Polypeptide, bioactive conjugate, and preparation method therefor and use thereof

Abstract

Disclosed in the present application are a polypeptide and polypeptide fragment, a linker containing the polypeptide and polypeptide fragment, a linker-drug, a drug conjugate, and the use of the linker-drug and drug conjugate in the field of disease treatment, comprising but not limited to the use in the treatment of tumor diseases.

Description

Polypeptides, bioactive conjugates, their preparation methods and applications Technical Field

[0001] This application relates to the field of biomedicine, specifically to a polypeptide, a linker, a linker drug, a bioactive conjugate, a method for preparing the same, and its application in the prevention / treatment of diseases. Background Technology

[0002] Abnormal cellular activity can lead to various diseases, such as cancer, autoimmune diseases, and neurodegenerative diseases. Cancer is characterized by abnormal cell proliferation, and intervening in this abnormal proliferation is a standard treatment method. Using small chemical molecules to treat cancer is currently a widely used approach. However, many cytotoxic drug molecules have limited clinical use due to issues such as lack of target selectivity, unsatisfactory pharmacokinetic properties, or excessive toxicity. This has led to the development of bioactive conjugates (BDCs). BDCs have played a significant role in improving targeting and overcoming the limitations of the drugs themselves, with antibody-drug conjugates (ADCs) becoming a leading example of BDCs in tumor treatment.

[0003] An antibody-drug conjugate (ADC) consists of three parts: an antibody, a linker, and a drug payload. This includes the connection methods between the antibody and the linker, and between the linker and the drug payload. The active component of an ADC is primarily the drug payload. Its mechanism of action involves the specific binding of the antibody and antigen to enrich the drug on the surface of tumor cells, followed by release at the tumor site using specific mechanisms. These release mechanisms include utilizing the tumor-specific environment outside the tumor cells to release the drug, or leveraging the endocytosis effect of tumor cells to internalize the ADC drug into lysosomes. Within the lysosomes, various enzymes release the active drug molecules, thereby exerting biological functions, such as inducing apoptosis and achieving an anti-tumor effect. Therefore, the key to ADC drug development lies in two aspects: firstly, maintaining the stability of the ADC in the general environment of the body, such as its stability in plasma circulation; and secondly, ensuring rapid and efficient release of the ADC upon reaching the disease site.

[0004] The release efficiency of the effective payload within tumor tissue or tumor cells is crucial for unlocking the therapeutic window of ADC drugs. However, balancing the targeted release efficiency of the effective payload within tumor tissue with the overall stability of the ADC drug in the body environment is a major challenge facing current technologies. Traditional ADC drugs, after accumulating in tumor tissue, require endocytosis to transport the drug to lysosomes. The effective payload is released and exerts its therapeutic effect only through the cleavage of the linker by lysosomal enzymes. During this process, the release efficiency is extremely low, typically only around 0.1% of the total drug, due to the influence of tumor cell endocytosis efficiency, endosome-to-lysosome transport efficiency, and endosome or lysosomal efflux. This limitation means that ADCs often select toxins with relatively high cellular activity as effective payloads, such as commonly used MMAE, DM1, and DX8951, whose cell-killing IC50 is usually in the single nM range. Some ADCs even use toxins with IC50 reaching the single pM level, such as PBD and doxorubicin analogs, as payloads.

[0005] On the other hand, employing relatively unstable linkers, allowing ADC drugs to accumulate in tumor tissue while simultaneously dropping off, is another strategy. The advantage of this design is that once the ADC reaches the tumor tissue, it can release its payload to kill tumor cells without undergoing endocytosis and intracellular transport, thus improving payload utilization. However, this design also faces the risk of toxicity due to non-specific payload drop-off in the body environment. Therefore, this type of ADC typically uses compounds with relatively low cytotoxic activity, such as SN38, as the payload in its development.

[0006] Therefore, developing linkers that can maintain relative stability in plasma and efficiently release drug-loaded substances in the tumor microenvironment and / or lysosomes is of great significance for further improving the therapeutic efficacy of bioactive conjugates and enhancing the patient's medication experience. Summary of the Invention

[0007] To improve the therapeutic efficacy of antibody-drug conjugates (ADCs) or other ligand-drug conjugates, reduce drug toxicity, and broaden the therapeutic window, it is crucial to discover linkers that maintain relative stability in plasma and efficiently release drug-loaded components in the tumor microenvironment and / or lysosomes.

[0008] This application designs a series of peptides that respond to both tumor tissue-specific MMP-2 and / or MMP-9 and intracellular Cathepsin B, as well as spacer structures connecting the peptides to antibodies and payloads.

[0009] This application provides a polypeptide or polypeptide fragment L' that is both tumor tissue-specific to MMP-2 and / or MMP-9 and responsive to Cathepsin B in tumor cells. The polypeptide or polypeptide fragment L' has the structure shown in -X1-P-X2-X3-Z-X4-, where P is a proline residue, X1 is selected from the absence of a single amino acid or a single amino acid derivative, X2, X3 and X4 are each independently selected from a single amino acid or a single amino acid derivative, and Z is selected from 1-2 amino acids or amino acid derivatives.

[0010] In some embodiments of this application, X1 is selected from those that do not exist, His (H), Lys (K), Arg (R), Ser (S), Thr (T), Cys (C), Ala (A), Gly (G), Pro (P) and their derivatives;

[0011] The X2 is selected from Arg(R), Cit, Orn, Asn(N), Glu(E), Asp(D), Ala(A), Dab, Lys(K) and their derivatives;

[0012] The X3 is selected from Gly (G), Pro (P), Asn (N), Ser (S), Glu (E), Ala (A), Gln (Q), Cys (C), Asp (D) and their derivatives;

[0013] Z is selected from Leu(L), Val(V), Leu-Val(LV), Ile(I), Ala(A), Phe(F), Dap, Dab, Orn and their derivatives;

[0014] The X4 is selected from Cit, Ala(A), Ser(S), Lys(K), Orn and their derivatives.

[0015] In other embodiments of this application, X1 is selected from non-existent, His, Ser, Gly and their derivatives (e.g., Ser(β-Glc));

[0016] The X2 is selected from Arg, Cit, Orn, Asn, Glu, Asp, Gla, Dab, Lys, Dab(Me2), Dab(Et2), Lys(Me2), Lys(Et2), Orn(Me2), Orn(Et2), Dap(Me2) or Dap(Et2);

[0017] The X3 is selected from Gly, Pro, Asn, Ser, Glu or Ser(β-Glc);

[0018] The X4 is selected from Cit, Ala, Ser(GlcA), Lys(Me2), Lys(NMPRZ), Orn(Me2), Orn(Pr2), Lys(Mor) or Lys(Pr2);

[0019] The Z is selected from Leu, Val, Leu-Val, Ile, Ala, Phe, Hof, N,N-dimethyl-3-aminopropionic acid (Dap(Me2)), N,N-diethyl-3-aminopropionic acid (Dap(Et2)), N,N-dimethyl-4-aminobutyric acid (Dab(Me2)), N,N-diethyl-4-aminobutyric acid (Dab(Et2)), N,N-dimethylornithine (Orn(Me2)), N,N-diethylornithine (Orn(Et2)), N,N-dimethyllysine (Lys(Me2)) or N,N-diethyllysine (Lys(Et2)).

[0020] In some embodiments of this application, X1 is selected from non-existent, His, Ser, Gly and their derivatives;

[0021] The X2 is selected from Arg, Cit, Orn, Asn, Glu, Asp, Gla, Dab, Lys, Dab(Me2), Dab(Et2), Lys(Me2), Lys(Et2), Orn(Me2), Orn(Et2), Dap(Me2), Dap(Et2) or Ala;

[0022] The X3 is selected from Gly, Pro, Asn, Ser, Glu or Ser(β-Glc);

[0023] The X4 is selected from Cit, Ala, Ser(GlcA), Lys(Me2), Lys(NMPRZ), Orn(Me2), Orn(Pr2), Lys(Mor) or Lys(Pr2);

[0024] Z is selected from Leu, Val, Leu-Val, Ile, Ala, Phe, Hof, Dap(Me2), Dap(Et2), Dab(Me2), Dab(Et2), Orn(Me2), Orn(Et2), Lys(Me2) or Lys(Et2).

[0025] In some embodiments of this application, X1 is selected from non-existent, His, Ser, Gly and their derivatives (e.g., Ser(β-Glc));

[0026] The X2 is selected from Arg, Cit, Orn, Asn, Glu, Asp, Gla, Dab, Lys, Dab(Me2), Dab(Et2), Lys(Me2), Lys(Et2), Orn(Me2), Orn(Et2), Dap(Me2), Dap(Et2) or Ala;

[0027] The X3 is selected from Gly, Pro, Asn, Ser, Glu and their derivatives (e.g., Ser(β-Glc));

[0028] The X4 is selected from natural or non-natural amino acids (e.g., Gly, Cit, Ala, Ser (GlcA), Lys (Me2), Lys (NMPR2), Orn (Me2), Orn (Pr2), Lys (Mor) or Lys (Pr2)).

[0029] Z is selected from Leu, Val, Leu-Val, Leu-Phe, Dap(Me2)-Val, Ile, Ala, Phe, Hof, Dap(Me2), Dap(Et2), Dab(Me2), Dab(Et2), Orn(Me2), Orn(Et2), Lys(Me2) or Lys(Et2);

[0030] In some embodiments, when X1 is absent, Z is Leu, Val, Leu-Val, Ile, Dap(Me2), Dap(Me2)-Val, or Hof; in some embodiments, when Z is Dap(Me2), X1 is absent; in some embodiments, when X4 is Ala, X2 is Arg; in some embodiments, when X1 is absent and Z is Leu-Val, X4 is Lys(Me2); in some embodiments, when X2 is Cit and X3 is Asn, Z is Val.

[0031] In some embodiments of this application, X1 is selected from non-existent, His, Ser, Gly and their derivatives;

[0032] The X2 is selected from Arg, Cit, Orn, Asn, Glu, Asp, Gla, Dab, Lys, Dab(Me2), Dab(Et2), Lys(Me2), Lys(Et2), Orn(Me2), Orn(Et2), Dap(Me2), Dap(Et2) or Ala;

[0033] The X3 is selected from Gly, Pro, Asn, Ser, Glu or Ser(β-Glc);

[0034] The X4 is selected from Cit, Ala, Ser(GlcA), Lys(Me2), Lys(NMPRZ), Orn(Me2), Orn(Pr2), Lys(Mor) or Lys(Pr2);

[0035] Z is selected from Leu, Val, Leu-Val, Dap(Me2)-Val, Ile, Ala, Phe, Hof, Dap(Me2), Dap(Et2), Dab(Me2), Dab(Et2), Orn(Me2), Orn(Et2), Lys(Me2), or Lys(Et2); wherein, in some embodiments, when X1 is absent, Z is Leu, Val, Leu-Val, Ile, Dap(Me2), Dap(Me2)-Val, or Hof; wherein, in some embodiments, when Z is Dap(Me2), X1 is absent; wherein, in some embodiments, when X4 is Ala, X2 is Arg; wherein, in some embodiments, when X1 is absent and Z is Leu-Val, X4 is Lys(Me2); wherein, in some embodiments, when X2 is Cit and X3 is Asn, Z is Val.

[0036] In some embodiments of this application, X1 is selected from non-existent, His, Ser, Gly, or Ser(β-Glc);

[0037] The X2 is selected from Arg, Cit, Glu, Lys, Lys(Me2) or Ala;

[0038] The X3 is selected from Gly, Pro, Asn, Ser, Glu or Ser(β-Glc);

[0039] The X4 is selected from Gly, Cit, Ala, Ser (GlcA), Lys (Me2), Lys (NMPRZ), Orn (Me2), Lys (Mor) or Lys (Pr2);

[0040] Z is selected from Leu, Val, Leu-Val, Leu-Phe, Dap(Me2)-Val, Ile, Hof, or Dap(Me2); wherein, in some embodiments, when X1 is absent, Z is Leu, Val, Leu-Val, Ile, Dap(Me2), Dap(Me2)-Val, or Hof; wherein, in some embodiments, when Z is Dap(Me2), X1 is absent; wherein, in some embodiments, when X4 is Ala, X2 is Arg; wherein, in some embodiments, when X1 is absent and Z is Leu-Val, X4 is Lys(Me2); wherein, in some embodiments, when X2 is Cit and X3 is Asn, Z is Val. In some embodiments of this application, X1 is selected from absent, Gly(G), His(H), Ser(S), or Ser(β-Glc);

[0041] The X2 is selected from Cit, Glu (E), Lys (K), Arg (R), and K (Me2);

[0042] The X3 is selected from Glu (E), Gly (G), Asn (N), Ser (S), Ser (β-Glc), and Pro (P);

[0043] Z is selected from Leu(L), Val(V), Leu-Val(LV), Dap(Me2), and Dap(Me2)-Val;

[0044] The X4 is selected from Cit, Ala(A), Lys(K), Lys(Me2), Lys(NMPRZ), Lys(Mor), Lys(Pr2), Orn(Me2), Ser(GlcA);

[0045] In some implementations, X1 does not exist when Z is Dap(Me2).

[0046] In some embodiments of this application, X1 is absent, His, Ser, or Ser(β-Glc);

[0047] X2 is selected from Arg, Lys(Me2), Cit, or Ala;

[0048] The X3 is selected from Gly, Pro, Asn, Ser or Ser(β-Glc);

[0049] Z is Leu-Val(LV); and

[0050] X4 is Cit.

[0051] In some embodiments of this application, X1 is Ser;

[0052] X2 is Cit;

[0053] The X3 is selected from Ser or Gly;

[0054] Z is Leu, Leu-Val, Hof, Dap(Me2), or Val; and

[0055] The X4 is selected from Ser (GlcA), Ala, Gly, Cit or Lys (Me2).

[0056] In some embodiments of this application, X1 is Ser;

[0057] X2 is Arg;

[0058] X3 is Gly;

[0059] Z is Leu-Val, Val, or Leu; and

[0060] The X4 is selected from Ala, Lys(Me2) or Cit.

[0061] In some embodiments of this application, X1 is absent, His, Arg, or Ser;

[0062] X2 is Arg;

[0063] X3 is Gly;

[0064] Z is Dap(Me2)-Val, Leu, or Val; and

[0065] X4 is either Cit or Lys(Me2).

[0066] In some embodiments of this application, X1 is either absent or Gly;

[0067] X2 is Arg;

[0068] X3 is Asn;

[0069] Z is Leu-Phe, Leu, Val, or Leu-Val; and

[0070] X4 is Arg, Cit, or Lys(Me2).

[0071] In some embodiments of this application, X1 is either absent or His;

[0072] X2 is Arg;

[0073] X3 is Ser or Ser(β-Glc);

[0074] Z is Leu, Dap(Me2), or Val; and

[0075] X4 is Cit, Ala, Lys(Me2), Lys(Pr2), Lys(NMPRZ), Orn(Me2), or Lys(Mor).

[0076] In some embodiments of this application, X1 is either absent or Gly;

[0077] X2 is Cit;

[0078] X3 is Asn;

[0079] Z is either Leu or Val; and

[0080] X4 is either Cit or Lys(Me2).

[0081] In some embodiments of this application, X1 is not present;

[0082] X2 is Cit;

[0083] X3 is Gly;

[0084] Z is either Leu or Val; and

[0085] X4 is either Cit or Lys(Me2).

[0086] In some embodiments of this application, X1 is not present;

[0087] X2 is Lys, Cit, Arg, or Glu;

[0088] X3 refers to Ser, Gly, and Glu.

[0089] Z is Leu; and

[0090] X4 is Cit.

[0091] In some embodiments of this application, X1 is not present;

[0092] X2 is Lys(Me2), Arg, or Cit;

[0093] X3 is Gly, Ser, or Glu;

[0094] Z is Leu; and

[0095] X4 is Lys(Me2).

[0096] In some embodiments of this application, X1 is not present;

[0097] X2 is Arg, Glu, Cit, Arg, or Lys(Me2);

[0098] X3 is Glu, Ser, or Pro;

[0099] Z is Val; and

[0100] X4 is Cit.

[0101] In some embodiments of this application, X1 is not present;

[0102] X2 is Lys(Me2), Arg, Glu, or Cit;

[0103] X3 is Ser, Glu, or Pro;

[0104] Z is Val; and

[0105] X4 is Lys(Me2).

[0106] In some embodiments of this application, X1 is not present;

[0107] X2 is Arg;

[0108] X3 is Ser;

[0109] Z is Val, Leu-Val, or Ile; and

[0110] X4 is Ala, Cit, Lys(Me2), or Lys(Mor).

[0111] In some embodiments of this application, X1 is either non-existent or Ser;

[0112] X2 and X3 are any natural or non-natural amino acids (for example, X2 is selected from Arg, Cit, Orn, Asn, Glu, Asp, Gla, Dab, Lys, Dab(Me2), Dab(Et2), Lys(Me2), Lys(Et2), Orn(Me2), Orn(Et2), Dap(Me2), Dap(Et2) or Ala, and X3 is selected from Gly, Pro, Asn, Ser, Glu, Ala, Gln, Asp, Cys or Ser(β-Glc)).

[0113] Z is Val, Leu, Leu-Val, or Dap(Me2);

[0114] X4 can be any natural or non-natural amino acid.

[0115] In some embodiments of this application, X1 is either non-existent or Ser;

[0116] X2 and X3 are any natural or non-natural amino acids (for example, X2 is selected from Arg, Cit, Orn, Asn, Glu, Asp, Gla, Dab, Lys, Dab(Me2), Dab(Et2), Lys(Me2), Lys(Et2), Orn(Me2), Orn(Et2), Dap(Me2), Dap(Et2) or Ala, and X3 is selected from Gly, Pro, Asn, Ser, Glu or Ser(β-Glc)).

[0117] Z is Val, Leu, Leu-Val, or Dap(Me2);

[0118] The X4 is Gly, Ala, Cit, Lys, Orn or its derivatives (e.g., Lys(Me2), Lys(Pr2), Lys(NMPRZ), Orn(Me2), or Lys(Mor)).

[0119] In some embodiments of this application, X1 is either non-existent or Ser;

[0120] X2 is Arg, Cit, or Lys(Me2);

[0121] X3 is Ser, Gly, or Asn;

[0122] Z is Val, Leu, Leu-Val, or Dap(Me2);

[0123] X4 is Gly, Ala, Cit, Lys(Me2), Lys(Pr2), Lys(NMPRZ), Orn(Me2), or Lys(Mor).

[0124] In some embodiments of this application, X1 is either non-existent or Ser;

[0125] X2 is Arg;

[0126] X3 is either Ser or Gly;

[0127] Z is Val, Leu, Leu-Val, or Dap(Me2);

[0128] X4 is Cit, Lys, Orn or its derivatives (e.g., Lys(Me2), Lys(Pr2), Lys(NMPRZ), Orn(Me2), or Lys(Mor)); wherein, in some embodiments, X3 is Ser when X1 is absent; wherein, in some embodiments, Z is Val, Leu, or Dap(Me2) when X1 is absent.

[0129] In some embodiments of this application, X1 is either non-existent or Ser;

[0130] X2 is Arg;

[0131] X3 is either Ser or Gly;

[0132] Z is Val, Leu, Leu-Val, or Dap(Me2);

[0133] X4 is Cit, Lys(Me2), Lys(Pr2), Lys(NMPRZ), Orn(Me2), or Lys(Mor); wherein, in some embodiments, when X1 is not present, X3 is Ser; wherein, in some embodiments, when X1 is not present, Z is Val, Leu, or Dap(Me2).

[0134] In some embodiments of this application, X1 is absent;

[0135] X2 is Arg;

[0136] X3 is Ser;

[0137] Z is either Leu or Val;

[0138] The X4 is Cit, Lys, Orn or its derivatives (e.g., Lys(Me2), Lys(Pr2), Lys(NMPRZ), Orn(Me2), or Lys(Mor)).

[0139] In some embodiments of this application, X1 is absent;

[0140] X2 is Arg;

[0141] X3 is Ser;

[0142] Z is either Leu or Val;

[0143] X4 is Cit, Lys(Me2), Lys(Pr2), Lys(NMPRZ), Orn(Me2), or Lys(Mor).

[0144] In some embodiments of this application, X1 is absent;

[0145] X2 is Arg;

[0146] X3 is Ser;

[0147] Z stands for Val;

[0148] X4 is Cit, Lys, Orn or its derivatives (e.g., Lys(Me2) or Lys(Mor)).

[0149] Specifically, in the embodiments of this application, the polypeptide or polypeptide fragment is selected from:

[0150] His-Pro-Arg-Gly-Leu-Val-Cit (SEQ ID NO: 1);

[0151] Ser-Pro-Arg-Gly-Leu-Val-Cit (SEQ ID NO: 2);

[0152] Ser-Pro-Arg-Pro-Leu-Val-Cit (SEQ ID NO: 3);

[0153] His-Pro-Arg-Asn-Leu-Val-Cit (SEQ ID NO: 4);

[0154] His-Pro-Arg-Ser-Leu-Val-Cit (SEQ ID NO: 5);

[0155] Ser-Pro-Lys(Me2)-Ser-Leu-Val-Cit (SEQ ID NO: 6);

[0156] Ser-Pro-Cit-Ser(β-Glc)-Leu-Val-Cit (SEQ ID NO:7);

[0157] Ser(β-Glc)-Pro-Cit-Ser-Leu-Val-Cit (SEQ ID NO: 8);

[0158] Ser(β-Glc)-Pro-Cit-Ser(β-Glc)-Leu-Val-Cit (SEQ ID NO: 9);

[0159] His-Pro-Cit-Gly-Leu-Val-Cit (SEQ ID NO: 10);

[0160] Ser-Pro-Cit-Ser-Leu-Val-Ser(GlcA)(SEQ ID NO:11);

[0161] Ser-Pro-Cit-Ser-Leu-Val-Ala(SEQ ID NO:12);

[0162] Ser-Pro-Arg-Gly-Leu-Val-Ala(SEQ ID NO:13);

[0163] Ser-Pro-Arg-Gly-Leu-Val-Lys(Me2)(SEQ ID NO:14);

[0164] Ser-Pro-Arg-Gly-Dap(Me2)-Val-Cit(SEQ ID NO:15);

[0165] His-Pro-Arg-Gly-Dap(Me2)-Val-Cit(SEQ ID NO:16);

[0166] Pro-Arg-Asn-Leu-Phe-Gly(SEQ ID NO:17);

[0167] Ser-Pro-Cit-Ser-Leu-Cit(SEQ ID NO:18);

[0168] Gly-Pro-Arg-Asn-Leu-Cit(SEQ ID NO:19);

[0169] Ser-Pro-Arg-Gly-Leu-Cit(SEQ ID NO:20);

[0170] His-Pro-Arg-Gly-Leu-Cit(SEQ ID NO:21);

[0171] His-Pro-Arg-Ser-Leu-Cit(SEQ ID NO:22);

[0172] Gly-Pro-Cit-Asn-Leu-Cit(SEQ ID NO:23);

[0173] Ser-Pro-Arg-Gly-Leu-Lys(Me2)(SEQ ID NO:24);

[0174] Ser-Pro-Cit-Gly-Hof-Gly(SEQ ID NO:25);

[0175] Ser-Pro-Cit-Ser-Dap(Me2)-Cit(SEQ ID NO:26);

[0176] Pro-Arg-Ser-Dap(Me2)-Cit(SEQ ID NO:27);

[0177] Pro-Arg-Ser-Leu-Cit(SEQ ID NO:28);

[0178] Pro-Cit-Gly-Leu-Cit(SEQ ID NO:29);

[0179] Pro-Arg-Ser(β-Glc)-Leu-Cit(SEQ ID NO:30);

[0180] Pro-Arg-Asn-Leu-Cit(SEQ ID NO:31);

[0181] Pro-Lys-Ser-Leu-Cit(SEQ ID NO:32);

[0182] Pro-Lys-Gly-Leu-Cit(SEQ ID NO:33);

[0183] Pro-Cit-Ser-Leu-Cit(SEQ ID NO:34);

[0184] Pro-Arg-Gly-Leu-Cit(SEQ ID NO:35);

[0185] Pro-Cit-Asn-Leu-Cit(SEQ ID NO:36);

[0186] Pro-Arg-Glu-Leu-Cit(SEQ ID NO:37);

[0187] Pro-Glu-Ser-Leu-Cit(SEQ ID NO:38);

[0188] Pro-Arg-Ser-Leu-Ala(SEQ ID NO:39);

[0189] Pro-Arg-Ser-Leu-Lys(Me2)(SEQ ID NO:40);

[0190] Pro-Cit-Gly-Leu-Lys(Me2)(SEQ ID NO:41);

[0191] Pro-Lys(Me2)-Gly-Leu-Lys(Me2)(SEQ ID NO:42);

[0192] Pro-Lys(Me2)-Ser-Leu-Lys(Me2)(SEQ ID NO:43);

[0193] Pro-Arg-Asn-Leu-Lys(Me2)(SEQ ID NO:44);

[0194] Pro-Arg-Glu-Leu-Lys(Me2)(SEQ ID NO:45);

[0195] Pro-Arg-Gly-Leu-Lys(Me2)(SEQ ID NO:46);

[0196] Pro-Cit-Ser-Leu-Lys(Me2)(SEQ ID NO:47);

[0197] Pro-Arg-Ser-Leu-Lys(Pr2)(SEQ ID NO:48);

[0198] Pro-Arg-Ser-Leu-Lys(NMPRZ)(SEQ ID NO:49);

[0199] Pro-Arg-Ser-Leu-Orn(Me2)(SEQ ID NO:50);

[0200] Pro-Arg-Ser-Leu-Lys(Mor)(SEQ ID NO:51);

[0201] Ser-Pro-Cit-Ser-Val-Cit(SEQ ID NO:54);

[0202] Gly-Pro-Arg-Asn-Val-Cit(SEQ ID NO:55);

[0203] Ser-Pro-Arg-Gly-Val-Cit(SEQ ID NO:56);

[0204] His-Pro-Arg-Ser-Val-Cit(SEQ ID NO:57);

[0205] His-Pro-Arg-Gly-Val-Cit(SEQ ID NO:58);

[0206] Ser-Pro-Arg-Gly-Val-Lys(Me2)(SEQ ID NO:59);

[0207] Ser-Pro-Cit-Ser-Val-Lys(Me2)(SEQ ID NO:60);

[0208] Gly-Pro-Arg-Asn-Val-Lys(Me2)(SEQ ID NO:61);

[0209] His-Pro-Arg-Ser-Val-Lys(Me2)(SEQ ID NO:62);

[0210] His-Pro-Arg-Gly-Val-Lys(Me2)(SEQ ID NO:63);

[0211] Pro-Arg-Ser-Val-Cit(SEQ ID NO:64);

[0212] Pro-Arg-Glu-Val-Cit(SEQ ID NO:65);

[0213] Pro-Arg-Asn-Val-Cit(SEQ ID NO:66);

[0214] Pro-Glu-Ser-Val-Cit(SEQ ID NO:67);

[0215] Pro-Cit-Gly-Val-Cit(SEQ ID NO:68);

[0216] Pro-Arg-Gly-Val-Cit(SEQ ID NO:69);

[0217] Pro-Cit-Ser-Val-Cit(SEQ ID NO:70);

[0218] Pro-Arg-Pro-Val-Cit(SEQ ID NO:71);

[0219] Pro-Cit-Pro-Val-Cit(SEQ ID NO:72);

[0220] Pro-Cit-Asn-Val-Cit(SEQ ID NO:73);

[0221] Pro-Lys(Me2)-Ser-Val-Cit(SEQ ID NO:74);

[0222] Pro-Arg-Ser-Val-Lys(Me2)(SEQ ID NO:75);

[0223] Pro-Lys(Me2)-Ser-Val-Lys(Me2) (SEQ ID NO:76);

[0224] Pro-Arg-Glu-Val-Lys(Me2)(SEQ ID NO:77);

[0225] Pro-Arg-Asn-Val-Lys(Me2)(SEQ ID NO:78);

[0226] Pro-Glu-Ser-Val-Lys(Me2)(SEQ ID NO:79);

[0227] Pro-Cit-Gly-Val-Lys(Me2)(SEQ ID NO:80);

[0228] Pro-Arg-Gly-Val-Lys(Me2)(SEQ ID NO:81);

[0229] Pro-Cit-Ser-Val-Lys(Me2)(SEQ ID NO:82);

[0230] Pro-Arg-Pro-Val-Lys(Me2)(SEQ ID NO:83);

[0231] Pro-Cit-Pro-Val-Lys(Me2)(SEQ ID NO:84);

[0232] Pro-Cit-Asn-Val-Lys(Me2)(SEQ ID NO:85);

[0233] Pro-Arg-Ser-Val-Lys(Pr2)(SEQ ID NO:86);

[0234] Pro-Arg-Ser-Val-Orn(Me2)(SEQ ID NO:87);

[0235] Pro-Arg-Ser-Val-Ala(SEQ ID NO:88);

[0236] Pro-Arg-Ser-Val-Cit(SEQ ID NO:89);

[0237] Pro-Arg-Ser-Ile-Cit (SEQ ID NO:90);

[0238] Pro-Arg-Ser-Ile-Ala (SEQ ID NO:91);

[0239] Pro-Arg-Ser-Ile-Lys(Me2) (SEQ ID NO:92);

[0240] Pro-Arg-Ser-Leu-Val-Lys(Me2) (SEQ ID NO:93);

[0241] Pro-Arg-Asn-Leu-Val-Lys(Me2) (SEQ ID NO:94);

[0242] Pro-Arg-Ser-Val-Lys(Mor)(SEQ ID NO:95); and

[0243] Pro-Ala-Ser-Leu-Val-Cit (SEQ ID NO: 132).

[0244] This application further provides a linker comprising the above-described polypeptide or polypeptide fragment, the linker having the following general formula:

[0245] C1-L1-L'-L2;

[0246] Wherein, C1 is a linker; L1 is an empty spacer or spacer; L2 is an empty spacer or spacer; and L' is the aforementioned polypeptide or polypeptide fragment. In some embodiments of this application, C1 is selected from the following structures:

[0247] in Indicates the location of the connection with the biological ligand unit. Indicates the position connected to L1 or L'.

[0248] Furthermore, the C1-L1- is

[0249] In some embodiments of this application, L2 is empty or selected from: n is any integer selected from 1-6, 1-5, 1-4, or 1-3;

[0250] Preferably, L2 is empty. (PAB) or -NHCH2-.

[0251] In some embodiments of this application, the connector is selected from:

[0252] -(succinimide-3-yl-N)-(CH2)5-C(=O)-L'-; -(succinimide-3-yl-N)-(CH2)5-C(=O)-L'-PAB-; -(succinimide-3-yl-N)-(CH2)5-C(=O)-L'-PAB-DMEDA-; -(succinimide-3-yl-N)-(CH2)5-C(=O)-L'-NMEDA- or -(succinimide-3-yl-N)-(CH2)5-C(=O)-L'-NHCH2-.

[0253] This application also provides a linker drug comprising the above-mentioned polypeptide or polypeptide fragment, having the following general formula:

[0254] C1'-L1-L'-L2-D;

[0255] Wherein, C1' is a functional group that reacts with biological ligands; L1 is an empty spacer or spacer; L2 is an empty spacer or spacer; D is a biologically active compound; and L' is the aforementioned polypeptide or polypeptide fragment.

[0256] In some implementations, C1' is selected from:

[0257] Among them, R x Selected from -OH, F, Cl, or Br; Indicates the connection position with L1 or L'; C1'-L1 is selected from MC, NHS-, Mal-PEG8- or DBCO-.

[0258] In some embodiments, D is selected from immunomodulators, protein degraders, and cytotoxic agents; preferably, D includes: amanitins, anthracyclines, auristatins, baccatins, calicheamicins, camptothecins, cemadotins, colchicine, colcimids, compbretastatins, cryptophycins, discormolides, duocarmycins, docetaxel, doxorubicin, duocarmycins, echinomycins, and enantoxamine. Elutherobins, epothilones, estramustines, lexitropsins, maytansines, maytansinoids, methotrexate, netropsins, pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), puromycins, rhizoxins, SN38, taxanes, tubulolysins, vincaalkaloids, tetrahydroisoquinoline alkaloids or their derivatives, protein degrading agents or their derivatives; more preferably, D includes DX8951, MMAE, Dxd or SN38.

[0259] In some specific embodiments of this application, the linker drug is selected from MC-L'-D; MC-L'-PAB-D; MC-L'-PAB-DMEDA-

[0260] D; MC-L'-NMEDA-D or MC-L'-NHCH2-D;

[0261] Wherein, the drug unit D is selected from DX8951, MMAE, Dxd or SN38.

[0262] In some specific embodiments of this application, the linker drug is selected from MC-L'-D, MC-L'-PAB-D, or MC-L'-NHCH2-

[0263] D; wherein the drug unit D is selected from DX8951, MMAE or Dxd.

[0264] This application also provides an anti-tissue factor (TF) antibody or its antigen-binding fragment, comprising:

[0265] The light chain and the heavy chain, wherein the light chain has the amino acid sequence shown in SEQ ID NO: 52, or an amino acid sequence having at least 80% (e.g., at least 90%, at least 95%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 52; and the heavy chain has the amino acid sequence shown in SEQ ID NO: 53, or an amino acid sequence having at least 80% (e.g., at least 90%, at least 95%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 53.

[0266] This application also provides a GPC1 antibody or its antigen-binding fragment, comprising:

[0267] The light chain and the heavy chain, wherein the light chain has the amino acid sequence shown in SEQ ID NO: 96, or an amino acid sequence having at least 80% (e.g., at least 90%, at least 95%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 96; and the heavy chain has the amino acid sequence shown in SEQ ID NO: 97, or an amino acid sequence having at least 80% (e.g., at least 90%, at least 95%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 97.

[0268] This application also provides a biological ligand-drug conjugate comprising the above-mentioned linker, having the following general formula:

[0269] Bp-(C1-L1-L′-L2-D) n

[0270] Wherein: Bp is a biological ligand; C1-L1-L'-L2 is the linker; D is the biologically active compound described above; n is any integer between 1 and 20.

[0271] In some embodiments, the biological ligand Bp targets cell surface receptors or tumor-associated antigens, wherein the cell surface receptors or tumor-associated antigens include: EGFR, HER2, HER3, c-Met, claudin18.2, TROP-2, folate receptor α (FRα), ROR2, BCMA, PSMA, CD19, CD20, CD22, CD30, CD33, CD37, CD46, CD48, CD56, CD79b, CD123, CD138, CD166, CD276, CEACAM5, SDC1, CD74, CD70, MUC1, MUC16, GUCY2C, MSLN, SCL34A2, FOLH1, TPBC, PVRL4, STEAP1, SCL44A4, NACM1, EDNRB, GPNMB, FGFR, SEZ6, Nectin-4, PD-L1, and Tissue. Factor(TF), B7-H3, B7-H4, LY6E, LIV-1, CDH4, CDH6, ITGB6, GPC1, GPC3, ENPP3, KAAG1, FZD7, SSTR2, DLK1, T MEFF1, TM4SF1, SLAMF7, DLL3, FLT3, TNFRSF10B, EPCAM, AXL, IL2RA, 5T4, FAP, ICAM-1, IGF-1R, TNFR1, GLP-1.

[0272] In some embodiments, the biological ligand includes an antibody or an antigen-binding fragment thereof, a modified antibody or an antigen-binding fragment thereof, an oligonucleotide, a DNA fragment, a nucleic acid aptamer, a polypeptide, a nanoparticle with targeting properties, or a chemically synthesized small molecule; preferably, the antigen-binding fragment includes Fab, Fab', Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb.

[0273] In some embodiments, the antibody or its antigen-binding fragment, or the modified antibody or its antigen-binding fragment, is: anti-EGFR, anti-HER2, anti-HER3, anti-c-Met, anti-claudin18.2, anti-TROP-2, anti-FRα, anti-ROR2, anti-BCMA, anti-PSMA, anti-CD19, anti-CD20, anti-CD22, anti-CD30, anti-CD33, anti-CD37, anti-CD... 46. ​​anti-CD48, anti-CD56, anti-CD79b, anti-CD123, anti-CD138, anti-CD166, anti-CD276, anti-CEACAM5, anti-SDC1, anti-C D74, anti-CD70, anti-MUC1, anti-MUC16, anti-GUCY2C, anti-MSLN, anti-SCL34A2, anti-FOLH1, anti-TPBC, anti-PVRL4, anti-S TEAP1, anti-SCL44A4, anti-NACM1, anti-EDNRB, anti-GPNMB, anti-FGFR, anti-SEZ6, anti-Nectin-4, anti-PD-L1, anti-TF, an ti-B7-H3, anti-B7-H4, anti-LY6E, anti-LIV-1, anti-CDH4, anti-CDH6, anti-ITGB6, anti-GPC1, anti-GPC3, anti-ENPP3, anti Antibodies or antigen-binding fragments of KAAG1, anti-FZD7, anti-SSTR2, anti-DLK1, anti-TMEFF1, anti-TM4SF1, anti-SLAMF7, anti-DLL3, anti-FLT3, anti-TNFRSF10B, anti-EPCAM, anti-AXL, anti-IL2RA, anti-5T4, anti-FAP, anti-ICAM-1, anti-IGF-1R, anti-TNFR1, and anti-GLP-1;

[0274] More preferably, the antibody or its antigen-binding fragment, or the modified antibody or its antigen-binding fragment, is an antibody or its antigen-binding fragment for anti-Her2, anti-TF, anti-GPC1, or anti-GPC3.

[0275] Alternatively, preferably, the antibody is an anti-TF antibody (preferably, the antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:53 and a light chain having the amino acid sequence shown in SEQ ID NO:52), Trastuzumab, Tisotumab, Zilovertamab, Codrituzumab, or an anti-GPC-1 antibody (preferably, the antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:97 and a light chain having the amino acid sequence shown in SEQ ID NO:96, or preferably Miltuximab);

[0276] More preferably, the antibody is Trastuzumab, Miltuximab, Tisotumab, Zilovertamab, or Codrituzumab; even more preferably, the antibody is Trastuzumab.

[0277] Alternatively, preferably, the antibody is an anti-TF antibody (preferably, the antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:53 and a light chain having the amino acid sequence shown in SEQ ID NO:52), Trastuzumab, or an anti-GPC-1 antibody (preferably, the antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:97 and a light chain having the amino acid sequence shown in SEQ ID NO:96).

[0278] In a specific embodiment of this application, the conjugate is an antibody-drug conjugate, and the antibody-drug conjugate is selected from:

[0279] Ab-((succinimide-3-yl-N)-(CH2)5-C(=O)-L'-D) n ;

[0280] Ab-((succinimide-3-yl-N)-(CH2)5-C(=O)-L'-PAB-D) n ;

[0281] Ab-((succinimide-3-yl-N)-(CH2)5-C(=O)-L'-PAB-DMEDA-D) n ;

[0282] Ab-((succinimide-3-yl-N)-(CH2)5-C(=O)-L'-NMEDA-D) n ;

[0283] Ab-((succinimide-3-yl-N)-(CH2)5-C(=O)-L'-NHCH2-D)n ;

[0284] Wherein, Ab represents a monoclonal antibody or its antigen-binding fragment, or a modified monoclonal antibody or its antigen-binding fragment, preferably an anti-Her2, anti-TF, anti-GPC1, or anti-GPC3 antibody or its antigen-binding fragment; L' is selected from the polypeptide or polypeptide fragment described above; D is selected from DX8951, MMAE, Dxd, or SN38; and n represents any value selected from 1 to 10.

[0285] More specifically, the Ab is selected from Trastuzumab, Miltuximab, Tisotumab, the antibody described above, or Codrituzumab; the D is selected from DX8951, MMAE, or Dxd.

[0286] Alternatively, preferably, the antibody is an anti-TF antibody (preferably, the antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:53 and a light chain having the amino acid sequence shown in SEQ ID NO:52), Trastuzumab, or an anti-GPC-1 antibody (preferably, the antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:97 and a light chain having the amino acid sequence shown in SEQ ID NO:96), and the D is DX8951, MMAE, or Dxd.

[0287] Another aspect of this application provides the use of the drug conjugates described herein in the preparation of medicaments for the treatment and / or prevention of diseases or conditions associated with abnormal cell activity.

[0288] This application also discloses a method for treating a disease or condition related to abnormal cellular activity in a subject in need, comprising administering an effective amount of the antibody-drug conjugate described herein to the subject.

[0289] This application also provides antibody-drug conjugates described herein for the treatment and / or prevention of diseases or conditions associated with abnormal cell activity.

[0290] This application also provides the use of the antibody-drug conjugates described herein for the treatment and / or prevention of diseases or conditions associated with abnormal cellular activity.

[0291] Furthermore, the disease or condition includes tumors; in some embodiments, the disease or condition includes solid tumors and hematomas.

[0292] Further, the disease or condition is selected from tumors associated with the expression of the following proteins: EGFR, HER2, HER3, c-Met, claudin18.2, TROP-2, folate receptor α (FRα), ROR2, BCMA, PSMA, CD19, CD20, CD22, CD30, CD33, CD37, CD46, CD48, CD56, CD79b, CD123, CD138, CD166, CD276, CEACAM5, SDC1, CD74, CD70, MUC1, MUC16, GUCY2C, MSLN, SCL34A2, FOLH1, TPBC, PVRL4, STEAP1, SCL44A4, NACM1, EDNRB, GPNMB, FGFR, SEZ6, Nectin-4, PD-L1, and Tissue. Factor (TF), B7-H3, B7-H4, LY6E, LIV-1, CDH4, CDH6, ITGB6, GPC1, GPC3, ENPP3, KAAG1, FZD7, SSTR2, DLK1, TMEFF1, TM4SF1, SLAMF7, DLL3, FLT3, TNFRSF10B, EPCAM, AXL, IL2RA, 5T4, FAP, ICAM-1, IGF-1R; preferably, selected from tumors associated with the expression of Her2, TF, GPC1, or GPC3.

[0293] More specifically, the disease or symptom is selected from esophageal cancer, brain cancer, lung cancer, epithelial cell carcinoma, bladder cancer, stomach cancer, ovarian cancer, pancreatic cancer, head and neck cancer, urothelial carcinoma, colon cancer, colorectal cancer, cervical cancer, endometrial cancer, rectal cancer, kidney cancer, prostate cancer, melanoma, liver cancer, gallbladder cancer, bile duct cancer, thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, and B-cell lymphoma; preferably, the tumor is selected from esophageal cancer, lung cancer, breast cancer, stomach cancer, urothelial carcinoma, bile duct cancer, prostate cancer, colorectal cancer, cervical cancer, and ovarian cancer.

[0294] This application also provides the use of the polypeptide or polypeptide fragment described herein, or a linker containing the polypeptide, in the preparation of an antibody-drug conjugate for treating tumors. Alternatively, this application provides the use of the polypeptide or polypeptide fragment described herein, or a linker containing the polypeptide, in the preparation of an antibody-drug conjugate for treating tumors. Alternatively, this application provides a method for preparing an antibody-drug conjugate, comprising linking an antibody to a payload via the polypeptide or polypeptide fragment described herein, or a linker containing the polypeptide, or a payload. Beneficial effects

[0295] This application designs a series of peptides that are both specifically responsive to MMP-2 and / or MMP-9 in tumor tissue and responsive to Cathepsin B in tumor cells, as well as spacer structures connecting peptides to antibodies and payloads, which can achieve at least one of the following technical effects.

[0296] 1. The polypeptide linker designed in this invention can respond to both extracellular proteases MMP-2 and MMP-9, which are widely and highly expressed in tumor tissues, and intracellular cathepsins such as Cathepsin B, thereby achieving the effect of dual extracellular / intracellular release and improving the drug utilization rate of ADC drugs after reaching tumor tissues.

[0297] 2. By controlling the response efficiency of the polypeptide linker to extracellular enzymes, the prepared ADC maintains relatively stable properties in terms of pharmacokinetics, that is, the prepared ADC is relatively stable in the in vivo environment. This reduces the occurrence of off-target toxicity, thereby making the effective payload cell activity range of this innovative linker relatively large.

[0298] 3. The peptide linker of this application can be widely used in various small molecule toxins, such as Exatecan and MMAE, to improve efficacy without increasing toxicity risk. Attached Figure Description

[0299] Figure 1 shows the rat PK test results of the Exatecan-ADC of this application.

[0300] Figure 2 shows the rat PK test results of the MMAE-ADC of this application.

[0301] Figure 3 shows the rat PK test results of MMAE-ADC and Exatecan-ADC of this application. Detailed Implementation

[0302] Unless otherwise stated, the terms “comprise”, “comprises”, and “comprising” or their equivalents (contain, contain, containing, include, include, including) used herein are open-ended expressions, meaning that they may cover other unspecified elements, components, and steps in addition to those listed.

[0303] Unless the context clearly indicates otherwise, singular terms in this document cover the plural referents, and vice versa.

[0304] For purposes of description and disclosure, all patents, patent applications, and other publications are expressly incorporated herein by reference. These publications are provided solely because their publication predates the filing date of this application. All statements regarding the dates of these documents or representations of their contents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the dates or contents of these documents. Furthermore, in any country, any reference to these publications herein does not constitute an endorsement that such publication is part of the general knowledge in the art.

[0305] The following description of specific embodiments further illustrates this application, but it is not intended to limit the scope of this disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of this application without departing from its basic ideas and scope. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0306] Table 1 Sequence List

[0307] Table 2. Structures and abbreviations of unconventional amino acids and other chemical substances.

[0308] Example 1: Synthesis of Linker Fragments or Linker Drugs

[0309] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 To determine this, use either nuclear magnetic resonance (NMR) or mass spectrometry (MS). 1 The H NMR (H2N) assay was performed using a Bruker AVANCE NEO 400MHz NMR spectrometer; the assay solvents were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0310] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below. s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuteride. δ values ​​are expressed in ppm.

[0311] The mass spectrometry (MS) measurements were performed using a SHIMADZU LCMS-2020 mass spectrometer.

[0312] 1.1 Synthesis of peptide MC-PRNL

[0313] The polypeptide was synthesized using standard Fmoc chemical methods.

[0314] 1) Resin preparation: In a container containing 2-CTC resin (3.00 mmol, 3.00 g, Sub = 1.00 mmol / g) and Fmoc-Leu-OH (1.06 g, 3.00 mmol, 1.00 eq.), DIEA (4.00 eq.) was added to DCM (80 mL) and mixed for 2 h. Then, MeOH (3.0 mL) was added to the resin bubbled with N2 for 30 min. The resin was washed with DMF (80 mL * 5), and then a DMF solution containing 20% ​​piperidine (80 mL) was added. The Fmoc protecting group was removed by bubbling with N2 at 25 °C for 12 min. The mixture was filtered, and the resin was washed with DMF (80 mL * 5) before proceeding to the next step.

[0315] 2) Coupling: A solution of Fmoc-Asn(Trt)-OH (3.00 eq.), HATU (2.85 eq.), and DIEA (6.00 eq.) from DMF (80 mL) was added to the resin, and the mixture was bubbled with N2 at 25 °C for 30 min. The coupling reaction was monitored using the ninhydrin method; if the reaction was colorless, the coupling was complete. The resin was then washed with DMF (80 mL * 5).

[0316] 3) Deprotection: Add 80 mL of DMF solution containing 20% ​​piperidine to the resin and bubble the mixture with N2 at 25 °C for 12 min. Then wash the resin with DMF (80 mL * 5). Monitor the deprotection reaction using the ninhydrin method; if the deprotection reaction is blue or brownish-red, the reaction is complete.

[0317] 4) Repeat steps 2) to 3) to obtain the following amino acid elongation rates: numbers #3-4, Table 3.

[0318] 5) Coupling: A solution of DIEA (12.00 eq.) and N-hydroxysuccinimide 6-maleimide hexanoate (6.00 eq.) in DMF (80 mL) was added to the resin, and the mixture was bubbled with N2 at 25 °C for 16 h. The coupling reaction was monitored using the chloroaniline method; if the reaction was colorless, the coupling was complete. The resin was then washed with DMF (80 mL * 5).

[0319] Table 3. List of amino acids and corresponding reagents used in SPP

[0320] Peptide cleavage and purification:

[0321] 1) The lysis mixture (TFA / Tis / H2O, 95 / 2.5 / 2.5, v / v / v, 100 mL) was added to a flask containing the side-chain protected peptide, and the mixture was stirred at 25 °C for 2 hours. LCMS showed detection of the desired MS.

[0322] 2) Filter the mixture. Precipitate the filtrate with cold isopropyl ether (500 mL) and centrifuge (3000 rpm for 2 min), then wash twice with isopropyl ether (500 mL * 2). Dry the residue under vacuum for 2 hours.

[0323] 3) The residue was purified by HPLC (A: 0.10% TFA in H2O, B: ACN) to obtain a white solid MC-PRNL (SEQ ID NO: 98) (1.15 g, yield 55.5%, purity 97.2%).

[0324] 1.2 Synthesis of Cit-PAB-P005

[0325] S1: EEDQ (622 mg, 2.50 mmol, 2.0 eq.) and PAB (309 mg, 2.51 mmol, 2.0 eq.) were added to a solution of compound Fmoc-Cit-COOH (500 mg, 1.20 mmol, 1.0 eq.) in DCM (5 mL) and MeOH (1.25 mL). The mixture was stirred at 25 °C for 16 hours. The reaction was confirmed by LCMS. The mixture was added dropwise to isopropyl ether (50 mL) and centrifuged to give a pale yellow solid compound 2-1 (578 mg, crude). ESI-MS (m / z): 503.2 [M+H]+.

[0326] S2: DIEA (271 mg, 2.10 mmol, 347 μL, 2.0 eq.) was added to a solution of compound 2-1 (528 mg, 1.05 mmol, 1.0 eq.) and PNP (639 mg, 2.10 mmol, 2.0 eq.) in DMF (5 mL). The mixture was stirred at 25 °C for 3.0 h. The reaction was confirmed by LCMS. DCM (3 mL) was added to the reaction mixture, and the mixture was then centrifuged with IPE (50 mL) to give a white solid compound 2-2 (575 mg, crude). ESI-MS (m / z): 668.3 [M+H]+.

[0327] S3: DIEA (222 mg, 1.72 mmol, 284 μL, 2.0 eq.) was added to a solution of compound 2-2 (575 mg, 861 μmol, 1.0 eq.), Exatecan (66.2 mg, 688 μmol, 0.8 eq.), and HOBt (139 mg, 1.03 mmol, 1.2 eq.) in DMF (5 mL). The mixture was stirred at 25 °C for 3.0 h. The reaction was confirmed by LCMS. The reaction mixture was centrifuged with IPE (50 mL) to give a yellow gelatinous compound 2-3 (830 mg, crude). ESI-MS (m / z): 964.4 [M+H]+.

[0328] S4: Compound 2-3 (830 mg, 860 μmol, 1.0 eq) was mixed with TEA (1.16 g, 11.4 mmol, 1.60 mL, 13.3 eq) in a mixture of DMF (6.4 mL). The mixture was stirred at 25 °C for 16 hours. The reaction was confirmed by LCMS. The reaction mixture was purified by preparative HPLC to give compound 2-4 (Cit-PAB-P005) as a yellow solid (116 mg, 140 μmol, yield 18.2%). ESI-MS (m / z): 742.4 [M+H]+.

[0329] 1.3 Synthesis of MC-PRNLCit (SEQ ID NO:31)-PAB-P005 (L096-P005)

[0330] To a solution of compound 1-1 (20.0 mg, 28.9 μmol, 1.0 eq.) and compound 2-4 (21.4 mg, 28.9 μmol, 1.0 eq.) in DMF (200 μL), TBTU (11.1 mg, 34.6 μmol, 1.2 eq.) and DIPEA (11.2 mg, 86.7 μmol, 14.3 μL, 3.0 eq.) were added. The mixture was stirred at 25 °C for 1.0 h. The reaction was confirmed by LC-MS. The mixture was purified by preparative HPLC to give a white solid compound L096-P005 (8.00 mg, 5.65 μmol, yield 19.5%). ESI-MS (m / z): 1415.9 [M+H]+.

[0331] 1.4 Synthesis of K(Me2)-PAB-P002

[0332] S1: EEDQ (792 mg, 3.2 mmol, 1.8 eq.) was added to a mixture of (S)-2-FmocNH-6-(dimethylamino)hexanoic acid (690 mg, 1.7 mmol, 1.0 eq.) and PAB (393 mg, 3.1 mmol, 1.8 eq.) in DCM (16 mL) and MeOH (4 mL), and the mixture was stirred at 25 °C for 16 h. The reaction was confirmed by LC-MS. The mixture was then ground with isopropyl ether (50 mL) in an ice bath and stirred at 0 °C for 10 min. The filtered cake was diluted with DCM (10 mL) and then concentrated to give a yellow colloidal compound 4-1 (670 mg, 1.1 mmol, yield 65.9%). ESI-MS (m / z): 502.3 [M+H] + .

[0333] S2: DIPEA (355.1 mg, 2.7 mmol, 1.0 eq.) was added to a solution of compound 4-1 (670 mg, 1.3 mmol, 1.0 eq.) and bis(4-nitrophenyl) carbonate (836.0 mg, 2.7 mmol, 2.0 eq.) in DMF (10 mL), and the mixture was stirred at 25 °C for 2.0 h. The reaction was confirmed by LC-MS. The mixture was then poured into isopropyl ether (500 mL) in an ice bath and stirred at 0 °C for 10 min. The filtered cake was diluted with DCM (50 mL) and concentrated to give a yellow gel-like crude product. The crude product was purified by HPLC to give a yellow solid compound 4-2 (162.7 mg, 207.6 μmol, yield 15.1%). ESI-MS (m / z): 667.4 [M+H] + .

[0334] S3: HOBt (39.5 mg, 292.4 μmol, 1.5 eq.) and DIPEA (75.6 mg, 584.9 μmol, 96.6 μL, 3.0 eq.) were added to a mixture of compound 4-2 (130 mg, 194.9 μmol, 1.0 eq.) and MMAE (111.9 mg, 155.9 μmol, 0.8 eq.) in DMF (1 mL). The mixture was stirred at 25 °C for 2.0 h. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give compound 4-3 (65.6 mg, 48.1 μmol, yield 24.7%) as a yellow solid. ESI-MS (m / z): 1245.6 [M+H] + .

[0335] S4: TEA (174.4 mg, 1.7 mmol, 240 μL, 35.3 eq.) was added to a DMF (360 μL) solution of compound 4-3 (65.6 mg, 48.7 μmol, 1.0 eq.). The mixture was stirred at 25 °C for 16 hours. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give a white solid compound 4-4 (40 mg, 30.9 μmol, yield 63.3%). ESI-MS (m / z): 1024.9 [M+H] + .

[0336] 1.5 Synthesis of MC-PRNLK(Me2)(SEQ ID NO:44)-PAB-P002

[0337] HOBT (4 mg, 0.03 mmol, 2.02 eq.) and EDCI (6 mg, 0.03 mmol, 2.14 eq.) were added to a DMF (1 mL) solution of compound 4-4 (20 mg, 0.03 mmol, 1.97 eq.). The mixture was stirred at 0 °C for 0.5 h. A DMF (1 mL) solution of compound 1-1 (15 mg, 0.01 mmol, 1.0 eq.) was added. The mixture was then stirred at 0 °C for another 1 h. The reaction was confirmed by LC-MS. The reaction mixture was purified by HPLC to give a white solid compound L115-P002 (7.04 mg, 0.01 mmol, yield 14.35%). ESI-MS (m / z): 849.2 [M / 2+H]+.

[0338] 1.6 Solid-phase synthesis of other peptides:

[0339] Other linker fragments were synthesized using the following raw materials according to the above-described solid-phase peptide synthesis method, as shown in Table 4 below:

[0340] Table 4 List of polypeptides

[0341] Example 2: Synthesis of Linker-Payload

[0342] 2.1 Synthesis of Linker-P005 (DX8951) intermediate

[0343] 1) Synthesis of K(Me2)-PAB-P005

[0344] S1: HOBt (97.3 mg, 720 μmol, 1.5 eq.) and DIEA (124 mg, 960 μmol, 159 μL, 2.0 eq.) were added to a DMF (3.2 mL) solution of compound 4-2 (320 mg, 480 μmol, 1.0 eq.) and Exatecan (255 mg, 480 μmol, 1.0 eq.). The mixture was stirred at 25 °C for 2.0 h. The reaction was confirmed by LC-MS. The reaction solution was added dropwise to isopropyl ether (5.00 mL * 3) and concentrated under reduced pressure to give a dark brown solid compound 6-1 (920 mg, crude product). ESI-MS (m / z): 963.7 [M + H] + .

[0345] S2: Piperidine (163 mg, 1.91 mmol, 189 μL, 2.0 eq.) was added to a DMF (9.2 mL) solution of compound 6-1 (920 mg, 955 μmol, 1.0 eq.). The mixture was stirred at 25 °C for 3.5 h. The reaction was confirmed by LC-MS. The reaction mixture was added dropwise to isopropyl ether (10.0 mL * 3) and purified by preparative HPLC to give the green solid compound 6-2 (K(Me2)-PAB-P005) (170 mg, 220 μmol, yield 23.1%). ESI-MS (m / z): 741.3 [M+H] + .

[0346] 2) Synthesis of Val-Cit-PAB-P005

[0347] S1: EEDQ (19.9 g, 80.5 mmol, 2.0 eq.) and PAB (9.92 g, 80.5 mmol, 2.0 eq.) were added to a solution of compound Fmoc-Val-Cit-OH (20.0 g, 40.3 mmol, 1.0 eq.) in DCM (200 mL) and MeOH (50.0 mL). The mixture was stirred at 25 °C for 16 hours. The reaction was detected by LC-MS. The reaction product was filtered, and the filtrate was added to 2000 mL of EtOAc / MTBE (8 / 1) and stirred twice for 10 min. The mixture was filtered, and the filtrate was washed with EtOAc to give a white solid compound 7-1 (19.8 g, crude product). ESI-MS (m / z): 602.3 [M+H] + .

[0348] S2: DIEA (8.59 g, 66.4 mmol, 1.0 eq.) was added to a solution of compound 7-1 (20.0 g, 33.2 mmol, 1.0 eq.) and PNP (20.2 g, 66.4 mmol, 2.0 eq.) in DMF (200 mL). The mixture was stirred at 25 °C for 3 hours. The reaction was confirmed by LC-MS. The reaction mixture was added dropwise to stirred ice-cold MTBE and filtered. The filtered solid was dissolved in DCM and then poured into MTBE / EtOAc (1:1) and stirred for 30 min. The mixture was then filtered and concentrated under reduced pressure to give a white solid compound 7-2 (21 g, crude). ESI-MS (m / z): 767.4 [M+H] + .

[0349] S3: Compound 7-2 (3.00 g, 3.91 mmol, 1.0 eq.), exatecan mesylate (1.87 g, 3.52 mmol, 0.8 eq.), and HOBt (635 mg, 4.70 mmol, 1.2 eq.) were dissolved in DMF (30 mL). DIEA (1.01 g, 7.83 mmol, 1.29 mL, 2.0 eq.) was added to the mixture. The mixture was stirred at 25 °C for 3.0 h under a nitrogen atmosphere. The reaction was confirmed by LC-MS. The mixture was ground with isopropyl ether (300 mL) to give a brown solid compound 7-3 (5.60 g, crude). ESI-MS (m / z): 1063.4 [M+H] + .

[0350] S4: 10 mL of TEA was added to a mixture of compound 7-3 (5.60 g, crude) in DMF (40 mL). The mixture was stirred at 25 °C for 16 hours. The reaction was confirmed by LC-MS. The reaction mixture was purified by HPLC to give compound 7-4 (2.75 g, 3.27 mmol, yield: 83.5%) as a yellow solid. ESI-MS (m / z): 841.6 [M+H] + .

[0351] 3) Synthesis of Gly-PAB-DX8951

[0352] Step 1:

[0353] EEDQ (54.9 g, 222 mmol, 2 eq.) was added to a solution of compound Fmoc-glycine (33 g, 111 mmol, 1.0 eq.) and PAB (20.50 g, 166 mmol, 1.5 eq.) in DCM (200 mL) and MeOH (60 mL). The mixture was stirred at 25 °C for 2.0 h. The reaction was confirmed by LC-MS. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was milled with MTBE for 20 min, then filtered and concentrated under reduced pressure to give a brown solid compound 8-1 (45.8 g, crude). ESI-MS (m / z): 403.0 [M+H] + .

[0354] Step Two:

[0355] DIEA (29.4 g, 228 mmol, 39.7 mL, 2 eq.) was added to a DMF (300 mL) solution of Fmoc-Gly-PAB (45.8 g, 114 mmol, 1 eq.) and PNP (69.2 g, 228 mmol, 2 eq.). The mixture was stirred at 25 °C for 2.0 h. The reaction was confirmed by LC-MS. The reaction solution was ground with isopropyl ether, filtered, and concentrated under reduced pressure to give a pink solid compound 8-2 (58 g, 102 mmol, crude). ESI-MS (m / z): 568.2 [M+H] + .

[0356] Step 3:

[0357] HOBt (80.9 mg, 599 μmol, 1.7 eq.) and DIEA (113 mg, 880 μmol, 145 μL, 2.5 eq.) were added to a DMF (1 mL) solution of compound 8-2 (200 mg, 352 μmol, 1.0 eq.) and Exatecan (243 mg, 458 μmol, 1.3 eq.). The mixture was stirred at 25 °C for 4.0 h. TEA (363 mg, 3.59 mmol, 0.5 mL, 10.1 eq.) was added to the solution, and the mixture was stirred at 25 °C for 16 h. The reaction was detected as complete by LC-MS. The reaction mixture was acidified to pH 6 with TFA. The reaction mixture was purified by preparative HPLC to give a yellow solid compound 8-3 (138 mg, 156 μmol, yield 44.4%). ESI-MS (m / z): 642.2 [M+H] + .

[0358] 4) Synthesis of Gly-NHCH2-Dxd

[0359] S1: Pyridine (13.9 g, 176.3 mmol, 1.0 eq.) was added to a mixture of Fmoc-Gly-Gly-OH (50 g, 141.1 mmol, 1.0 eq.) and lead triacetoxyacetate (78.2 g, 176.3 mmol, 1.2 eq.) in THF (450 mL). The mixture was degassed at 25 °C and purged three times with N2. The mixture was then stirred at 75 °C under N2 atmosphere for 1.0 h. Thin-layer chromatography (TLC) showed that the starting material had reacted completely. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with water (250 mL) and extracted with ethyl acetate (250 mL * 2). The combined organic layers were washed with brine (500 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was stirred with EA (50 mL) at 25 °C for 30 min. The filter cake was filtered and dried under high vacuum to give a white colloidal compound 9-1 (41.5 g, 112.6 mmol, yield 79.8%). 1 H NMR: (400MHz, DMSO-d6) δ: 1.99 (s, 3H), 3.64 (d, J = 6.13Hz, 2H), 4.24 (br d,J=6.88Hz,1H),4.26-4.31(m,2H),5.09(d,J=7.00Hz,2H),7.30-7.36(m,2H),7.39-7.45(m,2H),7.56(br t,J=6.00Hz,1H),7.71(d,J=7.50Hz,2H),7.84-7.94(m,1H),7.89(d,J=7.50Hz,1H),8.86-8.97(m,1H).ESI-MS(m / z):391.0.[M+Na] + .

[0360] S2: To a solution of compound 9-1 (5.0 g, 13.5 mmol, 1.0 eq.) and benzyl 2-hydroxyacetate (5.6 g, 33.9 mmol, 4.8 mL, 2.5 eq.) in DCM (47.5 mL), TFA (3.8 g, 33.6 mmol, 2.5 mL, 2.4 eq.) was added at 0 °C, and the mixture was stirred at 25 °C for 3.0 h. The reaction was detected by LC-MS. The reaction mixture was diluted with saturated NaHCO3 (50 mL) and extracted with DCM (80 mL * 3). The combined organic matter was washed with brine, dried over Na2SO4, filtered, and the filtrate was concentrated to obtain a residue. The residue was purified by ISCO and concentrated to give a yellow gelatinous crude product. The crude product was purified by preparative HPLC to give a white solid compound 9-2 (3.7 g, 7.2 mmol, yield 53.1%).

[0361] 1 H NMR: (400MHz, DMSO-d6) δ: 3.62 (br d, J=6.13Hz, 2H), 4.26-4.30 (m, 2H), 4.63 (br d,J=6.75Hz,2H),5.13-5.15(m,2H),5.75(s,1H),7.30-7.37(m,8H),7.39-7.43(m,2H),7.54-7. 60(m,1H),7.71(d,J=7.38Hz,2H),7.89(d,J=7.50Hz,2H),8.73(s,1H).ESI-MS(m / z):475.2[M+H] + .

[0362] S3: A solution of Pd / C (dry, 350 mg) in THF (25 mL) was purged three times with N2, and then a solution of compound 9-2 (3.73 g, 7.86 mmol) in THF (10 mL) was added, followed by degassing three times with H2. The mixture was stirred at 25 °C for 4.0 h under an H2 atmosphere (15 psi). The reaction was confirmed by LC-MS. The mixture was filtered and the filtrate was concentrated to give a white solid, compound 9-3 (2.3 g, 5.4 mmol, yield 76.2%). ESI-MS (m / z): 407.0 [M+Na] + .

[0363] S4: To a DMF (3 mL) solution of compound 9-3 (300.0 mg, 780.4 μmol, 1.0 eq.) and Exatecan (331.8 mg, 624.3 μmol, 0.8 eq.), HOBt (126.5 mg, 936.5 μmol, 1.2 eq.), EDCI (179.5 mg, 936.5 μmol, 1.2 eq.), and DIPEA (201.7 mg, 1.5 mmol, 257.9 μL, 2.0 eq.) were added. The mixture was stirred at 35 °C for 2.0 h. The reaction was detected by LC-MS. The reaction mixture was purified by preparative HPLC to give a yellow solid compound 9-4 (400 mg, 471.4 μmol, yield 60.4%). ESI-MS (m / z): 802.3 [M+H] + .

[0364] S5: TEA (581.6 mg, 5.7 mmol, 800 μL, 11.5 eq.) was added to a mixture of compound 9-4 (400 mg, 498.8 μmol, 1.0 eq.) in DMF (3.2 mL). The mixture was stirred at 25 °C for 4.0 h. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give a yellow solid compound 9-5 (Gly-NHCH2-Dxd) (123.7 mg, 213.4 μmol, yield 42.7%). ESI-MS (m / z): 580.3 [M+H] + .

[0365] 5) Synthesis of VA-PAB-DX8951

[0366] S1: DIPEA (1.07 g, 8.30 mmol, 1.37 mL, 2.0 eq.) was added to a DMF (20 mL) solution of compounds Fmoc-Val-Ala-PAB (2.00 g, 4.15 mmol, 1.0 eq.) and PNP (1.89 g, 6.22 mmol, 1.5 eq.). The mixture was stirred at 25 °C for 4.0 h. The reaction was detected by LC-MS to indicate completion. The reaction mixture was poured into MTBE (2 L) under ice bath conditions, and a large amount of yellow solid precipitated. The mixture was collected, filtered, and the filter cake was collected to give a pale yellow solid compound 10⁻¹ (2.5 g, crude product). ESI-MS (m / z): 681.8 [M+H] + .

[0367] S2: HOBT (476 mg, 3.525 mmol, 1.2 eq.) and DIPEA (759 mg, 5.87 mmol, 0.97 mL, 2.0 eq.) were added to a DMF (20 mL) solution of compound 10⁻¹ (2.5 g, 2.93 mmol, 1.0 eq.) and Exatecan (124 g, 2.35 mmol, 0.8 eq.). The mixture was stirred at 25 °C for 2.0 h. The reaction was confirmed by LC-MS. The reaction mixture was poured into isopropyl ether (2000 mL) under ice bath conditions, stirred for 10 min, filtered, and the filter cake was dissolved in DCM (250 mL). The solution was then concentrated under reduced pressure to give a black, colloidal compound 10⁻² (2.5 g, crude). ESI-MS (m / z): 977.2 [M+H] + .

[0368] S3: To a DMF (25 mL) solution of compound 10⁻² (2.50 g, 2.56 mmol, 1.0 eq.), TEA (4.07 g, 40.2 mmol, 5.5 mL, 14 eq.) was added, and the mixture was stirred at 25 °C for 3.0 h. The reaction was confirmed by LC-MS. The reaction mixture was purified by HPLC to give a yellow colloidal compound 10⁻³ (VA-PAB-DX8951) (800 mg, 1.02 mmol, 3.7% yield). ESI-MS (m / z): 755.4 [M+H] + .

[0369] 6) Synthesis of VS(β-GlcA)-PAB-DX8951

[0370] S1: A solution of compound Fmoc-O-benzyl-L-serine (10.0 g, 24.0 mmol, 1.0 eq.) and compound α-D-glucuronide methyl ester (28.5 g, 71.9 mmol, 3.0 eq.) and 4A MS (36.0 g) in DCM (360 mL) was stirred at 25 °C for 30 min, and then AgOTf (18.5 g, 71.9 mmol, 3.0 eq.) was added. The mixture was stirred at 25 °C for 16.0 h. The reaction was detected by LC-MS. The reaction mixture was filtered and diluted with ethyl acetate (200 mL). The combined organic layers were filtered and washed with NaHCO3 (300 mL), dried on Na2SO4, filtered and concentrated under reduced pressure to give the residue, which was then purified by silica gel column chromatography to give a yellow oily compound 11-1 (6.80 g, 6.67 mmol, yield 27.9%). ESI-MS (m / z): 734.1 [M+H] + .

[0371] S2: A solution of Pd / C (dry, 470 mg) in THF (45.0 mL) was degassed three times with N2, followed by the addition of a THF (45.0 mL) solution of compound 11-1 (4.7 g, 6.4 mmol, 1.0 eq.), followed by three degassed times with H2. The mixture was stirred at 25 °C for 0.5 h under an H2 atmosphere (15 psi). The reaction was confirmed by LC-MS. The mixture was concentrated to give a colorless colloidal compound 11-2 (2.1 g, 2.3 mmol, yield 37.1%). ESI-MS (m / z): 644.2 [M+H] + .

[0372] S3: EEDQ (1.6 g, 6.5 mmol, 2.0 eq.) was added to a solution of compound 11-2 (2.1 g, 3.2 mmol, 1.0 eq.) and PAB (602.7 mg, 4.8 mmol, 1.5 eq.) in DCM (40.0 mL). The mixture was stirred at 25 °C for 1.0 h. The reaction was detected as complete by LC-MS. The reaction was concentrated to give the residue. The residue was purified by preparative HPLC to give compound 11-3 (1.0 g, 1.2 mmol, yield 39.5%) as a white solid. ESI-MS (m / z): 749.5 [M+H] + .

[0373] S4: TEA (1.4 g, 14.3 mmol, 2.0 mL, 9.7 eq.) was added to a DMF (8.0 mL) solution of compound 11-3 (1.1 g, 1.4 mmol, 1.0 eq.). The mixture was stirred at 25 °C for 0.5 h. The reaction was detected by LC-MS. The mixture was acidified to pH 5 with TFA. The mixture was purified by preparative HPLC to give compound 11-4 as a white solid (652.9 mg, 1.2 mmol, yield 83.5%). ESI-MS (m / z): 527.2 [M+H] + .

[0374] S5: To a mixture of compound 11-4 (800.0 mg, 1.5 mmol, 1.0 eq.) and Fmoc-Val-OH (257.8 mg, 759.7 μmol, 0.5 eq.) in DMF (10.0 mL), TBTU (585.4 mg, 1.8 mmol, 1.2 eq.) and DIPEA (785.5 mg, 6.0 mmol, 1.0 mL, 4.0 eq.) were added. The mixture was stirred at 25 °C for 1.0 h. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give compound 11-5 (574.2 mg, 669.1 μmol, yield 44.0%) as a white solid. ESI-MS (m / z): 848.4 [M+H] + .

[0375] S6: DIPEA (204.2 mg, 1.5 mmol, 261.2 μL, 2.0 eq.) was added to a mixture of compound 11-5 (670 mg, 790.2 μmol, 1.0 eq.) and PNP (1.4 g, 4.7 mmol, 6.0 eq.) in DMF (10.0 mL). The mixture was stirred at 25 °C for 4.0 h. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give compound 11-6 (555.0 mg, 544.6 μmol, yield 68.9%) as a white solid. ESI-MS (m / z): 1013.3 [M+H] + .

[0376] S7: To a DMF (2 mL) solution of compound 11-6 (210 mg, 207 μmol, 1.0 eq.) and Exatecan (110 mg, 207 μmol, 1.0 eq.), DIPEA (32.1 mg, 249 μmol, 41.1 μL, 1.2 eq.) and HOBt (33.6 mg, 249 μmol, 1.2 eq.) were added. The mixture was stirred at 25 °C for 1.0 h. The reaction was detected by LC-MS. A DMF solution of compound 11-7 was obtained and used directly in the next step. ESI-MS (m / z): 1309.5 [M+H] + .

[0377] S8: Add 0.25 mL of TEA to a solution of compound 11-7 (271 mg, 207 μmol, 1.00 eq.) in DMF (2 mL). Stir the mixture at 25 °C for 16 hours. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give a yellow solid compound 11-8 (VS(β-GlcA)-PAB-DX8951) (60.1 mg, 50.5 μmol, yield 24.4%). ESI-MS (m / z): 1087.5 [M+H] + .

[0378] 7) Synthesis of Cit-NHCH2-Dxd

[0379] S1: EEDQ (9.33 g, 37.7 mmol, 3.0 eq) was added to a solution of compound Fmoc-L-citrulline (5.00 g, 12.5 mmol, 1.0 eq) and compound glycine tert-butyl ester (1.98 g, 15.1 mmol, 1.2 eq) in THF (50 mL) and DCM (50 mL). The mixture was stirred at 25 °C under a nitrogen atmosphere for 16 hours. The reaction was confirmed by LC-MS. The reaction mixture was concentrated under reduced pressure to remove the solvent, giving a residue. The residue was dissolved in DCM and then added to MTBE (10 mL x 2) to give a white solid compound 12-1 (6.40 g, 12.5 mmol, yield 99.6%, purity 94.3%). ESI-MS (m / z): 510.2 [M+H] + .

[0380] S2: TFA (600 μL) was added to a solution of compound 12-1 (5.80 g, 11.3 mmol, 1.0 eq.) in DCM (2.4 mL) at 0 °C. The mixture was stirred at 25 °C for 16 hours. The reaction was confirmed by LC-MS. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a residue. The residue was dissolved in DCM and then added to isopropyl ether (20 mL x 2) to give compound 12-2 (2.70 g, 5.94 mmol, yield 52.3%) as a white solid. ESI-MS (m / z): 455.1 [M+H] + .

[0381] S3: Pyridine (261 mg, 3.30 mmol, 1.0 eq.) was added to a solution of compound 12-2 (1.20 g, 2.64 mmol, 1.0 eq.) and lead triacetoxyacetate (1.46 g, 3.30 mmol, 1.25 eq.) in THF (18 mL) and toluene (6 mL). The mixture was stirred at 70 °C for 1.0 h. The reaction was detected as complete by LC-MS. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was dissolved in ethyl acetate, washed twice with saturated brine, and the organic layer was dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to obtain the residue. The residue was added to EtOAc (50 mL * 2) to give compound 12-3 (855 mg, crude product), a white solid. ESI-MS (m / z): 491.1 [M + Na] + .

[0382] S4: Add 4A to a solution of compound 12-3 (800 mg, 1.71 mmol, 1.0 eq.) and compound benzoyl glycolate (1.42 g, 8.54 mmol, 1.21 mL, 5.0 eq.) in THF (16 mL). MS (50.0 mg, 1.0 eq.) was added to the solution and stirred at 25 °C for 6 hours. Sc(CO3SF3)3 (1.09 g, 2.22 mmol, 1.3 eq.) was added to the solution, and the mixture was stirred at 25 °C for 6 hours. The reaction was confirmed by LC-MS. The residue was purified by preparative HPLC to give a white solid compound 12-4 (250 mg, 435 μmol, yield 25.4%, purity 98.2%). ESI-MS (m / z): 575.4 [M+H] + .

[0383] S5: Pd / C (dry, 4.00 mg, 10%, 15 psi) was added to a solution of compound 12-4 (20.0 mg, 34.8 μmol, 1.0 eq.) in methanol (1.5 mL) and DMF (500 μL), degassed, and purged three times with N2. The mixture was stirred at 25 °C for 1.5 h under H2 atmosphere. TLC showed that compound 12-4 reacted completely, and a new product was formed. The reaction mixture was filtered and concentrated under reduced pressure to give compound 12-5 (13.6 mg, crude) as a white solid. ESI-MS (m / z): 507.1 [M+Na] + .

[0384] S6: DIEA (8.00 mg, 61.9 μmol, 10.2 μL, 3.0 eq.) was added to a solution of compound 12-5 (10.0 mg, 20.6 μmol, 1.0 eq.) and TSTU (6.21 mg, 20.6 μmol, 1.0 eq.) in DMF (100 μL), and the mixture was stirred for 10 min. Then, Exatecan (10.9 mg, 20.6 μmol, 1.0 eq.) was added, and the mixture was stirred at 25 °C for 5.0 h. The reaction was confirmed by LC-MS. The residue was purified by HPLC to give a white solid compound 12-6 (7.00 mg, 7.76 μmol, yield 37.6%, purity 98.8%). ESI-MS (m / z): 902.4 [M+H] + .

[0385] S7: Add 50 μL of TEA to a solution of compound 12-6 (6.00 mg, 6.65 μmol, 1.0 eq.) in DMF (150 μL). Stir the mixture at 25 °C for 1.0 h. The reaction was confirmed by LC-MS. Add the mixture to isopropyl ether (1 mL * 2) to give a white solid compound 12-7 (216 mg, crude). ESI-MS (m / z): 680.3 [M + Na] + .

[0386] 8) Synthesis of K(Mor)-PAB-DX8951

[0387] S1: NaBH3OAc (8.626 g, 40.7 mmol, 3.0 eq) was added to a solution of compound 13-1 (5 g, 13.57 mmol, 1.0 eq) and compound 2,2'-oxydiacetaldehyde (4.156 g, 40.71 mmol, 3.0 eq) in DCE (200 mL). The mixture was stirred at 25 °C for 2 hours. The reaction was monitored by LC-MS to indicate completion. The reaction mixture was purified by HPLC to give compound 13-2 (2 g, 4.56 mmol, yield: 33.6%) as a pale yellow solid. ESI-MS (m / z): 439.2 [M+H] + .

[0388] S2: HATU (520 mg, 1.37 mmol, 1.5 eq), DIEA (236 mg, 1.83 mmol, 2.0 eq), and p-aminobenzyl alcohol (135 mg, 1.10 mmol, 1.2 eq) were added to a DMF (2 mL) solution of compound 13-2 (400 mg, 0.91 mmol, 1.0 eq), and the mixture was stirred at 25 °C for 2 h. The reaction was monitored by LC-MS to indicate completion. The reaction mixture was purified by HPLC to give a pale yellow solid compound 13-3 (450 mg, 0.83 mmol, yield: 90.7%). ESI-MS (m / z): 544.4 [M+H] + .

[0389] S3: PNP (378 mg, 1.24 mmol, 1.5 eq) was added to a DMF (5 mL) solution of compound 13-3 (450 mg, 0.83 mmol, 1.0 eq) and DIEA (216 mg, 1.66 mmol, 2.0 eq), and the mixture was stirred at 25 °C for 5 hours. The reaction was monitored by LC-MS to indicate completion. The reaction mixture was purified by HPLC to give a yellow solid compound 13-4 (450 mg, 0.63 mmol, yield: 76.71%). ESI-MS (m / z): 709.2 [M+H] + .

[0390] S4: HOBT (119 mg, 0.92 mmol, 2.50 eq.) and DIEA (119 mg, 0.92 mmol, 2.50 eq.) were added to a DMF (2 mL) solution of compound 13-4 (200 mg, 0.37 mmol, 1.0 eq.) and compound eczemab (234 mg, 0.37 mmol, 1.0 eq.). The mixture was stirred at 0 °C for 2 hours. The reaction was monitored for completion by LC-MS. The reaction mixture was purified by HPLC to give a yellow solid compound 13-5 (200 mg, 0.28 mmol, yield: 70.5%). ESI-MS (m / z): 1005.4 [M+H] + .

[0391] S5: Diethylamine (75 mg, 0.70 mmol, 5.0 eq.) was added to a 2 mL DMF solution of compound 13-5 (100 mg, 0.14 mmol, 1.0 eq.). The mixture was stirred at 0 °C for 0.5 h. The reaction was monitored by LC-MS to indicate completion. The reaction mixture was purified by HPLC to give compound 13-6 (100 mg, 0.1 mmol, yield: 100%) as a white solid. ESI-MS (m / z): 783.3 [M+H] + .

[0392] 2.2 Synthesis of Linker-P005

[0393] 1) Synthesis of L085-P005

[0394] S1: DIPEA (21.7 mg, 168 μmol, 27.8 μL, 3.00 eq.) was added to a DMF (0.6 mL) solution of compounds 11-8 (60.9 mg, 56.0 μmol, 1.00 eq.), 1-21 (43.8 mg, 56.0 μmol, 1.00 eq.), and TBTU (21.6 mg, 67.2 μmol, 1.2). The mixture was stirred at 25 °C for 0.5 h. The reaction was detected by LC-MS. The reaction mixture was purified by preparative HPLC to give a yellow solid compound 11-9 (61.1 mg, 32.8 μmol, yield 58.5%). ESI-MS (m / z): 1850.8 [M+H] + .

[0395] S2: Piperidine (5.6 mg, 66 μmol, 6.5 μL, 2.00 eq.) was added to a mixture of compound 11-9 (61.1 mg, 33.0 μmol, 1.00 eq.) in DMF (1.2 mL) at 0 °C, and the mixture was stirred at 25 °C for 0.5 h. The reaction was detected as complete by LC-MS. The reaction mixture was quenched at 0 °C by the addition of FA (6 μL). The residue was purified by HPLC to give compound 11-10 (37 mg, 23 μmol, yield 68.4%) as a white solid. ESI-MS (m / z): 1629.3 [M+H] + .

[0396] S3: LiOH (1M, 98.2μL, 4.0eq.) and H2O (100μL) were added to a solution of 11-10 (40.0 mg, 24.5 μmol, 1.0 eq.) in THF (100 μL) and MeOH (100 μL) at 0 °C. The mixture was stirred at 0 °C for 0.2 h. The reaction was detected by LC-MS. The mixture was acidified with AcOH to pH≈6, and then purified by preparative HPLC to give a yellow solid compound 11-11 (30.0 mg, 16.6 μmol, yield 67.7%). ESI-MS (m / z): 1488.9 [M+H] + .

[0397] S4: DIPEA (2.6 mg, 20.1 μmol, 3.3 μL, 1.5 eq.) was added to a solution of compound 11-11 (20.0 mg, 13.4 μmol, 1.0 eq.) and N-succinimidyl 6-maleimidohexanoate (4.1 mg, 13.4 μmol, 1.0 eq.) in DMF (200 μL). The mixture was stirred at 25 °C for 2.0 h. The reaction was confirmed by LC-MS. The mixture was purified by preparative HPLC to give a white solid compound L085-P005 (2.6 mg, 1.4 μmol, yield 11.0%, purity 95.9%). ESI-MS (m / z): 841.9 [M / 2+H] + .

[0398] 2) Synthesis of L068-P005

[0399] S1: To a solution of compounds 1-7 (83.7 mg, 53.5 μmol, 0.9 eq.) and 7-4 (50.0 mg, 59.4 μmol, 1.0 eq.) in DMF (500 μL), TBTU (22.9 mg, 71.3 μmol, 1.2 eq.) and DIPEA (30.7 mg, 237 μmol, 39.3 μL, 4.0 eq.) were added, and the reaction mixture was stirred at 25 °C for 3.0 h. The reaction was confirmed by LC-MS. The product was purified by preparative HPLC to give a yellow solid compound 7-5 (35 mg, 13.5 μmol, yield 22.8%). ESI-MS (m / z): 1195.9 [M+H] + .

[0400] S2: Compound 7-5 (30.0 mg, 12.5 μmol, 35.0 μL, 1.0 eq.) was added to a mixture of TFA (285 μL) and H2O (7.20 μL), followed by TIS (2.06 mg, 12.5 μmol, 7.50 μL, 1.0 eq.) at 0 °C. The reaction mixture was stirred at 0 °C for 6.0 h. The reaction was confirmed by LCMS. The reaction mixture was centrifuged with isopropyl ether (1.0 mL * 2) in a dry ice bath to give a yellow solid. The solid was purified by preparative HPLC to give a white solid compound L068-P005 (3.50 mg, 1.93 μmol, yield 15.4%, purity 91.3%). ESI-MS (m / z): 1651.9 [M+H] + .

[0401] 3) Other Linker-P005 synthesis list

[0402] The product can be obtained by direct condensation or by deprotection under acidic conditions after condensation, referring to the above-mentioned synthesis method L068-P005.

[0403] Table 5 Linker-P005 Synthesis List

[0404] 2.3 Synthesis of Linker-P002(MMAE) intermediate

[0405] 1) Synthesis of Cit-PAB-P002

[0406] Compound 2-2 (3.0 g, 4.49 mmol, 1.0 eq.) and MMAE (3.22 g, 4.48 mmol, 1.0 eq.) were mixed in DMF (30 mL) with HOBt (910 mg, 6.74 mmol, 1.5 eq.) and DIPEA (1.74 g, 13.4 mmol, 2.22 mL, 3.0 eq.), and stirred at 25 °C for 7.0 h. Product formation was detected by LC-MS. TEA (5.45 g, 53.8 mmol, 7.5 mL, 11.9 eq.) was added to the solution, and the mixture was stirred at 25 °C for 16 h. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give a white colloidal compound 14-1 (740 mg, 515 μmol, yield 11.4%). ESI-MS (m / z): 1024.6 [M+H] + .

[0407] 2) Synthesis of Dap(Me2)Cit-PAB-MMAE

[0408] S1: TBTU (56.4 mg, 175 μmol, 1.2 eq.) and DIPEA (56.7 mg, 439 μmol, 72.6 μL, 3.0 eq.) were added to a DMF (2 mL) solution of compound 14-1 (150 mg, 146 μmol, 1.0 eq.) and compound FMOC-AZA-L-LEUCINE (62.2 mg, 175 μmol, 1.2 eq.), and stirred at 25 °C for 1.0 h. The reaction was confirmed by LC-MS. The reaction product was purified by preparative HPLC to give a white solid compound 14-2 (30 mg, 21.8 μmol, yield 14.9%). ESI-MS (m / z): 1361.7 [M+H] + .

[0409] S2: TEA (145 mg, 1.43 mmol, 200 μL, 65.1 eq.) was added to a solution of compound 14-2 (30 mg, 22.0 μmol, 1.0 eq.) in DMF (800 μL), and the mixture was stirred at 25 °C for 2.0 h. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give a white solid compound 14-3 (Dap(Me2)Cit-PAB-MMAE) (13 mg, 10.7 μmol, yield 48.6%). ESI-MS (m / z): 1138.8 [M+H] + .

[0410] 3) Synthesis of Dap(Me2)VCit-PAB-MMAE

[0411] S1: DIEA (87.5 mg, 677 μmol, 112 μL, 3.0 eq.) was added to a DMF (3.0 mL) solution of Val-Cit-PAB-MMAE (254 mg, 226 μmol, 1.0 eq.), FMOC-AZA-L-LEUCINE (80.0 mg, 226 μmol, 1.0 eq.), and TBTU (87.0 mg, 271 μmol, 1.2 eq.). The mixture was stirred at 25 °C for 1.0 h. The reaction was confirmed by LC-MS. The mixture was added dropwise to isopropyl ether (30.0 mL) and centrifuged to give a brown oily compound 15-1 (361 mg, crude product). ESI-MS (m / z): 1459.9 [M+H] + .

[0412] S2: TEA (750 mg, 7.42 mmol, 1.03 mL, 30 eq.) was added to a DMF (4.10 mL) solution of compound 15-1 (361 mg, 248 μmol, 1.0 eq.). The mixture was stirred at 25 °C for 6.0 h. The reaction was confirmed by LC-MS. The mixture was purified by preparative HPLC to give a white solid compound 15-2 (Dap(Me2)VCit-PAB-MMAE) (143 mg, 116 μmol, yield 48.2%). ESI-MS (m / z): 1237.8 [M+H] + .

[0413] 4) Synthesis of VS(β-GlcA)-PAB-MMAE

[0414] HOBt (36.0 mg, 266.5 μmol, 1.2 eq.) and DIPEA (43.0 mg, 333.1 μmol, 55.0 μL, 1.5 eq.) were added to a mixture of compound 11-6 (225.0 mg, 222.1 μmol, 1.0 eq.) in DMF (2.0 mL), and the mixture was stirred at 25 °C for 4.0 h. Product formation was detected by LC-MS. TEA (290.8 mg, 2.8 mmol, 400.0 μL, 12.9 eq.) was added to the mixture, and the mixture was stirred at 25 °C for 16 h. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give compound 16-1 (142.7 mg, 102.2 μmol, yield 46.0%) as a white solid. ESI-MS (m / z): 1369.6 [M+H] + .

[0415] 5) Synthesis of VA-PAB-MMAE

[0416] S1: HOBt (297 mg, 2.2 mmol, 1.5 eq.) and DIPEA (569 mg, 4.4 mmol, 728 μL, 3.0 eq.) were added to a mixture of compound 10-1 (1 g, 1.4 mmol, 1.0 eq.) and MMAE (527 mg, 734 μmol, 0.5 eq.) in DMF, and the mixture was stirred at 25 °C for 2.0 h. The reaction was confirmed by LC-MS. The reaction mixture was purified by HPLC to give compound 17-1 (437.7 mg, 347 μmol, yield 23%) as a yellow solid. ESI-MS (m / z): 1259.6 [M+H] + .

[0417] S2: TEA (1.1 g, 11 mmol, 1.6 mL, 37.4 eq.) was added to a mixture of compound 17-1 (387 mg, 307 μmol, 1.0 eq.) in DMF (2.4 mL). The mixture was stirred at 25 °C for 16 hours. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give a yellow solid compound 17-2 (VA-PAB-MMAE) (300 mg, 270 μmol, yield 87.9%). ESI-MS (m / z): 1037.9 [M+H] + .

[0418] 6) Synthesis of VK(Me2)-PAB-MMAE

[0419] S1: To a mixture of compound 4-4 (80.5 mg, 78.6 μmol, 1.0 eq.) and Fmoc-L-Valine (21.3 mg, 62.9 μmol, 0.8 eq.) in DMF (1 mL), TBTU (30.3 mg, 94.3 μmol, 1.2 eq.) and DIPEA (40.6 mg, 314.6 μmol, 52.0 μL, 4.0 eq.) were added. The mixture was stirred at 25 °C for 2 hours. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give a yellow solid compound 18-1 (14.6 mg, 7.6 μmol, yield 9.7%). ESI-MS (m / z): 1344.8 [M+H] + .

[0420] S2: TEA (43.6 mg, 431.0 μmol, 60 μL, 18.1 eq.) was added to a mixture of compound 18-1 (32 mg, 23.7 μmol, 1 eq.) in DMF (240 μL). The mixture was stirred at 25 °C for 1.0 h. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give compound 18-2 (VK(Me2)-PAB-MMAE) as a white solid (15 mg, 12.6 μmol, yield 53.0%). ESI-MS (m / z): 1122.7 [M+H] + .

[0421] 7) Synthesis of Ala-PAB-MMAE

[0422] S1: EEDQ (2.3 g, 9.6 mmol, 2.0 eq.) was added to a mixture of Fmoc-Ala-OH (1.5 g, 4.8 mmol, 1.0 eq.) and PAB (1.1 g, 9.6 mmol, 2.0 eq.) in MeOH (10 mL) and DCM (40 mL). The mixture was stirred at 25 °C for 2.0 h. The reaction was confirmed by LC-MS. The mixture was precipitated with isopropyl ether (500 mL) and centrifuged. The mixture was filtered and the filter cake was dried under high vacuum to give a brown solid compound 19-1 (607 mg, 1.2 mmol, yield 26.8%). ESI-MS (m / z): 417.4 [M+H] + .

[0423] S2: DIPEA (376.7 mg, 2.9 mmol, 481.7 μL, 2.0 eq.) was added to a DMF (6 mL) solution of compound 19-1 (607 mg, 1.4 mmol, 1.0 eq.) and PNP (86.7 mg, 2.9 mmol, 2.0 eq.). The mixture was stirred at 25 °C for 1.0 h. The reaction was detected by LC-MS. The mixture was precipitated with isopropyl ether (60 mL) and centrifuged. The mixture was purified by preparative HPLC to give compound 19-2 (400 mg, 638.9 μmol, yield 43.8%) as a yellow solid. ESI-MS (m / z): 582.4 [M+H] + .

[0424] S3: HOBT (20.3 mg, 150.8 μmol, 1.5 eq.) and DIPEA (26.0 mg, 201.1 μmol, 33.2 μL, 2.0 eq.) were added to a mixture of compound 19-2 (58.5 mg, 100.5 μmol, 1.0 eq.) and MMAE (72.2 mg, 100.5 μmol, 1.0 eq.) in DMF (0.5 mL). The mixture was stirred at 25 °C for 4.0 h. LC-MS was used to determine the complete reaction of the starting materials. TEA (90.8 mg, 898.0 μmol, 125 μL, 8.9 eq.) was added to the solution, and the mixture was stirred at 25 °C for 16.0 h. LC-MS was used to determine the complete reaction. The mixture was acidified with TFA to pH ≈ 6. The mixture was purified by preparative HPLC to give a white solid compound 19-3 (Ala-PAB-MMAE) (54.6 mg, 56.7 μmol, yield 56.4%). ESI-MS (m / z): 938.7 [M+H] + .

[0425] 8) Synthesis of K(Mor)-PAB-MMAE

[0426] S1: HOBT (19 mg, 0.14 mmol, 1.0 eq.) and Py (12 mg, 0.15 mmol, 1.08 eq.) were added to a DMF (1 mL) solution of compound 13-4 (100 mg, 0.14 mmol, 1.0 eq.) and compound MMAE (101 mg, 0.14 mmol, 1.0 eq.). The mixture was stirred at 25 °C for 2 hours. The reaction was monitored by LC-MS to indicate completion. The reaction mixture was purified by HPLC to give a white solid compound 20-1 (100 mg, 0.04 mmol, yield: 27.5%). ESI-MS (m / z): 1288.6 [M+H] + .

[0427] S1: Diethylamine (30 mg, 0.20 mmol, 5.18 eq.) was added to a DMF (1 mL) solution of compound 20-1 (100 mg, 0.04 mmol, 1.0 eq.). The mixture was stirred at 0 °C for 0.5 h. The reaction was monitored by LC-MS to indicate completion. The reaction mixture was purified by HPLC to give compound 20-2 (38 mg, 0.04 mmol, yield 91.8%) as a white solid. ESI-MS (m / z): 1066.6 [M+H] + .

[0428] 2.4 Synthesis of Linker-P002

[0429] 1) Synthesis of L085-P002

[0430] S1: TBTU (14.0 mg, 43.8 μmol, 1.20 eq.) was added to a solution of compound 16-1 (50.0 mg, 36.5 μmol, 1.00 eq.) and compound 1-21 (28.5 mg, 36.5 μmol, 1.00 eq.) in DMF (1.0 mL). The mixture was stirred at 25 °C for 1.0 h. Product formation was detected by LC-MS. Then, TEA (181 mg, 1.79 mmol, 250 μL, 49.1 eq.) was added. The mixture was stirred at 25 °C for 3.0 h. The reaction was detected by LC-MS. The reaction mixture was purified directly by preparative HPLC to give compound 16-2 (27.2 mg, 14.1 μmol, yield 38.6%) as a white solid. ESI-MS (m / z): 956.4 [M / 2+H] + .

[0431] S2: To a solution of compound 16-2 (35.0 mg, 18.3 μmol, 1.00 eq.) in MeOH (233 μL), add H₂O (233 mL) and THF (233 μL), then add LiOH (1.0 M, 73.2 μL, 4.00 eq.). Stir the mixture at 0 °C for 10 min. The reaction was confirmed by LC-MS. The pH of the reaction mixture was adjusted to 5–6 by adding 1.0 M CH₃COOH (0.2 μL). The residue was purified by preparative HPLC to give compound 16-3 (19.0 mg, 10.2 μmol, yield 55.9%) as a white solid. ESI-MS (m / z): 886.5 [M + 2H] 2+ .

[0432] S3: DIPEA (2.08 mg, 16.0 μmol, 2.66 μL, 1.50 eq.) was added to a solution of compound 16-3 (19.0 mg, 10.7 μmol, 1.00 eq.) and compound 6-maleimide hexanoate N-succinimidyl 6-maleimidohexanoate (3.63 mg, 11.8 μmol, 1.10 eq.) in DMF (0.1 mL). The mixture was stirred at 25 °C for 2.0 h. The reaction was confirmed by LC-MS. The reaction mixture was purified by preparative HPLC to give compound 16-4 (L085-P002) as a white solid (9.00 mg, 4.53 μmol, yield 42.2%, purity 99.0%). ESI-MS (m / z): 983.1 [M / 2+H] + .

[0433] 2) Other Linker-P002 synthesis list

[0434] The product can be obtained by direct condensation or by deprotection under acidic conditions after condensation, referring to the above-mentioned synthesis method L068-P005.

[0435] Table 6 Linker-P002 Synthesis List

[0436] Example 3: Preparation and Physicochemical Property Analysis of ADC

[0437] 3.1 ADC Physicochemical Property Analysis Methods

[0438] 3.1.1 HIC-HPLC method for determining DAR

[0439] The DAR value of the coupled samples was determined using HIC-HPLC:

[0440] Sample preparation: Dilute the sample to 2.5 mg / mL with ultrapure water and inject 10 μL.

[0441] The average number of drug linkages was calculated using the HIC-HPLC formula:

[0442] Where A(total) represents the total peak area, and A(DAR2), A(DAR4), A(DAR6), and A(DAR8) represent the peak areas generated by antibody-drug conjugates with connection numbers of 2, 4, 6, and 8, respectively.

[0443] 3.1.2 PLRP-HPLC method for determining DAR

[0444] The DAR value of the coupled samples was determined by PLRP-HPLC.

[0445] Sample preparation: Take 20 μL (100 μg) of antibody-drug conjugate, add 5 μL of 1M Tris-HCl (pH 8.0) solution and 75 μL of 6M guanidine hydrochloride solution, mix well, then add 2 μL of 0.5M TCP solution, and incubate at 37℃ for 15 minutes to allow the antibody-drug conjugate to be fully reduced. The resulting solution is directly used for PLRP-HPLC detection.

[0446] The average number of drug linkages was calculated using the PLRP-HPLC formula:

[0447] After reduction treatment, the ADC is analyzed by PLRP-HPLC. Theoretically, the following components will appear: antibody light chain (LC0), antibody light chain coupled with 1 linker-payload (LC1), antibody heavy chain (HC0), antibody heavy chain coupled with 1 linker-payload (HC1), antibody heavy chain coupled with 2 linker-payloads (HC2), and antibody heavy chain coupled with 3 linker-payloads (HC3). In the formula, A represents the peak area, LC... total Represents all light chains, HC total This represents all heavy chains.

[0448] 3.1.3 Mass spectrometry method for determining DAR

[0449] The DAR value of the coupled sample was determined by mass spectrometry.

[0450] Sample preparation: 50 μg of sample was added to 2 μL of 0.5 M TCEP, and ultrapure water was added to dilute to approximately 100 μL to a concentration of about 0.5 mg / mL. The mixture was stirred and reduced at 37 °C for 30 min. The resulting sample was used for mass spectrometry analysis.

[0451] The average number of drug connections was calculated using the PLRP-MS formula:

[0452] After ADC reduction treatment, LC-MS analysis theoretically yields the following components: antibody light chain (LC0), antibody light chain conjugated to 1 linker-payload (LC1), antibody heavy chain (HC0), antibody heavy chain conjugated to 1 linker-payload (HC1), antibody heavy chain conjugated to 2 linker-payloads (HC2), and antibody heavy chain conjugated to 3 linker-payloads (HC3). In the formula, A represents the peak area, and LC... total Represents all light chains, HC total This represents all heavy chains.

[0453] 3.1.4 Determination of ADC purity using SEC-HPLC method

[0454] The purity of the coupled samples was determined by SEC-HPLC.

[0455] Sample preparation: Dilute the sample to 2 mg / mL with ultrapure water and inject 15 μL.

[0456] 3.2 ADC Preparation

[0457] 3.2.1 Preparation of mAb4-Linker-P005-DAR8 antibody conjugate

[0458] A solution of anti-TF antibody h1849-H3L3 (mAb4, prepared according to Example 2 of WO2025 / 082461A1) was replaced with 25 mM His-HCl (pH 5.5) or 50 mM PB / 150 mM NaCl (pH 7.4) solution, and 5 mM tris(2-carboxyethyl)phosphine (TCEP) was added, with a TCEP to mAb4 molar ratio of 6 or 7:1. The mixture was then reduced at 37°C for 2 hours. After 2 hours, N,N-dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO) and Linker-P005 (15 mg / mL), a compound soluble in DMA or DMSO, were added to the mixture. The final concentration of the organic reagent was 10%, and the molar ratio of drug to mAb4 was between 13:1 and 22:1. Continue reacting this solution at 22°C for 1 hour, 22°C for 18 hours, or 30°C for 1 hour to ensure complete coupling. After the reaction, use 40 kDa MWCO Zeba [reactor / product name - likely a specific brand or product name] to react. TM A one-step purification was performed using a centrifugal desalting column, and the coupled product was then stored in a 25 mM His-HCl (pH 5.5) solution.

[0459] List of obtained ADC structures:

[0460] The antibody h1849-H3L3 (mAb4) solution was replaced with 25 mM His-HCl (pH 5.5) solution, and 5 mM tris(2-carboxyethyl)phosphine (TCEP) was added, with a TCEP to mAb4 molar ratio of 6:1. This mixture was then incubated at 37°C for 2 hours for reduction. After 2 hours, excess TCEP was removed by ultrafiltration using a 30 kDa MWCO ultrafiltration centrifuge tube. Then, N,N-dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO) and Linker-P005 (15 mg / mL), a compound soluble in DMA or DMSO, were added to the mixture to bring the final organic reagent concentration to 10% and the drug to mAb4 molar ratio to 13:1. This solution was then incubated at 22°C for 1 hour to ensure complete coupling. After the reaction, the solution was purified using a 40 kDa MWCO Zeba... TM A one-step purification was performed using a centrifugal desalting column, and the coupled product was then stored in a 25 mM His-HCl (pH 5.5) solution.

[0461] List of obtained ADC structures:

[0462] 3.2.2 Preparation of mAb4-Linker-P002-DAR4 antibody conjugate

[0463] The antibody h1849-H3L3 (mAb4) solution was replaced with 25 mM His-HCl (pH 5.5) solution, and 5 mM tris(2-carboxyethyl)phosphine (TCEP) was added. The molar ratio of TCEP to mAb4 was 2.8:1-3.3:1. The mixture was then reduced at 37°C for 2 hours. After 2 hours, dimethyl sulfoxide (DMSO) and Linker-P002 (15 mg / mL), a compound dissolved in DMSO, were added to the mixture to bring the final organic reagent content to 10% and the drug-to-mAb4 molar ratio to 8:1. The mixture was then further reacted at 22°C for 1 hour or 18 hours to ensure complete coupling. After the reaction, 40 kDa MWCO Zeba was used. TM A one-step purification was performed using a centrifugal desalting column, and the coupled product was then stored in a 25 mM His-HCl (pH 5.5) solution.

[0464] List of obtained ADC structures:

[0465] The antibody h1849-H3L3 (mAb4) solution was replaced with 50 mM PB and 150 mM NaCl (pH 7.4) solution, and 5 mM tris(2-carboxyethyl)phosphine (TCEP) was added. The molar ratio of TCEP to mAb4 was 2.9:1-3.22:1. The mixture was then reduced at 37°C for 2 hours. After 2 hours, N,N-dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO) and Linker-P002 (15 mg / mL), a compound dissolved in DMA or DASO, were added to the mixture to bring the final organic reagent content to 10% and the drug-to-mAb4 molar ratio to 7:1 or 8:1. The solution was then reacted at 22°C for 1 hour or 18 hours to ensure complete coupling. After the reaction, 40 kDa MWCO Zeba was used. TM A one-step purification was performed using a centrifugal desalting column, and the coupled product was then stored in a 25 mM His-HCl (pH 5.5) solution.

[0466] List of obtained ADC structures:

[0467] 3.2.3 Preparation of Trastuzumab-Linker-P002-DAR4 antibody conjugate

[0468] The anti-HER2 antibody Trastuzumab solution was replaced with 25 mM His-HCl (pH 5.5) solution, and 5 mM tris(2-carboxyethyl)phosphine (TCEP) was added. The molar ratio of TCEP to Trastuzumab was 2.8:1–3.3:1. The mixture was then incubated at 37°C for 2 hours for reduction. After 2 hours, dimethyl sulfoxide (DMSO) and Linker-P002 (15 mg / mL), a compound dissolved in DMSO, were added to the mixture to bring the final concentration of the organic reagent to 10% and the molar ratio of drug to Trastuzumab to 8:1. The mixture was then incubated at 22°C for 1 hour or 18 hours to ensure complete coupling. After the reaction, 40 kDa MWCO Zeba was used. TM A one-step purification was performed using a centrifugal desalting column, and the coupled product was then stored in a 25 mM His-HCl (pH 5.5) solution.

[0469] 3.2.4 Preparation of 2H8L4-Linker-P002-DAR4 antibody-drug conjugate

[0470] The anti-GPC-1 antibody 2H8L4 solution was replaced with 25 mM His-HCl (pH 5.5) solution, and 5 mM tris(2-carboxyethyl)phosphine (TCEP) was added. The molar ratio of TCEP to 2H8L4 was 2.8:1–3.3:1. The mixture was then incubated at 37°C for 2 hours for reduction. After 2 hours, dimethyl sulfoxide (DMSO) and Linker-P002 (15 mg / mL), a compound dissolved in DMSO, were added to the mixture to bring the final concentration of the organic reagent to 10% and the molar ratio of drug to 2H8L4 to 8:1. The mixture was then incubated at 22°C for 1 hour or 18 hours to ensure complete coupling. After the reaction, 40 kDa MWCO Zeba was used. TM A one-step purification was performed using a centrifugal desalting column, and the coupled product was then stored in a 25 mM His-HCl (pH 5.5) solution.

[0471] List of obtained ADC structures:

[0472] Table 6 Purity and DAR value of the ADC in this application

[0473] Experimental Example 1: Enzymatic digestion test of matrix metalloproteinase (MMP) and cathepsin B in the Linker payload

[0474] Experimental Protocol 1.1 Enzymatic digestion test of Linker-payload using MMP2

[0475] MMP2 to a final concentration of 200 μg / mL was mixed with 4 mM APMA at a 1:1 ratio and incubated in a 37°C water bath for 24 h to activate the MMP2 enzyme solution. Then, a linker-payload to a final concentration of 100 μmol was incubated with the activated MMP2 solution to a final concentration of 1 μg / mL in a 37°C water bath for 24 h. After the reaction was complete, LC-MS analysis was performed.

[0476] LC-MS results (Table 7) show that all Linker-payloads can be digested by MMP2 under these conditions, thereby releasing active molecules.

[0477] Table 7. Enzymatic digestion results of Linker-payload using MMP2.

[0478] Experimental Protocol 1.2 Enzymatic Digestion Test of Linker-payload using MMP9

[0479] MMP9 to a final concentration of 200 μg / mL was mixed with 4 mM APMA at a 1:1 ratio and incubated in a 37°C water bath for 24 h to activate the MMP9 enzyme solution. Then, a linker-payload to a final concentration of 100 μmol was incubated with the activated MMP9 solution to a final concentration of 0.2 μg / mL in a 37°C water bath for 24 h. After the reaction was complete, LC-MS analysis was performed.

[0480] LC-MS results (Table 8) show that all Linker-payloads can be digested by MMP9 under these conditions, thereby releasing active molecules.

[0481] Table 8. Enzymatic digestion results of Linker-payload using MMP9

[0482] Experimental Protocol 1.3 Enzymatic Digestion Test of Linker-payload with Cathepsin B

[0483] A catepsin B solution with a final concentration of 10 μg / mL was incubated at room temperature for 15 min in a 25 mM acetate-sodium acetate (pH 5.5) solution containing 15 mM EDTA and 30 mM DTT to activate catepsin B. Then, a linker-payload with a final concentration of 25 μg / mL was mixed with the activated catepsin B enzyme solution with a final concentration of 2 μg / mL in a 25 mM acetate-sodium acetate + 1 mM EDTA (pH 6.0) solution and incubated at 37 °C and 220 rpm for 4 h. After the reaction was complete, LC-MS analysis was performed.

[0484] LC-MS results showed that the above linker-payload could be enzymatically hydrolyzed to varying degrees by Cathepsin B under these conditions, thereby releasing the active molecules.

[0485] Table 9. Results of Cathepsin B digestion of the Linker-payload

[0486] Experimental Example 2: Determination of the hydrophilicity of Linker-DX8951-ADC and Linker-Dxd-ADC by HIC-HPLC method

[0487] As shown in Table 10, the HIC-HPLC results (the determination method is the same as described in Section 3.1.1 of Example 3) show that, under the same payload conditions, namely DX8951 or Dxd, the linkers prepared in the embodiments of this application can have comparable or even stronger hydrophilicity compared to Derextecan.

[0488] Table 10

[0489] Experimental Example 3: Determination of the hydrophilicity of Linker-MMAE-ADC by HIC-HPLC method

[0490] As shown in Table 11, the HIC-HPLC results (the determination method is the same as described in Section 3.1.1 of Example 3) show that, under the same payload, i.e., MMAE, the linkers prepared in the embodiments of this application can have comparable or even stronger hydrophilicity compared to vcMMAE.

[0491] Table 11

[0492] Experiment 4: Killing effect of ADC drugs on DU145 cells and SK-OV-3 cells

[0493] The inhibitory activity of bioactive molecules on relevant tumor cells was detected in vitro. DU145 cells (human prostate cancer cells) were purchased from Nanjing Kebai.

[0494] 4.1 In vitro killing experiment of Exatecan-ADC on DU145 tumor cells

[0495] DU145 cells in logarithmic growth phase were digested with trypsin and then RPMI-1640 medium was added to adjust the viable cell density to 3 × 10⁻⁶ cells / year. 4 Cells / mL were added to 100 μL / well of a 96-well cell culture plate and incubated overnight (37°C, 5% CO2) to ensure complete cell adhesion. The Exatecan-ADC sample was prepared as a standard curve solution using RPMI-1640 medium, starting at 200 nM and diluted 5-fold for a total of 9 concentration gradients, with replicates. The overnight culture medium was discarded, and 100 μL / well of the prepared Exatecan-ADC sample solution was added to the 96-well cell culture plate. The plate was incubated for 3 days (37°C, 5% CO2). After incubation, the 96-well cell culture plate was removed and incubated at room temperature for 10-15 min. Cell viability was then tested using a CGT kit. Luminescent Cell Viability Assay (Promega, Cat: G7573), 50 μL / well, shaken at 1000 rpm for 2 min, incubated at room temperature in the dark for 10 min; after color development, the chemiluminescence was measured using an ELISA reader.

[0496] The formula for calculating the growth inhibition rate of conjugated drugs on tumor cells is: Inhibition rate % = (Fluorescence reading of cell administration wells - Fluorescence reading of culture medium control wells) / (Fluorescence reading of cell wells - Fluorescence reading of culture medium control wells) × 100%.

[0497] Based on the cell viability inhibition results and the concentration of the added drug, a four-parameter fitting method was used to calculate the half-maximal inhibitory concentration (IC50) of the drug on cell growth.

[0498] The IC50 of Exatecan-ADC in this application inhibits the in vitro proliferation of DU145 cells. 50 Value and I max The values ​​are shown in Table 12.

[0499] As shown in Table 12, the Exatecan-ADC in this application exhibits significant inhibitory activity against the proliferation of DU145 cells. All the bioactive molecules in this application possess similar inhibitory activity against tumor proliferation.

[0500] Table 12 Summary of test results of Exatecan-ADC killing DU145 cells

[0501] 4.2 In vitro killing experiment of MMAE-ADC on DU145 tumor cells

[0502] DU145 cells in logarithmic growth phase were digested with trypsin and then RPMI-1640 medium was added to adjust the viable cell density to 3 × 10⁻⁶ cells / year. 4 Cells / mL were added to 100 μL / well of a 96-well cell culture plate and incubated overnight (37°C, 5% CO2) to ensure complete cell adhesion. The MMAE-ADC sample was prepared as a standard curve solution using RPMI-1640 medium, starting at 1 μM and diluted 5-fold for a total of 9 concentration gradients, with replicates. The overnight culture medium was discarded, and the prepared MMAE-ADC sample solution was added to the 96-well cell culture plate at 100 μL / well. The plate was incubated for 3 days (37°C, 5% CO2). After incubation, the 96-well cell culture plate was removed and incubated at room temperature for 10-15 min. Cell viability was then tested using a CGT kit. Luminescent Cell Viability Assay (Promega, Cat: G7573), 50 μL / well, shaken at 1000 rpm for 2 min, incubated at room temperature in the dark for 10 min; after color development, the chemiluminescence was measured using an ELISA reader.

[0503] The formula for calculating the growth inhibition rate of conjugated drugs on tumor cells is: Inhibition rate % = (Fluorescence reading of cell administration wells - Fluorescence reading of culture medium control wells) / (Fluorescence reading of cell wells - Fluorescence reading of culture medium control wells) × 100%.

[0504] Based on the cell viability inhibition results and the concentration of the added drug, a four-parameter fitting method was used to calculate the half-maximal inhibitory concentration (IC50) of the drug on cell growth.

[0505] The IC50 of MMAE-ADC in this application inhibits the in vitro proliferation of DU145 cells. 50 Value and I max The values ​​are shown in Table 13.

[0506] As shown in Table 13, the MMAE-ADC in this application exhibits significant inhibitory activity against the proliferation of DU145 cells. All the bioactive molecules in this application possess similar inhibitory activity against tumor proliferation.

[0507] Table 13 Summary of MMAE-ADC killing assay results against DU145 cells

[0508] 4.3 Killing effect of ADC drugs on SK-OV-3 cells

[0509] The inhibitory activity of bioactive molecules on relevant tumor cells was detected in vitro. SK-OV-3 were human ovarian cancer cells, purchased from Nanjing Kebai.

[0510] SK-OV-3 cells in logarithmic growth phase were digested with trypsin and then added to McCoy's 5a medium to adjust the viable cell density to 3 × 10⁻⁶ cells / year. 4 Cells / mL were added to 100 μL / well of a 96-well cell culture plate and incubated overnight (37°C, 5% CO2) to ensure complete cell adhesion. The ADC sample was prepared as a standard curve solution using McCoy's 5a medium, starting at 500 nM and diluted 5-fold for a total of 9 concentration gradients, with replicates. The overnight medium was discarded, and the prepared ADC sample solution was added to the 96-well cell culture plate at 100 μL / well. The plate was incubated for 3 days (37°C, 5% CO2). After incubation, the 96-well cell culture plate was removed and incubated at room temperature for 10-15 min. Cell viability was then tested using a CGT kit. Luminescent Cell Viability Assay (Promega, Cat: G7573), 50 μL / well, shaken at 1000 rpm for 2 min, incubated at room temperature in the dark for 10 min; after color development, the chemiluminescence was measured using an ELISA reader.

[0511] The formula for calculating the growth inhibition rate of conjugated drugs on tumor cells is: Inhibition rate % = (Fluorescence reading of cell administration wells - Fluorescence reading of culture medium control wells) / (Fluorescence reading of cell wells - Fluorescence reading of culture medium control wells) × 100%.

[0512] Based on the cell viability inhibition results and the concentration of the added drug, a four-parameter fitting method was used to calculate the half-maximal inhibitory concentration (IC50) of the drug on cell growth.

[0513] The IC50 of the bioactive molecules in this application inhibits the in vitro proliferation of SK-OV-3 cells. 50 Value and I max The values ​​are shown in Table 14.

[0514] As shown in Table 14, the bioactive molecules in this application exhibit significant inhibitory activity against the proliferation of SK-OV-3 cells. All bioactive molecules in this application possess similar inhibitory activity against tumor proliferation.

[0515] Table 14 Summary of ADC drug killing test results on SK-OV-3 cells

[0516] 4.4 Killing effect of ADC drugs on breast cancer and gastric cancer cells

[0517] The inhibitory activity of bioactive molecules on relevant tumor cells was detected in vitro. HCC1954, JIMT-1, and MDA-MB-231 cells were human breast cancer cells, and NCI-N87 cells were human gastric cancer cells, purchased from Nanjing Kebai.

[0518] The cells in the logarithmic growth phase were digested with trypsin and then RPMI-1640 medium was added to adjust the viable cell density to 3 × 10⁴ cells / year. 4 Cells / mL were added to 100 μL / well of a 96-well cell culture plate and incubated overnight (37°C, 5% CO2) to ensure complete cell adhesion. The ADC sample was prepared as a standard curve using RPMI-1640 medium, starting at 200 nM and diluted 5-fold for a total of 9 concentration gradients, with replicates. The overnight culture medium was discarded, and the prepared ADC sample solution was added to the 96-well cell culture plate at 100 μL / well. The plate was incubated for 3 days (37°C, 5% CO2). After incubation, the 96-well cell culture plate was removed and allowed to stand at room temperature for 10-15 minutes. Cell viability was then tested using a CGT kit. Luminescent Cell Viability Assay (Promega, Cat: G7573), 50 μL / well, shaken at 1000 rpm for 2 mins, incubated at room temperature in the dark for 10 min; after color development, the chemiluminescence was measured using an ELISA reader.

[0519] The formula for calculating the growth inhibition rate of conjugated drugs on tumor cells is: Inhibition rate % = (Fluorescence reading of cell administration wells - Fluorescence reading of culture medium control wells) / (Fluorescence reading of cell wells - Fluorescence reading of culture medium control wells) × 100%.

[0520] Based on the cell viability inhibition results and the concentration of the added drug, a four-parameter fitting method was used to calculate the half-maximal inhibitory concentration (IC50) of the drug on cell growth.

[0521] The IC50 of the bioactive molecules in this application on the above-mentioned in vitro cell proliferation inhibition is... 50 Value and I max The values ​​are shown in Table 15.

[0522] As shown in Table 15, the bioactive molecule Tras-L128-P002 in this application exhibits significant inhibitory activity against proliferation in all four cell types. All bioactive molecules in this application possess similar inhibitory activity against tumor proliferation.

[0523] Table 15 Summary of ADC drug killing test results on HCC1954, JIMT-1, MDA-MB-231 and NCI-N87 cells

[0524] 4.5 Killing effect of ADC drugs on lung squamous cell carcinoma cells

[0525] The inhibitory activity of bioactive molecules on relevant tumor cells was detected in vitro. HCC95, EBC-1, and NCI-H1703 cells were human lung squamous cell carcinoma cells, purchased from Nanjing Kebai.

[0526] The cells in the logarithmic growth phase were digested with trypsin and then RPMI-1640 medium was added to adjust the viable cell density to 3 × 10⁶ cells / year. 4 Cells / mL were added to 100 μL / well of a 96-well cell culture plate and incubated overnight (37°C, 5% CO2) to ensure complete cell adhesion. The ADC sample was prepared as a standard curve using RPMI-1640 medium, starting at 200 nM and diluted 5-fold for a total of 9 concentration gradients, with replicates. The overnight culture medium was discarded, and the prepared ADC sample solution was added to the 96-well cell culture plate at 100 μL / well. The plate was incubated for 3 days (37°C, 5% CO2). After incubation, the 96-well cell culture plate was removed and allowed to stand at room temperature for 10-15 minutes. Cell viability was then tested using a CGT kit. Luminescent Cell Viability Assay (Promega, Cat: G7573), 50 μL / well, shaken at 1000 rpm for 2 mins, incubated at room temperature in the dark for 10 min; after color development, the chemiluminescence was measured using an ELISA reader.

[0527] The formula for calculating the growth inhibition rate of conjugated drugs on tumor cells is: Inhibition rate % = (Fluorescence reading of cell administration wells - Fluorescence reading of culture medium control wells) / (Fluorescence reading of cell wells - Fluorescence reading of culture medium control wells) × 100%.

[0528] Based on the cell viability inhibition results and the concentration of the added drug, a four-parameter fitting method was used to calculate the half-maximal inhibitory concentration (IC50) of the drug on cell growth.

[0529] The IC50 of the bioactive molecules in this application on the above-mentioned in vitro cell proliferation inhibition is...50 Value and I max The values ​​are shown in Table 16.

[0530] As shown in Table 16, the bioactive molecule 2H8L4-L128-P2 in this application exhibits significant inhibitory activity against proliferation in all four cell types. All bioactive molecules in this application possess similar inhibitory activity against tumor proliferation.

[0531] Table 16 Summary of the killing effect of ADC drugs on HCC95, EBC-1, and NCI-H1703 cells

[0532] Experimental Example 5: Rat PK Detection Method for ADC

[0533] The concentrations of antibodies against all antibodies and conjugate drugs in rat plasma were detected using the ELISA method. The reagents and consumables used in the ELISA experiment are shown in Table 17.

[0534] Table 17 Information on Main Reagents and Consumables

[0535] 5.1 ELISA method for detecting the concentration of total mAb in rat plasma

[0536] The concentration of total antibodies in rat plasma was detected using an ELISA method. The specific procedure was as follows: The corresponding antigen (Goat F(ab')2 Anti-Human IgG-(Fab')2, pre-adsorbed, Abcam, Cat#AB98597) was diluted to 500 ng / mL with coating buffer, mixed well, and transferred to a 96-well ELISA plate, 100 μL / well, incubated overnight at 4°C; the antigen coating solution was discarded, and the plate was washed with PBST, 300 μL / well, repeated 5 times; blocking buffer (PBS buffer containing 3% BSA) was added, 100 μL / well, and the plate was blocked at room temperature for 1 h; the ADC standard curve was prepared using reagent dilution buffer containing 0.05% monkey plasma, with an initial concentration of 300 ng / mL, 3-fold dilution, for a total of 10 concentration gradients. A secondary antibody control group and a blank control group were also included. Rat plasma samples were diluted with reagent diluent and replicates were set up. After blocking, the plate was washed repeatedly, and the test sample and ADC standard curve were added at 100 μL / well, incubating at room temperature for 1 h. After primary antibody incubation, the plate was washed repeatedly, and secondary antibody prepared at a 1:5000 dilution ratio with reagent diluent was added at 100 μL / well, incubating at room temperature for 1 h. After secondary antibody incubation, the plate was washed repeatedly, and TMB chromogenic solution was added at 100 μL / well, incubating for approximately 15 min. Finally, 100 μL / well of stop solution was added to stop the chromogenic reaction. The OD value at 450 nm was read using a microplate reader for concentration calculation.

[0537] 5.2 ELISA method for detecting P005-Conjugated mAb concentration in rat plasma

[0538] The concentration of P005-Conjugated mAb in rat plasma was detected using an ELISA method. The specific procedure was as follows: The corresponding antigen (Monoclonal Anti-Dxd Antibody, Acro, Cat#DXD-S222) was diluted to 500 ng / mL with coating buffer, mixed well, and transferred to a 96-well ELISA plate, 100 μL / well, incubated overnight at 4°C; the antigen coating solution was discarded, and the plate was washed with PBST, 300 μL / well, repeated 5 times; blocking buffer was added, 100 μL / well, and the plate was blocked at room temperature for 1 h; an ADC standard curve was prepared using reagent diluent containing 0.05% monkey plasma, with an initial concentration of 2000 ng / mL, 3-fold dilution, for a total of 10 concentration gradients. A secondary antibody control well and blank wells were also included. Rat plasma samples were diluted with reagent diluent and replicates were set up. After blocking, the plate was washed repeatedly, and the test sample and ADC standard curve were added at 100 μL / well, incubating at room temperature for 1 h. After primary antibody incubation, the plate was washed repeatedly, and secondary antibody prepared at a 1:5000 dilution ratio with reagent diluent was added at 100 μL / well, incubating at room temperature for 1 h. After secondary antibody incubation, the plate was washed repeatedly, and TMB chromogenic solution was added at 100 μL / well, incubating for approximately 15 min. Finally, 100 μL / well of stop solution was added to stop the chromogenic reaction. The OD value at 450 nm was read using a microplate reader for concentration calculation.

[0539] 5.3 ELISA method for detecting P002-Conjugated mAb concentration in rat plasma

[0540] The concentration of P002-Conjugated mAb in rat plasma was detected using an ELISA method. The specific procedure was as follows: The corresponding antigen (Monoclonal Anti-MMAE Antibody, Acro, Cat#MME-M5252) was diluted to 500 ng / mL with blocking buffer, mixed well, and transferred to a 96-well ELISA plate, 100 μL / well, incubated overnight at 4°C; the antigen coating solution was discarded, and the plate was washed with PBST, 300 μL / well, repeated 5 times; blocking buffer was added, 100 μL / well, and the plate was blocked at room temperature for 1 h; an ADC standard curve was prepared using reagent diluent containing 0.05% monkey plasma, with an initial concentration of 1000 ng / mL, 4-fold dilutions, for a total of 10 concentration gradients. Secondary antibody control wells and blank wells were also included. Rat plasma samples were diluted with reagent diluent and replicates were set up. After blocking, the plate was washed repeatedly, and the test sample and ADC standard curve were added at 100 μL / well, incubating at room temperature for 1 h. After primary antibody incubation, the plate was washed repeatedly, and secondary antibody prepared at a 1:5000 dilution ratio with reagent diluent was added at 100 μL / well, incubating at room temperature for 1 h. After secondary antibody incubation, the plate was washed repeatedly, and TMB chromogenic solution was added at 100 μL / well, incubating for approximately 15 min. Finally, 100 μL / well of stop solution was added to stop the chromogenic reaction. The OD value at 450 nm was read using a microplate reader for concentration calculation.

[0541] Experimental Example 6: ADC Rat PK Test

[0542] SD rats weighing 180–220 g were purchased from Chengdu Vital River Pharmaceuticals. Before administration, the rats were randomly divided into two groups of two (half male and half female) according to their body weight. The prepared ADC samples were injected into the rats via the tail vein (Exatecan-ADC (mAb4-DX8951) at a dose of 10 mg / kg, and MMAE-ADC (mAb4-MMAE) at a dose of 5 mg / kg). mAb4-Dxd and mAb4-vc-MMAE were used as positive controls. Blood was collected from the jugular sinus of the rats at 5 min, 6 h, 1 d, 3 d, 5 d, and 7 d after injection, with 0.2 mL of blood collected per rat at each time point. The collected blood samples were placed in EDTA-K2 anticoagulant tubes (temporarily stored on ice for centrifugation) and centrifuged at 1800 g and 4℃ for 10 min. All plasma was separated into EP tubes for analysis, and the processed plasma samples were frozen at -80℃. The rats were euthanized after the animal experiment. The Yangshen ADC was treated in the same way.

[0543] The results of the rat PK property test of ADC are shown in Figures 1, 2 and 3.

[0544] Conclusion: As can be seen from the ELISA data in Figures 1-3, the ADC in this application exhibits relatively stable pharmacokinetic activity in rats, which is generally consistent with typical rat pharmacokinetic characteristics.

Claims

1. A polypeptide or polypeptide fragment L' having the structure -X1-P-X2-X3-Z-X4- wherein, P is a proline residue, X1 is selected from the absence of a single amino acid or a single amino acid derivative, X2, X3 and X4 are each independently selected from a single amino acid or a single amino acid derivative; Z is selected from 1-2 amino acids or amino acid derivatives, and the polypeptide or polypeptide fragment L' is a substrate of matrix metalloproteinase 2 or matrix metalloproteinase 9, and / or cathepsin B.

2. The polypeptide or polypeptide fragment L' according to claim 1, wherein, X1 is selected from those that do not exist, His(H), Lys(K), Arg(R), Ser(S), Thr(T), Cys(C), Ala(A), Gly(G), Pro(P) and their derivatives; The X2 is selected from Arg(R), Cit, Orn, Asn(N), Glu(E), Asp(D), Ala(A), Dab, Lys(K) and their derivatives; The X3 is selected from Gly(G), Pro(P), Asn(N), Ser(S), Glu(E), Ala(A), Gln(Q), Asp(D), Cys(C), Asp(D) and their derivatives; Z is selected from Leu(L), Val(V), Leu-Val(LV), Ile(I), Ala(A), Phe(F), Dap, Dab, Orn and their derivatives; The X4 is selected from Cit, Ala(A), Ser(S), Lys(K), Orn and their derivatives; Preferably, X1 is selected from non-existent, His, Ser, Gly and their derivatives; The X2 is selected from Arg, Cit, Orn, Asn, Glu, Asp, Gla, Dab, Lys, Dab(Me2), Dab(Et2), Lys(Me2), Lys(Et2), Orn(Me2), Orn(Et2), Dap(Me2) or Dap(Et2); The X3 is selected from Gly, Pro, Asn, Ser, Glu or Ser(β-Glc); X4 is selected from Cit, Ala, Ser(GlcA), Lys(Me2), Lys(NMPRZ), Orn(Me2), Orn(Pr2), Lys(Mor), or Lys(Pr2); Z is selected from Leu, Val, Leu-Val, Ile, Ala, Phe, Hof, N,N-dimethyl-3-aminopropionic acid (Dap(Me2)), N,N-diethyl-3-aminopropionic acid (Dap(Et2)), N,N-dimethyl-4-aminobutyric acid (Dab(Me2)), N,N-diethyl-4-aminobutyric acid (Dab(Et2)), N,N-dimethylornithine (Orn(Me2)), N,N-diethylornithine (Orn(Et2)), N,N-dimethyllysine (Lys(Me2)), or N,N-diethyllysine (Lys(Et2)). Alternatively, X1 is selected from non-existent, His, Ser, Gly and their derivatives; The X2 is selected from Arg, Cit, Orn, Asn, Glu, Asp, Gla, Dab, Lys, Dab(Me2), Dab(Et2), Lys(Me2), Lys(Et2), Orn(Me2), Orn(Et2), Dap(Me2), Dap(Et2) or Ala; The X3 is selected from Gly, Pro, Asn, Ser, Glu and their derivatives; The X4 is selected from Gly, Cit, Ala, Ser (GlcA), Lys (Me2), Lys (NMPRZ), Orn (Me2), Orn (Pr2), Lys (Mor) or Lys (Pr2); Z is selected from Leu, Val, Leu-Val, Leu-Phe, Dap(Me2)-Val, Ile, Ala, Phe, Hof, Dap(Me2), Dap(Et2), Dab(Me2), Dab(Et2), Orn(Me2), Orn(Et2), Lys(Me2) or Lys(Et2); Alternatively, X1 is selected from non-existent, His, Ser, Gly, or Ser(β-Glc); The X2 is selected from Arg, Cit, Glu, Lys, Lys(Me2) or Ala; The X3 is selected from Gly, Pro, Asn, Ser, Glu or Ser(β-Glc); The X4 is selected from Gly, Cit, Ala, Ser (GlcA), Lys (Me2), Lys (NMPRZ), Orn (Me2), Lys (Mor) or Lys (Pr2); Z is selected from Leu, Val, Leu-Val, Leu-Phe, Dap(Me2)-Val, Ile, Hof or Dap(Me2); Alternatively, X1 may be non-existent or Ser; The X2 is selected from Arg, Cit, Orn, Asn, Glu, Asp, Gla, Dab, Lys, Dab(Me2), Dab(Et2), Lys(Me2), Lys(Et2), Orn(Me2), Orn(Et2), Dap(Me2), Dap(Et2) or Ala; The X3 is selected from Gly, Pro, Asn, Ser, Glu, Ala, Gln, Asp, Cys or Ser(β-Glc); Z is Val, Leu, Leu-Val, or Dap(Me2); The X4 is Gly, Ala, Cit, Lys, Orn, Lys(Me2), Lys(Pr2), Lys(NMPRZ), Orn(Me2), or Lys(Mor)); Alternatively, X1 may be non-existent or Ser; X2 is Arg, Cit, or Lys(Me2); X3 is Ser, Gly, or Asn; Z is Val, Leu, Leu-Val, or Dap(Me2); Said X4 is Gly, Ala, Cit, Lys(Me2), Lys(Pr2), Lys(NMPRZ), Orn(Me2), or Lys(Mor).

3. The polypeptide or polypeptide fragment L' according to claim 1 or 2, wherein, Said polypeptide or polypeptide fragment L' is selected from: His-Pro-Arg-Gly-Leu-Val-Cit (SEQ ID NO:1); Ser-Pro-Arg-Gly-Leu-Val-Cit (SEQ ID NO:2); Ser-Pro-Arg-Pro-Leu-Val-Cit (SEQ ID NO:3); His-Pro-Arg-Asn-Leu-Val-Cit (SEQ ID NO:4); His-Pro-Arg-Ser-Leu-Val-Cit (SEQ ID NO:5); Ser-Pro-Lys(Me2)-Ser-Leu-Val-Cit (SEQ ID NO:6); Ser-Pro-Cit-Ser(β-Glc)-Leu-Val-Cit (SEQ ID NO:7); Ser(β-Glc)-Pro-Cit-Ser-Leu-Val-Cit (SEQ ID NO:8); Ser(β-Glc)-Pro-Cit-Ser(β-Glc)-Leu-Val-Cit (SEQ ID NO:9); His-Pro-Cit-Gly-Leu-Val-Cit (SEQ ID NO:10); Ser-Pro-Cit-Ser-Leu-Val-Ser(GlcA) (SEQ ID NO:11); Ser-Pro-Cit-Ser-Leu-Val-Ala (SEQ ID NO:12); Ser-Pro-Arg-Gly-Leu-Val-Ala (SEQ ID NO:13); Ser-Pro-Arg-Gly-Leu-Val-Lys(Me2) (SEQ ID NO:14); Ser-Pro-Arg-Gly-Dap(Me2)-Val-Cit (SEQ ID NO:15); His-Pro-Arg-Gly-Dap(Me2)-Val-Cit (SEQ ID NO:16); Pro-Arg-Asn-Leu-Phe-Gly (SEQ ID NO:17); Ser-Pro-Cit-Ser-Leu-Cit (SEQ ID NO:18); Gly-Pro-Arg-Asn-Leu-Cit (SEQ ID NO:19); Ser-Pro-Arg-Gly-Leu-Cit (SEQ ID NO:20); His-Pro-Arg-Gly-Leu-Cit (SEQ ID NO:21); His-Pro-Arg-Ser-Leu-Cit(SEQ ID NO:22); Gly-Pro-Cit-Asn-Leu-Cit(SEQ ID NO:23); Ser-Pro-Arg-Gly-Leu-Lys(Me2)(SEQ ID NO:24); Ser-Pro-Cit-Gly-Hof-Gly(SEQ ID NO:25); Ser-Pro-Cit-Ser-Dap(Me2)-Cit(SEQ ID NO:26); Pro-Arg-Ser-Dap(Me2)-Cit(SEQ ID NO:27); Pro-Arg-Ser-Leu-Cit(SEQ ID NO:28); Pro-Cit-Gly-Leu-Cit(SEQ ID NO:29); Pro-Arg-Ser(β-Glc)-Leu-Cit(SEQ ID NO:30); Pro-Arg-Asn-Leu-Cit(SEQ ID NO:31); Pro-Lys-Ser-Leu-Cit(SEQ ID NO:32); Pro-Lys-Gly-Leu-Cit(SEQ ID NO:33); Pro-Cit-Ser-Leu-Cit(SEQ ID NO:34); Pro-Arg-Gly-Leu-Cit(SEQ ID NO:35); Pro-Cit-Asn-Leu-Cit(SEQ ID NO:36); Pro-Arg-Glu-Leu-Cit(SEQ ID NO:37); Pro-Glu-Ser-Leu-Cit(SEQ ID NO:38); Pro-Arg-Ser-Leu-Ala(SEQ ID NO:39); Pro-Arg-Ser-Leu-Lys(Me2)(SEQ ID NO:40); Pro-Cit-Gly-Leu-Lys(Me2)(SEQ ID NO:41); Pro-Lys(Me2)-Gly-Leu-Lys(Me2)(SEQ ID NO:42); Pro-Lys(Me2)-Ser-Leu-Lys(Me2)(SEQ ID NO:43); Pro-Arg-Asn-Leu-Lys(Me2)(SEQ ID NO:44); Pro-Arg-Glu-Leu-Lys(Me2)(SEQ ID NO:45); Pro-Arg-Gly-Leu-Lys(Me2)(SEQ ID NO:46); Pro-Cit-Ser-Leu-Lys(Me2)(SEQ ID NO:47); Pro-Arg-Ser-Leu-Lys(Pr2)(SEQ ID NO:48); Pro-Arg-Ser-Leu-Lys(NMPRZ)(SEQ ID NO:49); Pro-Arg-Ser-Leu-Orn(Me2)(SEQ ID NO:50); Pro-Arg-Ser-Leu-Lys(Mor)(SEQ ID NO:51); Ser-Pro-Cit-Ser-Val-Cit(SEQ ID NO:54); Gly-Pro-Arg-Asn-Val-Cit(SEQ ID NO:55); Ser-Pro-Arg-Gly-Val-Cit(SEQ ID NO:56); His-Pro-Arg-Ser-Val-Cit(SEQ ID NO:57); His-Pro-Arg-Gly-Val-Cit(SEQ ID NO:58); Ser-Pro-Arg-Gly-Val-Lys(Me2)(SEQ ID NO:59); Ser-Pro-Cit-Ser-Val-Lys(Me2)(SEQ ID NO:60); Gly-Pro-Arg-Asn-Val-Lys(Me2)(SEQ ID NO:61); His-Pro-Arg-Ser-Val-Lys(Me2)(SEQ ID NO:62); His-Pro-Arg-Gly-Val-Lys(Me2)(SEQ ID NO:63); Pro-Arg-Ser-Val-Cit(SEQ ID NO:64); Pro-Arg-Glu-Val-Cit(SEQ ID NO:65); Pro-Arg-Asn-Val-Cit(SEQ ID NO:66); Pro-Glu-Ser-Val-Cit(SEQ ID NO:67); Pro-Cit-Gly-Val-Cit(SEQ ID NO:68); Pro-Arg-Gly-Val-Cit(SEQ ID NO:69); Pro-Cit-Ser-Val-Cit(SEQ ID NO:70); Pro-Arg-Pro-Val-Cit(SEQ ID NO:71); Pro-Cit-Pro-Val-Cit(SEQ ID NO:72); Pro-Cit-Asn-Val-Cit(SEQ ID NO:73); Pro-Lys(Me2)-Ser-Val-Cit(SEQ ID NO:74); Pro-Arg-Ser-Val-Lys(Me2)(SEQ ID NO:75); Pro-Lys(Me2)-Ser-Val-Lys(Me2)(SEQ ID NO:76); Pro-Arg-Glu-Val-Lys(Me2) (SEQ ID NO:77); Pro-Arg-Asn-Val-Lys(Me2) (SEQ ID NO:78); Pro-Glu-Ser-Val-Lys(Me2) (SEQ ID NO:79); Pro-Cit-Gly-Val-Lys(Me2)(SEQ ID NO:80); Pro-Arg-Gly-Val-Lys(Me2) (SEQ ID NO:81); Pro-Cit-Ser-Val-Lys(Me2)(SEQ ID NO:82); Pro-Arg-Pro-Val-Lys(Me2) (SEQ ID NO:83); Pro-Cit-Pro-Val-Lys(Me2) (SEQ ID NO:84); Pro-Cit-Asn-Val-Lys(Me2)(SEQ ID NO:85); Pro-Arg-Ser-Val-Lys(Pr2) (SEQ ID NO:86); Pro-Arg-Ser-Val-Orn(Me2)(SEQ ID NO:87); Pro-Arg-Ser-Val-Ala (SEQ ID NO:88); Pro-Arg-Ser-Val-Cit (SEQ ID NO:89); Pro-Arg-Ser-Ile-Cit (SEQ ID NO:90); Pro-Arg-Ser-Ile-Ala (SEQ ID NO:91); Pro-Arg-Ser-Ile-Lys(Me2) (SEQ ID NO:92); Pro-Arg-Ser-Leu-Val-Lys(Me2) (SEQ ID NO:93); Pro-Arg-Asn-Leu-Val-Lys(Me2) (SEQ ID NO:94); Pro-Arg-Ser-Val-Lys(Mor)(SEQ ID NO:95); and Pro-Ala-Ser-Leu-Val-Cit (SEQ ID NO: 132).

4. A linker comprising the polypeptide or polypeptide fragment L' of any one of claims 1-3, wherein, The connector has the following general formula: C1-L1-L'-L2; Wherein, C1 is a linker; L1 is an empty spacer or spacer; L2 is an empty spacer or spacer; and L' is a polypeptide or polypeptide fragment as described in any one of claims 1-3.

5. The connector according to claim 4, wherein, said C1 is selected from the following structures: wherein denotes the position of attachment to the biological ligand unit, Indicates the position connected to L1 or L'.

6. The connector according to claim 4 or 5, wherein, said C1-L1- is and / or L2 is null or selected from the group consisting of: n is any integer selected from 1-6, 1-5, 1-4, or 1-3; Preferably, L2 is empty. (PAB) or -NHCH2-.

7. The linker of any one of claims 4-6, wherein, The connector is selected from: -(succinimide-3-yl-N)-(CH2)5-C(=O)-L'-; -(succinimide-3-yl-N)-(CH2)5-C(=O)-L'-PAB-; -(succinimide-3-yl-N)-(CH2)5-C(=O)-L'-PAB-DMEDA-; -(succinimide-3-yl-N)-(CH2)5-C(=O)-L'-NMEDA-; -(succinimide-3-yl-N)-(CH2)5-C(=O)-L'-NHCH2-.

8. A linker drug comprising the polypeptide or polypeptide fragment L' of any one of claims 1-3, wherein, The linker drug has the following general formula: C1'-L1-L'-L2-D; Wherein, C1' is a functional group that reacts with biological ligands; L1 is an empty spacer or a spacer; L2 is an empty spacer or a spacer; D is a biologically active compound; and L' is a polypeptide or polypeptide fragment as described in any one of claims 1-3.

9. The linker drug according to claim 8, wherein, The C1' is selected from: wherein R x is selected from: -OH, F, Cl or Br; Indicates the position where it connects to L1 or L'; The C1'-L1 is selected from MC, NHS-, Mal-PEG8-, or DBCO-; and / or The D is selected from immunomodulators, protein degraders, and cytotoxic agents; preferably, the D includes: amanitins, anthracyclines, auristatins, baccatins, calicheamicins, camptothecins, cemadotins, colchicine, colcimids, compbretastatins, cryptophycins, discormolides, duocarmycins, docetaxel, doxorubicin, duocarmycins, echinomycins, and elixelosin. Eutherobins, epothilones, estramustines, lexitropsins, maytansines, maytansinoids, methotrexate, netropsins, pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), puromycins, rhizoxins, SN38, taxanes, tubulolysins, vincaalkaloids, tetrahydroisoquinoline alkaloids or their derivatives, protein degrading agents or their derivatives; more preferably, D includes DX8951, MMAE, Dxd or SN38.

10. The Linker Drug according to claim 8 or 9, wherein, The linker drug is selected from: MC-L'-D; MC-L'-PAB-D; MC-L'-PAB-DMEDA-D; MC-L'-NMEDA-D; MC-L'-NHCH2-D; The D is selected from DX8951, MMAE, Dxd or SN38.

11. A bioligand-drug conjugate comprising the linker of any one of claims 4-7, wherein, The bio-ligand-drug conjugate has the following general formula: Bp-(C1-L1-L'-L2-D) n Wherein: Bp is a biological ligand; C1-L1-L'-L2 is the linker; D is a biologically active compound as described in any one of claims 9 or 10; n is any integer between 1 and 20.

12. The bio-ligand-drug conjugate of claim 11, wherein, The biological ligand Bp targets cell surface receptors or tumor-associated antigens, wherein the cell surface receptors or tumor-associated antigens include: EGFR, HER2, HER3, c-Met, claudin18.2, TROP-2, folate receptor α (FRα), ROR2, BCMA, PSMA, CD19, CD20, CD22, CD30, CD33, CD37, CD46, CD48, CD56, CD79b, CD123, CD138, CD166, CD276, CEACAM5, SDC1, CD74, CD70, MUC1, MUC16, GUCY2C, MSLN, SCL34A2, FOLH1, TPBC, PVRL4, STEAP1, SCL44A4, NACM1, EDNRB, GPNMB, FGFR, SEZ6, Nectin-4, PD-L1, and Tissue. Factor(TF), B7-H3, B7-H4, LY6E, LIV-1, CDH4, CDH6, ITGB6, GPC1, GPC3, ENPP3, KAAG1, FZD7, SSTR2, DLK1, T MEFF1, TM4SF1, SLAMF7, DLL3, FLT3, TNFRSF10B, EPCAM, AXL, IL2RA, 5T4, FAP, ICAM-1, IGF-1R, TNFR1, GLP-1; Preferably, the biological ligand includes an antibody or its antigen-binding fragment, a modified antibody or its antigen-binding fragment, an oligonucleotide, a DNA fragment, a nucleic acid aptamer, a polypeptide, a nanoparticle with targeting properties, or a chemically synthesized small molecule; preferably, the antigen-binding fragment includes Fab, Fab', Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb; More preferably, the antibody or its antigen-binding fragment, or the modified antibody or its antigen-binding fragment, is: anti-EGFR, anti-HER2, anti-HER3, anti-c-Met, anti-claudin18.2, anti-TROP-2, anti-FRα, anti-ROR2, anti-BCMA, anti-PSMA, anti-CD19, anti-CD20, anti-CD22, anti-CD30, anti-CD33, anti-CD37, anti-CD46, anti-CD48, or anti-CD56. anti-CD79b, anti-CD123, anti-CD138, anti-CD166, anti-CD276, anti-CEACAM5, anti-SDC1, anti-CD74, anti-CD70, anti-MUC1, anti-MUC1 6. anti-GUCY2C, anti-MSLN, anti-SCL34A2, anti-FOLH1, anti-TPBC, anti-PVRL4, anti-STEAP1, anti-SCL44A4, anti-NACM1, anti-EDNRB, a nti-GPNMB, anti-FGFR, anti-SEZ6, anti-Nectin-4, anti-PD-L1, anti-TF, anti-B7-H3, anti-B7-H4, anti-LY6E, anti-LIV-1, anti-CDH4, a nti-CDH6, anti-ITGB6, anti-GPC1, anti-GPC3, anti-ENPP3, anti-KAAG1, anti-FZD7, anti-SSTR2, anti-DLK1, anti-TMEFF1, anti-TM4SF1, Antibodies or antigen-binding fragments thereof for anti-SLAMF7, anti-DLL3, anti-FLT3, anti-TNFRSF10B, anti-EPCAM, anti-AXL, anti-IL2RA, anti-5T4, anti-FAP, anti-ICAM-1, anti-IGF-1R, anti-TNFR1, and anti-GLP-1; preferably, the antibodies or antigen-binding fragments thereof, or modified antibodies or antigen-binding fragments thereof, are antibodies or antigen-binding fragments for anti-Her2, anti-TF, anti-GPC1, and anti-GPC3.More preferably, the antibody is Trastuzumab, an anti-TF antibody or its antigen-binding fragment, or an anti-GPC1 antibody or its antigen-binding fragment, wherein the anti-TF antibody comprises a light chain having the amino acid sequence shown in SEQ ID NO: 52 and a heavy chain having the amino acid sequence shown in SEQ ID NO: 53; the anti-GPC1 antibody comprises a light chain having the amino acid sequence shown in SEQ ID NO: 96 and a heavy chain having the amino acid sequence shown in SEQ ID NO:

97.

13. The biological ligand-drug conjugate according to claim 11 or 12, wherein, The conjugate is an antibody-drug conjugate, and the antibody-drug conjugate is selected from: Ab-((succinimid-3-yl-N)-(CH2)5-C(=0)-L'-D) n ; Ab-((succinimid-3-yl-N)-(CH2)5-C(=0)-L'-PAB-D) n ; Ab-((succinimid-3-yl-N)-(CH2)5-C(=0)-L'-PAB-DMEDA-D) n ; Ab-((succinimid-3-yl-N)-(CH2)5-C(=0)-L'-NMEDA-D) n ; Ab-((succinimid-3-yl-N)-(CH2)5-C(=0)-L'-NHCH2-D) n ; The Ab represents a monoclonal antibody or its antigen-binding fragment, or a modified monoclonal antibody or its antigen-binding fragment, preferably an anti-Her2, anti-TF, anti-GPC1, or anti-GPC3 antibody or its antigen-binding fragment; the L' is selected from any one of claims 1-3, the D is selected from DX8951, MMAE, Dxd, or SN38; and the n represents any value selected from 1-10.

14. A biological ligand-drug conjugate according to any one of claims 11-13 for the treatment and / or prevention of diseases or conditions associated with abnormal cellular activity; Preferably, the disease or condition includes a tumor.

15. The biological ligand-drug conjugate as described in claim 14 for treating and / or preventing diseases or conditions associated with abnormal cellular activity, wherein, The disease or condition described is selected from tumors associated with the expression of the following proteins: EGFR, HER2, HER3, c-Met, claudin18.2, TROP-2, folate receptor α (FRα), ROR2, BCMA, PSMA, CD19, CD20, CD22, CD30, CD33, CD37, CD46, CD48, CD56, CD79b, CD123, CD138, CD166, CD276, CEACAM5, SDC1, CD74, CD70, MUC1, MUC16, GUCY2C, MSLN, SCL34A2, FOLH1, TPBC, PVRL4, STEAP1, SCL44A4, NACM1, EDNRB, GPNMB, FGFR, SEZ6, Nectin-4, PD-L1, and Tissue. Factor(TF), B7-H3, B7-H4, LY6E, LIV-1, CDH4, CDH6, ITGB6, GPC1, GPC3, ENPP3, KAAG1, FZD7, SSTR2, DLK1, TMEFF1, TM4SF1, SLAMF7, DLL3, FLT3, TNFRSF10B, EPCAM, AXL, IL2RA, 5T4, FAP, ICAM-1, IGF-1R; More preferably, the disease or symptom is selected from: esophageal cancer, brain cancer, lung cancer, epithelial cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, pancreatic cancer, head and neck cancer, urothelial carcinoma, colon cancer, colorectal cancer, cervical cancer, endometrial cancer, rectal cancer, kidney cancer, prostate cancer, melanoma, liver cancer, gallbladder cancer, bile duct cancer, thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, and B-cell lymphoma; preferably, the tumor is selected from esophageal cancer, lung cancer, breast cancer, gastric cancer, urothelial carcinoma, bile duct cancer, prostate cancer, colorectal cancer, cervical cancer, and ovarian cancer.

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