Antibody-drug conjugate, method for producing the same, and use thereof

Abstract

The present invention discloses an antibody-drug conjugate, its preparation method and use, in particular a conjugate of an anti-PD-L1 antibody with a TLR7 and / or TLR8 agonist, its pharmaceutical composition, preparation method and use. The present invention obtains a modified anti-PD-L1 antibody with a mutated cysteine ​​by gene editing, which substantially maintains the structure of the original antibody and is useful for constructing an antibody-drug conjugate. Antitumor experiments have found that the obtained antibody-drug conjugate has excellent activity, such as high antitumor activity, can significantly increase the survival rate of cancer-bearing animals, and has significantly reduced toxicity and less burden on the body of experimental animals. The minimum effective dose of the small molecule drugs used individually is greatly reduced, and the therapeutic window is expanded, which is promising for the development of therapeutic drugs for multiple diseases (such as tumors and viral diseases such as hepatitis B), and has promising application prospects and value.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical pharmaceuticals, and specifically relates to antibody-drug conjugates (ADCs), particularly conjugates of antibodies for immunotherapy and immunomodulators, such as conjugates of anti-PD-L1 antibodies and TLR7 and / or TLR8 agonists, pharmaceutical compositions thereof, manufacturing methods, and uses.

Background Art

[0002] Toll-like receptors are a family of receptors conserved during evolution, including at least 13 members. Ten types (TLR1-10) have been discovered in humans. TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 are expressed on the cell surface and rapidly recognize the products of bacterial metabolism. TLR3, TLR7, TLR8, and TLR9 are expressed intracellularly and mainly monitor and recognize viral nucleic acids. TLR3 recognizes double-stranded RNA, TLR7 and TLR8 recognize single-stranded RNA, TLR9 recognizes unmethylated CpG coenzyme I, and regulates the reaction of bacterial DNA with certain viruses.

[0003] TLRs specifically recognize pathogen-associated molecular patterns (PAMPs) and play important roles in both innate and adaptive immunity, serving as a bridge connecting innate and adaptive immunity. Among them, TLR7 recognizes single-stranded RNA that binds to viruses or artificially synthesized small molecule purine compounds, and then recruits specific linker proteins, activates a series of signal cascade reactions, initiates a high-level systemic adaptive immune response, and kills virus-infected cells to completely remove the virus. Clinically, TLR7 agonists have been used to initiate the treatment of chronic viral infections such as hepatitis B and hepatitis C. Furthermore, TLR7 agonists can induce more rapid and effective immune protection as an adjuvant for influenza vaccines. In anti-tumor treatment, TLR7 agonists not only directly stimulate pDCs to secrete IFN-α, but also enhance the co-stimulatory and antigen-presenting abilities of pDCs. Activated pDCs promote the proliferation of CD4+ T cells, further activate CD8+ T cells, and kill tumor cells. Therefore, the role of TLR7 agonists as immune adjuvants in the processes of biorecognition and killing has been increasingly emphasized.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Based on the results of previous research and development, the inventors continued their research and, as a result, developed an antibody-drug conjugate (ADC) that has a better therapeutic effect, significantly reduces toxicity and side effects, and expands the therapeutic window of immunomodulators (such as TLR7 and / or TLR8 agonists). In particular, they developed a conjugate of an antibody for immunotherapy and an immunomodulator, such as a conjugate of an anti-PD-L1 antibody and a TLR7 and / or TLR8 agonist.

Means for Solving the Problems

[0005] Specifically, the above-mentioned antibody-drug conjugate has the following structure. TIFF0007703050000001.tif17170 (However, Ab is an antibody or its antigen-binding fragment, D is a small molecule drug, L is a linking unit that connects Ab and D, and n is an integer from 1 to 100.)

[0006] Specifically, the above antibody is a monoclonal antibody.

[0007] Specifically, the above antibody may be in the form of, for example, a chimeric antibody, a humanized antibody, a fully human antibody, etc.

[0008] Specifically, the above antigen-binding fragment includes a Fab fragment, an F(ab')2 fragment, an Fd fragment, an Fv fragment, a dAb fragment, a single-chain Fv (scFv), an Fv linked by a disulfide bond (sdFv), a fragment containing a CDR, or an isolated CDR, etc.

[0009] Specifically, the above-mentioned antibody is reactive against related antigens or their epitopes such as tumors, infectious microorganisms, or autoimmune diseases. Specifically, the antigens targeted by the above-mentioned antibody are, for example, Claudin18.2, GPC3, HER-2 / neu, carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM-6, alpha-fetoprotein (AFP), VEGF, ED-B fibronectin, EGP-1, EGP-2, EGF receptor (ErbB1), ErbB2, ErbB3, factor H, FHL-1, Flt-3, folate receptor, Ga 733, GROB, HMGB-1, hypoxia-inducible factor (HIF), HM1.24, insulin-like growth factor (ILGF), IFN-γ, IFN-α, IFN-β, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24. Gangliosides, HCG, HLA-DR, CD66a-d, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration inhibitory factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5, PD-1, PD-L1, placental growth factor (PIGF), PSA, PSMA, PSMA dimer, PAM4 antigen, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le(y), mesothelin, S100, tenascin, TAC, Tn antigen, Thomas-Friedenreich antigen, tumor necrosis antigen, tumor angiogenesis antigen, TNF-α, TRAIL receptors (R1 and R2), VEGFR, RANTES, T101, cancer stem cell antigen, complement factor C3, C3a, C3b, C5a, C5, and oncogene products, etc.

[0010] In one embodiment of the present invention, the above antibody is an anti-PD-L1 antibody.

[0011] Specifically, the above antibody has reactive groups (e.g., mercapto, amino, carboxy, amide, halogen, ester, acyl halide, anhydride, epoxy, maleimide, aminooxy, azide, alkynyl, TIFF0007703050000002.tif33170, where R may be selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl). The above antibody may itself have those reactive groups, or may be obtained by mutating (e.g., site-directed mutagenesis) one or more amino acid residues of a conventional antibody into amino acids (including natural and non-natural amino acids, etc.) containing the same or different desired reactive groups. In one embodiment of the present invention, the above antibody has a cysteine residue inserted / substituted at a specific position in the amino acid sequence of the antibody by mutation (e.g., Thiomab technology).

[0012] In one embodiment of the present invention, the above-mentioned antibody is a modified anti-PD-L1 antibody obtained by mutating one or more amino acid residues of a conventional anti-PD-L1 antibody (for example, Atezolizumab, Durvalumab, Avelumab, Cemiplimab, KN035, CS1001, BGB-A333, KL-A167, SHR-1316, STI-A1014, etc.) into cysteine by genetic engineering techniques.

[0013] Specifically, the above-mentioned antibody includes a heavy chain and a light chain, The amino acid sequence of the heavy chain is shown in SEQ ID NO:4, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:4, and / or, The amino acid sequence of the light chain is shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:9.

[0014] More specifically, the above-mentioned antibody has a light chain sequence shown in SEQ ID NO:9.

[0015] In one embodiment of the present invention, the above-mentioned antibody has a heavy chain sequence shown in SEQ ID NO:4 and a light chain sequence shown in SEQ ID NO:9.

[0016] Specifically, the above-mentioned n (that is, the drug / antibody ratio (DAR)) can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, for example, 1 to 50, 1 to 40, 1 to 20, 1 to 10, 1 to 6, 1 to 4, and in some embodiments of the present invention, n = 2 or 4.

[0017] In one embodiment of the present invention, in the above antibody-drug conjugate, the linking site of the antibody to L is the free mercapto group at the cysteine residue in the amino acid sequence of the antibody.

[0018] In one example of the present invention, in the above antibody-drug conjugate, the antibody has a heavy chain sequence shown in SEQ ID NO:4 and a light chain sequence shown in SEQ ID NO:9, and the linking site of the antibody to L is the free mercapto group at the cysteine residue at position 226 of the amino acid sequence shown in SEQ ID NO:9, and preferably, n = 2.

[0019] In another example of the present invention, in the above antibody-drug conjugate, the antibody has a heavy chain sequence shown in SEQ ID NO:4 and a light chain sequence shown in SEQ ID NO:3, and n is from 1 to 10, particularly from 1 to 6.

[0020] Specifically, the above small molecule drug may be an immunomodulator, for example, a Toll-like receptor agonist (Toll-like receptors, TLR), specifically, a TLR7 and / or TLR8 agonist, for example, the pyridopyrimidine derivative and its salt described in Patent Application Publication No. WO2019 / 095455A1.

[0021] In one embodiment of the present invention, the D moiety in general formula I has the following structure. TIFF0007703050000003.tif32170 (however, L' is a linking group selected from a single bond or C1-C6 alkylene, C1-C6 alkenylene, C3-C6 cycloalkylene, where all of the above groups may be optionally substituted by C1-C4 alkyl, R1 is selected from a single bond, C1-C6 alkylene, C1-C6 alkenylene, C1-C6 alkyleneoxy, where all of the above groups may be optionally substituted by C1-C4 alkyl, X is TIFF0007703050000004.tif17170, -NR4-, -O-, -S-, single bond, -OCO-, -COO-, -NR4-(C1-C6 alkylene)-NR5-, heterocyclylene (especially nitrogen-containing heterocyclylene), where R4 and R5 are independently H, C1-C6 alkyl, C1-C6 alkoxy, amino acid residue, oligopeptide residue, haloalkyl, carboxy-substituted alkyl, ester-substituted alkyl, TIFF0007703050000005.tif21170selected from, where a is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), and R8 and R9 are independently selected from H, C1-C6 alkyl, or R8 and R9, together with the atoms in between them TIFF0007703050000006.tif18170form, or R4 and R5, together with the nitrogen atom to which they are both linked, form a heterocyclyl, R2 is selected from -NR6R7, -OR6, -SR6, where R6 and R7 are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxyalkyl, or R6 and R7, together with the nitrogen atom to which they are both linked, form a heterocyclyl, or R6, together with the oxygen atom to which it is linked, forms a heterocyclyl, or R6, together with the sulfur atom to which it is linked, forms a heterocyclyl, R3 is one or more independent substituents on the benzene ring, selected from H, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxyalkyl, m is an integer from 0 to 4 (e.g., 0, 1, 2, 3, 4).)

[0022] Specifically, Formula II has the following structure. TIFF0007703050000007.tif55170

[0023] In one embodiment of the present invention, the above L' is C1-C6 alkylene, for example, C1-C3 alkylene.

[0024] In one embodiment of the present invention, the above L' has the following structure TIFF0007703050000008.tif16170, where R a and R b are independently selected from H, C1-C3 alkyl (such as methyl, ethyl, n-propyl, isopropyl), or R a and R b together with the carbon atom to which they are attached form C3-C6 cycloalkylene (such as cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene).

[0025] Specifically, L' may be selected from TIFF0007703050000009.tif11170.

[0026] In one embodiment of the present invention, the above R1 is C1-C6 alkylene, such as C1-C3 alkylene, such as -CH2-.

[0027] In another embodiment of the present invention, the above R1 is a single bond.

[0028] In one embodiment of the present invention, the above R1 is C1-C6 alkyleneoxy, such as C1-C3 alkyleneoxy, such as -CH2O-, -CH2CH2O-, -CH2CH2CH2O-.

[0029] Specifically, R3 is one or more independent substituents on the benzene ring and is selected from H, methyl, and methoxy.

[0030] In the examples of the present invention, m is 0 or 1.

[0031] Specifically, a is an integer from 1 to 5, such as 1, 2, 3, 4, 5.

[0032] Specifically, R8 and R9 are independently selected from H, methyl, ethyl, n-propyl, and isopropyl.

[0033] Specifically, the above R4 and R5 are independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, amino acid residue, oligopeptide residue, -CF3, -CH2CF3, selected from TIFF0007703050000010.tif52170, or R4 and R5 together with the nitrogen atom to which they are both attached form a substituted or unsubstituted heterocyclyl.

[0034] Specifically, the above substituted or unsubstituted heterocyclyl may be selected from TIFF0007703050000011.tif19170.

[0035] Specifically, the above amino acid residues and the amino acid residues in the oligopeptide are independently one or more selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine residues. In one embodiment of the present invention, the above amino acid residue is a leucine residue. In one embodiment of the present invention, the above oligopeptide residue is -asparagine-alanine-alanine-leucine-.

[0036] In some embodiments of the present invention, X is TIFF0007703050000012.tif17170, and R4 and R5 are independently selected from C1-C6 alkyl. For example, both R4 and R5 are methyl.

[0037] In some other embodiments of the present invention, X is -NR4-, and R4 is selected from H, C1-C6 alkyl. For example, R4 is H or methyl.

[0038] In some other embodiments of the present invention, X is -NR4-(C1-C6 alkylene)-NR5-, and R4 and R5 are independently selected from C1-C6 alkyl. For example, both R4 and R5 are methyl.

[0039] In some other embodiments of the present invention, X is a heterocyclylene, particularly a nitrogen-containing heterocyclylene, for example TIFF0007703050000013.tif20170, the A ring is a 4- to 10-membered nitrogen-containing heterocyclic ring, and the A ring may be a monocyclic, bicyclic or tricyclic ring (including fused rings, spiro rings, bridged rings). Specifically, X may be TIFF0007703050000014.tif41170.

[0040] In one embodiment of the present invention, R2 is selected from -NHR6, -OR6, -SR6, where R6 is C1-C6 alkyl or C1-C6 alkoxyalkyl, for example C1-C4 alkyl, such as butyl, particularly n-butyl, for example C1-C4 alkoxyalkyl, such as methoxyethyl. In some embodiments of the present invention, R2 is selected from TIFF0007703050000015.tif11170.

[0041] In another embodiment of the present invention, R2 is selected from -NR6R7, where R6 and R7 together with the nitrogen atom to which they are both linked form a heterocyclyl, for example, TIFF0007703050000016.tif18170.

[0042] Specifically, the D moiety in general formula I is selected from the following structures. TIFF0007703050000017.tif210170TIFF0007703050000018.tif224170

[0043] In one embodiment of the present invention, the D moiety in general formula I has the following structure. TIFF0007703050000019.tif23170

[0044] In one embodiment of the present invention, the above conjugate has the following structure. TIFF0007703050000020.tif32170(where Ab, n, m, R1, R2, R3, R4, R5, R a 、R b have the above definitions in the present invention.)

[0045] Specifically, the linking unit in the above conjugate may be in the form of a chemically unstable linking unit (e.g., hydrazone, disulfide), an enzyme-catalyzed linking unit (e.g., peptide residue, carbonate residue unstable to esterase), etc.

[0046] Specifically, the linking unit L that links Ab and D is a group Y that links to a reactive group of Ab (e.g., mercapto, amino, carboxy, etc., especially mercapto), such as a single bond, TIFF0007703050000021.tif24170, -S-, -CO-, -NH-, -CONH-, TIFF0007703050000022.tif12170(aminooxy), and in some embodiments of the present invention, Y is -S- or TIFF0007703050000023.tif24170.

[0047] Furthermore, L further includes a group Q that is a saturated or unsaturated divalent linear or branched C1-50 hydrocarbon chain, where 0 to 6 methylene units are independently -Cy-, -O-, -NR 10 -, -S-, -OC(O)-, -C(O)O-, -C(O)-, -S(O)-, -S(O)2-, -NR 10 S(O)2-, -S(O)2-NR 10 -, -NR 10 -C(O)-, -C(O)NR 10 -, -OC(O)NR 10 -, -NR 10 -C(O)O-, TIFF0007703050000024.tif is substituted by 18170 amino acid residues and oligopeptide residues, where k is selected from integers of 1 to 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), and -Cy- is independently selected from optionally substituted divalent rings of arylene, cycloalkylene, and heterocyclylene, and R 10 is selected from H, -OH, C1-C6 alkyl, C3-C6 cycloalkyl, and heterocycloalkyl.

[0048] Specifically, -Cy- is independently selected from optionally substituted divalent rings of phenylene, bicyclic arylene, monocyclic cycloalkylene, bicyclic cycloalkylene, monocyclic heteroarylene, bicyclic heteroarylene, monocyclic heterocycloalkylene, and bicyclic heterocycloalkylene, particularly from optionally substituted divalent rings of phenylene, monocyclic cycloalkylene, monocyclic heteroarylene, and monocyclic heterocycloalkylene.

[0049] More specifically, -Cy- is independently selected from TIFF0007703050000025.tif149170, where R 11 is one or more independent substituents on the ring and is selected from H, halogen, -CN, -NO2, -CF3, -OCF3, -NH2, -OH, C1-C6 alkyl, -O(C1-C6 alkyl), -NH(C1-C6 alkyl), -N(C1-C6 alkyl)(C1-C6 alkyl), residues of monosaccharides or their derivatives, and R 12 is selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted heterocyclyl, and substituted or unsubstituted heterocyclylalkyl.

[0050] Specifically, the monosaccharide or its derivative may be selected from arabinose, xylose, ribose, glucose, mannose, galactose, fructose, glucuronic acid, and galacturonic acid. In some embodiments of the present invention, the residue of the monosaccharide or its derivative is a galacturonic acid residue, for example is TIFF0007703050000026.tif27170.

[0051] In some embodiments of the present invention, -Cy- is independently selected from TIFF0007703050000027.tif57170.

[0052] Specifically, R 10 may be selected from H and C1-C6 alkyl. In some embodiments of the present invention, R 10 is H or methyl.

[0053] Specifically, in the above definition of Q, the amino acid residues in the amino acid residue and the oligopeptide residue are independently one or more selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, citrulline, ornithine residues. In particular, they are one or more selected from valine, citrulline, alanine, asparagine, aspartic acid, glutamic acid, proline, glycine residues. In particular, the amino acid residues in the amino acid residue and the oligopeptide residue are residues of L-amino acids.

[0054] Specifically, in the above definition of Q, the oligopeptide residue consists of 2 to 10 amino acid residues, particularly 2 to 6 amino acid residues, such as 2, 3, 4, or 5 amino acid residues.

[0055] Specifically, in the above definition of Q, the oligopeptide residue is selected from a valine-citrulline residue, an alanine-alanine-asparagine residue, an aspartic acid-valine residue, a glutamic acid-valine residue, a glycine-proline residue, and the like.

[0056] In some embodiments of the present invention, the oligopeptide residue is TIFF0007703050000028.tif49170.

[0057] In one embodiment of the present invention, the L moiety in general formula I has the following structure. TIFF0007703050000029.tif39170 (where Y is a group that links to a reactive group of Ab (for example, mercapto, amino, carboxy, etc., especially mercapto), R L1 、R L3 and R L4 are, independently, TIFF0007703050000030.tif17170, -(CH2) j O-, -(CH2) j N(R L9 )-, -(CH2) j CO-, -(CH2) j OCO-, -(CH2) j OCON(R L9 )-, -(CH2) j N(R L9 )CON(R L10 )-, -(CH2) j N(R L9 )CO-, -O(CH2) j COO-, -(CH2) j COO-, -(CH2) j CON(R L9 )-, -(CH2) j S-S-, -(CH2) j N(R L9 )-NH=, TIFF0007703050000031.tif56170Selected from one or more combinations of cycloalkylene and arylene, and j is an integer from 0 to 10, R L2 is a single bond, -(CH2) i OCO-, -(CH2) i OCOO-, -(CH2) i NH-COO-, an amino acid residue, an oligopeptide residue, where i is an integer from 0 to 10, R L5 R L6 R L7 and R L8 are independently H, substituted or unsubstituted alkyl, halogen, nitro, cyano, -OR L9 -NR L9 R L10 -S(O) t R L9 -C(O)OR L9 -C(O)R L9 and -C(O)NR L9 R L10 selected from, where t is 0, 1 or 2, each R L9 and R L10 are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl.)

[0058] Specifically, Y is a single bond, TIFF0007703050000032.tif21170, -S-, -CO-, -NH-, -CONH-, TIFF0007703050000033.tif12170(aminooxy). In some embodiments of the present invention, Y is -S- or TIFF0007703050000034.tif20170.

[0059] Specifically, the above R L2In the definition, the amino acid residues in the amino acid residues and oligopeptide residues are independently one or more selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, citrulline, ornithine residues. In particular, the amino acid residues in the amino acid residues and oligopeptide residues are residues of L-amino acids. Specifically, the above R L2 In the definition, the oligopeptide residue consists of 2 to 10 amino acid residues, particularly 2 to 6 amino acid residues, for example, 2, 3, 4 or 5 amino acid residues.

[0060] Specifically, the above R L2 In the definition, the oligopeptide residue may be selected from valine-citrulline residue, alanine-alanine-asparagine residue, aspartic acid-valine residue, glutamic acid-valine residue, glycine-proline residue, etc.

[0061] In one embodiment of the present invention, R L2 is -(CH2) i OCO-, and i is an integer from 0 to 6, particularly 1.

[0062] In one embodiment of the present invention, R L2 is -(CH2) i and i is an integer from 1 to 10, particularly 2.

[0063] In another embodiment of the present invention, R L2 is an oligopeptide residue selected from valine-citrulline residue, alanine-alanine-asparagine residue, aspartic acid-valine residue, glutamic acid-valine residue, etc., particularly TIFF0007703050000035.tif49170.

[0064] Specifically, R L1 , R L3 and R L4is, independently, -(CH2) j -, -(CH2) j O-, -(CH2) j NH-, -(CH2) j CO-, -(CH2) j OCOO-, -(CH2) j OCONH-, -(CH2) j NHCONH-, -(CH2) j NHCO-, -O(CH2) j COO-, -(CH2) j COO-, and -(CH2) j CONH- and is a combination of one or more selected therefrom, and j is an integer from 0 to 10.

[0065] Specifically, each of the above j may independently be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0066] In one embodiment of the present invention, R L1 is TIFF0007703050000036.tif17170.

[0067] In another embodiment of the present invention, R L1 is -(CH2) j CO-, and j is an integer from 0 to 6, particularly 5.

[0068] In one embodiment of the present invention, R L3 is -(CH2) j NH-, and j is an integer from 0 to 6, particularly 0.

[0069] In one embodiment of the present invention, R L4 is -(CH2) j -, and j is an integer from 0 to 5, particularly 1.

[0070] Specifically, R L5 , R L6 , R L7 and R L8 are independently H, C1-6 alkyl, C1-6 haloalkyl, halogen, nitro, cyano, -OR L9selected from, R L8 is selected from H, C1-6 alkyl, and more specifically, R L5 , R L6 , R L7 and R L8 are independently selected from H, halogen.

[0071] In one embodiment of the present invention, R L5 is H.

[0072] In one embodiment of the present invention, R L6 is H.

[0073] In one embodiment of the present invention, R L7 is H.

[0074] In one embodiment of the present invention, R L8 is H.

[0075] In one embodiment of the present invention, the above L moiety has the following structure. TIFF0007703050000037.tif29170 (where Y, R L1 , R L2 each have the above definitions in the present invention.)

[0076] In one embodiment of the present invention, the above L moiety may have the following structure. TIFF0007703050000038.tif24170

[0077] In another embodiment of the present invention, the above L moiety may further have the following structure. TIFF0007703050000039.tif27170 (where R L2 is an oligopeptide residue and has the above definition in the present invention.)

[0078] Specifically, the above L moiety has the following structure. TIFF0007703050000040.tif240170TIFF0007703050000041.tif237170TIFF0007703050000042.tif62170

[0079] In some other embodiments of the present invention, the L moiety has the following structure. TIFF0007703050000043.tif152170

[0080] k1 and k2 are independently selected from integers from 1 to 10.

[0081] In one embodiment of the present invention, the conjugate described above has the following structure. TIFF0007703050000044.tif27170(wherein Ab, n, and D have the above definitions in the present invention, respectively.)

[0082] In some embodiments of the present invention, the conjugate described above has the following structure. TIFF0007703050000045.tif199170TIFF0007703050000046.tif229170TIFF0007703050000047.tif204170(wherein Ab and n have the above definitions in the present invention, respectively.)

[0083] The present invention further provides stereoisomers (shown above) of the conjugate or mixtures thereof.

[0084] The present invention further provides pharmaceutically acceptable salts, solvates, and prodrugs of the conjugate.

[0085] Specifically, the above-mentioned pharmaceutically acceptable salts may include one or more organic or inorganic salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetate, pantothenate, succinate, citrate, tartrate, fumarate, maleate, gluconate, glucuronate, saccharate, benzoate, lactate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, arginine salt, aspartate, glutamate, pantothenate, and ascorbate.

[0086] The present invention further provides a method for producing the above conjugate, which includes a step of binding a conjugate (L-D) of a small molecule drug and a linking unit to an antibody.

[0087] Specifically, before the binding step, the above production method further includes a step of reducing and oxidizing the disulfide bonds of the antibody.

[0088] Specifically, the above production method includes the following steps. (1) Incubate the antibody with a disulfide bond reducing agent. (2) Incubate the antibody obtained in step (1) with an oxidizing agent. (3) Incubate the antibody obtained in step (2) with the above conjugate L-D.

[0089] Specifically, step (1) may further include removing the excess disulfide bond reducing agent (for example, by ultrafiltration).

[0090] Specifically, in step (1), the disulfide bond reducing agent may be tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), β-mercaptoethanol, etc.

[0091] Specifically, in step (1), the equivalent ratio of the antibody to the disulfide bond reducing agent is 1:8 to 15 (specifically, for example, 1:8, 1:9, 1:10, 1:11, 1:12).

[0092] Specifically, the temperature of incubation in step (1) is 35 to 40 °C (specifically, for example, 35, 36, 37, 38, 39, 40 °C). In one embodiment of the present invention, the temperature of incubation is 37 °C.

[0093] Specifically, the incubation time in step (1) is 0.5 to 6 hours (specifically, for example, 0.5, 1, 2, 3, 4, 5, 6 hours). In one embodiment of the present invention, the incubation time is 3 hours.

[0094] Specifically, step (2) may further include removing excess oxidizing agent (for example, by ultrafiltration).

[0095] Specifically, the oxidizing agent in step (2) may be dehydroascorbic acid (DHAA), Cu(II), etc.

[0096] Specifically, in step (2), the equivalent ratio of the antibody to the oxidizing agent is 1:40 to 60 (specifically, for example, 1:40, 1:45, 1:50, 1:55, 1:60).

[0097] Specifically, the temperature of incubation in step (2) is 20 to 30 °C (specifically, for example, 20, 22, 24, 25, 26, 28, 30 °C). In one embodiment of the present invention, the temperature of incubation is room temperature.

[0098] Specifically, the incubation time in step (2) is 1 to 6 hours (specifically, for example, 1, 2, 3, 4, 5, 6 hours). In one embodiment of the present invention, the incubation time is 3 hours.

[0099] Specifically, in step (3), the equivalent ratio of the antibody to the small molecule L-D is 1:10 to 20 (specifically, for example, 1:10, 1:12, 1:14, 1:15, 1:16, 1:18, 1:20).

[0100] Specifically, the temperature of incubation in step (3) is 20 to 30 °C (specifically, for example, 20, 22, 24, 25, 26, 28, 30 °C). In one embodiment of the present invention, the temperature of incubation is room temperature.

[0101] Specifically, the incubation time in step (3) is 6 to 48 hours (specifically, for example, 6, 12, 18, 20, 22, 24, 26, 28, 30, 36, 42, 48 hours). In one embodiment of the present invention, the incubation time is 24 hours.

[0102] In one embodiment of the present invention, the above manufacturing method further includes a step of manufacturing a conjugate (L-D) of a small molecule drug and a linking unit.

[0103] The present invention further provides a modified anti-PD-L1 antibody obtained by mutating one or more amino acid residues of a conventional anti-PD-L1 antibody (for example, Atezolizumab, Durvalumab, Avelumab, Cemiplimab, KN035, CS1001, BGB-A333, KL-A167, SHR-1316, and STI-A1014, etc.) into cysteine respectively by genetic engineering techniques (for example, Thiomab technology), for example, by mutating the amino acids in the heavy chain and / or light chain of Avelumab into cysteine.

[0104] Specifically, the above antibody includes a heavy chain and a light chain. The amino acid sequence of the heavy chain is shown in SEQ ID NO:4, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:4, and / or The amino acid sequence of the light chain is shown in SEQ ID NO:9 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:9.

[0105] Specifically, the above antibody has a light chain sequence shown in SEQ ID NO:9.

[0106] In one embodiment of the present invention, the above antibody has a heavy chain sequence shown in SEQ ID NO:4 and a light chain sequence shown in SEQ ID NO:9.

[0107] The present invention further provides the use of the above modified anti-PD-L1 antibody in the manufacture of an antibody-drug conjugate (ADC).

[0108] The present invention further provides a small molecule compound having the following structure, and a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug thereof. TIFF0007703050000048.tif33170 (wherein m, X, L', R1, R2, R3, Q each have the above definitions in the present invention, and Z is a reactive group.)

[0109] In one embodiment of the present invention, the small molecule compound has the following structure. TIFF0007703050000049.tif40170 (wherein m, X, L', R1, R2, R3, R L1 , R L2 , R L3 , R L4 , R L5 , R L6 , R L7 , R L8 each have the above definitions in the present invention, and Z is a reactive group.)

[0110] Specifically, Z is a reactive linking group with a reactive group of an amino acid (for example, mercapto, amino, carboxy, etc., especially mercapto). Specifically, for example, the mercapto-reactive group may be maleimide, carboxy, amide, halogen, disulfide, etc., the amino-reactive group may be ester, acyl halide, anhydride, carboxy, epoxy, etc., and the carboxyamino-reactive group may be hydroxy, amino, halogen, acyl halide, etc.

[0111] In some embodiments of the present invention, Z is TIFF0007703050000050.tif22170.

[0112] Specifically, the compound represented by Formula VII has the following structure. TIFF0007703050000051.tif30170 (wherein Z, R L1 , R L2 , R L3 , R L4 , R1, R2, R3, R4, R5, R a , R b , and m each have the above definitions in the present invention.)

[0113] More specifically, the compound represented by Formula VII has the following structure. TIFF0007703050000052.tif30170 (wherein Z, R L1 , R L2 , R1, R2, R3, R4, R5, R a , R b , and m have the above definitions in the present invention.)

[0114] Even more specifically, the compound represented by Formula VII has the following structure. TIFF0007703050000053.tif32170 (wherein Z, R L1 , R L2 have the above definitions in the present invention.)

[0115] In some embodiments of the present invention, the compound represented by Formula VII has the following structure. TIFF0007703050000054.tif216170TIFF0007703050000055.tif228170TIFF0007703050000056.tif216170TIFF0007703050000057.tif181170

[0116] The present invention further provides a compound having the following structure, which is useful as a linking unit moiety of an antibody-drug conjugate, and a pharmaceutically acceptable salt and stereoisomer thereof. TIFF0007703050000058.tif42170(wherein Z, R L1 , R L2 , R L3 , R L4 , R L5 , R L6 , R L7 , R L8 have the above definitions in the present invention, and Z' is a reactive group.)

[0117] Specifically, Z' may be a halogen (e.g., chlorine), carboxy, ester, hydroxy, epoxy, amine group, etc.

[0118] Specifically, the compound represented by Formula IX has the following structure. TIFF0007703050000059.tif20170(wherein Z, R L1 , R L2 , Z' have the above definitions in the present invention.)

[0119] In some embodiments of the present invention, the compound represented by Formula IX has the following structure. TIFF0007703050000060.tif231170TIFF0007703050000061.tif88170

[0120] In some embodiments of the present invention, the above Z' is a halogen (e.g., chlorine).

[0121] In some other embodiments of the present invention, the above Z' is a carbonate ester TIFF0007703050000062.tif14170.

[0122] The present invention further provides the use of the compound represented by the above formula IX in the manufacture of an antibody-drug conjugate (ADC).

[0123] The present invention further provides a pharmaceutical composition comprising the above conjugate and a pharmaceutically acceptable auxiliary material.

[0124] Specifically, the above pharmaceutically acceptable auxiliary materials are, for example, diluents such as water, excipients; fillers such as starch, sucrose; adhesives such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; wetting agents such as glycerin; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorbent carriers such as kaolin and soap clay; lubricants such as talc, calcium stearate, magnesium, polyethylene glycol, etc., which are ordinary pharmaceutical auxiliary materials in the pharmaceutical field. In addition, other auxiliary materials such as flavoring agents, sweetening agents, stabilizers, etc. can also be added to the pharmaceutical composition.

[0125] Specifically, depending on the desired administration method, the above pharmaceutical composition contains about 1 to about 99% by weight (specifically, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, etc.) of the above conjugate of the present invention, and the remainder is an appropriate auxiliary material.

[0126] Specifically, the above pharmaceutical composition is useful for administration by any route of administration, and may be oral administration or parenteral administration such as pulmonary, nasal, rectal and / or intravenous administration. Therefore, the preparation according to the present invention is suitable for local or systemic administration, particularly for administration to the skin, subcutaneous, muscle, intra-articular, intraperitoneal, lung, oral, sublingual, nasal, percutaneous puncture, vagina, oral or parenteral. The form of rectal administration is preferably a suppository.

[0127] Dosage forms suitable for oral administration include tablets, pills, chewing gum agents, capsules, granules, drip agents, syrups, etc. Dosage forms suitable for parenteral administration include solutions, suspensions, rehydratable dry agents, sprays, etc.

[0128] The composition of the present invention may be manufactured into a deposit or patch in a dissolved form for transdermal administration. Examples of skin applications include ointments, gels, creams, lotions, suspensions, emulsions, etc.

[0129] The various dosage forms of the pharmaceutical composition of the present invention can be manufactured according to conventional manufacturing methods in the pharmaceutical field, for example, by mixing the active ingredient with one or more auxiliary materials to form the desired dosage form.

[0130] Specifically, in the above pharmaceutical composition, the conjugate of the present invention can be used as the sole active ingredient or in combination with one or more other active ingredients for the same indication, where the conjugate of the present invention can be manufactured for simultaneous, separate or sequential administration together with the other active ingredients.

[0131] The present invention further provides a composition containing the antibody and small molecule drug described in the present invention as active ingredients.

[0132] Specifically, in the above composition, the antibody is, for example, an anti-PD-L1 antibody such as atezolizumab, durvalumab, avelumab, cemiplimab, KN035, CS1001, BGB-A333, KL-A167, SHR-1316, and STI-A1014, and a modified anti-PD-L1 antibody obtained by mutating one or more amino acid residues of a conventional anti-PD-L1 antibody (the above) into cysteine by genetic engineering techniques. For example, it is obtained by mutating amino acids in the heavy chain and / or light chain of atezolizumab into cysteine by genetic engineering techniques.

[0133] Specifically, the above antibody includes a heavy chain and a light chain. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 4, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4, and / or The amino acid sequence of the light chain is shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 9.

[0134] Specifically, the above antibody has the light chain sequence shown in SEQ ID NO: 9.

[0135] In one embodiment of the present invention, the above antibody includes the heavy chain sequence shown in SEQ ID NO: 4 and the light chain sequence shown in SEQ ID NO: 9.

[0136] Specifically, in the above composition, the small molecule drug is an immunomodulator, for example, a Toll-like receptor agonist (Toll-like receptors, TLR), specifically, for example, a TLR7 and / or TLR8 agonist, such as the pyridopyrimidine derivative and its salts described in Patent Application Publication No. WO2019 / 095455A1.

[0137] Specifically, the small molecule drug in the above composition has the following structure. TIFF0007703050000063.tif34170(However, R1, R2, R3, m, L' have the above definitions in the present invention, X' is H, halogen, -NR4R5, -N + R4R5, -OR4, -SR4, azide, phosphonic acid group, diethylphosphonate group, haloalkyl, carboxy, ester, -CN, where R4 and R5 are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, amino acid residue, oligopeptide residue, haloalkyl, or R4 and R5 together with the nitrogen atom to which they are both linked form a heterocyclyl. )

[0138] More specifically, the small molecule drug in the above composition may be selected from the following structures. TIFF0007703050000064.tif225170TIFF0007703050000065.tif239170TIFF0007703050000066.tif180170

[0139] In one embodiment of the present invention, in the above composition, the antibody is avelumab, and the small molecule drug is TIFF0007703050000067.tif24170 having the structure.

[0140] The present invention further provides the use of the above conjugate, or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer thereof, the above antibody of the present invention, the compound represented by formula VII, and the composition in the manufacture of a drug for preventing and / or treating a disease.

[0141] Specifically, the above-mentioned disease may be a disease related to PD-L1 expression or a disease related to TLR7 and / or TLR8 activity.

[0142] Specifically, the above-mentioned disease may be one or more of respiratory diseases, immune diseases, viral diseases, and tumors.

[0143] Specifically, the above-mentioned respiratory diseases include, but are not limited to, asthma, chronic obstructive pulmonary disease, and adult respiratory distress syndrome.

[0144] Specifically, the above-mentioned immune diseases are autoimmune diseases including, but not limited to, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, Sjogren's syndrome, polymyositis, vasculitis, Wegener's granulomatosis, sarcoidosis, ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis, and Behcet's syndrome.

[0145] Specifically, the pathogens of the above viral diseases include, but are not limited to, Adenoviridae (such as adenovirus), Herpesviridae (such as HSV1 (oral herpes), HSV2 (genital herpes), VZV (varicella), EBV (Epstein-Barr virus), CMV (cytomegalovirus), etc.), Poxviridae (such as smallpox virus, vaccinia virus, etc.), Papillomaviridae (such as papillomavirus, etc.), Parvoviridae (such as B19 virus, etc.), Hepadnaviridae (such as hepatitis B virus, etc.), Polyomaviridae (such as polyomavirus, etc.), Reoviridae (such as reovirus, rotavirus, etc.), Picornaviridae (such as enterovirus, foot-and-mouth disease virus, etc.), Caliciviridae (such as norovirus, hepatitis E virus, etc.), Togaviridae (such as rubella virus, etc.), Arenaviridae (such as lymphocytic choriomeningitis virus, etc.), Retroviridae (HIV-1, HIV-2, HTLV-1), Flaviviridae (such as dengue virus, Zika virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus, etc.), Orthomyxoviridae (such as influenza virus (such as influenza A virus, influenza B virus, influenza C virus, etc.)), Paramyxoviridae (such as human parainfluenza virus type 1 (HPV), type 2 HPV, type 3 HPV, type 4 HPV, Sendai virus, mumps virus, measles virus, RS virus, Newcastle disease virus, etc.), Bunyaviridae (such as California encephalitis virus, hantavirus, etc.), Rhabdoviridae (such as rabies virus, etc.), Filoviridae (such as Ebola virus, Marburg virus, etc.), Coronaviridae (such as HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, etc.), Astroviridae (such as astrovirus, etc.), Bornaviridae (such as Borna virus, etc.). More specifically, the above viral diseases may be influenza, SARS, COVID-19, viral hepatitis (such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, etc.), AIDS, rabies, dengue fever, Ebola virus, etc.

[0146] Specifically, the above-mentioned tumor is a malignant tumor, and malignant tumors include lymphoma, granuloma, medulloblastoma, retinoblastoma, liposarcoma, synovial sarcoma, neuroendocrine tumor, carcinoid tumor, gastrinoma, pancreatic islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancy, squamous cell carcinoma, epidermoid carcinoma, lung cancer (small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, Merkel cell carcinoma, esophageal cancer, biliary tract tumor, head and neck cancer, and hematological malignancies, but are not limited thereto. In particular, colon cancer, bladder cancer, melanoma, meningioma, lung cancer, and pancreatic cancer.

[0147] The present invention further provides a method for preventing and / or treating a disease, comprising administering a therapeutically effective amount of the above conjugate of the present invention, or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer thereof, the above antibody of the present invention, the compound represented by Formula VII, or the composition to a system or an individual in need thereof.

[0148] Specifically, in the above method, the disease has the above definition in the present invention.

[0149] The present invention further provides a method for improving a positive immune response of a living body, comprising administering a therapeutically effective amount of the above conjugate of the present invention, or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer thereof, the above antibody of the present invention, the compound represented by Formula VII, or the composition to a system or an individual in need thereof.

[0150] The present invention further provides a method for enhancing a chemotherapy effect, comprising administering a therapeutically effective amount of the above conjugate of the present invention, or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer thereof, the above antibody of the present invention, the compound represented by Formula VII, or the composition to a system or an individual in need thereof.

[0151] The present invention further provides a method for enhancing the effect of immunotherapy, which comprises administering a therapeutically effective amount of the above conjugate of the present invention, or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer thereof, the above antibody of the present invention, the compound represented by Formula VII, or the composition to a system or an individual in need thereof.

[0152] Specifically, the above therapeutically effective amount can be changed according to factors such as the administration route, the age, weight, gender of the patient, and the type and severity of the disease to be treated, and the present invention does not particularly limit this.

[0153] The present invention obtains a modified anti-PD-L1 antibody having two mutant cysteines that substantially maintain the structure of the original antibody and are useful for the construction of ADCs by gene editing. Experiments confirmed that this modified antibody and the obtained ADC maintain the antigen recognition affinity of the original anti-PD-L1 antibody and internalize into cells with good selectivity by binding to cell surface PD-L1. Anti-tumor experiments showed that the obtained ADC has high anti-tumor activity, can significantly increase the survival rate of tumor-bearing animals, and moreover, its toxicity is significantly reduced compared to the case of using the small molecule drug individually or in combination with the antibody, and the burden on the body of the experimental animals is small. The ADC described in the present invention significantly reduces the minimum effective dose in the case of using the small molecule drug therein individually, expands its therapeutic window, and is promising for the development of therapeutic drugs for multiple diseases (such as tumors, viral diseases such as hepatitis B), and the future potential and value of its application can be expected.

Brief Description of the Drawings

[0154]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Mode for Carrying Out the Invention

[0155] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art of the present invention.

[0156] In the context of the present invention, the following terms have the meanings detailed below.

[0157] "Alkyl" refers to a straight-chain or branched-chain hydrocarbon radical that does not contain an unsaturated bond, and the hydrocarbon radical is linked to other parts of the molecule by a single bond. Representative alkyl groups include those containing 1 to 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, t-pentyl, n-hexyl, isohexyl, etc. When alkyl is substituted with cycloalkyl, correspondingly, "cycloalkylalkyl" radicals such as cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, etc. are formed. When alkyl is substituted with aryl, correspondingly, "aralkyl" radicals such as benzyl, diphenylmethyl, phenethyl, etc. are formed. When alkyl is substituted with heterocyclyl, correspondingly, "heterocyclylalkyl" radicals are formed.

[0158] "Alkenyl" refers to a straight-chain or branched-chain hydrocarbon radical that contains at least two carbon atoms and at least one unsaturated bond, and the hydrocarbon radical is linked to other parts of the molecule by a single bond. Representative alkenyls include those containing 1 to 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms such as vinyl, 1-methyl-vinyl, 1-propenyl, 2-propenyl or butenyl, etc.

[0159] "Alkoxy" refers to a substituent in which the hydrogen in hydroxy is substituted by alkyl. Representative alkoxys include those containing 1 to 12 carbon atoms (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), such as methoxy, ethoxy, propoxy, butoxy, etc.

[0160] "Aryl" means a monocyclic or polycyclic radical, including a monocyclic aryl group and / or a polycyclic radical containing a condensed aryl group. Representative aryls include those containing 1 to 3 monocyclic or condensed rings and 6 to about 18 carbon ring atoms, preferably 6 to about 14 carbon ring atoms, such as phenyl, naphthyl, biphenyl, indenyl, phenanthrenyl or anthracenyl, etc.

[0161] "Heterocyclyl" includes heteroaromatic and heteroalicyclic groups containing 1 to 3 monocyclic and / or fused rings and 3 to about 18 ring atoms. Preferred heteroaromatic and heteroalicyclic groups contain 5 to about 10 ring atoms. Suitable heteroaryl in the compounds of the present invention contains 1, 2, or 3 heteroatoms selected from N, O, or S atoms, and the heteroaryl includes, for example, coumarin (including 8-coumarin), quinolyl (including 8-quinolyl, isoquinolyl), pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, phthalazinyl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuryl, benzofuranyl, benzenethiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridyl, etc. Suitable heteroalicyclic groups in the compounds of the present invention contain 1, 2, or 3 heteroatoms selected from N, O, or S atoms, and the heteroalicyclic groups include, for example, pyrrolidinyl, tetrahydrofuranyl, dihydrofuran, tetrahydrothiophenyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, oxathianyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxiranyl, thiaranyl, azepinyl, oxazepanyl, diazepinyl, triazepinyl, 1,2,3,6-tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl, indolyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3H-indolyl, and quinolyl, etc.

[0162] "Halogen" or "halo" refers to bromo, chloro, iodo or fluoro.

[0163] Unless otherwise indicated, the compounds of the present invention further include isotopically labeled forms, i.e., the only difference is the presence of one or more compounds of atoms enriched in isotopes. For example, substituting only at least one hydrogen atom with deuterium or tritium, or substituting at least one carbon with 13 C or 14 carbon enriched in 15 C, or substituting at least one nitrogen with nitrogen enriched in

[0164] The term "PD-L1" includes mammalian isotypes such as human PD-L1, homologs of the species of human PD-L1, and analogs containing at least one epitope common to PD-L1. The amino acid sequence of PD-L1, for example human PD-L1, is known in the art. In the present invention, "anti-PD-L1 antibody" refers to an antibody that can specifically bind to PD-L1.

[0165] The term "antibody" refers to an immunoglobulin molecule containing four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains interconnected via disulfide bonds (i.e., "whole antibody molecule"), and multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain includes a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (including regions CH1, CH2, CH3). Each light chain includes a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The VH and VL regions are further subdivided into hypervariable regions called complementarity determining regions (CDRs), and relatively conserved regions called framework regions (FRs) are interspersed therebetween. Each VH and VL consists of three CDRs and four FRs, and are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus.

[0166] As used herein, terms such as "antigen-binding portion" and "antibody fragment" of an antibody include any naturally occurring polypeptide or glycoprotein that specifically binds to an antigen to form a complex, and that can be obtained by enzymatic methods, synthesized, or genetically engineered polypeptides or glycoproteins. As used herein, the term "antigen-binding portion" or "antibody fragment" of a PD-L1 antibody refers to one or more fragments of an antibody that maintain the ability to specifically bind to PD-L1. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs)). As used in the expressions herein, "antigen-binding fragments" also include other engineered molecules such as bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and microantibodies. Antigen-binding fragments of an antibody typically include at least one variable domain.

[0167] The term "therapeutically effective amount" refers to an amount that can achieve in a subject the treatment, prevention, alleviation, and / or mitigation of a disease or condition described in the present invention.

[0168] Terms such as "patient", "subject", "individual", etc. can be used interchangeably herein and refer to any animal or its cells that follow the methods described herein, whether in vitro or in situ, and in some non-limiting embodiments are mammals such as humans, monkeys, dogs, rabbits, mice, etc.

[0169] The term "treatment" includes eradicating, removing, reversing, remitting, changing, or controlling a disease and / or condition after its onset.

[0170] The term "prevention" refers to the ability to avoid, minimize, or make difficult the onset or progression of a disease and / or condition through treatment before the onset of the disease and / or condition.

[0171] The term "disease" refers to the physical state of a subject associated with the diseases described in the present invention.

[0172] The disclosures of the various publications, patents, and patent specifications cited herein are hereby incorporated by reference in their entirety.

[0173] Hereinafter, with reference to the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. It is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the present invention.

[0174] Example 1: Production of Small Molecule Drugs 1. Production of (R)-2-((5-nitropyridin-2-yl)dithiocyclohexyl)prop-1-ol (Compound 24) TIFF0007703050000068.tif221700 °C, under nitrogen protection, SOCl2 (316 μL, 3.94 mmol) was added dropwise to a solution of 5-nitropyridine-2-thiol (560 mg, 3.59 mmol) in anhydrous DCM (8 mL). The resulting mixture was stirred at room temperature for 2 h. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to afford Intermediate 24 as a yellow powder. To a solution of Intermediate 4 in dry DCM (10 mL) was added dropwise a solution of methyl (R)-2-mercaptoprop-1-ol (prepared according to the procedure described in WO2013055987, 360 mg, 3.88 mmol) in dry DCM (5 mL) drop by drop. The reaction mixture was stirred overnight at room temperature under nitrogen protection. After completion of the reaction, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a yellow solid. The residue was suspended in water, alkalized with sodium hydrogen carbonate solution, and then extracted with DCM (3 × 100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (PE:EA = 10:3) to afford Compound 24. Yield: 76%. 1H NMR (400 MHz, chloroform-d) δ 9.23 (dd, J = 2.7, 0.8 Hz, 1H), 8.33 (dd, J = 8.9, 2.6 Hz, 1H), 7.71 (dd, J = 8.8, 0.7 Hz, 1H), 4.48 (t, J = 6.7 Hz, 1H), 3.62 (ddd, J = 11.8, 7.1, 4.3 Hz, 1H), 3.38 (ddd, J = 11.9, 7.3, 4.4 Hz, 1H), 3.11 (pd, J = 7.0, 4.4 Hz, 1H), 1.28 (d, J = 6.9 Hz, 3H). 13C NMR (101 MHz, chloroform-d) δ 167.95, 145.12, 142.45, 131.48, 120.85, 64.00, 49.68, 16.73. C8H 11 N2O3S2MS (ESI) m / z [M + H] + 。 Calculated value 247.0, found value 247.2. 2. Preparation of (R)-2-((5-nitropyridin-2-yl)dithiocyclohexyl)propyl (4-(hydroxymethyl)phenyl)carbamate (Compound 26) Compound 24 (246 mg, 1 mmol) and pyridine (79 mg, 1 mmol) were dissolved in dry DCM (5 mL) and stirred at 0 °C. Under nitrogen protection, a solution of triphosgene (145 mg, 0.5 mmol) in anhydrous DCM (5 mL) was added dropwise to the mixture at 0 °C one drop at a time, and stirring was continued for 1 h. After the reaction was completed, the resulting reaction solution was rotary evaporated under reduced pressure to obtain (R)-2-((5-nitropyridin-2-yl)dithioalkyl)propyl carbon chloride (Compound 25), which was used as it was without further purification. Next, Compound 25 (308 mg, 1.0 mmol) was dissolved in dry DCM (5 mL), and a mixed DCM solution (5 mL) of (4-aminophenyl)methanol (148 mg, 1.2 mmol) and pyridine (79 mg, 1.0 mmol) was slowly added dropwise. The reaction mixture was stirred at room temperature for 2 h under nitrogen protection. After the reaction was completed, the resulting solution was transferred to a saturated ammonium chloride solution and extracted with DCM (3 × 50 mL). The organic layer was washed with water and brine, then dried over anhydrous Na2SO4, filtered, and rotary evaporated under reduced pressure to obtain a crude product. The crude product was purified by silica gel flash chromatography (PE:EA = 2:1) to obtain Compound 26 as a white solid. Yield: 70%. 1H NMR (400 MHz, Chloroform-d) δ 9.23 (d, J = 2.6 Hz, 1H), 8.33 (dd, J = 8.8, 2.6 Hz, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.34 (m, 4H), 6.82 (s, 1H), 4.67 (s, 2H), 4.31 - 4.23 (m, 2H), 3.37 (h, J = 6.8 Hz, 1H), 1.43 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, Chloroform-d) δ 168.63, 152.87, 145.03, 142.06, 136.88, 136.37, 131.61, 128.04, 119.47, 118.73, 67.26, 64.86, 45.57, 17.10. Mass (ESI): C 16 H 18 N3O5S2 m / z [M+H] + . Calculated value 396.1, measured value 396.2. 3. Preparation of (R)-2-((5-Nitropyridin-2-yl)dithiocyclohexyl)propyl (4-(benzyl chloride)phenyl)carbamate (Compound 19) TIFF0007703050000070.tif22170At 0 °C, thionyl chloride (22 μL, 0.304 mmol) was added dropwise to a solution of Compound 26 (100 mg, 0.253 mmol) in anhydrous DCM (5 mL). The reaction mixture was taken out at 0 °C and stirred at room temperature for 30 minutes. After completion of the reaction, excess thionyl chloride was removed by rotary evaporation under reduced pressure to obtain Compound 19, the target product, as a white solid. Yield: 96%. 1H NMR (400 MHz, chloroform-d) δ 9.24 (d, J = 2.6 Hz, 1H), 8.35 (dd, J = 8.9, 2.7 Hz, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.35 (m, 4H), 6.75 (s, 1H), 4.56 (s, 2H), 4.27 (qd, J = 11.5, 6.1 Hz, 2H), 3.35 (h, J = 6.7 Hz, 1H), 1.41 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, chloroform-d) δ 168.59, 152.71, 145.06, 142.11, 137.56, 132.88, 131.60, 129.59, 119.47, 118.71, 67.34, 45.90, 45.51, 17.07. C 16 H17ClN3O4S2 MS(ESI): m / z [M+H] + . Calculated value: 414.0, Measured value: 414.2. 4. Preparation of Compound 20 TIFF0007703050000071.tif41170Compound N 4A solution of 6-(4-((dimethylamino)methyl)benzyl)-1-butylpyrido[3,2-d]pyrimidine-2,4-diamine (40 mg, 0.11 mmol) in DMF (0.5 mL) was added with Compound 19 (50 mg, 0.12 mmol), TBAB (8 mg, 0.022 mmol), and DIEA (18 μL, 0.11 mmol). The reaction mixture was stirred at room temperature and monitored by LC-MS. After completion of the reaction, DMF was removed by rotary evaporation under reduced pressure. The residue was redissolved in a 10% methanol solution and stirred at room temperature for 2 h. The resulting solution was concentrated by rotary evaporation and then purified by reversed-phase high-performance liquid chromatography (RPHPLC) to obtain Compound 20 as a formate salt. Yield: 68.0%. 1H NMR (400 MHz, methanol-d4) δ 9.09 (d, J = 2.6 Hz, 1H), 8.32 (s, 1H), 8.31 (dd, J = 8.7, 2.6 Hz, 1H), 8.09 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 8.6 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.62 - 7.44 (m, 8H), 4.69 (s, 2H), 4.57 (s, 2H), 4.35 (s, 2H), 4.26 - 4.16 (m, 2H), 3.70 (t, J = 7.3 Hz, 2H), 3.44 (pd, J = 6.9, 4.8 Hz, 1H), 2.97 (s, 6H)), 1.73 (p, J = 7.5 Hz, 2H), 1.50 - 1.40 (m, 5H), 1.00 (t, J = 7.5 Hz, 3H). 13 C NMR (101 MHz, methanol-d4) δ 168.46, 165.88, 160.00, 157.48, 155.18, 153.27, 144.32, 142.10, 141.94, 141.18, 133.68, 133.63, 133.31, 131.74, 129.62, 129.31, 126.30, 125.85, 125.76, 121.22, 120.11, 118.01, 68.23, 67.23, 67.20 43.08, 40.56, 30.66, 19.77, 15.98, 12.78. C 37 H 44 N9O4S2MS (ESI): m / z [M] + . Calculated value 742.3, measured value 742.3. HRMS (ESI) [M] + :. Calculated value 742.295 8, measured value 742.2968. 5. Preparation of MC-Val-Cit-PAB-Cl (Compound 29) TIFF0007703050000072.tif At 341700 °C, thionyl chloride (8.7 μL, 0.304 mmol) was added dropwise to a solution of MC-Val-Cit-PAB-OH (57 mg, 0.1 mmol) in anhydrous DCM (5 mL). The reaction mixture was removed at 0 °C and stirred at room temperature for 12 hours. After completion of the reaction, the excess thionyl chloride was removed by rotary evaporation under reduced pressure, and the desired product was obtained as a brown solid 29 and used as it was without purification. 6. Preparation of Compound 30 TIFF0007703050000073.tif40170 Compound N 4 -butyl-6-(4-((dimethylamino)methyl)benzyl)pyridine[3,2-d]pyrimidine-2,4-diamine (40 mg, 0.11 mmol) in DMF (1 mL) was added with compound 29 (71 mg, 0.12 mmol), TBAB (8 mg, 0.022 mmol), and DIEA (18 μL, 0.11 mmol). The reaction mixture was stirred at room temperature and monitored by LC-MS. After completion of the reaction, DMF was removed by rotary evaporation under reduced pressure. The residue was redissolved in a 10% methanol solution and stirred at room temperature for 2 h. The resulting solution was concentrated by rotary evaporation and then purified by reversed-phase high-performance liquid chromatography (RPHPLC) to obtain compound 30 as a formate. Yield: 55.0%. 11H NMR (400 MHz, Chloroform) δ 10.34 (bs, 1H), 9.40 (bs, 1H), 8.13 (d, J = 8.6 Hz, 1H), 7.82 - 7.68 (m, 4H), 7.61 - 7.39 (m, 4H), 7.25 - 6.96 (m, 3H), 4.51 - 4.14 (m, 5H), 4.25 (s, 2H), 4.00 (m, 2H), 3.46 (t, J = 6.8 Hz, 2H), 3.27 (s, 6H), 2.95 (m, 2H), 2.73 (m, 1H), 2.49 (m, 1H), 2.10 - 1.85 (m, 6H), 1.74 - 1.53 (m, 4H), 1.60 - 1.38 (m, 2H), 1.41 - 1.19 (m, 6H), 1.14 - 0.80 (m, 9H). MS(ESI) [M] + : Calculated value 920.15, measured value 920.30. 7. (S)-N 1 -(4-(Chloromethyl)phenyl)-2-((R)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propionamido)propionamido)succinamide (Compound 31) Production TIFF0007703050000074.tif At 271700 °C, (S)-2-((R)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propionamido)propionamido)-N 1 To a solution of (S)-2-((R)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propionamido)propionamido)-N-(4-(hydroxymethyl)phenyl)succinamide (100 mg, 0.174 mmol) in anhydrous DCM (5 mL) was added dropwise thionyl chloride (15 μL, 0.210 mmol). The reaction mixture was removed at 0 °C and stirred at room temperature for 30 minutes. After completion of the reaction, the excess thionyl chloride was removed by rotary evaporation under reduced pressure to obtain the desired product as a brown solid. The product was used as it was without purification. 8. Production of Compound 32 TIFF0007703050000075.tif27170 Compound N 4A solution of 6-(4-((dimethylamino)methyl)benzyl)-3-butylpyrido[3,2-d]pyrimidine-2,4-diamine (40 mg, 0.11 mmol) in DMF (1 mL) was added with compound 31 (71 mg, 0.12 mmol), TBAB (8 mg, 0.022 mmol), and DIEA (18 μL, 0.11 mmol). The reaction mixture was stirred at room temperature and monitored by LC-MS. After completion of the reaction, DMF was removed by rotary evaporation under reduced pressure. The residue was redissolved in a 10% methanol solution and stirred at room temperature for 2 h. The resulting solution was concentrated by rotary evaporation and then purified by reversed-phase high-performance liquid chromatography (RPHPLC) to obtain compound 32 formate. Yield: 48.0%. LC-MS (ESI) [M] + :. Calculated value 920.10, measured value 920.25. 9. Preparation of Compound 33 The commercially available compound (2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triol triacetate (455 mg, 1 mmol) was dissolved in DMF (5 mL), and N-succinimidyl 6-maleimidohexanoate (340 mg, 1.1 mmol), 1-hydroxybenzotriazole (135 mg, 1 mmol), and N,N-diisopropylethylamine (210 μL, 1.2 mmol) were sequentially added, followed by stirring at room temperature for 2 h. The reaction solution was extracted with EA / H2O, and the organic phase was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (DCM:MeOH = 10:1) to obtain 278 mg of the target product. LC-MS (ESI) [M] + : 649.3. 10. Preparation of Compound 34 TIFF0007703050000077.tif At 411700 °C, thionyl chloride (8 μL, 0.12 mmol) was added dropwise to a solution of (2S,3R,4S,5S,6S)-2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacyltriacetate (Compound 33) (65 mg, 0.1 mmol) in anhydrous DCM (2 mL). The reaction mixture was stirred at 0 °C for 30 minutes. After completion of the reaction, the organic solvent was removed by rotary evaporation under reduced pressure to obtain the target product. The product was used as it was without purification. 11. Preparation of Compound 35 TIFF0007703050000078.tif To a solution of the compound (2S,3R,4S,5S,6S)-2-(4-(chloromethyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacyltriacetate (67 mg, 0.1 mmol) (Compound 34) in DMF (1 mL) was added N 4 -butyl-6-(4-((dimethylamino)methyl)benzyl)pyrido[3,2-d]pyrimidine-2,4-diamine (40 mg, 0.11 mmol), TBAB (8 mg, 0.022 mmol), and DIEA (18 μL, 0.11 mmol). The reaction mixture was stirred at room temperature and monitored by LC-MS. After completion of the reaction, the reaction solution was filtered and then purified by reversed-phase high-performance liquid chromatography (RPHPLC) to obtain 30 mg of the target product, with a yield of 28%. LC-MS (ESI) [M] + : 995.4. 12. Preparation of Compound 36 Compound 35 (30 mg, 0.03 mmol) was dissolved in a mixed solvent (THF:H2O = 2:1), lithium hydroxide monohydrate (15 mg, 0.3 mmol) was added, and the mixture was stirred at room temperature for 30 min. Then acetic acid was added to adjust the pH to 7. The mixture was concentrated under reduced pressure to remove the organic solvent, methanol was added to dissolve it, and 12 mg of the target product was produced by reverse-phase chromatography with a yield of 46%. LC-MS (ESI) [M] + : 855.4. 13. Preparation of Compound 37 TIFF0007703050000080.tif20170N 4 -Butyl-6-(4-(chloromethyl)benzyl)pyrido[3,2-d]pyrimidine-2,4-diamine (356 mg, 1 mmol, for the synthesis method, refer to the preparation method of Compound 10 in Example of Chinese Patent CN 108069963 B) was dissolved in DMF, 2-Boc-2,6-diazaspiro[3.3]heptane hemioxalate (980 mg, 0.2 mmol), and potassium carbonate (420 mg, 0.3 mmol) were added, and the mixture was stirred at 35 °C overnight. After the reaction was completed, the reaction solution was extracted with ethyl acetate and saturated brine, and washed three times with water. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by silica gel column chromatography (DCM-MeOH system) to obtain 340 mg of the target product with a yield of 65%. LC-MS (ESI) [M+H] + : 518.3. 14. Preparation of Compound 38 TIFF0007703050000081.tif201706-(4-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)methyl)benzyl)-2,6-diazaspiro[3.3]heptane-2-t-butyl carboxylate (Compound 37) (260 mg, 0.5 mmol) was dissolved in a mixed solvent (DCM:TFA = 10:1). The reaction solution was stirred at 0 °C for 1 h. The organic solvent was removed by concentration under reduced pressure to obtain a crude product. Ethyl ether was added to the crude product, sonicated, and filtered to obtain the target product. LC-MS (ESI) [M+H] + : 418.3. 15. Preparation of Compound 39 TIFF0007703050000082.tif331704 - ((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-uridine valeramide)benzyl(4-nitrobenzene) carbonate (81.0 mg, 0.11 mmol) was dissolved in DMF (0.5 mL), and 6-(4-((2,6-diazaspiro[3.3]heptan-2-yl)methyl)benzyl)-N 4 -butylpyridine[3,2-d]pyrimidine-2,4-diamine (Compound 38) (42.0 mg, 0.10 mmol) and N,N-diisopropylethylamine (21 μL, 0.12 mmol) were added, and the mixture was stirred at room temperature, and the reaction was detected by LCMS. After the reaction was completed, the target product (20 mg) was obtained directly by RP-HPLC, and the yield was 20%. LC-MS (ESI) [M+H] + : 1016.4 1 H NMR (400 MHz, DMSO-d6) δ 7.78 (d, J = 8.5 Hz, 1H), 7.60 - 7.55 (m, 4H), 7.29 - 7.17 (m, 5H), 4.00 (s, 2H), 3.66 (s, 2H), 3.46 (t, J = 13.2 Hz, 2H), 3.07 (s, 2H), 2.95 (t, J = 12.4 Hz, 1H), 2.76 - 2.67 (m, 1H), 2.08 - 2.01 (m, 2H), 1.75 - 1.59 (m, 6H), 1.50 - 1.43 (m, 3H), 1.38 - 1.27 (m, 6H), 1.01 - 0.87 (m, 9H). 16. Preparation of Compound 40 TIFF0007703050000083.tif 4-((S)-4-Amino-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propionamido)-4-oxobutylamido)benzyl (4-nitrobenzene) carbonate (81.0 mg, 0.11 mmol) was dissolved in DMF (0.5 mL), and 6-(4-((2,6-diazaspiro[3.3]heptan-2-yl)methyl)benzyl)-N 4 -butylpyrido[3,2-d]pyrimidine-2,4-diamine (Compound 38) (42.0 mg, 0.10 mmol), and N,N-diisopropylethylamine (21 μL, 0.12 mmol) were added, and the mixture was stirred at room temperature, and the reaction was detected by LCMS. After completion of the reaction, the desired product (15 mg) was obtained directly by RP-HPLC, and the yield was 16%. LC-MS (ESI) [M+H] + : 1016.4. 17. Preparation of Compound 41 TIFF0007703050000084.tif (2S,3R,4S,5S,6S)-2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (260 mg, 0.4 mmol) was dissolved in DMF (2 mL), and di(p-nitrobenzene) carbonate (182 mg, 0.6 mmol) and N,N-diisopropylethylamine (105 μL, 0.6 mmol) were sequentially added, and the mixture was reacted for 6 hours. The reaction solution was extracted 3 times with EA / H2O, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (DCM:MeOH = 15:1) to obtain the desired product (250 mg, yield 78%). LC-MS (ESI) [M+H] + : 814.4. 18. Preparation of Compound 42 TIFF0007703050000085.tif36170(2S,3R,4S,5S,6S)-2-(2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-4-(((4-nitrophenoxy)carbonyl)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (Compound 41) (82 mg, 0.1 mmol) was dissolved in DMF (1 mL), and 6-(4-((2,6-diazaspiro[3.3]heptan-2-yl)methyl)benzyl)-N 4 -butylpyrido[3,2-d]pyrimidine-2,4-diamine (Compound 38) (42.0 mg, 0.10 mmol), and N,N-diisopropylethylamine (21 μL, 0.12 mmol) were added, and the mixture was stirred at room temperature, and the reaction was detected by LCMS. After the reaction was completed, the desired product (30 mg) was obtained directly by RP-HPLC, and the yield was 27%. LC-MS (ESI) [M+H] + : 1092.4 19. Preparation of Compound 43 TIFF0007703050000086.tif33170Compound 42 (30 mg, 0.027 mmol) was dissolved in a mixed solvent (THF:H2O = 2:1), lithium hydroxide monohydrate (15 mg, 0.27 mmol) was added, and the mixture was stirred at room temperature for 30 min, and then acetic acid was added to adjust the pH to 7. The organic solvent was removed by concentration under reduced pressure, methanol was added to dissolve it, and the desired product (15 mg) was obtained by reverse phase chromatography, and the yield was 51%. LC-MS (ESI) [M+H] + : 952.3 1 H NMR (400 MHz, CD3OD ) 8.06 (bs, 1H), 7.82(d, 1H), 7.31 - 7.18 (m, 5H), 7.15- 7.03 (m, 2H), 3.98 (td, J = 13.4, 4.8 Hz, 2H), 3.63 (s, 2H), 3.46 (t, J = 13.2 Hz, 2H), 3.05 (s, 2H), 2.37 (m, 2H), 1.77-1.25 (m, 12H), 0.89 (t, J = 7.4 Hz, 3H). 20. Preparation of Compound 44 TIFF0007703050000087.tif23170 Compound 38 (41.8 mg, 1.0 mmol) was dissolved in DMF (2 mL), and N-succinimidyl 6-maleimidohexanoate (34 mg, 0.11 mmol) was added, followed by reaction at room temperature for 3 hours. The reaction solution was extracted with ethyl acetate / saturated brine, and the organic phase was washed 3 times with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the organic phase, and a crude product was obtained. The crude product was purified by silica gel column chromatography (DCM:MeOH = 5:1) to obtain 28 mg of the target product, with a yield of 48%. LC-MS (ESI) [M+H] + : 611.4 1 H NMR (400 MHz, Chloroform) δ 7.67 (d, J = 8.4Hz, 1H), 7.62 (br, 2H), 7.31 - 7.16 (m, 5H), 4.21 (s, 2H), 3.89 (t, J = 10.4 Hz, 2H), 3.62 (s, 2H), 3.45 (t, J = 13.4 Hz, 2H), 3.01 (s, 2H), 2.23 (td, J = 13.2, 4.8 Hz, 2H), 1.63 - 1.27 (m, 12H), 0.89 (t, J = 7.4 Hz, 3H). 21. Preparation of Compound 45 TIFF0007703050000088.tif22170 Compound 38 (21 mg, 0.5 mmol) was dissolved in methanol (1 mL), 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetaldehyde (28 mg, 2 mmol) was added, and after stirring at room temperature for 30 min, sodium cyanoborohydride (8 mg, 13 mmol) was added batchwise, and the reaction was continued for 1 hour. The reaction solution was filtered, and 6 mg was directly prepared by reverse-phase chromatography, with a yield of 23%. LC-MS (ESI) [M+H] + : 541.3 11H NMR (400 MHz, CDCl3): δ 7.62 (br, 2H), 7.56 (d, J = 8.6 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.43 - 7.32 (m, 4H), 4.24 (s, 2H), 4.70 (s, 4H), 4.12 (s, 2H), 3.53 (td, J = 13.2, 5.9 Hz, 2H), 3.46 - 3.33 (s, 4H), 3.33 (s, 4H), 2.50 (t, 3H), 1.70 - 1.65 (m, 2H), 1.53 - 1.44 (m, 2H), 1.00 (t, J = 7.3 Hz, 3H). 22. Preparation of Compound 46 TIFF0007703050000089.tif 21170 Compound 38 (210 mg, 0.5 mmol) was dissolved in acetonitrile (5 mL), and (2-(2-(2-iodoethoxy)ethoxy)ethoxy)ethyl) t-butylcarbamate (305 mg, 0.75 mmol) and potassium carbonate (216 mg, 1.5 mmol) were sequentially added. The reaction solution was heated to 65 °C and reacted for 5 hours. After completion of the reaction, filtration was carried out, and the filter residue was washed with acetonitrile. The filtrate was collected and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (DCM:MeOH = 4:1) to obtain 218 mg of the target product, and the yield was 62%. LC-MS (ESI) [M+H] + : 693.5. 23. Preparation of Compound 47 TIFF0007703050000090.tif 21170 Compound 46 (218 mg) was dissolved in a mixed solvent (DCM:TFA = 20:1, 10 mL), and the reaction solution was reacted at 0 °C for 1 hour. After completion of the reaction, the organic solvent was removed by concentration under reduced pressure to obtain the target product. The product was used as it was without purification. 24. Preparation of Compound 48 Compound 47 (184 mg, 0.3 mmol) was dissolved in DMF (1 mL), and 2,5-dioxopyrrolidin-1-yl 3-(2-(2-azidoethoxy)ethoxy)ethoxy)propionate (106 mg, 0.3 mmol) and triethylamine (135 μL, 0.9 mmol) were sequentially added. The reaction mixture was reacted at room temperature for 3 hours. After completion of the reaction, dichloromethane and water were added for extraction. The organic phase was washed with saturated aqueous ammonium chloride solution and water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The obtained crude product was used as it was without purification. LC-MS (ESI) [M+H] + : 822.3. 25. Preparation of Compound 49 Compound 48 (100 mg, 0.12 mmol) was dissolved in a mixed solvent (DMSO:H2O = 1:1, 1 mL), and 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl)cyclohexane-1-carboxamide (66 mg, 0.24 mmol) and cuprous bromide (51 mg, 0.36 mmol) were added. The reaction mixture was reacted at room temperature for 1 hour. After completion of the reaction, it was filtered, and the filtrate was directly prepared by reverse-phase chromatography to obtain 22 mg of the target compound, and the yield was 16%. LC-MS (ESI) [M+H] + : 1140.4. 11H NMR (400 MHz, DMSO) δ 9.43 (t, J = 5.8 Hz, 1H), 8.21 (t, J = 5.6 Hz, 1H), 7.91 (t, 1H), 7.82 (bs, 2H), 7.79 (d, J = 8.6 Hz, 1H), 7.68 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.01 (s, 1H), 4.48 (t, J = 5.2 Hz, 2H), 4.26 (m, 4H), 3.82 - 3.76 (m, 4H), 3.67 (t, J = 6.4 Hz, 1H), 3.62 - 3.55 (m, 6H), 3.55 - 3.43 (m, 22H), 3.39 (t, J = 5.0 Hz, 4H), 3.33 (t, J = 6.3 Hz, 2H), 3.20 (m, 7H), 2.31 (t, J = 6.4 Hz, 4H), 2.06 (t, J = 11.3 Hz, 2H), 1.67 (m, 8H), 1.34 (m, 6H), 0.91 (t, J = 7.3Hz, 3H). 26. Preparation of Compound 50 TIFF0007703050000093.tif18170 Compound 50 was prepared with reference to the synthesis method of Compound 38. 27. Preparation of Compound 51 TIFF0007703050000094.tif22170 Compound 51 was prepared with reference to the synthesis method of Compound 45, and the yield was 56%. LC-MS(ESI) [M+H] + : 599.4. 28. Preparation of Compound 52 TIFF0007703050000095.tif20170 Compound 52 was prepared with reference to the synthesis method of Compound 38. 29. Preparation of Compound 53 TIFF0007703050000096.tifCompound 53 was prepared by referring to the synthesis method of compound 45, with 6-maleimidohexanoic acid N-succinimidyl ester changed to N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and the yield was 42%. LC-MS (ESI) [M+H] + : 655.3. 30. Preparation of Compound 54 TIFF0007703050000097.tifMethyl (2-(methylamino)ethyl) tert-butylcarbamate (1 g, 5.3 mmol) was dissolved in dichloromethane (10 mL). At 0 °C, chloromethyl chloroformate (0.95 mL) and pyridine (0.86 mL) were successively added to the reaction solution, and the reaction was continued at 0 °C for 2 hours. After the reaction was completed, dichloromethane and water were added for separation. The organic phase was washed with a saturated aqueous sodium chloride solution, then dried over anhydrous sodium sulfate, and the organic layers were combined and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA = 5:1) to obtain the target product as a colorless oil. LC-MS (ESI) [M+H] + : 281.3. 31. Preparation of Compound 55 TIFF0007703050000098.tif21170N 4 -Butyl-6-(4-((dimethylamino)methyl)benzyl)pyrido[3,2-d]pyrimidine-2,4-diamine (400 mg, 1.1 mmol) in chloroform (10 mL), compound 54 (1080 mg, 3.9 mmol) and TBAI (1420 mg, 3.9 mmol) were added. The reaction solution was heated to 60 °C and reacted for 6 hours. After the reaction was completed, the reaction solution was concentrated and then directly purified by column chromatography (DCM:MeOH = 4:1) to obtain 480 mg of the target compound, and the yield was 72%. LC-MS (ESI) [M] + : 609.3. 32. Preparation of Compound 56 Compound 55 (480 mg) was dissolved in a mixed solvent (DCM:TFA = 10:1), and the reaction solution was reacted at 0 °C for 1 hour. After the reaction was completed, the organic solvent was removed by concentration under reduced pressure to obtain the trifluoroacetate salt of the target product. Ethyl ether was added to the yellow oil and sonicated to precipitate a solid, which was then filtered. The filter residue was the trifluoroacetate salt of the target product. LC-MS (ESI) [M] + : 509.3. 33. Preparation of Compound 57 4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-uridinevaleramido)benzyl (4-nitrobenzene) carbonate (MC-VC-PAB-NPC) (81.0 mg, 0.11 mmol) was dissolved in DMF (0.5 mL), and Compound 56 (51 mg, 0.10 mmol), HOBT (13.5 mg, 0.10 mmol), and N,N-diisopropylethylamine (53 μL, 0.3 mmol) were added. The mixture was stirred at room temperature, and the reaction was detected by LCMS. After the reaction was completed, it was directly prepared by RP-HPLC to obtain 30 mg of the target product, and the yield was 24%. LC-MS (ESI) [M] + : 1107.4. 1 H NMR (400 MHz, DMSO) δ 10.16 (s, 1H), 8.38 (bs, 2H), 8.24 (bs, 1H), 7.90 (d, J = 7.7 Hz, 1H), 7.75 (t, 1H), 7.66 - 7.37 (m, 7H), 7.26 (m, 2H), 7.00 (s, 2H), 6.24 (s, 2H), 5.50 (s, 2H), 5.26 - 5.06 (m, 2H), 5.04 - 4.87 (m, 2H), 4.61 - 4.12 (m, 8H), 3.60 - 3.27 (m, 8H), 2.89 (m, 12H), 2.25 - 2.05 (m, 2H), 1.97 (m, 1H), 1.79 - 1.09 (m, 13H), 1.00 - 0.71 (m, 8H). 34. Preparation of Compound 58 TIFF0007703050000101.tif25170 Compound 58 was prepared by referring to the synthesis method of Compound 57 and changing MC-VC-PAB-NPC to MC-AAN-PAB-NPC. LC-MS(ESI) [M] + : 1107.4 1 H NMR (400 MHz, DMSO) δ 9.75 (s, 1H), 8.33 (bs, 2H), 8.24 (s, 1H), 8.08 (d, 1H), 7.76 (t, 1H), 7.63 (d, J = 7.6 Hz, 2H), 7.55 - 7.38 (m, 7H), 7.27 (m, 2H), 7.00 (s, 2H), 6.94 (s, 1H), 6.26 (s, 2H), 5.14 (m, 2H), 4.96 (m, 2H), 4.65 - 4.42 (m, 3H), 4.32 - 4.14 (m, 5H), 3.50 - 3.35 (m, 10H), 3.04 - 2.77 (m, 12H), 2.58 (d, J = 6.3 Hz, 2H), 2.09 (t, J = 7.5 Hz, 3H), 1.67 - 1.55 (m, 2H), 1.53 - 1.27 (m, 6H), 1.24 - 1.18 (m, 10H), 0.93 (t, J = 7.3 Hz, 3H). 35. Preparation of Compound 59 TIFF0007703050000102.tif39170 Compound 59 was prepared by referring to the synthesis method of Compound 57 and changing MC-VC-PAB-NPC to Compound 41. LC-MS(ESI) [M] + : 1183.5 36. Preparation of Compound 60 TIFF0007703050000103.tif 35170 Compound 59 (35 mg, 0.03 mmol) was dissolved in a mixed solvent (THF:H2O = 2:1), lithium hydroxide monohydrate (16 mg, 0.3 mmol) was added, and the mixture was stirred at room temperature for 30 min. Then acetic acid was added to adjust the pH to 7. The organic solvent was removed by concentration under reduced pressure, methanol was added to dissolve it, and 5 mg of the target product was produced by reverse-phase chromatography, with a yield of 15%. LC-MS (ESI) [M+H] + : 1043.4. Example 2: Production of anti-PD-L1 THIOMAB

[0175] 1. Gene editing of anti-PD-L1 THIOMAB Avelumab (MSB0010718C, trade name Bavencio) is a fully humanized IgG1 monoclonal anti-PD-L1 antibody drug developed by Merck KGaA and Pfizer. It was approved by the FDA as a therapeutic drug for Merkel-cell carcinoma in 2017 (Apolo et al., 2017; Shirley, 2018). The nucleotide sequences encoding its light chain and heavy chain are shown in SEQ ID NO:1 and 2 respectively, and the amino acid sequences of the light chain and heavy chain are shown in SEQ ID NO:3 and 4 respectively. In addition to binding to human PD-L1, Avelumab can cross-react with mouse-derived PD-L1 (Deng et al., 2016). Therefore, developing an anti-PD-L1 ADC drug using Avelumab as a raw material ensures that the antibody itself has sufficient excellent biological activity and drug discovery potential, and makes the obtained ADC drug applicable to the previous mouse model tests and subsequent human clinical trials during development. The expression plasmid of Avelumab consists of two parts, the light chain 159-pFuse-αPDL1-LC plasmid and the heavy chain 162-pFuse-αPDL1-hIgG-Fc2 plasmid, and its nucleotide sequence is shown in SEQ ID NO:5 and 6. These two types of plasmids were co-transfected into cells for expression and purified by protein A resin to obtain the anti-PD-L1 antibody Avelumab. The inventors constructed a THIOMAB expression plasmid by the PCR site-directed mutagenesis method. A pair of completely complementary and reverse primers containing the mutation site was designed, and using the complete plasmid to be mutated as a template, a high-fidelity PCR enzyme was used to replicate and amplify the entire plasmid sequence with the mutation site. Subsequently, the methylated template plasmid was removed with DpnI restriction endonuclease, and the non-methylated PCR product was maintained. Furthermore, through plasmid transformation and culture, the complete plasmid with the mutation site was amplified. After the site-directed mutagenesis experiment was completed, the obtained plasmid was extracted and sequenced to identify whether the mutation was successful. The plasmid obtained by site-specific mutagenesis of the light chain 159-pFuse-αPDL1-LC plasmid was named αPD-L1 THIOMAB LC-V205C, and its nucleotide sequence is shown in SEQ ID NO:7. The results of the sequencing comparison are shown in Figure 1. When aligned with the template plasmid, the sequence at the predetermined mutation site mutated from GTG to TGC, but the other bases did not change. Therefore, the amino acid at position 205 of the translated light chain mutated from valine to cysteine. These two artificially introduced cysteines are used in the subsequent binding reaction of the ADC drug. For the anti-PD-L1 THIOMAB antibody obtained by mutation, the nucleotide sequence encoding the light chain is shown in SEQ ID NO:8, and the amino acid sequence of the light chain is shown in SEQ ID NO:9. 2. Expression of Anti-PD-L1 Antibody and THIOMAB The purified anti-PD-L1 antibody was expressed and identified by reducing and non-reducing SDS PAGE analysis. As shown in Figure 2, this antibody was reduced to the light chain (~25KD) and heavy chain (~50KD) in reducing SDS PAGE, and remained as the complete antibody (~150KD) in non-reducing SDS PAGE. After expression and purification, anti-PD-L1 THIOMAB was obtained and identified by reducing and non-reducing SDS PAGE analysis. As shown in Figure 3, the SDS staining results of anti-PD-L1 THIOMAB (shown in Figure 3) were almost identical to those of the anti-PD-L1 antibody (shown in Figure 2), that is, the antibody was reduced to a light chain (~25KD) and a heavy chain (~50KD) in reducing SDS PAGE, and remained as a complete antibody (~150KD) in non-reducing SDS PAGE. Experiments suggested that the anti-PD-L1 THIOMAB obtained by gene editing basically maintained the original structure of the antibody, and that the expression process and self-assembly process of THIOMAM were not affected by artificial mutations. Also, under the same conditions, there was no difference in the expression levels of the antibody and THIOMAB, suggesting that there was no significant impact on intracellular antibody expression and the assembly process. To further identify the obtained THIOMAB, the inventors treated the complete THIOMAB in the sequence of "TCEP reduction - purification - DHAA reoxidation - purification" to obtain THIOMAB with free mercapto (THIOMAB-S2). The change in its molecular weight was detected by Exactive Plus EMROrbitrap LC-MS. The results are shown in Figure 4. The molecular weight of the complete anti-PD-L1 THIOMAB was approximately 143791.75, while the molecular weight of THIOMAB-S2 with free mercapto was approximately 143544.31, and the difference in molecular weight was 247.44. Theoretically, generally one free cysteine is modified at the mercapto of the complete anti-PD-L1 THIOMAB, so the molecular weight is only more than that of THIOMAB-S2 by the molecular weight of the residue where two cysteines and a mercapto are condensed, which is approximately 238. This error is acceptable for antibody macromolecules. Therefore, it was determined that the change in the molecular weight of the obtained THIOMAB was as expected experimentally. From this result, it was suggested that the inventors successfully expressed a homogeneous anti-PD-L1 THIOMAB with two mutant cysteines by gene editing, which could be used for the construction of subsequent homogeneous ADC drugs. In subsequent experiments, the inventors further needed to examine whether this artificial mutation affected the antigen-binding ability and other activities of the antibody by comparing the results of its in vitro and in vitro experiments with those of the natural anti-PD-L1 antibody. As shown in Figure 4, compared with the original anti-PD-L1 THIOMAB coated with cysteine, the THIOMAB after reduction dialysis lost two cysteines and exposed two free mercaptos. As a result, the expected molecular weight of THIOMAB at this time decreased by 238 to 143553.75, but the actually detected molecular weight of THIOMAB was 143544.31, a decrease of 247.44. Example 3: Synthesis Route and Identification of ADC Based on THIOMAB

[0176] 1. Synthesis and Identification of ADC HE-S2 The schematic diagram of the synthesis of ADC HE-S2 is shown in Figure 5. The inventors used anti-PD-L1 THIOMAB and compound 20 (Example 1) as raw materials to synthesize a conjugate of an anti-PD-L1 antibody with a disulfide bond linker named ADC HE-S2 and N 4 -butyl-6-(4-((dimethylamino)methyl)benzyl)pyridine[3,2-d]pyrimidine-2,4-diamine (hereinafter abbreviated as compound D18). Here, the conjugation site on the antibody is the cysteine at position 226 of the amino acid sequence (light chain sequence) shown in SEQ ID NO:9. For the ADC HE-S2 obtained by the reaction, after measuring the concentration and purity by Nonadrop, the yield (about 30% - 50%) was estimated. A part of the sample was sent to the National Protein Center, and the results of measuring the change in antibody molecular weight by high molecular weight MS are shown in Figure 6. Theoretically, when the mercapto in ADC HE-S2 is modified with S2-D18, the change in the molecular weight of the whole ADC with a DAR of 2 increases by ~934 compared to THIOMAB. The molecular weight of the ADC obtained by MS detection was 144721.08, while the molecular weight of THIOMAB was 143791.75, and the difference in molecular weight was 929.33. This error is acceptable in the proteospectrum analysis of ~147K. Therefore, it was determined that the ADC drug ADC HE-S2 (ADC1) with a uniform DAR of 2 was successfully manufactured and is shown below. The mass spectrometry results of TIFF0007703050000104.tif42170ADC HE-S2 are shown in Figure 6. The molecular weight of the ADC obtained by MS was 144721.08, and the molecular weight of THIOMAB was 143791.75. 2. Synthesis and identification of other ADC drugs After the experimental protocol for synthesizing ADC HE-S2 matured, the inventors utilized this conjugation strategy (using anti-PD-L1 THIOMAB and compounds 30, 32, 57 (manufactured in Example 1) as raw materials) to synthesize two types of ADCs with different linkers: ADC VC (ADC2) and ADC Legumain (ADC3), ADC4. The synthetic routes are shown in Figures 7 and 8 respectively. By MS identification (shown in Figure 9), it was calculated and verified by conventional calculation analysis methods that the theoretically molecular weight of ADC VC is approximately 1600 larger than that of THIOMAB. As a result of MS, the difference between the two was 145399.23 - 143791.75 = 1607.48, indicating that it is very close to the theoretical value. Similarly, it was also verified that the theoretically molecular weight of ADC Legumain should increase by approximately 1600 compared to THIOMAB. As a result of MS, the difference between the two was 145391.09 - 143791.75 = 1599.34, also indicating that it is very close to the result of theoretical calculation. Therefore, it can be concluded that two types of homogeneous ADCs with a DAR of 2, ADC VC and ADC Legumain, were obtained. Example 4: Synthetic route and identification of ADC based on wild-type PD-L1 antibody

[0177] 1. ADC conjugation step and method a. Preparation of reaction raw materials and reagents: The antibody was prepared in a PBS solution with a concentration of 5 - 10 mg / mL (Avelumab described in Example 2). It was prepared in an aqueous TCEP solution with a concentration of 10 mM. The conjugated small molecule (compounds 39, 40, 43, 44, 51, 45, 49, 53, 57, 58, 60, manufactured in Example 1) was prepared in a DMSO solution with a concentration of 10 mM. It was prepared in a cysteine PBS solution with a concentration of 20 mM. b. Antibody reduction: 10 or 20 equivalents of TCEP (10 mM) were added, and the mixture was shaken at 37 °C at a rotation speed of 220 rpm / min for 2 - 3 h. c. Binding reaction: After antibody reduction, 10 - 20 equivalents of the small molecule were added, and the mixture was shaken at room temperature at a rotation speed of 220 rpm / min for 2 - 4 h. d. Quenching reaction: A cysteine solution equivalent to the small molecule was added, and the reaction was stopped by incubating at 4 or 10 °C for 30 min. e. Desalting: After the reaction was completed, desalting was performed using a 5 ml prepackaged desalting column from Cytiva. 2. Measurement of the DAR value of ADC Calculation method of DAR value by tandem mass spectrometry (MS) using ultra-high performance liquid chromatography (UHPLC) a. Equipment: Waters Acquity-Class ultra-high performance liquid chromatography (UHPLC) tandem Waters Synapt G2-Si Q-TOF high-resolution mass spectrometry system was adopted. High-purity nitrogen gas was used as the nebulizing gas, and high-purity argon gas was used as the collision gas. b. Liquid chromatography conditions: The column was an ACQUITY UPLC Protein BEH SEC column (200 Å, 2.1 mm × 150 mm, 1.7 μm), the column temperature was 25 °C, and the sample injection volume was 15 μL. Isocratic elution: The mobile phase was 50 mM ammonium acetate, the flow rate was 0.065 mL / min, the collection time was 10 min, and the ultraviolet detection wavelength was 280 nm. Mass spectrometry conditions: The capillary voltage was set to 3.0 kV, and ESI source positive ionization mode scanning was performed. The cone voltage was 120 V, and the source temperature and desolvation temperature were set to 120 °C and 500 °C, respectively. The flow rate of the cone gas was 20 L / H, the flow rate of the desolvation gas was 600 L / H, and the primary mass spectrometry range was m / z 400 - m / z 8000. c. Data analysis: The data was analyzed using Waters Masslynx software, and the mass spectrometry data was deconvoluted using the MaxEnt 1 plugin. The ADC DAR value was measured by analyzing the mass spectrometry deconvolution map. First, the peaks were integrated to calculate the peak area ratio, and the sum of the peak area ratios was set to 100. The weighted average drug conjugation ratio of the ADC was calculated using the formula: DAR = Σ weighted peak area / 100. 3. Structural information of specific ADC products TIFF0007703050000106.tif224170TIFF0007703050000107.tif228170TIFF0007703050000108.tif87170Example 5: Research on biological activity

[0178] 1. Detection of the antigen-binding ability of ADC HE-S2 (ADC1) Sufficient specific antigen affinity is the basis of antibody biological activity and is extremely important for the activity and therapeutic effect of antibody drugs. During the production process of ADC drugs, due to antibody modification, chemical treatment, and small molecule binding, all of these may disrupt or affect the original antigen affinity and stability of the antibody and may impair the antibody antigen-binding ability. Therefore, in the process of treating the antibody, the usage conditions should be as mild as possible. When selecting the modification and binding sites, the antigenic determinants of the original antibody should be avoided so as not to impair the antigen-binding ability of the antibody. Furthermore, the antigen affinity of the obtained ADC drug should be verified to ensure that the ADC drug maintains sufficient targeting and specificity. The antibody antigen affinity of ADC HE-S2 designed and manufactured in the present invention is an important basis for achieving its anti-tumor activity. ADC HE-S2 needs to achieve targeted transport of the immunomodulator D18 by specifically binding the antibody to PD-L1 highly expressed in tumors. In addition, ADC HE-S2 can block the tumor PD-1 / PD-L1 immune inhibitory signaling pathway through PD-L1 binding on the cell surface and induce the anti-tumor reaction of T cells. Therefore, after successfully manufacturing ADC HE-S2, the inventors first verified its ability to recognize and bind the PD-L1 antigen in vitro. Using PD-L1-positive MC38 as a platform, the inventors verified whether ADC HE-S2 and the genetically modified anti-PD-L1 THIOMAB maintained their original antibody-antigen binding ability by fluorescence-activated cell sorting (FACS). In the experiment, ADC HE-S2, an anti-PD-L1 antibody (i.e., Avelumab of Example 2), and anti-PD-L1 THIOMAB (i.e., the mutant antibody produced in Example 2) were incubated with MC38 cells as primary antibodies so that the antibodies could sufficiently contact and bind to PD-L1 on the cell surface. After washing with PBS, the cells were treated with an FITC-labeled goat anti-human secondary antibody and incubated for 30 minutes in the dark to allow sufficient binding of the secondary antibody to the primary antibody. Also, two groups of negative controls were performed. One was used as a negative control (NC), and cells were treated by adding only isotype control IgG without staining to show the fluorescence background of the cells themselves. The other was used as a secondary antibody. After adding isotype control IgG, it was stained with an FITC-labeled secondary antibody together with other experimental groups to characterize the false-positive fluorescence signal due to the non-specific binding of the secondary antibody to the cells. The stained MC38 cells were analyzed with a BD FACSAria (登録商標) III flow cytometer. By detecting and comparing the intensity of the FITC fluorescence signal on MC38 cells treated with different antibody-drugs, the affinity of ADC, THIOMBA, and the antibody for PD-L1 was characterized. The results of processing all the results with FlowJo software (TreeStar Inc.) are shown in Figure 10. The results of FACS are shown in Figure 10. Based on the blank control group (NC) using IgG isotype antibody, the false positive signal offset rate of the control group using fluorescent secondary antibody staining was only 1.7%. As a result of calculating the mean fluorescence intensity (MFI), the MFI of the NC group was 142.00, while the mean fluorescence intensity of MC38 after secondary staining was 166.75, showing a significant difference (p < 0.0001), but the difference was small. Such a significant but weak difference was a false positive signal produced by the non-specific binding of the FITC secondary antibody used in the experiment to MC38 cells. The experiment proved that this phenomenon of false positive binding was weak and did not affect the results. Therefore, the antibody-antigen binding ability of anti-PD-L1 antibody drugs was evaluated based on the NC control group. In MC38 cells treated with anti-PD-L1 antibody, anti-PD-L1 THIOMAB, and ADC HE-S2, the inventors observed similar FITC positive signals. The fluorescence signal offset rates for the NC group were 35.7%, 36.6%, and 34.2% respectively, much higher than the false positive signal (1.7%) due to the non-specific binding of the fluorescent secondary antibody. The calculated MFI values for anti-PD-L1 antibody, anti-PD-L1 THIOMAB, and ADC HE-S2 were 445.3, 449.0, and 392.5 respectively, also shown to be significantly higher than the two control groups (p < 0.0001). It was shown that all three types of antibodies could specifically bind to PD-L1. Also, by analysis using the Student's t method, it was proved that anti-PD-L1 THIOMAB that had experienced gene editing and ADC HE-S2 that had been further chemically modified had no significant difference in the detected MFI compared to the natural anti-PD-L1 antibody. From this, it was also shown that the gene editing and chemical modification by the inventors did not impair the original antigen recognition affinity of the antibody nor cause the appearance of non-specific target off-target phenomena. Therefore, the inventors were able to confirm that ADC HE-S2 maintained the antigen recognition affinity of the original anti-PD-L1 antibody and could be further applied to subsequent experiments and research and development. 2. Internalization ability of ADC HE-S2 (ADC1) The drug release of ADCs is widely considered to depend on the internalization ability of the corresponding antibodies. The antibody-antigen complex formed by the binding of an ADC to an antigen protein on the cell surface enters the target cells along with the internalization process of the antigen protein and releases the conjugated drug under the action of intracellular enzymes or other intracellular physiological environments. Therefore, the internalization ability of the complex formed by the selected antigen binding to the ADC is essential for the achievement of the function of the ADC drug. Since ADC HE-S2 designed by the inventors of the present invention should theoretically act both inside cancer cells and inside DC cells, the inventors verified the ability of anti-PD-L1 antibody drugs to internalize into tumor cells and DC cells through in vitro experiments. pHrodo TM The red dye is known to be a pH-sensitive fluorescent dye, and its molecular fluorescence intensity rapidly increases when the surrounding environment becomes acidic (Lehrman et al., 2018). When this dye internalizes into cells together with the antigen-antibody complex, the pH around the dye immediately becomes acidic, thereby generating an observable fluorescent signal. This property enables TM monitoring of the internalization process into antibodies using the pHrodo red dye. Using the IncuCyte (登録商標) live cell analysis system, the inventors were able to perform image-based monitoring and quantitative analysis of the internalization process of the anti-PD-L1 antibody labeled with the pHrodo TM red dye. In 0.1 M sodium carbonate (pH 8.3) buffer, 1 mg / mL of anti-PD-L1 THIOMAB and 3 - 5 equivalents of pHrodo TM red dye were incubated together for about 1 h, and residual small molecules were removed by dialysis to obtain the fluorescent dye-labeled anti-PD-L1 THIOMAB. The labeled antibody should be stored at 4°C in the dark. For long-term storage, it should be stored at -20°C in the dark and repeated freeze-thaw cycles should be avoided. To characterize the internalization process of the PD-L1 / anti-PD-L1 antibody complex, the inventors used pHrodo TMA red dye was used. As described above, since the present inventors verified that the anti-PD-L1 THIOMAB has the same antigen recognition ability as ADC HE-S2, the internalization ability of the anti-PD-L1 THIOMAB can also reflect the internalization ability of ADC HE-S2. (1) Experiment on the internalization of anti-PD-L1 THIOMAB in cancer cells Using two types of PD-L1-positive tumor cells, MC38 and B16, as materials, it was verified that the internalization of the PD-L1 / anti-PD-L1 antibody complex was characterized. After adding the fluorescently labeled anti-PD-L1 THIOMAB into the cells, they were incubated for 24 h in the dark, and then, with the IncuCyte (登録商標) live cell analysis system, the distribution of the red fluorescence signal inside the cells was photographed, and the results are shown in Fig. 11A. When the labeled THIOMAB was added, it was clearly observed that a significant red signal occurred inside the cells, while in the control group without adding the antibody, almost no red fluorescence was observed. From the synthesized images, it was found that almost no red fluorescence occurred outside the cells, proving the relevance between the fluorescence signal and the internalization of the antibody. Therefore, as shown in Fig. 11B, the present inventors (登録商標) used the software to analyze the total integrated intensity of the obtained total red objects, and thus the internalization level of the fluorescently labeled THIOMAB could be evaluated. After adding the labeled THIOMAB, the total integrated intensity of the total red objects in MC38 was 13701.63, which was significantly higher than 84.88 of the control group. The signal intensity in B16 reached 283613.60, which was also significantly higher than 98.53 of the control group. The fluorescence signal in B16 cells was stronger than that in MC38 cells, which may be due to differences in the PD-L1 internalization rate and the intracellular pH value by the cells. (2) Experiment on the internalization and selectivity of anti-PD-L1 THIOMAB into DC cells To verify the internalization of anti-PD-L1 THIOMAB into DC cells, the inventors obtained two types of DC cells, Cd274 knockout DC 2.4 cells and wild-type DC 2.4 cells, from Mr. Haidong Tang of the School of Pharmacy, Tsinghua University. The Cd274 gene is the PD-L1 coding gene, and after knockout, DC 2.4 could not express the PD-L1 protein, so it was a PD-L1 negative DC cell. On the other hand, wild-type DC 2.4 expressed PD-L1 and was a PD-L1 positive DC cell (shown in Figure 12). According to the hypothesis, the inventors should be able to observe that anti-PD-L1 THIOMAB internalizes into PD-L1 positive DC cells but not into PD-L1 negative Cd274 knockout DC 2.4 cells. Therefore, through the anti-PD-L1 THIOMAB internalization experiments on these two types of cells, the inventors can simultaneously verify the internalization ability of anti-PD-L1 THIOMAB into DC cells and the selectivity of the antibody. In preliminary experiments, the inventors added 10% heat-inactivated fetal bovine serum (FBS) as a blocking agent to the medium of DC 2.4 cells according to normal procedures, incubated for 8 h, and then used FACS to detect the red fluorescence signal inside the cells. The results are shown in Figure 13. Surprisingly, as a result of FACS detection, the same proportion of red fluorescence positive cells, 53.6% and 52.9% respectively, were observed in both PD-L1 positive wild-type DC 2.4 cells and PD-L1 negative CD274 knockout DC 2.4 cells. In the negative control group without adding fluorescently labeled anti-PD-L1 THIOMAB, almost no red fluorescence positive cells (1.57% and 0.92%) were observed. Similar results were obtained even after repeated use, suggesting that the anti-PD-L1 THIOMAB labeled with a fluorescent dye smoothly internalizes not only into PD-L1 positive DC 2.4 cells but also into PD-L1 negative Cd274 knockout DC 2.4 cells. This unexpected result is contrary to the inventors' hypothesis. Through literature research, the inventors found that the ability of the Fc segment of human IgG to bind to mouse Fcγ receptors is very similar to its ability to bind to human orthologous Fcγ receptors (Dekkers et al., 2017). Therefore, the inventors speculated that the highly expressed and highly active mouse-derived Fcγ receptors on the surface of DC 2.4 cells could similarly bind to the humanized anti-PD-L1 antibody and internalize it, enabling both cells to internalize the anti-PD-L1 antibody by internalization. To verify this speculation, the inventors added 10% mouse serum and 10% FBS to the cell culture medium for each group as blocking agents. Compared with FBS, the mouse-derived IgG abundantly contained in mouse serum can more effectively block the Fcγ receptors on the surface of DC 2.4, thereby inhibiting the antibody internalization process mediated by the Fcγ receptors of DC cells. Before adding THIOMAB, the DC cells were first incubated at 4°C for 30 minutes to pause the internalization process of cell surface receptors and at the same time fully bind the IgG in mouse serum to the Fcγ receptors on the surface of DC cells. Then, fluorescently labeled anti-PD-L1 THIOMBA was added to the cells of each group. Next, light-avoiding culture was performed at 4°C for 30 min to allow the antibody and antigen to fully contact and bind under the condition where cell internalization was stopped. The total integrated object intensity-time change curves obtained from the internalization experiment of anti-PD-L1 THIOMAB are shown in Figures 14A and B. As can be seen from the results in Figure 14A, when blocked by adding 10% FBS, similar fluorescence intensity change curves can be observed in PD-L1-positive wild-type DC 2.4 cells and PD-L1-negative CD274 knockout DC 2.4 cells. However, in the negative control group without adding fluorescently labeled anti-PD-L1 THIOMAB, no change in the fluorescence signal was observed. As shown in Fig. 14B, in the experimental group with 10% mouse serum added, in PD-L1-positive wild-type DC cells, the enhancement of the total red object integrated intensity progressed significantly more than in PD-L1-negative Cd274 knockout DC cells. In particular, at the detection start stage (0 - 60 min), in PD-L1-negative cells, there was almost no generation of fluorescence signal, showing a change curve similar to the negative control. After that, in PD-L1-negative DC cells, a fluorescence signal by the antibody began to appear, but the rate of signal enhancement (slope) was slower than that of wild-type DC 2.4 cells, and finally a significant difference was recognized by t-test (p < 0.0001). After 6 hours of culture, IncuCyte (登録商標) The experimental groups with mouse serum blockade were photographed using live cells, and the distribution of the red fluorescence signal inside the cells of each group was observed. As a result, as shown in Fig. 14A, the red fluorescence signal inside wild-type DC 2.4 cells was significantly stronger than that in Cd274 knockout DC cells. This is interpreted as follows: in the 4°C incubation 30 minutes before detection, the internalization process of cell receptors almost stopped and the antigen-antibody binding was not affected, so PD-L1 on the surface of DC cells was fully bound to the anti-PD-L1 antibody, and the Fcγ receptor was also fully blocked by homologous IgG in mouse serum. When detection started, as the culture temperature recovered to 37°C, the internalization process of cell surface receptors also recovered, and the anti-PD-L1 THIOMAB that had been fully bound to PD-L1 on the surface of wild-type DC cells until then was rapidly internalized, but PD-L1-negative cells did not internalize. On the other hand, as the incubation time became longer, PD-L1-negative DC cells internalized anti-PD-L1 THIOMAB via recycled Fcγ receptors to generate a fluorescence signal. However, due to the action of mouse homologous IgG in the medium, such an internalization process was competitively inhibited. Therefore, in Cd274 knockout DC cells, the internalization rate of THIOMAB was slower than that of wild-type DC 2.4 cells that could internalize THIOMAB by binding to PD-L1. This result verified the speculation of the inventors and demonstrated the ability of the anti-PD-L1 antibody to internalize into cells by binding to cell surface PD-L1. In addition, the difference in the expression of the internalization rate between the Cd274 knockout type and wild-type DC 2.4 cells of the anti-PD-L1 THIOMAB after blocking with mouse serum also demonstrated that the anti-PD-L1 antibody has good selectivity. Example 6: Research on antitumor activity

[0179] 1. Research on the antitumor activity of ADC HE-S2 (ADC1) To verify the anti-cancer activity of ADC HE-S2, the inventors first established a mouse model using MC38. After tumor implantation, tumors were observed on the body surface of the mice in about one week. After the average tumor tissue volume of the mice reached 100 mm 3 ³, the mice were randomly divided into groups according to the final administration type, in order, into an IgG isotype control antibody group (abbreviated as IgG), an anti-PD-L1 antibody group (anti-PD-L1), an anti-PD-L1 THIOMAB group (Thiomab), a D18 group (D18), a combined treatment group of D18 and anti-PD-L1 antibody (anti-PD-L1 & D18), and an ADC HE-S2 group (ADC HE-S2), with 7 - 8 mice in each group. In the IgG isotype control antibody group, the anti-PD-L1 antibody (i.e., Avelumab in Example 2) group, and the anti-PD-L1 THIOMAB (i.e., the mutant antibody produced in Example 2) group, 150 μg each was intraperitoneally injected twice a week. In the D18 group, 25 μg each was intraperitoneally injected twice a week. In the combined treatment group of D18 / anti-PD-L1 antibody, the anti-PD-L1 antibody was administered 150 μg each twice a week, and D18 was administered 25 μg once. In the ADC HE-S2 group, ADC HE-S2 was intraperitoneally injected 150 μg each once a week, and the anti-PD-L1 antibody was injected intraperitoneally 150 μg each once a week at a time shifted from ADC. The experimental results are shown in Fig. 15A. For MC38 tumors, the effects of the three types of single-drug treatments with anti-PD-L1 antibody, anti-PD-L1 THIOMAB, and D18 were all very similar. There was no significant difference in their inhibitory activities against tumor growth, but all were slightly superior to the therapeutic effect of the IgG isotype antibody as a negative control. This result indicates that both D18 and the anti-PD-L1 antibody have certain anti-cancer activities, and anti-PD-L1 THIOMAB also maintains the biological function of the original antibody. On the other hand, the combination treatment of D18 and the anti-PD-L1 antibody showed very good tumor growth inhibitory activity, and its effect was far superior to that of any group of single-drug treatments. This result re-verified the synergistic effect between D18 and PD-1 / PD-L1 blockade antibody treatment. ADC HE-S2 showed extremely surprising anti-cancer activity, and its tumor growth inhibitory activity was significantly superior to that of the combination treatment group (P<0.001). When the experiment was conducted until the 24th day, the tumor volume of the mice in the combination treatment group began to show a relatively obvious increase, while in the ADC treatment group, the tumor size of the mice was maintained at a low level. To further study the survival rate of mice after long-term administration, the inventors conducted a survival experiment on mice. The experiment was finally conducted until the 70th day (see Fig. 15B). The survival rate of the mice in the ADC HE-S2 group was 6 / 8, and almost no tumors were detected in the last 6 surviving mice. The survival rate of the mice in the D18 and PD-L1 combination administration group was 4 / 8. The survival rate of the mice in the D18 group was 1 / 8. On the other hand, all the mice in the other two single-administration groups and the negative control group died. This result also proves the strong anti-cancer activity of ADC HE-S2. Furthermore, in order to further confirm the therapeutic effect of ADC HE-S2, the inventors used a B16 melanoma mouse model (see Figure 15C). The dosage and time were the same as those shown in Table 1. B16 melanoma is a tumor with a low PD-L1 level and is somewhat resistant to PD-1 / PD-L1 blockade therapy. As a result, as expected by the inventors, compared with the MC38 tumor, the inhibitory effect of various drugs on B16 melanoma decreased. The therapeutic effects of the single administration groups of three drugs, anti-PD-L1 antibody, anti-PD-L1 THIOMAB, and D18, were almost the same as the effect of the IgG isotype antibody. The combination therapy of anti-PD-L1 antibody + D18 and ADC HE-S2 alone showed a certain tumor inhibitory effect. The mice after ADC HE-S2 treatment had a significantly slower growth rate of B16 tumors in their bodies compared with the combination therapy (P<0.05). This result demonstrated that in the B16 tumor model, the antitumor activity of ADC HE-S2 was stronger than the combination therapy of two drugs, anti-PD-L1 antibody and D18. Table 1 Drug administration schedule for mouse treatment TIFF0007703050000109.tif46170Dosage: For antibody drugs, 150 μg is administered per mouse. For D18, 25 μg is administered per mouse. - indicates that it is not administered on that day. ※ indicates that only the mice in the MC38 tumor model progress until day 24, and the mice in the B16 tumor model are not administered after day 21. 2. Expansion of the D18 treatment window by the ADC conjugation strategy Note that the DAR of ADC HE-S2 is 2, the molecular weight is about 150K, and the D18 content in the ADC is 0.748 μg per single administration (150 μg / 150000×2×364), which can be easily estimated to be much less than the dosage at the time of combined administration (25 μg per mouse per single administration). Therefore, we want to study the effect of the change in D18 dosage on the synergistic anticancer effect between D18 and the anti-PD-L1 antibody. Therefore, the inventors combined different doses of D18 (0.25 μg, 2.5 μg, and 25 μg) with 150 μg of anti-PD-L1 antibody to treat MC38 tumors (see Figure 16). Mice were divided into groups according to the administration type: IgG isotype antibody control group (abbreviated as IgG), anti-PD-L1 antibody group (anti-PD-L1), anti-PD-L1 antibody + D18 (25 μg) group (anti-PD-L1 + D18 (25 μg)), anti-PD-L1 antibody + D18 (2.5 μg) group (anti-PD-L1 + D18 (2.5 μg)), and anti-PD-L1 antibody + D18 (0.25 μg) group (anti-PD-L1 + D18 (0.25 μg)). Administration started after the average tumor volume per group reached 100 mm 3 3 . IgG isotype antibody and anti-PD-L1 antibody were intraperitoneally injected at a dose of 150 μg per mouse twice a week. D18 was administered once a week, and each administration was intraperitoneally injected at 25 μg per mouse, 2.5 μg per mouse, and 0.25 μg per mouse, respectively, according to the group. Mice in the IgG isotype antibody group were intraperitoneally injected with the same amount of DMSO as that at the time of D18 administration every week as a control. The anti-tumor results of gradient administration of D18 combined with anti-PD-L1 antibody are shown in Figure 16. From the experimental results, D18 showed the function of enhancing the anti-tumor activity of anti-PD-L1 antibody and showed dose-dependent characteristics. The combined treatment with anti-PD-L1 antibody showed significant tumor inhibitory activity when the dose of D18 per administration was 25 μg. As the D18 dose decreased, the anti-tumor effect of the combined treatment of D18 and anti-PD-L1 antibody also weakened. In fact, when the dose decreased to 2.5 μg or 0.25 μg per mouse, the tumor inhibitory effect was not significantly different from that of anti-PD-L1 antibody alone administration. In addition, the D18 loading amount per administration of ADC HE-S2 (~0.75 μg) is much smaller than 2.5 μg. Therefore, it was judged that ADC targeted administration obtained a more significant tumor inhibitory effect with a D18 dose (~0.75 μg) that was insufficient to exert activity in combination than when 25 μg of D18 was combined with anti-PD-L1 antibody. Subsequently, in a mouse model, the effects of the drugs on the body weights of each group of mice were examined, and the results of judging the toxicity and side effects of the drugs used are shown in Fig. 17. Healthy mice were divided into five mice each in the ADC group, anti-PD-L1 antibody + D18 group, anti-PD-L1 antibody group, D18 group, and IgG group according to the administration. The administration methods and dosages of the mice in each group were the same as those in the mouse tumor model test. In the ADC group, ADC HE-S2 was administered once a week, and anti-PD-L1 antibody was injected once, 150 μg each time. D18 was administered at 25 μg once a week. The anti-PD-L1 antibody and IgG isotype antibody were administered at 150 μg twice a week. The change in the body weight of the mice in the anti-PD-L1 antibody group was almost the same as that of the blank control IgG group, proving that the toxicity and side effects of the anti-PD-L1 antibody itself on the mice were not significant. In the anti-PD-L1 antibody + D18 group and D18 group, each time the administration of D18 ended, the body weight of the mice showed a certain degree of decrease. Compared with the administration of D18 alone, the degree of body weight loss of the mice in the anti-PD-L1 antibody + D18 group was particularly significant (p < 0.05). This result proved the previous hypothesis of the present inventors that the anti-PD-L1 antibody, an immune checkpoint inhibitor, increases the toxicity and side effects of the TLR7 / 8 agonist D18. In the ADC group, each time ADC HE-S2 was injected, it brought about a certain degree of body weight loss, but the range was significantly smaller than that of the D18 group (p < 0.05) and the combination administration group (p < 0.01). After administration, the body weight of the mice also quickly returned to normal, and finally, there was no significant difference in the body weight change of the mice in the ADC group compared with the control group. From these results, it was revealed that ADC HE-S2 also showed certain toxicity, but its toxicity was significantly lower than that of the administration of D18 alone or in combination, and the burden on the mice was small. It is considered that the main reason for the reduction of the toxicity of D18 is the small amount of D18 actually loaded on the ADC. Finally, it was concluded that ADC HE-S2 can show better tumor inhibitory activity than when used in combination by using less actual D18, and at the same time, significantly reduce the toxicity and side effects of the combination treatment. By the ADC strategy proposed by the present inventors, the minimum effective dose when using D18 was significantly reduced, and the therapeutic window of D18 was expanded. 2. Anti-tumor experiment of antibody-conjugated drug ADC10 with wild-type PD-L1 antibody in mice MC38 cells (1×106 cells / mouse) were inoculated subcutaneously into the right flank of mice. When the average tumor tissue volume of the mice reached 100 mm 3 After that, according to the final administration type, the mice were randomly divided into a PBS control group and an ADC10 group, 5 - 6 mice in each group. They were intraperitoneally injected with 10 mg / kg twice a week. The body weight change of the mice was measured daily (Figure 18A), and the tumor volume of the mice was measured every three days (Figure 18B). As shown in Figure 18B, the tumor inhibition rate of ADC was 70%.

[0180] The above are only preferred embodiments of the present invention and do not limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0181] The foregoing embodiments and methods described in the present invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0182] In the present invention, the steps of the method are listed in a certain order, but there is no limitation on the order of the steps of the method.

Claims

1. An antibody-drug conjugate having the following structure, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein: Herein, Ab is an antibody or an antigen-binding fragment thereof, D is a small molecule drug, L is a linking unit that links Ab and D, the D moiety in General Formula I has the following structure, Herein, L' is the following structure having, where R ａ and R ｂ are each independently selected from H, C １ to C ３ alkyl, or R ａ and R ｂ together with the carbon atom to which they are attached form C ３ to C ６ cycloalkylene, R １ is C １ to C ６ alkylene, or R １ is a single bond, or R １ is C １ to C ６ alkyleneoxy, X is 、 -NR ４ -,-NR ４ -(C １ ~C ６ alkylene)-NR ５ - (Ring A is a 4- to 10-membered nitrogen-containing heterocyclic ring) selected from, where R ４ and R ５ are, independently, H, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, amino acid residue, oligopeptide residue, -CF ３ , -CH ２ CF ３ , selected from, or R ４ and R ５ together with a nitrogen atom linked to both of them form a substituted or unsubstituted heterocyclyl, and the substituted or unsubstituted heterocyclyl is selected from, R ２ is -NHR ６ -OR ６ -SR ６ selected from, where R ６ is C １ to C ６ alkyl or C １ to C ６ alkoxyalkyl, R ３ is one or more independent substituents on the benzene ring, selected from H, C １ to C ６ alkyl, C １ to C ６ alkoxy, C １ to C ６ alkoxyalkyl, m is an integer from 0 to 4, the L moiety has the following structure and Y is a single bond, selected from, R Ｌ１ is or R Ｌ１ is -(CH ２ ) ｊ CO-, j is an integer from 0 to 6, R Ｌ２ is -(CH ２ ) ｉ OCO-, i is an integer from 0 to 6, or R Ｌ２ is an oligopeptide residue, and the oligopeptide residue is selected from, the antibody is an anti-PD-L1 antibody and has the heavy chain sequence shown in SEQ ID NO: 4 and the light chain sequence shown in SEQ ID NO: 9, n is an integer from 1 to 10, an antibody-drug conjugate.

2. The antibody-drug conjugate according to claim 1, wherein n is an integer from 1 to 6.

3. The antibody-drug conjugate according to claim 1, wherein n = 2 or 4.

4. The antibody-drug conjugate according to claim 1, wherein the linking site of the antibody to L is the free mercapto at the cysteine residue at position 226 of the amino acid sequence shown in SEQ ID NO:

9.

5. L' is selected from,

6. R ３ is one or more independent substituents on the benzene ring, independently selected from H, methyl, and methoxy, and the antibody-drug conjugate according to claim 1, characterized in that.

7. The antibody-drug conjugate according to claim 1, wherein m is 0 or 1.

8. X is and R ４ and R ５ are each independently selected from C １ -C ６ alkyl, and the antibody-drug conjugate according to claim 1, characterized in that.

9. R ４ and R ５ are both methyl, and the antibody-drug conjugate according to claim 8, characterized in that.

10. X is -NR ４ -, and R ４ is selected from H, C １ ~C ６ alkyl, and the antibody-drug conjugate according to claim 1, characterized in that.

11. R ４ The antibody-drug conjugate according to claim 10, wherein R is H or methyl.

12. X is -NR ４ -(C １ ~C ６ alkylene)-NR ５ -, and R ４ and R ５ are, independently, selected from C １ ~C ６ alkyl, and the antibody-drug conjugate according to claim 1, characterized in that.

13. R ４ and R ５ are both methyl, the antibody-drug conjugate according to claim 12, characterized in that.

14. X is selected from,

15. R ２ is selected from,

16. The antibody-drug conjugate according to claim 1, wherein the D moiety in General Formula I is selected from the following structures.

17. The antibody-drug conjugate according to claim 1, wherein the D moiety in General Formula I has the following structure.

18. The antibody-drug conjugate according to claim 1, wherein the L moiety has the following structure.

19. The antibody-drug conjugate according to claim 1, wherein the L moiety is selected from the following structures.

20. An antibody-drug conjugate selected from the following structures, wherein: Herein, Ab is an antibody or an antigen-binding fragment thereof, and the antibody has a heavy chain sequence shown in SEQ ID NO: 4 and a light chain sequence shown in SEQ ID NO: 9, or has a heavy chain sequence shown in SEQ ID NO: 4 and a light chain sequence shown in SEQ ID NO: 3, n is an integer from 1 to 10, An antibody-drug conjugate characterized by the above.

21. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 20 and a pharmaceutically acceptable adjuvant.

22. A medicament for preventing and / or treating a disease, comprising the antibody-drug conjugate according to any one of claims 1 to 20, wherein the disease is selected from respiratory diseases, immune diseases, viral diseases, and tumors.

23. The medicament according to claim 22, wherein the respiratory disease is selected from asthma, chronic obstructive pulmonary disease, and adult respiratory distress syndrome.

24. The medicament according to claim 22, wherein the immune disease is an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, Sjogren's syndrome, polymyositis, vasculitis, Wegener's granulomatosis, sarcoidosis, ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis, and Behcet's syndrome.

25. The medicament according to claim 22, wherein the viral disease is selected from influenza, SARS, COVID-19, hepatitis A, hepatitis B, hepatitis C, hepatitis D, AIDS, rabies, dengue fever, and Ebola hemorrhagic fever.

26. The medicament according to claim 22, wherein the tumor is selected from lymphoma, granuloma, medulloblastoma, retinoblastoma, liposarcoma, synovial sarcoma, neuroendocrine tumor, carcinoid tumor, gastrinoma, pancreatic islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancy, squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, Merkel cell carcinoma, esophageal cancer, biliary tract tumor, head and neck cancer, and hematological malignancy.

27. The medicament according to claim 26, wherein the lung cancer is selected from small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma.

Images