry; PEG represents polyethylene glycol; RT represents retention time; SEC represents volume exclusion chromatography; TFA represents trifluoroacetic acid; TSTU represents N,N,N',N'-tetramethyl-O-(N-succinimide)urea tetrafluoroborate.

[0307] General chromatographic analysis method (1): Mobile phase A: water (0.01% TFA) B: acetonitrile (0.01% TFA); gradient: from 5% to 95% B within 1.3 min; flow rate: 0.8 mL / min; column: Poroshell 120EC-C18, 2.1*50 mm, 1.9 μm; column temperature: 45℃. Analytical method (2): Mobile phase A: water (0.01% TFA) B: acetonitrile (0.01% TFA); gradient: from 5% to 95% B within 3 min; flow rate: 0.8 mL / min; column: Poroshell 120EC-C18, 2.1*50 mm, 1.9 μm A-RP-749; column temperature: 45℃. Analytical Method (3): Mobile phase A: Water (0.01% TFA) B: Acetonitrile (0.01% TFA); Gradient: from 5% to 95% B over 4 min; Flow rate: 0.8 mL / min; Column: Poroshell 120EC-C18, 2.1*50 mm, 1.9 μm; Column temperature: 45℃. Analytical Method (4): Mobile phase A: Water B: Acetonitrile; Gradient: from 5% to 95% B over 0.6 min; Flow rate: 0.8 mL / min; Column: Poroshell 120EC-C18, 2.1*50 mm, 1.9 μm A-RP-530; Column temperature: 45℃.

[0308] 1.1 Synthesis of LP1

[0309] 1.1.1 Synthesis of CPT24

[0310] 10,11-Methylenedioxycamptothecin (CAS: 135415-73-5) (3 g, 7.65 mmol, 1.0 eq) was dissolved in methanol (80 mL) and water (70 mL). The reaction solution was cooled to 0 °C, and 75% H2SO4 (60 mL) was added, followed by FeSO4·7H2O (6.38 g, 22.95 mmol, 3.0 eq). 30% H2O2 (15 mL) was slowly added dropwise at 0 °C. The reaction solution was stirred at room temperature for 16 h. LCMS analysis showed that the reaction was complete, and the reaction was stopped.

[0311] The reaction solution was poured directly into ice water, filtered, and the filter cake was dried to obtain 3.1 g of crude yellow-brown solid. The crude product was slurried with DMF to obtain 1.2 g of yellow solid compound CPT24, with a yield of 37%. Analytical method (1): RT = 0.80 min. ESI-MS (+) m / z = 423.1 [M+H].

[0312] 1.1.2 Synthesis of A1

[0313] CPT24 (500 mg, 1.184 mmol, 1.0 eq) and (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazaundecane-11-ylacetate (CAS: 2505045-86-1) (683 mg, 1.421 mmol, 1.2 eq) were dissolved in 10 mL DMSO, and BF3Et2O (500 mg, 3.552 mmol, 3.0 eq) was added. The reaction mixture was allowed to react overnight at room temperature. The reaction solution was quenched in an ice-cold aqueous solution of NaHCO3, resulting in the formation of a large amount of solid. The mixture was filtered, the filter cake was washed with water, and the filter cake was lyophilized to obtain 500 mg of crude yellow solid compound A1. The crude product was used directly in the next reaction. Analysis method (1): RT = 1.11 min. ESI-MS (+) m / z = 844.2 [M+H].

[0314] 1.1.3 Synthesis of A2

[0315] Compound A1 (3.0 g, 3.55 mmol, 1.0 eq) was dissolved in DMF (6 mL), and triethylenediamine (3.99 g, 35.55 mmol, 10 eq) was added. After addition, the mixture was stirred at room temperature for 3 h. LCMS showed that the reaction was complete. The reaction solution was directly and rapidly separated by column chromatography (RP flash chromatography) (ACN in H2O 30%, 0.1% TFA). The solution was lyophilized to obtain 220 mg of yellow solid compound A2. The overall yield of the two steps was 29%. Analytical method (2): RT = 0.98 min. ESI-MS (+) m / z = 622.2 [M+H].

[0316] 1.1.4 Synthesis of LP1

[0317] Compound A2 (100 mg, 94.15 μmol, 1.0 eq) was dissolved in DMF (6 mL). DBCO-PEG4-COOH (CAS: 1537170-85-6) (35 mg, 0.053 mmol, 1.2 eq), DIEA (61 mg, 0.47 mmol, 5 eq), and HATU (47 mg, 0.122 mmol, 1.3 eq) were added under ice bath conditions. The reaction was allowed to proceed at room temperature for 1 h. LC-MS indicated the reaction was complete. The reaction solution was filtered and purified by high-performance liquid chromatography (Ultimate XB-C18, 50*250 mm, 10 μm, 0.1% FA, ACN in H2O 35%–65%). The prepared solution was lyophilized to obtain a yellow solid compound LP1 (55 mg), yield 50%, purity 98%. Analysis method (3): RT = 2.29 min. ESI-MS (+) m / z = 1156.4 [M+H].

[0318] 1 H NMR (400MHz, DMSO): δ8.83(t,J=6.3Hz,1H),8.13(d,J=6.5Hz,1H),7.80(d,J=8.9Hz,1H),7.74(t,J=5.9Hz,1H),7.69–7.64(m,1H),7.60(d,J=6.7Hz,1 H),7.52(s,1H),7.48(d,J=6.2Hz,2H),7.46–7.43(m,1H),7.39–7.30(m,2H ),7.30–7.26(m,1H),7.25(s,1H),6.48(s,1H),6.29(s,2H),5.42(s,2H),5 .29(d,J=5.2Hz,2H),5.10–4.97(m,3H),4.82–4.72(m,2H),4.24–4.15(m,2 H),3.61–3.54(m,3H),3.50–3.40(m,12H),3.27(d,J=6.2Hz,1H),3.12–2.9 7(m,2H),2.62–2.52(m,2H),2.46–2.34(m,2H),2.27–2.17(m,1H),2.08–1. 59(m,5H),1.18(d,J=7.2Hz,3H),0.90–0.82(m,6H),0.79(d,J=6.8Hz,3H).

[0319] 1.2 Synthesis of LP2

[0320] 1.2.1 Synthesis of I2-1

[0321] DBCO-NHS ester (CAS: 1353016-71-3) (400 mg, 1.0 mmol, 1.0 eq) and NH2-PEG8-COOH (CAS: 756526-04-2) (440 mg, 1.0 mmol, 1.0 eq) were dissolved in DMF. Then, DIEA (390 mg, 3.0 mmol, 3.0 eq) was added to the reaction solution. The reaction was carried out at room temperature for 2 hours. LCMS showed that the reaction was complete. The solution was rapidly filtered through a column (0.1% formic acid system, mobile phase: acetonitrile / water). The prepared solution was lyophilized to obtain 500 mg of colorless oily compound I2-1, with a yield of 68.7%. Analytical method (1): RT = 1.00 min. ESI-MS (+) m / z = 729.3 [M+H].

[0322] 1.2.2 Synthesis of I2-2

[0323] Compound I2-1 (100 mg, 0.137 mmol, 1.0 eq) and TSTU (83 mg, 0.275 mmol, 2.0 eq) were dissolved in 2 mL of DMF. DIEA (53 mg, 0.411 mmol, 3.0 eq) was added at room temperature, and the reaction was allowed to proceed for 2 hours at room temperature. LC-MS showed that the reaction was complete. The solution was rapidly passed through a column (0.1% formic acid system, mobile phase: acetonitrile / water), and lyophilized to give 80 mg of white solid compound I2-2, yield 70.8%. Analytical method (1): RT = 1.05 min. ESI-MS (+) m / z = 826.3 [M+H].

[0324] 1.2.3 Synthesis of LP2

[0325] Compound A2 (40 mg, 0.064 mmol, 1.0 eq) and compound I2-2 (52.8 mg, 0.064 mmol, 1.0 eq) were dissolved in 1 mL DMF, and DIEA (25 mg, 0.193 mmol, 3.0 eq) was added. The mixture was reacted at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was purified by high performance liquid chromatography (Xtimate C18, 21.2*250 mm, 5 μm, 0.1% TFA, ACN in H2O from 30% to 70%). The prepared solution was lyophilized to give 31 mg of white solid compound LP2, yield: 36%. Analytical method (1): RT = 1.08 min. ESI-MS (+) m / z = 1332.5 [M+H].

[0326] 1H NMR (400MHz, DMSO) δ8.85(t,J=6.7Hz,1H),8.16(d,J=6.9Hz,1H),7.83(d,J=8.8Hz,1H),7.77(t,J=5.7Hz,1H),7.70-7.68(m,1H),7.63(d,J= 6.4Hz,1H),7.54(s,1H),7.53–7.44(m,4H),7.21-7.30(m,3H),7.27(s ,1H),6.51(s,1H),6.31(s,2H),5.44(s,2H),5.40–5.23(m,2H),5.13–4 .98(m,3H),4.83-4.76(m,2H),4.25-4.20(m,2H),3.63-3.58(m,3H),3 .52–3.44(m,28H),3.15–3.05(m,2H),2.65-2.55(m,1H),2.48-2.43(m, 1H),2.43-2.33(m,2H),2.29-2.21(m,1H),2.06–1.93(m,2H),1.92-1. 84(m,2H),1.82-1.73(m,1H),1.20(d,J=7.1Hz,3H),0.92–0.79(m,9H).

[0327] 1.3 Synthesis of LP3

[0328] 1.3.1 Synthesis of I3-1

[0329] Compound I1-2 (CAS: 1427004-19-0) (400 mg, 0.616 mmol, 1.0 eq) and glycyl-glycyl-L-phenylalanine (CAS: 6234-26-0) (172 mg, 0.616 mmol, 1.0 eq) were dissolved in 6 mL of DMF. Then, DIEA (159 mg, 1.232 mmol, 2.0 eq) was added to the reaction solution. The reaction was carried out at room temperature for 1 hour. LC-MS showed that the reaction was complete. The solution was rapidly filtered through a column (0.1% formic acid system, mobile phase: acetonitrile / water). The solution was lyophilized to obtain 200 mg of white solid I3-1, yield: 40%. Analytical method (1): RT = 1.00 min. ESI-MS (+) m / z = 814.3 [M+H].

[0330] 1.3.2 Synthesis of A3

[0331] CPT24 (500 mg, 1.184 mmol, 1.0 eq) and methyl 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl)acetyl) 500 mg,"" 523 mg, 1.421 mol, 1.2 eq) ester) was dissolved in 10 mL DMF. BF3 Et2O (1 mg, 7.104 mmol, 6.0 eq) was added at room temperature and the reaction was allowed to proceed overnight at room temperature. The reaction solution was then quenched in an ice-cold NaHCO3 aqueous solution, resulting in the formation of a large amount of solid. The solid was filtered, the filter cake was washed with water, and the filter cake was lyophilized to give 400 mg of a yellow solid compound A3, with a yield of 40%. Analytical method (1): RT = 1.02 min. ESI-MS (+) m / z = 731.2 [M+H].

[0332] 1.3.3 Synthesis of A4

[0333] Compound A3 (500 mg, 0.685 mmol, 1.0 eq) was dissolved in 10 mL of DMF, and triethylenediamine (767 mg, 6.85 mmol, 10 eq) was added. After the addition was complete, the mixture was stirred at room temperature for 16 h. LC-MS showed that the reaction was complete. The solution was then rapidly passed through a column (0.1% TFA system, mobile phase: water / methanol), and the prepared solution was lyophilized to obtain 60 mg of compound A4. Analytical method (1): RT = 0.70 min. ESI-MS (+) m / z = 509.1 [M+H].

[0334] 1.3.4 Synthesis of LP3

[0335] Compound A4 (25 mg, 0.049 mmol, 1.0 eq), compound I3-1 (40 mg, 0.049 mmol, 1.0 eq), and COMU (CAS: 1075198-30-9) (23 mg, 0.054 mmol, 1.1 eq) were dissolved in 1 mL of DMF. 2,6-Dimethylpyridine (16 mg, 0.147 mmol, 3.0 eq) was added, and the reaction was carried out at room temperature for 1 hour. LC-MS showed that the reaction was complete. The solution was purified by high-performance liquid chromatography (Xtimate C18, 21.2*250 mm, 5 μm, 0.1% FA, ACN in H2O from 30% to 70%). The prepared solution was lyophilized to obtain 12 mg of white solid, yield: 18.8%. Analytical method (4): RT = 0.69 min. ESI-MS (+) m / z = 1304.4 [M+H].

[0336] 1H NMR (400MHz, DMSO) δ8.77(t,J=6.8Hz,1H),8.40(t,J=5.9Hz,1H),8.19-8.15(m,2H ),8.03(t,J=5.7Hz,1H),7.77(t,J=5.4Hz,1H),7.71–7.66(m,1H),7.63(d,J=7.1H z,1H),7.55(s,2H),7.53–7.43(m,4H),7.42–7.28(m,4H),7.27(s,1H),7.25–7.19 (m,4H),7.18–7.10(m,1H),6.51(s,1H),6.29(s,2H),5.48–5.37(m,2H),5.35–5.2 0(m,2H),5.09–5.00(m,3H),4.79(d,J=6.5Hz,2H),4.59–4.50(m,1H),3.85–3.74( m,3H),3.71(d,J=5.7Hz,2H),3.67-3.58(m,4H),3.51–3.43(m,14H),3.14–3.01(m ,3H),2.85-2.79(m,1H),2.59-2.51(m,1H),2.39(t,J=6.5Hz,2H),2.2-2.21(m,1H ),2.06–1.95(m,1H),1.91-1.74(m,2H),1.83–1.72(m,1H),0.90(t,J=7.3Hz,3H).

[0337] Furthermore, LP4 was prepared according to the process described in patent application CN113264983A:

[0338] 2. Preparation and characterization of antibodies and ADCs

[0339] 2.1 Construction of eukaryotic expression vectors

[0340] Based on the Duligotuzumab (MEHD7945A, hereinafter referred to as MEHD, EGFR×HER3 bispecific antibody) sequence published in WHO Drug Information, Vol. 28, No. 3, 2014 Recommended INN: List 72 and the KN026 (HER2×HER2 bispecific antibody) sequence published in patent WO2016110267, genes were synthesized and cloned into the eukaryotic expression vector pCDNA3.4 to obtain the eukaryotic expression vectors pCDNA3.4-MEHD and pCDNA3.4-KN026, respectively. The variable region of the negative control antibody (anti-HEL antibody, VL and VH as shown in SEQ ID NO: 29 and 30, respectively) was fused with the human IgG1 constant region and then cloned into the eukaryotic expression vector pCDNA3.4 to obtain the eukaryotic expression vector of Iso-IgG1, namely pCDNA3.4-Iso-IgG1.

[0341] 2.2 Antibody Expression and Purification

[0342] For a 100ml transient transduction system, the CHO cell density was first adjusted to 6×10⁶ cells / mL using expression culture medium. 6 Cells / mL were prepared for use. PEI and plasmid (150ug) were mixed at a mass ratio of 6:1 and allowed to react at room temperature for 5 minutes to form a complex. This complex was then added to the prepared cell suspension and incubated at 36.5±0.5℃, 7%±3% CO2, and 100±10rpm for 4 hours. The incubation speed was then adjusted to 130±10rpm and continued for 24 hours. After that, the temperature was lowered to 32℃ and incubated for another 10 days, with inhibitors and feed added as needed.

[0343] After transient cell culture, the cells were centrifuged at 4000 rpm for 10 min at 4℃, and the cell supernatant was collected. Supernatant particles were removed using a 0.22 μm filter membrane. The treated supernatant was then loaded onto a Protein A affinity column (Mabselect SuRe) equilibrated with 10 mM PB (pH 6.0). TM Unadsorbed impurities were removed by rinsing with the same buffer solution, and weakly adsorbed impurities were washed off with a high-salt buffer (25 mM PB, 500 mM NaCl, pH 7.0). The target protein was eluted with elution buffer (20 mM citrate buffer, pH 3.6), and the pH of the eluted protein was adjusted to 7.0 with neutralization buffer (2 M Tris-HCl, pH 9.5). The purified fractions were then analyzed by electrophoresis.

[0344] 2.3 Preparation and Characterization of ADC

[0345] Mix the antibody (mainly the G0 / G0F glycoform), the substrate UDP-GalNAz (purchased from Qingdao SugarWise Medical Technology Co., Ltd.), GalT1 (expressed and purified by adding a His tag to the N-terminus of the sequence shown in SEQ ID NO: 26), and MnCl2 into Tris-HCl buffer, adjust the pH to between 7.30 ± 0.1, and react overnight at 25 - 30 °C to obtain the intermediate product antibody-(N3)4. Then, mix it with DMSO, LP1 / LP2 / LP3 / LP4, adjust the pH to 5.1 ± 0.2, and react overnight at 25 - 30 °C to obtain the ADC sample. Taking ADC1 as an example, the preparation process is shown in Figure 1; the structural compositions of each ADC sample are shown in Table 1.

[0346] SEC detection: After diluting the sample with phosphate solution, perform isocratic separation using a TOSOH G3000 SWxl chromatographic column. At a wavelength of 280 nm, calculate the sample purity by the peak area normalization method. The purity of each ADC sample is above 90%.

[0347] Average DAR value detection: After diluting the sample with ultrapure water, perform separation using a Waters / ACQUITY UPLC Protein BEH C4 chromatographic column. At a wavelength of 280 nm, detect the molecular weight by retention time and peak area, calculate the coupling number, calculate the percentage content of each component according to the percentage content calculation formula using the response, and then calculate the DAR value based on the content of each component and the coupling number. As shown in Table 1, the DAR values of each ADC sample are above 3.6.

[0348] Table 1. Results of the structural composition and average DAR value of ADC molecules

[0349] 2.4 ADC hydrophobicity detection

[0350] After diluting the sample with buffer (about 0.4 M ammonium sulfate), perform gradient separation using a TOSOH TSKgel HIC-ADC Butyl chromatographic column, detect at 280 nm, and confirm the hydrophobicity of the sample by retention time. The results are shown in Figure 2. Taking the MEHD naked antibody as a reference, the hydrophobicity from weak to strong is: ADC1 < ADC3 < ADC4, and the peak shape of ADC3 is not good.

[0351] 3. Killing effect of ADC on tumor cells

[0352] (1) HCC827 (purchased from Nanjing Kebai Biotechnology Co., Ltd., growth medium: RPMI-1640 + 10% FBS) and MDA-MB-468 (purchased from Beina Chuanglian Biotechnology Co., Ltd., growth medium: Leibovitz's L-15 + 10% FBS) tumor cells were digested with trypsin. Cells were collected in centrifuge tubes, resuspended in the corresponding growth medium, and 2000-5000 cells / well were added to 96-well plates and cultured overnight. The next day, the corresponding growth medium was used to dilute ADC, and 50 μL was added to the 96-well cell plate (the final concentration started from 100 nM, with 4-fold serial dilutions (8 concentration gradients plus zero point)). The cells were cultured at 37℃ and 5% CO2 for 96 h. The cell culture plate was then removed, and each well was added with... The Luminescent Cell Viability Assay (Promega, G7571) reagent (50 μL) was used to detect fluorescence intensity and calculate the ADC's killing ability against tumor cells. As shown in Table 2, the two ADCs showed comparable killing effects on the two types of tumor cells.

[0353] Table 2. Killing effects of ADCs on HCC827 and MDA-MB-468 cells

[0354] The sub-nanomolar IC50 value of ADC1 is surprising, as the ADC molecule (Ag1-Ex_6-1) with CPT24 as the toxin molecule, as disclosed in patent publication WO2019195665, exhibits a weaker cytotoxic effect. To verify whether this difference in activity is due to the choice of linker unit, we selected ADCs with different PEG chain lengths and different enzyme digestion units to test their cytotoxic activity. The results are shown in Table 3. The PEG chain length has no effect on ADC activity, and the in vitro activity of ADC1 linked with VA is an order of magnitude higher than that of ADC3 linked with GGFG.

[0355] Table 3. Killing effect of different ADCs on MDA-MB-468 cells

[0356] (2) Using a method similar to that in (1), the killing effect of ADC molecules on A431 tumor cells (purchased from Beinan Chuanglian Biotechnology Co., Ltd., with a growth medium of 90% DMEM-H + 10% FBS) was tested. The results are shown in Figure 6. In this tumor cell, the killing effect of ADC1 was stronger than that of ADC4, indicating that the ADC molecules disclosed herein can still play a role in some Dxd-insensitive tumor cell lines.

[0357] 4. The spectator damage effect of ADCs

[0358] (1) MDA-MB-468 tumor cells and 293T-GFP cells (a GFP-overexpressing pCDNA3.4 plasmid was constructed, and the plasmid was stably transfected into 293T cells using Lipo3000 transfection reagent. Single clones were obtained by limiting dilution and then identified by flow cytometry before library construction) were digested with trypsin. Cells were collected in centrifuge tubes and resuspended in culture medium (DMEM + 10% FBS). 96-well plates were seeded with 6000 MDA-MB-468 cells and 3000 293T-GFP cells per well and cultured overnight. The next day, ADC was diluted with culture medium (DMEM + 10% FBS) to a final concentration of 50 nM and 5 nM, and 50 μL was added to the 96-well cell plate. A zero-concentration control was also set up. Cells were cultured at 37℃ and 5% CO2 for 72 h. Afterward, the cell culture plates were removed, cells were digested with trypsin, counted, and analyzed by flow cytometry. Flow cytometry was used to distinguish MDA-MB-468 cells and 293T-GFP cells by displaying positive and negative GFP signals. The cell counts of each cell type were calculated based on the total cell count and the percentage of each type. Finally, the bystander killing ability of ADCs against 293T-GFP cells was calculated. The results, shown in Figure 3, indicate that ADC1 exhibited significantly stronger bystander killing activity against 293T-GFP cells than ADC4 with the GGFG-Dxd combination.

[0359] (2) MDA-MB-468 tumor cells and 293T-Luc2 cells (constructed using a similar method, but with the fluorescent element replaced by Luc2) were digested with trypsin. Cells were collected in centrifuge tubes and resuspended in culture medium (DMEM + 10% FBS). 96-well plates containing 6000 MDA-MB-468 cells and 3000 293T-Luc2 cells per well were seeded and cultured overnight. The next day, the ADC was diluted with culture medium (DMEM + 10% FBS) and 50 μL was added to each well of the 96-well cell plate (starting at 200 nM, with 4-fold serial dilutions (8 concentration gradients plus a zero point)). The plates were incubated at 37°C and 5% CO2 for 72 h. The cell culture plates were then removed, and 50 μL of Bio-Glo luciferase Assay system (Promega, G7940) reagent was added to each well. Fluorescence intensity was measured, and the bystander killing ability of different ADCs on 293T-Luc2 cells was calculated. As shown in Figure 4, ADC1 and ADC2 exhibited strong bystander killing effects on 293T-Luc2, while ADC3 and ADC4 showed weaker killing effects at all concentrations than the former two.

[0360] 5. Antitumor effect of ADC on NCI-N87 human gastric cancer xenograft model

[0361] The human gastric cancer NCI-N87 xenograft model in nude mice was established by subcutaneously inoculating nude mice (Vitalliwa, Certificate No.: 20240223Abzz0619000214) with NCI-N87 cells (purchased from Beinan Chuanglian Biotechnology Co., Ltd., growth medium: RPMI-1640 + 10% FBS) at a concentration of 5 × 10⁻⁶ cells. 6 Cells were suspended in PBS solution, mixed with matrix gel at a 1:1 ratio, and subcutaneously in the right scapular region of nude mice. The long and short diameters of the xenograft were measured using calipers, and the result was calculated as V = 1 / 2 × long diameter × short diameter. 2 Calculate the tumor volume V, and wait until the tumor grows to 200 mm. 3 The animals were randomly divided into two groups of six mice each. One group received an intraperitoneal injection of ADC5, while the other group received a negative control (group 2). The dosage was 10 mg / kg, and the administration volume was 200 μL. A single administration was given, and tumor volume and mouse weight were measured twice weekly. The results are shown in Figure 5. Even after administration to the negative control (group 2), tumor volume continued to increase, indicating that ADC5 had a significant inhibitory effect on tumor growth.

[0362] 6. Identification of ADC metabolites in tumor cells

[0363] ADC1 (10 μg / mL) was incubated with HCC827 tumor cells at 37°C for 24 and 48 hours, respectively, and the cell supernatant and cell samples were collected after incubation. Simultaneously, ADC1 (10 μg / mL) was incubated with a culture system without HCC827 cells at 37°C for 24 and 48 hours, and the culture supernatant samples were collected as negative controls. The incubated samples were analyzed using LC-UV-MS. Structural determination and identification were performed by comparing the primary and secondary mass spectrometry signals of each metabolite with those of the reference compound.

[0364] The results showed that one metabolite, CPT24 (m / z = 423.1188), was detected in HCC827 cells after 24 and 48 hours of incubation, with a relative abundance of 100.00% in both cases. Simultaneously, CPT24 was also detected in the cell supernatant after 24 and 48 hours of incubation, with a relative abundance of 100.00% in both cases; compared to the negative control sample, the mass spectrometry peak area of ​​CPT24 in the cell supernatant was more than 47 times larger. These results indicate that CPT24 is a metabolite of ADC1 in HCC827 tumor cells, and is further released into the cell supernatant.

[0365] 7. Stability study of ADC in serum of different species

[0366] Mouse, rat, cynomolgus monkey, and human serum, as well as phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA), were mixed with ADC1 and incubated in a 37°C water bath for 0, 3, 7, 14, and 21 days, respectively. The incubation concentration of ADC1 was 100 μg / mL. The release rate of CPT24 loading was determined by LC-MS / MS for all analytes.

[0367] As shown in Figure 7, after prolonged incubation, the release rate of CPT24 in the serum of various species was very low, indicating that the ADC disclosed in this invention has excellent cyclic stability in vivo.

Claims

1. A coupling having the structure shown in Formula I: in, LU is the ligand unit, SP1 is the first spacer unit, L1 is the dipeptide linker, SP2 is the second spacer unit, L2 is the linker that connects to LU, and n = 1 to 20.

2. The coupling according to claim 1, wherein SP1 is absent, or SP1 is selected from: R1 is independently selected from hydrogen, C 1-6 Alkyl, hydroxyl, amino, halogen, nitro, cyano d is an integer from 1 to 20, and e is an integer from 1 to 20; R2 and R3 are each independently selected from hydrogen and C. 1-6 alkyl; The alkyl group may optionally be substituted with hydroxyl, amino, halogen, nitro, and cyano groups.

3. The coupling according to claim 2, wherein SP1 is selected from:

4. The conjugate according to any one of claims 1-3, wherein L1 is a dipeptide linker that can be cleaved by cathepsins.

5. The coupling according to any one of claims 1-4, wherein L1 is AA1 and AA2 are each an amino acid residue, and AA1 is more hydrophobic than AA2.

6. The conjugate according to claim 5, wherein AA1 is selected from Val, Phe, Leu, Ile and Trp, and AA2 is selected from Ala, Lys, Cit, Gly, Arg, Gln, Asn, Ser, His, Glu, Thr and Asp.

7. The conjugate according to any one of claims 1-6, wherein L1 is selected from the following dipeptide residues: SP2 —Val—Ala— SP1 , SP2 —Val—Lys— SP1 , SP2 —Val—Cit— SP1 , SP2 —Val—Gly— SP1 , SP2 —Val—Arg— SP1 , SP2 —Val—Glu— SP1 , SP2 —Val—Asp— SP1 , SP2 —Phe—Lys— SP1 , SP2 —Phe—Cit— SP1 , SP2 —Phe—Ala— SP1 , SP2 —Phe—Arg— SP1 , SP2 —Leu—Ala— SP1 , SP2 —Leu—Lys— SP1 , SP2 —Leu—Cit— SP1 , SP2 —Ile—Ala— SP1 , SP2 —Ile—Lys— SP1 , SP2 —Ile—Cit— SP1 and SP2 —Trp—Cit— SP1 .

8. The coupling according to any one of claims 1-7, having the structure shown in Formula II: in, AA'2 is selected from Ala, Lys, Cit, Gly, Arg, Gln, Asn, Ser, His, Glu, Thr and Asp, preferably from Ala, Lys, Cit, Gly, Arg and Glu, more preferably from Ala, Lys and Cit, and even more preferably from Ala.

9. The coupling according to any one of claims 1-8, wherein the structure of SP2 is as shown in Formula III: in, a1 = 0 or 1, a2 = an integer from 0 to 6, b1 = 0 or 1, b2 = an integer from 0 to 16, b3 = an integer from 0 to 16, c = an integer from 0 to 6, and at least one of b2 and b3 is 0.

10. The coupling according to claim 9, wherein: (1) a1 = 0, a2 = 2, 3, 4, 5 or 6, b1 = 0, b2 = 0, b3 = 0, c = 0; (2) a1 = 0, a2 = 0, b1 = 0, b2 = 0, b3 = 0, c = 2, 3, 4, 5 or 6; (3) a1 = 1, a2 = 2, 3, 4, 5 or 6, b1 = 1, b2 = 2, 3, 4, 5, 6, 7 or 8, b3 = 0, c = 0; (4) a1 = 0, a2 = 2, 3, 4, 5 or 6, b1 = 1, b2 = 2, 3, 4, 5, 6, 7 or 8, b3 = 0, c = 0; (5) a1 = 1, a2 = 0, b1 = 0, b2 = 0, b3 = 2, 3, 4, 5, 6, 7 or 8, c = 2, 3, 4, 5 or 6; or (6) a1 = 0, a2 = 0, b1 = 1, b2 = 2, 3, 4, 5, 6, 7 or 8, b3 = 0, c = 0.

11. The coupling according to any one of claims 1-10, wherein L2 is selected from: in, Ar represents C 6-10 Aryl groups, which are optionally coated with halogens, C 1-6 Alkyl substitution; R4 is selected from hydrogen, halogen, and C. 1-6 Alkyl group; Z is selected from straight bond, C 2-6 Ethyne group, C 2-6 imidene group, C 6-10 aryl, 5-10 heteroaryl, amide, sulfonamide, imino, and CF2; m is an integer from 1 to 6.

12. The conjugate according to any one of claims 1-11, wherein the ligand unit comprises an antibody or an antigen-binding fragment thereof.

13. The conjugate according to claim 12, wherein L2 is connected to the ligand unit via an oligosaccharide.

14. The conjugate according to claim 13, wherein the oligosaccharide is an N-glycan chain.

15. The conjugate according to claim 13 or 14, wherein the oligosaccharide has the structure shown in formula IV-a or IV-b: in, LU* represents the ligand unit, GlcNAc represents N-acetylglucosamine, Fuc represents fuc, Man represents mannose, f represents 0 or 1, and j represents 1 to 20; Gal* is a modified galactose selected from the following structures: The oligosaccharide is connected to LU* via the core GlcNAc.

16. The conjugate according to claim 15, wherein the modified galactose is linked to GlcNAc via a β-1,4-glycosidic bond.

17. The conjugate according to any one of claims 13-16, wherein the oligosaccharide is linked to the Fc fragment of the ligand unit; preferably linked to the CH2 domain of the Fc fragment; more preferably linked to the Asn297 (according to the EU index number of Kabat) of the Fc fragment.

18. A pharmaceutical composition comprising the conjugate according to any one of claims 1-17, and a pharmaceutically acceptable carrier.

19. A method of treating and / or preventing cancer, comprising administering to a subject the conjugate of any one of claims 1-17 or the pharmaceutical composition of claim 18.

20. The compound shown in formula V, and its salts and solvates: in, SP1 is a first spacer unit as defined in claim 2 or 3, L1 is a dipeptide linker as defined in any one of claims 4-7, SP2 is a second spacer unit as defined in claim 9 or 10, and L2' is a linker unit used to connect with the ligand unit.

21. The compound according to claim 20, and its salts and solvates, having the structure shown in Formula VI: in, AA'2 is as defined in claim 7.

22. The compound according to claim 20 or 21, and its salts and solvates, wherein L2' is selected from: in, Ar represents C 6-10 Aryl groups, which are optionally coated with halogens, C 1-6 Alkyl substitution; R4 is selected from hydrogen, halogen, and C. 1-6 Alkyl group; Z is selected from straight bond, C 2-6 Ethyne group, C 2-6 imidene group, C 6-10 aryl, 5-10 heteroaryl, amide, sulfonamide, imino, and CF2; m is an integer from 1 to 6; X is a halogen.

23. The compounds shown in Formula VIII, and their salts and solvates: in, R5 is selected from hydrogen or a protecting group; preferably, the protecting group is selected from fluoreneoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), p-methoxybenzyl (PMB), benzyl (Bn), triphenylmethyl (Trt), p-toluenesulfonyl (Tos), phthaloyl (Pht), and allyloxycarbonyl (Alloc).

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