Antibody-drug conjugate and its preparation method and application

Abstract

The present invention discloses an antibody-drug conjugate and a preparation method and application thereof, in particular, a conjugate of an anti-PD-L1 antibody and a TLR7 and / or TLR8 agonist and a pharmaceutical composition, preparation method and application thereof. The present invention obtains a modified anti-PD-L1 antibody with a mutant cysteine ​​by gene editing, which basically retains the structure of the original antibody and can be used for the construction of an antibody-drug conjugate. Through anti-tumor experiments, it was found that the obtained antibody-drug conjugate has better activity, such as strong anti-tumor activity, can significantly improve the survival rate of tumor-bearing animals, and has significantly reduced toxicity, less burden on the body of experimental animals, greatly reduces the minimum effective dose of small molecule drugs when used alone, expands its therapeutic window, and is expected to be used in the development of therapeutic drugs for various diseases (such as tumors, viral diseases such as hepatitis B, etc.), with good application prospects and value.

Description

Technical Field

[0001] The present invention relates to the field of biochemical medicine technology, and specifically to an antibody-drug conjugate (ADC), in particular a conjugate of an antibody and an immunomodulator for immunotherapy, such as a conjugate of an anti-PD-L1 antibody and a TLR7 and / or TLR8 agonist, as well as pharmaceutical compositions, preparation methods, and uses thereof. Background Art

[0002] Toll-like receptors are a relatively conserved receptor family in evolution, including at least 13 members, 10 of which (TLR1-10) have been found in humans. TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 are expressed on the cell surface and quickly identify the products of bacterial metabolism. TLR3, TLR7, TLR8, and TLR9 are expressed inside the cell and are mainly used to monitor and identify viral nucleic acids. TLR3 recognizes double-stranded RNA, while TLR7 and TLR8 recognize single-stranded RNA. TLR9 recognizes unmethylated CG coenzyme I and regulates the response to bacterial DNA and certain viruses.

[0003] TLRs specifically recognize pathogen-associated molecular patterns (PAMPs) and play a crucial role in both innate and adaptive immunity, serving as a bridge between these two pathways. Specifically, upon recognizing virally bound single-stranded RNA or synthetic small purine compounds, TLR7 recruits specific adaptor proteins, activating a series of signaling cascades and initiating a high-level systemic adaptive immune response that kills virus-infected cells, ultimately eliminating the virus. TLR7 agonists have been clinically used to treat chronic viral infections such as hepatitis B and C. Furthermore, TLR7 agonists can be used as influenza vaccine adjuvants to induce more rapid and effective immune protection. In the anti-tumor context, TLR7 agonists not only directly stimulate pDCs to secrete IFN-α but also enhance their costimulatory and antigen presentation abilities. Activated pDCs promote the proliferation of CD4+ T cells, further activating CD8+ T cells to kill tumor cells. Therefore, the role of TLR7 agonists as immune adjuvants in tumor recognition and killing is increasingly recognized. Summary of the Invention

[0004] Based on their previous research and development results, the inventors of the present invention continued to further develop an antibody-drug conjugate (ADC) (particularly a conjugate of an antibody and an immunomodulator for immunotherapy, such as a conjugate of an anti-PD-L1 antibody and a TLR7 and / or TLR8 agonist), which has better therapeutic effects, significantly reduces toxic side effects, and expands the therapeutic window of immunomodulators (such as TLR7 and / or TLR8 agonists).

[0005] Specifically, the above-mentioned antibody drug conjugate has the following structure:

[0006]

[0007] in,

[0008] Ab is an antibody or an antigen-binding fragment thereof;

[0009] D is a small molecule drug;

[0010] L is a linker unit connecting Ab and D;

[0011] n is an integer from 1 to 100.

[0012] Specifically, the above-mentioned antibody is a monoclonal antibody.

[0013] Specifically, the antibody may be in the form of, for example, a chimeric antibody, a humanized antibody, a fully human antibody, or the like.

[0014] Specifically, the above-mentioned antigen-binding fragments include Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, single-chain Fv (scFv), disulfide-linked Fv (sdFv), CDR-containing fragments or isolated CDRs, etc.

[0015] Specifically, the above antibodies are reactive to antigens or epitopes thereof related to tumors, infectious microorganisms or autoimmune diseases; specifically, the antigens targeted by the above antibodies 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, Ga733, GROB, HMGB-1, hypoxia-inducible factor (HI F), 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, ganglioside, 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 receptor (R1 and R2), VEGFR, RANTES, T101, cancer stem cell antigen, complement factors C3, C3a, C3b, C5a, C5 and oncogene products, etc.

[0016] In one embodiment of the present invention, the above-mentioned antibody is an anti-PD-L1 antibody.

[0017] Specifically, the above antibodies contain reactive groups (e.g., sulfhydryl, amino, carboxyl, amide, halogen, ester, acyl halide, anhydride, epoxy, maleimide, aminooxy, azide, alkynyl, Wherein R can 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). It can inherently carry these reactive groups, or it can be obtained by mutation (such as site-directed mutagenesis) by mutating one or more amino acid residues of a known antibody to amino acids (including natural amino acids and non-natural amino acids) containing the same or different desired reactive groups. In one embodiment of the present invention, the above-mentioned antibody is mutated (for example, by Thiomab technology) to insert / substitute a cysteine ​​residue at a specific position in the antibody amino acid sequence.

[0018] In one embodiment of the present invention, the above-mentioned antibody is a modified anti-PD-L1 antibody, which is obtained by genetic engineering by mutating one or more amino acid residues of known anti-PD-L1 antibodies (for example, atezolizumab, durvalumab, avelumab, cemiplimab, KN035, CS1001, BGB-A333, KL-A167, SHR-1316 and STI-A1014, etc.) to cysteine.

[0019] Specifically, the above-mentioned antibodies comprise heavy chains and light chains;

[0020] wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4; and / or

[0021] The amino acid sequence of the light chain is as shown in SEQ ID NO:9, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.

[0022] More specifically, the antibody has a light chain sequence as shown in SEQ ID NO:9.

[0023] In one embodiment of the present invention, the antibody has a heavy chain sequence as shown in SEQ ID NO: 4 and a light chain sequence as shown in SEQ ID NO: 9.

[0024] Specifically, the above n (i.e., 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, 1-50, 1-40, 1-20, 1-10, 1-6, 1-4; in some embodiments of the present invention, n=2 or 4.

[0025] In one embodiment of the present invention, in the above-mentioned antibody-drug conjugate, the linking site between the antibody and L is the free thiol group on the cysteine ​​residue in the amino acid sequence of the antibody.

[0026] In one embodiment of the present invention, in the above-mentioned antibody-drug conjugate, the antibody has a heavy chain sequence as shown in SEQ ID NO: 4 and a light chain sequence as shown in SEQ ID NO: 9, and the connection site between the antibody and L is the free thiol group on the cysteine ​​residue at position 226 of the amino acid sequence shown in SEQ ID NO: 9; preferably, n=2.

[0027] In another embodiment of the present invention, in the above antibody-drug conjugate, the antibody has a heavy chain sequence as shown in SEQ ID NO: 4 and a light chain sequence as shown in SEQ ID NO: 3, and n is 1-10, particularly 1-6.

[0028] Specifically, the above-mentioned small molecule drugs can be immunomodulators, for example, Toll-like receptor agonists (TLR), specifically TLR7 and / or TLR8 agonists, such as the pyridopyrimidine derivatives and salts thereof described in patent application publication number WO2019 / 095455A1.

[0029] In one embodiment of the present invention, the D portion in Formula I has the following structure:

[0030]

[0031] in,

[0032] L' is a linking group selected from: a single bond or a C1-C6 alkylene group, a C1-C6 alkenylene group, a C3-C6 cycloalkylene group, wherein said groups may be optionally substituted with a C1-C4 alkyl group;

[0033] R1 is selected from the group consisting of: a single bond, C1-C6 alkylene, C1-C6 alkenylene, C1-C6 alkyleneoxy, wherein said groups are optionally substituted with C1-C4 alkyl;

[0034] X is selected from: -NR4-, -O-, -S-, single bond, -OCO-, -COO-, -NR4-(C1-C6 alkylene)-NR5-, heterocyclic group (especially nitrogen-containing heterocyclic group), wherein R4 and R5 are independently selected from: H, C1-C6 alkyl, C1-C6 alkoxy, amino acid residue, oligopeptide residue, haloalkyl, carboxyl substituted alkyl, ester substituted alkyl, wherein a is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), R8 and R9 are independently selected from: H, C1-C6 alkyl, or R8 and R9 together with the atoms between them form Or R4 and R5 together with the nitrogen atom to which they are attached form a heterocyclic group;

[0035] R2 is selected from: -NR6R7, -OR6, -SR6, wherein 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 attached form a heterocyclic group, or R6 together with the oxygen atom to which it is attached form a heterocyclic group, or R6 together with the sulfur atom to which it is attached form a heterocyclic group;

[0036] R3 is one or more independent substituents on the benzene ring selected from: H, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxyalkyl;

[0037] m is an integer of 0-4 (e.g., 0, 1, 2, 3, 4).

[0038] Specifically, Formula II has the following structure:

[0039]

[0040] In one embodiment of the present invention, the above L' is a C1-C6 alkylene group, such as a C1-C3 alkylene group.

[0041] In one embodiment of the present invention, the above L' has the following structure: Among them, R a and R b 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 it is attached, it forms a C3-C6 cycloalkylene group (eg, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene).

[0042] Specifically, L' may be selected from: -CH2-,

[0043] In one embodiment of the present invention, the above R1 is C1-C6 alkylene, such as C1-C3 alkylene, such as -CH2-.

[0044] In another embodiment of the present invention, the above R1 is a single bond.

[0045] In one embodiment of the present invention, the above-mentioned R1 is a C1-C6 alkyleneoxy group, such as a C1-C3 alkyleneoxy group, such as -CH2O-, -CH2CH2O-, -CH2CH2CH2O-.

[0046] Specifically, R3 is one or more independent substituents on the benzene ring selected from: H, methyl, methoxy.

[0047] In an embodiment of the present invention, m is 0 or 1.

[0048] Specifically, a is an integer of 1-5, for example, 1, 2, 3, 4, 5.

[0049] Specifically, R8 and R9 are independently selected from: H, methyl, ethyl, n-propyl, isopropyl.

[0050] Specifically, the above R4 and R5 are independently selected from: H, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, amino acid residue, oligopeptide residue, -CF3, -CH2CF3, Alternatively, R4 and R5 together with the nitrogen atom to which they are attached form a substituted or unsubstituted heterocyclic group.

[0051] Specifically, the above-mentioned substituted or unsubstituted heterocyclic group can be selected from:

[0052] Specifically, the amino acid residues and the amino acid residues in the oligopeptide are independently selected from one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine residues. In one embodiment of the present invention, the amino acid residue is a leucine residue. In one embodiment of the present invention, the oligopeptide residue is -asparagine-alanine-alanine-leucine-.

[0053] In some embodiments of the present invention, X is R4 and R5 are independently selected from C1-C6 alkyl groups, for example, R4 and R5 are both methyl groups.

[0054] In 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.

[0055] In 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, R4 and R5 are both methyl.

[0056] In other embodiments of the present invention, X is a heterocyclic group, especially a nitrogen-containing heterocyclic group, for example Ring A is a 4- to 10-membered nitrogen-containing heterocyclic ring, which can be a monocyclic, bicyclic, or tricyclic ring (including fused rings, spiro rings, and bridged rings); specifically, X can be

[0057] In one embodiment of the present invention, R2 is selected from: -NHR6, -OR6, -SR6, wherein R6 is C1-C6 alkyl or C1-C6 alkoxyalkyl, such as C1-C4 alkyl, such as butyl, especially n-butyl, such as C1-C4 alkoxyalkyl, such as methoxyethyl. In some embodiments of the present invention, R2 is selected from:

[0058] In another embodiment of the present invention, R2 is selected from: -NR6R7, wherein R6 and R7 together with the nitrogen atom to which they are attached form a heterocyclic group, for example,

[0059] Specifically, the D portion in Formula I can be selected from the following structures:

[0060]

[0061]

[0062]

[0063] In one embodiment of the present invention, the D portion in Formula I has the following structure:

[0064]

[0065] In one embodiment of the present invention, the conjugate has the following structure:

[0066]

[0067] Among them, Ab, n, m, R1, R2, R3, R4, R5, R a 、R b The present invention has the above definition.

[0068] Specifically, the linking unit in the above-mentioned conjugate can be in the form of a chemically unstable linking unit (such as containing a hydrazone or a disulfide group), an enzyme-catalyzed linking unit (such as containing a peptide residue or a carbonate residue that is unstable to esterase), etc.

[0069] Specifically, the linking unit L connecting Ab and D comprises a linking group Y, such as a single bond, -S-、-CO-、-NH-、-CONH-、 (aminooxy); In some embodiments of the present invention, Y is -S- or

[0070] Furthermore, L further comprises a group Q, wherein Q is a divalent saturated or unsaturated linear or branched C1-50 hydrocarbon chain, wherein 0-6 methylene units are independently substituted by: -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-, An amino acid residue or oligopeptide residue, wherein k is selected from an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), and each -Cy- is independently an optionally substituted bivalent ring selected from the following: an arylene group, a cycloalkylene group, and a heterocyclylene group; R 10 Selected from: H, -OH, C1-C6 alkyl, C3-C6 cycloalkyl, heterocycloalkyl.

[0071] Specifically, each -Cy- is independently an optionally substituted bivalent ring selected from: phenylene, bicyclic arylene, monocyclic cycloalkylene, bicyclic cycloalkylene, monocyclic heteroarylene, bicyclic heteroarylene, monocyclic heterocycloalkylene, bicyclic heterocycloalkylene; in particular, phenylene, monocyclic cycloalkylene, monocyclic heteroarylene, monocyclic heterocycloalkylene.

[0072] More specifically, each -Cy- can be independently selected from the following: Among them, 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), a residue of a monosaccharide or its derivative, R 12 is selected from the group consisting of: 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.

[0073] Specifically, the monosaccharide or its derivative can be selected from: arabinose, xylose, ribose, glucose, mannose, galactose, fructose, glucuronic acid, galacturonic acid. In some embodiments of the present invention, the residue of the monosaccharide or its derivative is a galacturonic acid residue, for example

[0074] In some embodiments of the invention, each -Cy- is independently selected from the following:

[0075] Specifically, R 10 Can be selected from: H, C1-C6 alkyl; In some embodiments of the present invention, R 10 is H or methyl.

[0076] Specifically, in the above definition of Q, the amino acid residues and the amino acid residues in the oligopeptide residues are independently selected from: one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, citrulline, and ornithine residues, in particular selected from: one or more of valine, citrulline, alanine, asparagine, aspartic acid, glutamic acid, proline, and glycine residues; in particular, the amino acid residues and the amino acid residues in the oligopeptide residues are residues of L-type amino acids.

[0077] Specifically, in the above definition of Q, the oligopeptide residue consists of 2-10 amino acid residues, in particular consists of 2-6 amino acid residues, for example consists of 2, 3, 4 or 5 amino acid residues.

[0078] Specifically, in the above definition of Q, the oligopeptide residue is selected from: valine-citrulline residue, alanine-alanine-asparagine residue, aspartic acid-valine residue, glutamic acid-valine residue, glycine-proline residue, etc.

[0079] In some embodiments of the invention, the oligopeptide residue is

[0080] In one embodiment of the present invention, the L portion in Formula I has the following structure:

[0081]

[0082] in,

[0083] Y is a linking group to the active group of Ab (such as sulfhydryl, amino, carboxyl, etc., especially sulfhydryl);

[0084] R L1 、R L3 and R L4 Independently selected from: -(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 SS-, -(CH2) j N(R L9 )-NH=、 a combination of one or more of cycloalkylene and arylene, wherein j is an integer of 0-10;

[0085] R L2 Selected from: single bond, -(CH2)i OCO-, -(CH2) i OCOO-、-(CH2) i NH-COO-, amino acid residue, oligopeptide residue, wherein i is an integer from 0 to 10;

[0086] R L5 、R L6 、R L7 and R L8 Independently selected from: 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 , where t is 0, 1 or 2;

[0087] Each R L9 and R L10 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.

[0088] Specifically, Y is selected from: a single bond, -S-、-CO-、-NH-、-CONH-、 (aminooxy). In some embodiments of the present invention, Y is -S- or

[0089] Specifically, the above R L2 In the definition of , the amino acid residues and the amino acid residues in the oligopeptide residues are independently selected from one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, citrulline, and ornithine residues. In particular, the amino acid residues and the amino acid residues in the oligopeptide residues are residues of L-amino acids.

[0090] Specifically, the above R L2 In the definition of , the oligopeptide residue consists of 2-10 amino acid residues, in particular consists of 2-6 amino acid residues, such as consists of 2, 3, 4 or 5 amino acid residues.

[0091] Specifically, the above R L2In the definition of , the oligopeptide residue can be selected from: valine-citrulline residue, alanine-alanine-asparagine residue, aspartic acid-valine residue, glutamic acid-valine residue, glycine-proline residue, etc.

[0092] In one embodiment of the present invention, R L2 -(CH2) i OCO-, i is an integer from 0 to 6, especially i is 1.

[0093] In one embodiment of the present invention, R L2 -(CH2) i , i is an integer from 1 to 10, in particular i is 2.

[0094] In another embodiment of the present invention, R L2 is an oligopeptide residue selected from the group consisting of: valine-citrulline residues, alanine-alanine-asparagine residues, aspartic acid-valine residues, glutamic acid-valine residues, etc., in particular

[0095] Specifically, R L1 、R L3 and R L4 Independently selected from: -(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 A combination of one or more of CONH-, j is an integer from 0 to 10.

[0096] Specifically, each j mentioned above can independently be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0097] In one embodiment of the present invention, R L1 for

[0098] In another embodiment of the present invention, R L1 -(CH2) j CO-, j is an integer from 0 to 6, especially j is 5.

[0099] In one embodiment of the present invention, R L3 -(CH2) j NH-, j is an integer from 0 to 6, especially j is 0.

[0100] In one embodiment of the present invention, R L4 -(CH2) j -, j is an integer from 0 to 5, in particular j is 1.

[0101] Specifically, R L5 、R L6 、R L7 and R L8 Independently selected from: H, C1-6 alkyl, C1-6 haloalkyl, halogen, nitro, cyano, -OR L9 ; R L8 Selected from: H, C1-6 alkyl; More specifically, R L5 、R L6 、R L7 and R L8 Independently selected from: H, halogen.

[0102] In one embodiment of the present invention, R L5 For H.

[0103] In one embodiment of the present invention, R L6 For H.

[0104] In one embodiment of the present invention, R L7 For H.

[0105] In one embodiment of the present invention, R L8 For H.

[0106] In one embodiment of the present invention, the L portion has the following structure:

[0107]

[0108] Among them, Y, R L1 、R L2 It has the corresponding definitions of the present invention mentioned above.

[0109] In one embodiment of the present invention, the L portion may have the following structure:

[0110]

[0111] In another embodiment of the present invention, the L portion may also have the following structure:

[0112]

[0113] Among them, R L2 is an oligopeptide residue having the above definition of the present invention.

[0114] Specifically, the above L part has the following structure:

[0115]

[0116]

[0117]

[0118] In other embodiments of the present invention, the L portion has the following structure:

[0119]

[0120]

[0121] k1 and k2 are independently selected from integers between 1-10.

[0122] In one embodiment of the present invention, the conjugate has the following structure:

[0123]

[0124] Wherein, Ab, n and D have the corresponding definitions of the present invention mentioned above.

[0125] In some embodiments of the present invention, the conjugate has the following structure:

[0126]

[0127]

[0128]

[0129]

[0130] Wherein, Ab and n have the corresponding definitions of the present invention as described above.

[0131] The present invention also provides a stereoisomer of the conjugate (as shown above) or a mixture thereof.

[0132] The present invention also provides a pharmaceutically acceptable salt, solvate and prodrug of the conjugate.

[0133] Specifically, the above-mentioned pharmaceutically acceptable salts may include organic salts or inorganic salts, for example, one or more of 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, aspartate, glutamate, pantothenate and ascorbate.

[0134] The present invention also provides a method for preparing the above-mentioned conjugate, which may include the step of conjugating a conjugate (LD) of a small molecule drug and a linker unit with an antibody.

[0135] Specifically, before the coupling step, the above preparation method further includes the steps of reducing and oxidizing the disulfide bonds of the antibody.

[0136] Specifically, the above preparation method comprises the following steps:

[0137] (1) incubating the antibody with a disulfide bond reducing agent;

[0138] (2) incubating the antibody obtained in step (1) with an oxidizing agent;

[0139] (3) Incubate the antibody obtained in step (2) with the above-mentioned conjugate LD.

[0140] Specifically, step (1) may further comprise removing excess disulfide bond reducing agent (eg, by ultrafiltration).

[0141] Specifically, the disulfide bond reducing agent in step (1) can be tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), β-mercaptoethanol, etc.

[0142] Specifically, in step (1), the equivalent ratio of the antibody to the disulfide bond reducing agent is 1:8-15 (specifically 1:8, 1:9, 1:10, 1:11, 1:12).

[0143] Specifically, the incubation temperature in step (1) is 35-40°C (specifically 35, 36, 37, 38, 39, 40°C); in one embodiment of the present invention, the incubation temperature is 37°C.

[0144] Specifically, the incubation time in step (1) is 0.5-6 hours (specifically 0.5, 1, 2, 3, 4, 5, 6 hours); in one embodiment of the present invention, the incubation time is 3 hours.

[0145] Specifically, step (2) may further comprise removing excess oxidant (eg, by ultrafiltration).

[0146] Specifically, the oxidant in step (2) can be dehydroascorbic acid (DHAA), Cu(II), etc.

[0147] Specifically, in step (2), the equivalent ratio of the antibody to the oxidant is 1:40-60 (specifically 1:40, 1:45, 1:50, 1:55, 1:60).

[0148] Specifically, the incubation temperature in step (2) is 20-30°C (specifically 20, 22, 24, 25, 26, 28, 30°C); in one embodiment of the present invention, the incubation temperature is room temperature.

[0149] Specifically, the incubation time in step (2) is 1-6 hours (specifically 1, 2, 3, 4, 5, 6 hours); in one embodiment of the present invention, the incubation time is 3 hours.

[0150] Specifically, in step (3), the equivalent ratio of the antibody to the small molecule LD is 1:10-20 (specifically 1:10, 1:12, 1:14, 1:15, 1:16, 1:18, 1:20).

[0151] Specifically, the incubation temperature in step (3) is 20-30°C (specifically 20, 22, 24, 25, 26, 28, 30°C); in one embodiment of the present invention, the incubation temperature is room temperature.

[0152] Specifically, the incubation time in step (3) is 6-48 hours (specifically 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.

[0153] In one embodiment of the present invention, the above preparation method further comprises the step of preparing a conjugate (LD) of a small molecule drug and a linker unit.

[0154] The present invention also provides a modified anti-PD-L1 antibody, which is obtained by mutating one or more amino acid residues of known anti-PD-L1 antibodies (e.g., atezolizumab, durvalumab, avelumab, cemiplimab, KN035, CS1001, BGB-A333, KL-A167, SHR-1316 and STI-A1014, etc.) to cysteine ​​by genetic engineering means (e.g., Thiomab technology), for example, by mutating amino acids in the heavy chain and / or light chain of avelumab to cysteine.

[0155] Specifically, the above-mentioned antibody comprises a heavy chain and a light chain;

[0156] wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4; and / or

[0157] The amino acid sequence of the light chain is as shown in SEQ ID NO:9, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.

[0158] Specifically, the above antibody has a light chain sequence as shown in SEQ ID NO:9.

[0159] In one embodiment of the present invention, the antibody has a heavy chain sequence as shown in SEQ ID NO: 4 and a light chain sequence as shown in SEQ ID NO: 9.

[0160] The present invention also provides a use of the modified anti-PD-L1 antibody in the preparation of an antibody-drug conjugate (ADC).

[0161] The present invention also provides a small molecule compound and a pharmaceutically acceptable salt, stereoisomer, solvate, and prodrug thereof, wherein the compound has the following structure:

[0162]

[0163] Wherein, m, X, L', R1, R2, R3, and Q have the corresponding definitions of the present invention above,

[0164] Z is an active group.

[0165] In one embodiment of the present invention, the small molecule compound has the following structure:

[0166]

[0167] Among them, m, X, L', R1, R2, R3, R L1 、R L2 、R L3 、R L4 、R L5 、R L6 、R L7 、R L8 With the corresponding definitions of the present invention, Z is an active group.

[0168] Specifically, Z is a reactive linking group with an amino acid active group (such as a sulfhydryl group, an amino group, a carboxyl group, etc., especially a sulfhydryl group); specifically, for example, a sulfhydryl reactive group can be a maleimide group, a carboxyl group, an amide group, a halogen group, a disulfide group, etc.; an amino reactive group can be an ester group, an acyl halide group, an acid anhydride group, a carboxyl group, an epoxy group, etc.; a carboxylamino reactive group can be a hydroxyl group, an amino group, a halogen group, an acyl halide group, etc.

[0169] In some embodiments of the present invention, Z is

[0170] Specifically, the compound represented by formula VII has the following structure:

[0171]

[0172] Among them, Z, R L1 、R L2 、R L3 、R L4 、R1、R2、R3、R4、R5、R a 、R b , m have the corresponding definitions as defined above in the present invention.

[0173] More specifically, the compound represented by formula VII has the following structure:

[0174]

[0175] Among them, Z, R L1 、R L2 、R1、R2、R3、R4、R5、R a 、R b , m have the corresponding definitions as defined above in the present invention.

[0176] More specifically, the compound represented by formula VII has the following structure:

[0177]

[0178] Among them, Z, R L1 、R L2 It has the corresponding definitions of the present invention mentioned above.

[0179] In some embodiments of the present invention, the compound represented by formula VII has the following structure:

[0180]

[0181]

[0182]

[0183]

[0184] The present invention also provides a compound and a pharmaceutically acceptable salt and stereoisomer thereof, which can be used as a linker unit portion of an antibody drug conjugate, and has the following structure:

[0185]

[0186] Among them, Z, R L1 、R L2 、R L3 、R L4 、R L5 、R L6 、R L7 、R L8 With the above corresponding definitions of the present invention,

[0187] Z' is a reactive group.

[0188] Specifically, Z' can be a halogen (eg, chlorine), a carboxyl group, an ester group, a hydroxyl group, an epoxy group, an amine group, or the like.

[0189] Specifically, the compound represented by formula IX has the following structure:

[0190]

[0191] Among them, Z, R L1 、R L2 , Z' have the corresponding definitions in the present invention.

[0192] In some embodiments of the present invention, the compound represented by Formula IX has the following structure:

[0193]

[0194]

[0195] In some embodiments of the present invention, the aforementioned Z' is a halogen (eg, chlorine).

[0196] In other embodiments of the present invention, the above Z' is a carbonate group (eg ).

[0197] The present invention also provides a use of the compound represented by the above formula IX in the preparation of an antibody-drug conjugate (ADC).

[0198] The present invention also provides a pharmaceutical composition comprising the above-mentioned conjugate and pharmaceutically acceptable excipients.

[0199] Specifically, the pharmaceutically acceptable excipients mentioned above are conventional pharmaceutical excipients in the pharmaceutical field, such as diluents and excipients such as water; fillers such as starch and sucrose; binders such as cellulose derivatives, alginates, gelatin, and polyvinyl pyrrolidone; humectants such as glycerol; disintegrants such as agar, calcium carbonate, and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium and magnesium stearate, and polyethylene glycol. In addition, other excipients such as flavoring agents, sweeteners, and stabilizers may also be added to the pharmaceutical composition.

[0200] Specifically, depending on the desired route of administration, the pharmaceutical composition will contain about 1 to about 99% by weight (specifically, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%) of the conjugate of the present invention, with the remainder being suitable excipients.

[0201] Particularly, the above-mentioned pharmaceutical composition can be used for administration by any route of administration, and can be oral or parenteral administration, such as administration through the lungs, nose, rectum and / or intravenous injection. Therefore, the preparation according to the present invention is suitable for topical or systemic administration, particularly for skin, subcutaneous, intramuscular, intra-articular, intraperitoneal, lung, oral cavity, sublingual, nose, percutaneous puncture, vagina, oral or parenteral administration. The preferred form for rectal administration is a suppository.

[0202] The dosage forms suitable for oral administration include tablets, pills, chewable gums, capsules, granules, drops or syrups, etc. The dosage forms suitable for parenteral administration include solutions, suspensions, reconstituted dry preparations or sprays, etc.

[0203] The compositions of the present invention can be formulated into deposits or patches in dissolved form for transdermal administration. Skin applications include ointments, gels, creams, lotions, suspensions or emulsions.

[0204] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field, for example, by mixing the active ingredient with one or more excipients and then preparing the mixture into the desired dosage form.

[0205] Specifically, in the above-mentioned pharmaceutical composition, the above-mentioned conjugate of the present invention can be used as the sole active ingredient, or can be used in combination with one or more other active ingredients for the same indication, wherein the above-mentioned conjugate of the present invention and the other active ingredients can be formulated for simultaneous, separate or sequential administration.

[0206] The present invention also provides a composition comprising the antibody of the present invention and a small molecule drug as active ingredients.

[0207] Specifically, in the above-mentioned composition, the antibody is an anti-PD-L1 antibody, for example, atezolizumab, durvalumab, avelumab, cemiplimab, KN035, CS1001, BGB-A333, KL-A167, SHR-1316 and STI-A1014, etc., as well as a modified anti-PD-L1 antibody obtained by genetic engineering means by mutating one or more amino acid residues of a known anti-PD-L1 antibody (as described above) to cysteine, for example, mutating the amino acid in the heavy chain and / or light chain of avelumab to cysteine.

[0208] Specifically, the above-mentioned antibody comprises a heavy chain and a light chain;

[0209] wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4; and / or

[0210] The amino acid sequence of the light chain is as shown in SEQ ID NO:9, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.

[0211] Specifically, the above antibody has a light chain sequence as shown in SEQ ID NO: 9

[0212] In one embodiment of the present invention, the antibody has a heavy chain sequence as shown in SEQ ID NO: 4 and a light chain sequence as shown in SEQ ID NO: 9.

[0213] Specifically, in the above-mentioned composition, the small molecule drug can be an immunomodulator, for example, a Toll-like receptor agonist (TLR), specifically a TLR7 and / or TLR8 agonist, such as the pyridopyrimidine derivatives and salts thereof described in patent application publication number WO2019 / 095455A1.

[0214] Specifically, the small molecule drug in the above composition has the following structure:

[0215]

[0216] Wherein, R1, R2, R3, m, and L' have the corresponding definitions of the present invention;

[0217] X' is selected from: H, halogen, -NR4R5, -N + R4R5, -OR4, -SR4, azido, phosphonic acid, diethyl phosphonate, haloalkyl, carboxyl, ester, -CN; wherein, 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 connected form a heterocyclic group.

[0218] More specifically, the small molecule drug in the above composition can be selected from the following structures:

[0219]

[0220]

[0221]

[0222]

[0223] in particular:

[0224]

[0225] In one embodiment of the present invention, in the above composition, the antibody is Avelumab, and the small molecule drug has the following structure:

[0226] The present invention also provides use of the above-mentioned conjugate or its pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, the above-mentioned antibody of the present invention, the compound represented by formula VII, and the composition in the preparation of drugs for preventing and / or treating diseases.

[0227] Specifically, the above-mentioned disease may be a disease related to PD-L1 expression, or a disease related to TLR7 and / or TLR8 activity.

[0228] Specifically, the above-mentioned diseases may be one or more of respiratory diseases, immune diseases, viral diseases, and tumors.

[0229] Specifically, the above-mentioned respiratory diseases include but are not limited to: asthma, chronic obstructive pulmonary disease, and adult respiratory distress syndrome.

[0230] 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, Behçet's syndrome, etc.

[0231] 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 (external genital herpes), VZV (varicella), EBV (Epstein-Barr virus), CMV (cytomegalovirus)), Poxviridae (such as smallpox virus, cowpox virus), Papovaviridae (such as papillomavirus), Parvoviridae (such as B19 virus), Hepadnaviridae (such as hepatitis B virus), Polyomaviridae (such as polyomavirus), Reoviridae (such as variola virus, cowpox virus), Ornaviridae (such as reovirus, rotavirus), Picornaviridae (such as enterovirus, foot-and-mouth disease virus), Caliciviridae (such as Norwalk virus, hepatitis E virus), Togaviridae (such as rubella virus), Arenaviridae (such as lymphocytic choriomeningitis virus), 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 viruses (such as influenza A virus, influenza B virus, influenza C virus, etc.)), Paramyxoviridae (such as human parainfluenza virus (HPV) type 1, HPV type 2, HPV type 3, HPV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, Newcastle disease virus, etc.), Bunyaviridae (such as California encephalitis virus, Hantavirus), Rhabdoviridae (such as rabies virus), Filoviridae (such as Ebola virus, Marburg virus), Coronaviridae (such as HCoV -229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, etc.), Astroviridae (such as astrovirus), Bornaviridae (such as Borna virus); more specifically, the above-mentioned viral diseases can be influenza, SARS, COVID-19, viral hepatitis (such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, etc.), AIDS, rabies, dengue fever, Ebola virus disease, etc.

[0232] Specifically, the above tumors are malignant tumors, including but not limited to: lymphoma, blastoma, medulloblastoma, retinoblastoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumor, carcinoid tumor, gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancies, squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer (small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung cancer, squamous cell lung cancer), abdominal pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, ovarian 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, liver cancer, anal cancer, penile cancer, Merkel cell carcinoma, esophageal cancer, biliary tract tumors, head and neck cancer, and hematological malignancies; in particular, colon cancer, bladder cancer, melanoma, meningioma, lung cancer, and pancreatic cancer.

[0233] The present invention also provides a method for preventing and / or treating a disease, comprising the step of administering a therapeutically effective amount of the above-mentioned conjugate of the present invention or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer thereof, the above-mentioned antibody of the present invention, the compound represented by formula VII, or the combination thereof to a system or individual in need thereof.

[0234] Specifically, the disease in the above method has the above definition of the present invention.

[0235] The present invention also provides a method for enhancing the body's positive immune response, comprising the step of administering a therapeutically effective amount of the above-mentioned conjugate of the present invention or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer thereof, the above-mentioned antibody of the present invention, the compound represented by formula VII, or the combination thereof to a system or individual in need thereof.

[0236] The present invention also provides a method for enhancing the effect of chemotherapy, comprising the step of administering a therapeutically effective amount of the above-mentioned conjugate of the present invention or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer thereof, the above-mentioned antibody of the present invention, the compound represented by formula VII, or the combination thereof to a system or individual in need thereof.

[0237] The present invention also provides a method for improving the effect of immunotherapy, comprising the step of administering a therapeutically effective amount of the above-mentioned conjugate of the present invention or a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer thereof, the above-mentioned antibody of the present invention, the compound represented by formula VII, or the combination thereof to a system or individual in need.

[0238] Specifically, the therapeutically effective amount may vary depending on factors such as the route of administration, the patient's age, weight, gender, the type and severity of the disease being treated, etc., and the present invention does not impose any specific limitations on this.

[0239] The present invention obtains a modified anti-PD-L1 antibody with two mutated cysteines through gene editing, which basically retains the structure of the original antibody and is used for the construction of ADC. Experimental verification shows that the modified antibody and the resulting ADC maintain the antigen recognition affinity of the original anti-PD-L1 antibody, can be internalized into the cell by binding to cell surface PD-L1, and have good selectivity. Anti-tumor experiments have shown that the resulting ADC has strong anti-tumor activity, can significantly improve the survival rate of tumor-bearing animals, and has significantly lower toxicity than the toxicity of small molecule drugs administered alone and in combination with antibodies, and has less burden on the body of experimental animals. The ADC described in the present invention greatly reduces the minimum effective dose of small molecule drugs when used alone, expands its therapeutic window, and is expected to be used in the development of therapeutic drugs for a variety of diseases (such as tumors, viral diseases such as hepatitis B, etc.), and has good application prospects and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0240] Figure 1 Shown are the sequencing results of the plasmid αPD-L1 THIOMAB LC-V205C, where A. The sequencing sequence of αPD-L1 THIOMAB LC-V205C (top) is aligned with the sequence of the template light chain 159-pFuse-αPDL1-LC plasmid (bottom). The GTG site on the template plasmid is mutated to TGC; B. The amino acid encoded at the mutation site of αPD-L1 THIOMAB LC-V205C is cysteine ​​(Cys); C. The amino acid encoded at the mutation site of the template plasmid is valine (Val).

[0241] Figure 2 Shown are the results of anti-PD-L1 antibody expression analysis identified by reducing and non-reducing SDS PAGE.

[0242] Figure 3 Shown are the expression results of anti-PD-L1 THIOMAB identified by reducing and non-reducing SDS PAGE analysis.

[0243] Figure 4 Shown are the mass spectrometry analysis results of THIO-S2.

[0244] Figure 5 Shown is the conjugation route for ADC HE-S2.

[0245] Figure 6 Shown are the mass spectrometry detection results of ADC HE-S2.

[0246] Figure 7 Shown is the conjugation route for ADC VC.

[0247] Figure 8 Shown is the conjugation route of the ADC Legumain.

[0248] Figure 9 Shown are the mass spectrometry detection results of ADC VC and ADC Legumain.

[0249] Figure 10 Shown are the results of characterization of the antibody-antigen affinity of ADC HE-S2 and anti-PD-L1 THIOMAB. MC38 cells were treated with anti-PD-L1 antibody, anti-PD-L1 THIOMAB, and ADC HE-S2, then stained with FITC-conjugated goat anti-human secondary antibody. The FITC signal intensity on the MC38 surface was analyzed by FACS. A. The histograms (from top to bottom) represent the fluorescence signals measured for the negative control (NC), secondary antibody, anti-PD-L1 antibody, anti-PD-L1 THIOMAB, and ADC HE-S2, respectively. B. The mean fluorescence intensity (MFI) measured on MC38 cells after treatment with different antibody drugs. NC group: negative control with only isotype antibody added, without secondary antibody staining; secondary antibody group: control group with isotype antibody added and stained with fluorescent secondary antibody. Significant differences between groups were determined using the Student's t test, and **** indicates p < 0.0001.

[0250] Figure 11 The results show the internalization of anti-PD-L1 THIOMAB into cancer cells. MC38 and B16 cells were treated with pHrodo TM After 24 hours of treatment with red dye-labeled anti-PD-L1 THIOMAB, A. The living cell system acquires high-definition phase images, red fluorescence images, and combined images of cells; B. The histogram of total red object integrated intensity obtained by software analysis was plotted with the vertical axis taking the logarithm of 10. The Student's t test was used to compare the significant differences between the groups. **** Indicates p < 0.0001.

[0251] Figure 12 The results show the results of FACS analysis of PD-L1 levels on the surface of Cd274 knockout and wild-type DC 2.4 cells. KO: Cd274 knockout DC 2.4 cells, WT: wild-type DC 2.4 cells. The Student's t test was used to compare the significant differences between the groups. ** Indicates P < 0.01.

[0252] Figure 13The results are shown as preliminary experiments on the internalization of anti-PD-L1 THIOMAB in DC cells. PD-L1 positive wild-type DC 2.4 cells and PD-L1 negative CD274 knockout DC 2.4 cells were cultured in 10% FBS medium. After the cells were stable, pHrodo was added. TM Red dye-labeled anti-PD-L1 THIOMAB was incubated overnight, and the intensity of intracellular red fluorescence was analyzed by FACS to characterize the internalization level of THIOMAB.

[0253] Figure 14 The results show the internalization capacity of PD-L1 / anti-PD-L1 antibody complex in DC cells. TM The red dye labeled anti-PD-L1 THIOMAB in the wild-type DC 2.4 cell line and the CD274 knockout DC 2.4 cell line shows the overall red fluorescence object integrated intensity-time change curve. The cells were incubated at 4 ° C for 30 minutes to temporarily inhibit the internalization process of the cell-receptor. After adding the labeled anti-PD-L1 antibody, it was incubated at 4 ° C for 30 minutes to allow the antigen and antibody to fully bind. Then, the cells were incubated at 4 ° C for 30 minutes. Live cell system analysis of antibody internalization. A. Cells were cultured in medium containing 10% FBS. B. Cells were cultured in medium containing 10% mouse serum. C. After 6 hours, High-definition phase images, red fluorescence images, and combined images of DC 2.4 cells collected by the live cell system. The Student's t test was used to compare the significant differences between the groups. **** Indicates P < 0.0001.

[0254] Figure 15 Shown are the experimental results of validating the anti-tumor activity of ADC HE-S2 using a mouse tumor model. A. Inhibitory activity of ADC HE-S2 treatment, anti-PD-L1 antibody alone, D18 alone, anti-PD-L1 THIOMAB alone, and anti-PD-L1 antibody / D18 combination therapy on the growth of MC38 tumors in mice. B. Overall animal survival study. The vertical axis of the curve represents the percentage of tumor-bearing mice that are still alive after different days, and the horizontal axis represents the number of experimental days. C. Inhibitory activity of ADC HE-S2 treatment, anti-PD-L1 antibody alone, D18 alone, anti-PD-L1 THIOMAB alone, and anti-PD-L1 antibody / D18 combination therapy on the growth of MC38 tumors in mice. The dosage and administration time of each group of mice are shown in Table 1. The Student's t test was used to compare the significant differences between the groups. *** Indicates P < 0.001.

[0255] Figure 16 The results show that gradient doses of D18 combined with anti-PD-L1 antibodies have different tumor inhibitory abilities. D18 was administered once a week, and anti-PD-L1 antibodies or IgG were administered twice a week. When antibody drugs were administered, each mouse in each group was administered 150 μg. According to the combination groups, the anti-PD-L1+D18 (25 μg) group received an injection of D18 25 μg per mouse each time; the anti-PD-L1+D18 (2.5 μg) group received an injection of D18 2.5 μg per mouse each time; the anti-PD-L1+D18 (0.25 μg) group received an injection of D18 0.25 μg per mouse each time. The Student's t test was used to compare the significant differences between the groups. **** Indicates P < 0.0001.

[0256] Figure 17 The results show the effect of ADC HE-S2 on mouse body weight. Mice were treated with ADC HE-S2, anti-PD-L1 antibody + D18 combination, anti-PD-L1 antibody alone, D18 alone, and isotype IgG, and their body weight changes were recorded. The administration time of each drug is marked with arrows. The Student's t test was used to compare the significant differences between the groups. * Indicates P < 0.05, ** Indicates P < 0.01, and ns indicates no significant difference.

[0257] Figure 18 Shown are the results of ADC10's anti-tumor experiment in mice. DETAILED DESCRIPTION

[0258] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0259] In the context of the present invention, the following terms have the meanings detailed below.

[0260] "Alkyl" refers to a straight or branched hydrocarbon chain radical that does not contain unsaturated bonds and is connected to the rest of the molecule by a single bond. Typical alkyl groups contain 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, etc. If an alkyl group is substituted with a cycloalkyl group, it is a "cycloalkylalkyl" radical, such as cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, etc. If an alkyl group is substituted with an aryl group, it is a "aralkyl" radical, such as benzyl, benzhydryl, or phenethyl. If an alkyl group is substituted with a heterocyclyl group, it is a "heterocyclylalkyl" radical.

[0261] "Alkenyl" refers to a straight or branched hydrocarbon radical containing at least two carbon atoms, at least one unsaturated bond, and attached to the rest of the molecule by a single bond. Typical alkenyl groups contain 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, such as ethenyl, 1-methyl-ethenyl, 1-propenyl, 2-propenyl, or butenyl.

[0262] "Alkoxy" refers to a substituent formed by replacing the hydrogen of a hydroxy group with an alkyl group. Typical alkoxy groups contain 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, such as methoxy, ethoxy, propoxy, butoxy, etc.

[0263] "Aryl" refers to a monocyclic or polycyclic free radical, including polycyclic free radicals containing a monocyclic aryl group and / or a fused aryl group. Typical aryl groups contain 1 to 3 monocyclic or fused 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 radicals.

[0264] "Heterocyclic" 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 groups in the compounds of the present invention contain 1, 2 or 3 heteroatoms selected from N, O or S atoms and include, for example, coumarin (including 8-coumarin), quinolinyl (including 8-quinolinyl, isoquinolinyl), pyridinyl, pyrazinyl, pyrazolyl, pyrimidinyl, furanyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl and furopyridinyl, and the like. 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, dihydrofuranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, oxathianyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxirane, thiirane, azepine, oxazepine, diazepine indole, indole, triazepanyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindole, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothiophenyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3H-indolyl, and quinolizinyl, among others.

[0265] "Halogen" or "halo" refers to bromo, chloro, iodo or fluoro.

[0266] Unless otherwise stated, the compounds of the present invention also include isotopically labeled forms, i.e. compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having at least one hydrogen atom replaced solely by deuterium or tritium, or by using an isotopically enriched 13 C or 14 C to replace at least one carbon, or use a carbon-rich 15 Compounds in which at least one nitrogen in an existing structure is replaced by a nitrogen in the form of N are all within the scope of the present invention.

[0267] The term "PD-L1" includes isoforms, mammalian, such as human PD-L1, species homologs of human PD-L1, and analogs that share at least one common epitope with PD-L1. The amino acid sequence of PD-L1, such as human PD-L1, is known in the art. As used herein, an "anti-PD-L1 antibody" refers to an antibody that specifically binds to PD-L1.

[0268] The term "antibody" means an immunoglobulin molecule comprising four polypeptide chains, namely two heavy (H) chains and two light (L) chains interconnected by disulfide bonds (i.e., a "full antibody molecule"), as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain comprises a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (comprising domains CH1, CH2, and CH3). Each light chain comprises a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0269] As used herein, the terms "antigen-binding portion" and "antibody fragment" of an antibody include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. 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 retains the ability to specifically bind to PD-L1. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, CDR-containing fragments, 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 identification units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementary determining regions (CDRs)). As used herein, the expression "antigen-binding fragment" also encompasses other engineered molecules, such as bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and miniantibodies. An antigen-binding fragment of an antibody will typically comprise at least one variable domain.

[0270] The term "therapeutically effective amount" refers to a dose that can achieve treatment, prevention, alleviation and / or relief of the diseases or conditions described herein in a subject.

[0271] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or its cells amenable to the methods described herein, whether in vitro or in situ, and in some non-limiting embodiments, is a mammal, such as a human, monkey, dog, rabbit, mouse, and the like.

[0272] The term "treating" includes eradicating, removing, reversing, alleviating, modifying or controlling a disease and / or condition after onset.

[0273] The term "prevent" refers to the ability to avoid, minimize, or make the onset or development of a disease and / or condition difficult by treating the disease and / or condition before it occurs.

[0274] The term "disease" refers to a physical condition of the subject, which is associated with the disease of the present invention.

[0275] The disclosures of various publications, patents, and published patent specifications cited herein are incorporated by reference in their entirety.

[0276] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0277] Example 1: Preparation of small molecule loading

[0278] 1. Preparation of (R)-2-((5-nitropyridin-2-yl)dithiocyclohexyl)propan-1-ol (Compound 24)

[0279]

[0280] At 0 DEG C, under argon protection, SOCl2 (316 μL, 3.94mmol) was added dropwise to a solution of 5-nitropyridine-2-thiol (560mg, 3.59mmol) in anhydrous DCM (8mL). The resulting mixture was stirred at room temperature for 2h. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to provide an intermediate 24 as a yellow powder. A solution of methyl (R) -2-mercaptopropan-1-ol (prepared according to the procedure described in WO2013055987, 360mg, 3.88mmol) in dry DCM (5mL) was added dropwise to a solution of intermediate 4 in dry DCM (10mL). The reaction mixture was stirred at room temperature overnight under argon protection. After the reaction was completed, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain a yellow solid. The residue was suspended in water and alkalized with sodium bicarbonate solution, then extracted with DCM (3 × 100mL). The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (PE:EA=10:3) to give 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] + The calculated value is 247.0 and the measured value is 247.2.

[0281] 2. Preparation of (R)-2-((5-nitropyridin-2-yl)dithiocyclohexyl)propyl(4-(hydroxymethyl)phenyl)carbamate (Compound 26)

[0282]

[0283] 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 argon protection, a solution of anhydrous DCM (5 mL) with triphosgene (145 mg, 0.5 mmol) was added dropwise to the mixture at 0 ° C and stirred for 1 h. After the reaction was completed, the resulting reaction solution was evaporated under reduced pressure to obtain (R)-2-((5-nitropyridin-2-yl)disulfanyl)propylcarbochloridate (compound 25), which was used directly without further purification. Subsequently, compound 25 (308 mg, 1.0 mmol) was dissolved in dry DCM (5 mL), and a DCM mixed 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 argon protection. After the reaction was complete, 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 evaporated under reduced pressure to give a crude product. The crude product was purified by flash chromatography on silica gel (PE: EA = 2: 1) to give compound 26 as a white solid. Yield: 70%. 1H NMR (400MHz, Chloroform-d) δ9.23 (d, J=2.6Hz, 1H), 8.33 (dd, J=8.8, 2.6Hz, 1H), 7.91 (d, J=8.8Hz, 1H), 7 .34(m, 4H), 6.82(s, 1H), 4.67(s, 2H), 4.31–4.23(m, 2H), 3.37(h, J=6.8Hz, 1H), 1.43(d, J=7.0Hz, 3H).13C NMR (101MHz, 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] + The calculated value is 396.1 and the measured value is 396.2.

[0284] 3. Preparation of (R)-2-((5-nitropyridin-2-yl)dithiocyclohexyl)propyl(4-(benzylchloro)phenyl)carbamate (Compound 19)

[0285]

[0286] To a solution of compound 26 (100 mg, 0.253 mmol) in anhydrous DCM (5 mL) at 0°C was added thionyl chloride (22 μL, 0.304 mmol) dropwise. The reaction mixture was removed from 0°C and stirred at room temperature for 30 minutes. After the reaction was complete, excess thionyl chloride was removed by rotary evaporation under reduced pressure to afford the desired product, compound 19, as a white solid. Yield: 96%. 1H NMR (400MHz, chloroform-d) δ9.24 (d, J=2.6Hz, 1H), 8.35 (dd, J=8.9, 2.7Hz, 1H), 7.89 (d, J=8.9Hz, 1H), 7.35 (m, 4 H), 6.75 (s, 1H), 4.56 (s, 2H), 4.27 (qd, J=11.5, 6.1Hz, 2H), 3.35 (h, J=6.7Hz, 1H), 1.41 (d, J=7.0Hz, 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. 16 H17ClN3O4S2 MS(ESI): m / z[M+H] + The calculated value is 414.0 and the measured value is 414.2.

[0287] 4. Preparation of Compound 20

[0288]

[0289] To compound N 4Compound 19 (50 mg, 0.12 mmol), TBAB (8 mg, 0.022 mmol) and DIEA (18 μL, 0.11 mmol) were added to a solution of butyl-6-(4-((dimethylamino)methyl)benzyl)pyridino[3,2-d]pyrimidine-2,4-diamine (40 mg, 0.11 mmol) in DMF (0.5 mL). The reaction mixture was stirred at room temperature and monitored by LC-MS. When the reaction was complete, DMF was removed by rotary evaporation under reduced pressure. The residue was redissolved in 10% methanol solution and stirred at room temperature for 2 h. The resulting solution was concentrated by rotary evaporation and purified by phase preparative high performance liquid chromatography (RPHPLC) to give compound 20 as formate salt. Yield: 68.0%. 1H NMR (400MHz, methanol-d4) δ9.09 (d, J=2.6Hz, 1H), 8.32 (s, 1H), 8.31 (dd, J=8.7, 2.6Hz, 1H), 8.0 9(d, J=8.8Hz, 1H), 7.79(d, J=8.6Hz, 1H), 7.66(d, J=8.5Hz, 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=7.3Hz, 2H), 3.44(pd, J=6.9, 4.8Hz, 1H), 2.97 (s, 6H)), 1.73 (p, J=7.5Hz, 2H), 1.50-1.40 (m, 5H), 1.00 (t, J=7.5Hz, 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. 37 H 44 N9O4S2MS(ESI):m / z[M] + , calculated value is 742.3, found value is 742.3. HRMS(ESI)[M] + : The calculated value is 742.2958, and the measured value is 742.2968.

[0290] 5. Preparation of MC-Val-Cit-PAB-Cl (Compound 29)

[0291]

[0292] To a solution of MC-Val-Cit-PAB-OH (57 mg, 0.1 mmol) in anhydrous DCM (5 mL) was added thionyl chloride (8.7 μL, 0.304 mmol) dropwise at 0°C. The reaction mixture was removed from 0°C and stirred at room temperature for 12 hours. After the reaction was complete, the excess thionyl chloride was removed by rotary evaporation under reduced pressure to afford the desired product 29 as a brown solid, which was used directly without further purification.

[0293] 6. Preparation of Compound 30

[0294]

[0295] To compound N 4 To a solution of butyl-6-(4-((dimethylamino)methyl)benzyl)pyridino[3,2-d]pyrimidine-2,4-diamine (40 mg, 0.11 mmol) in DMF (1 mL) was added 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. When the reaction was complete, DMF was removed by rotary evaporation under reduced pressure. The residue was redissolved in 10% methanol solution and stirred at room temperature for 2 h. The resulting solution was concentrated by rotary evaporation and purified by phase preparative high performance liquid chromatography (RPHPLC) to give compound 30 as a formate salt. Yield: 55.0%. 1 H NMR(400MHz,Chloroform)δ10.34(bs,1H),9.40(bs,1H),8.13(d,J=8.6Hz,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=6.8Hz,2H),3.27(s,6H),2.95(m,2H),2.73(m,1H),2.49(m,1H),2.10–1.85(m,6 H),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] + : The calculated value is 920.15, and the measured value is 920.30.

[0296] 7. (S)-N 1Preparation of -(4-(chloromethyl)phenyl)-2-((R)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)propionamide)propionamide)succinamide (Compound 31)

[0297]

[0298] At 0°C, (S)-2-((R)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)propionamide)propionamide)-N 1 To a solution of -(4-(hydroxymethyl)phenyl)succinamide (100 mg, 0.174 mmol) in anhydrous DCM (5 mL) was added thionyl chloride (15 μL, 0.210 mmol) dropwise. The reaction mixture was removed from 0°C and stirred at room temperature for 30 minutes. After the reaction was complete, 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 directly without purification.

[0299] 8. Preparation of Compound 32

[0300]

[0301] To compound N 4 Compound 31 (71 mg, 0.12 mmol), TBAB (8 mg, 0.022 mmol) and DIEA (18 μL, 0.11 mmol) were added to a solution of butyl-6-(4-(dimethylamino)methyl)benzyl)pyridinium[3,2-d]pyrimidine-2,4-diamine (40 mg, 0.11 mmol) in DMF (1 mL). The reaction mixture was stirred at room temperature and monitored by LC-MS. When the reaction was complete, DMF was removed by rotary evaporation under reduced pressure. The residue was redissolved in 10% methanol solution and stirred at room temperature for 2 h. The resulting solution was concentrated by rotary evaporation and purified by phase preparative high performance liquid chromatography (RPHPLC) to give compound formate 32. Yield: 48.0%. LC-MS (ESI) [M] + : The calculated value is 920.10, and the measured value is 920.25.

[0302] 9. Preparation of Compound 33

[0303]

[0304] Commercially available (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). 6-(maleimido)hexanoic acid succinimidyl ester (340 mg, 1.1 mmol), 1-hydroxybenzotriazole (135 mg, 1 mmol), and N,N-diisopropylethylamine (210 μL, 1.2 mmol) were added sequentially, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was extracted with EA / H2O, and the organic phase was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM:MeOH = 10:1) to obtain 278 mg of the desired product. LC-MS (ESI) [M] + :649.3.

[0305] 10. Preparation of Compound 34

[0306]

[0307] To a solution of (2S,3R,4S,5S,6S)-2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (Compound 33) (65 mg, 0.1 mmol) in anhydrous DCM (2 mL) was added thionyl chloride (8 μL, 0.12 mmol) dropwise at 0°C. The reaction mixture was stirred at 0°C for 30 minutes. After completion of the reaction, the organic solvent was removed by rotary evaporation to obtain the desired product. The product was used directly without purification.

[0308] 11. Preparation of Compound 35

[0309]

[0310] To a DMF (1 mL) solution of (2S,3R,4S,5S,6S)-2-(4-(chloromethyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (67 mg, 0.1 mmol) (Compound 34) was added N 46-(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) were added. The reaction mixture was stirred at room temperature and monitored by LC-MS. When the reaction was complete, the reaction solution was filtered and purified by preparative high performance liquid chromatography (RPHPLC) to obtain 30 mg of the desired product in a 28% yield. LC-MS (ESI) [M] + :995.4.

[0311] 12. Preparation of Compound 36

[0312]

[0313] Compound 35 (30 mg, 0.03 mmol) was dissolved in a mixed solvent (THF:H2O = 2:1), and lithium hydroxide monohydrate (15 mg, 0.3 mmol) was added. After stirring at room temperature for 30 minutes, acetic acid was added to adjust the pH to 7. The organic solvent was removed by concentration under reduced pressure, and methanol was added for dissolution. Reverse-phase chromatography was performed to obtain 12 mg of the desired product in a 46% yield. LC-MS (ESI) [M] + :855.4.

[0314] 13. Preparation of Compound 37

[0315]

[0316] Take N 4 2-Butyl-6-(4-(chloromethyl)benzyl)pyrido[3,2-d]pyrimidine-2,4-diamine (356 mg, 1 mmol, synthesis method refers to the preparation method of compound 10 in Example B of Chinese patent CN 108069963) 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 stirred at 35°C overnight. After completion of the reaction, the reaction solution was extracted with ethyl acetate and saturated brine, and washed with water three times. The organic phase was dried over anhydrous sodium sulfate and spin-dried 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.

[0317] 14. Preparation of Compound 38

[0318]

[0319] Tert-butyl 6-(4-((2-amino-4-(butylamino)pyridino[3,2-d]pyrimidin-6-yl)methyl)benzyl)-2,6-diazaspiro[3.3]heptane-2-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 hour. The organic solvent was removed by concentration under reduced pressure to obtain a crude product. Diethyl ether was added to the crude product, sonicated, and filtered to obtain the desired product. LC-MS (ESI) [M+H] + :418.3.

[0320] 15. Preparation of Compound 39

[0321]

[0322] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-3-methylbutanamide)-5-uridinepentanamide)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]heptane-2-yl)methyl)benzyl)-N 4 1-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 stirred at room temperature and monitored by LCMS. After completion of the reaction, 20 mg of the desired product was obtained directly by RP-HPLC with a yield of 20%. LC-MS (ESI) [M+H] + :1016.4. 1 H NMR (400MHz, DMSO-d6) δ7.78(d,J=8.5Hz,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=13.2Hz,2H),3.07(s, 2H),2.95(t,J=12.4Hz,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).

[0323] 16. Preparation of Compound 40

[0324]

[0325] Commercially available 4-((S)-4-amino-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propionamido)-4-oxobutanamido)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-nitrophenyl)carbonate was added. 4 1-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 stirred at room temperature and monitored by LCMS. After completion of the reaction, 15 mg of the desired product was obtained directly by RP-HPLC with a yield of 16%. LC-MS (ESI) [M+H] + :1016.4.

[0326] 17. Preparation of Compound 41

[0327]

[0328] (2S,3R,4S,5S,6S)-2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (260 mg, 0.4 mmol) was dissolved in DMF (2 mL). Di(p-nitrobenzene) carbonate (182 mg, 0.6 mmol) and N,N-diisopropylethylamine (105 μL, 0.6 mmol) were added sequentially and the mixture was allowed to react for 6 hours. The reaction solution was extracted three times with EA / H2O. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM:MeOH = 15:1) to obtain 250 mg of the desired product (yield 78%). LC-MS (ESI) [M+H] + :814.4.

[0329] 18. Preparation of Compound 42

[0330]

[0331] (2S,3R,4S,5S,6S)-2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-4-((((4-nitrophenoxy)carbonyl)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Compound 41) (82 mg, 0.1 mmol) was dissolved in DMF (1 mL), and 6-(4-((2,6-diazaspiro[3.3]heptane-2-yl)methyl)benzyl)-N 4 1-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 stirred at room temperature and monitored by LCMS. After completion of the reaction, 30 mg of the desired product was obtained directly by RP-HPLC with a yield of 27%. LC-MS (ESI) [M+H] + :1092.4.

[0332] 19. Preparation of Compound 43

[0333]

[0334] Compound 42 (30 mg, 0.027 mmol) was dissolved in a mixed solvent (THF:H2O = 2:1), and lithium hydroxide monohydrate (15 mg, 0.27 mmol) was added. After stirring at room temperature for 30 minutes, acetic acid was added to adjust the pH to 7. The organic solvent was removed by concentration under reduced pressure, and methanol was added for dissolution. Reverse-phase chromatography was performed to obtain 15 mg of the desired product in a 51% yield. LC-MS (ESI) [M+H] + :952.3. 1 H NMR(400MHz, 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=13.4,4.8Hz, 2H),3.63(s,2H),3.46(t,J=13.2Hz,2H),3.05(s,2H),2.37(m,2H),1.77–1.25(m,12H),0.89(t,J=7.4Hz,3H).

[0335] 20. Preparation of Compound 44

[0336]

[0337] Compound 38 (41.8 mg, 1.0 mmol) was dissolved in DMF (2 mL), and succinimidyl 6-(maleimido)hexanoate (34 mg, 0.11 mmol) was added. The mixture was allowed to react at room temperature for 3 hours. The reaction solution was extracted with ethyl acetate / saturated brine, and the organic phase was washed three times with water, 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 = 5:1) to obtain 28 mg of the desired product in a 48% yield. LC-MS (ESI) [M+H] + :611.4. 1 H NMR(400MHz,Chloroform)δ7.67(d,J=8.4Hz,1H),7.62(br,2H),7.31–7.16(m,5H),4.21(s,2H),3.89(t,J=10.4Hz,2H),3 .62(s,2H),3.45(t,J=13.4Hz,2H),3.01(s,2H),2.23(td,J=13.2,4.8Hz,2H),1.63–1.27(m,12H),0.89(t,J=7.4Hz,3H).

[0338] 21. Preparation of Compound 45

[0339]

[0340] Compound 38 (21 mg, 0.5 mmol) was dissolved in methanol (1 mL), and 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetaldehyde (28 mg, 2 mmol) was added. After stirring at room temperature for 30 min, sodium cyanoborohydride (8 mg, 13 mmol) was added portionwise and the reaction continued for 1 hour. The reaction solution was filtered and directly subjected to reverse phase chromatography to prepare 6 mg of the product with a yield of 23%. LC-MS (ESI) [M+H] + :541.3. 1 H NMR (400MHz, CDCl3): δ7.62(br,2H),7.56(d,J=8.6Hz,1H),7.45(d,J=8.6Hz,1H),7.43-7.32(m,4H),4.24(s,2H),4.70(s,4H),4.12(s,2H ),3.53(td,J=13.2,5.9Hz,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=7.3Hz,3H).

[0341] 22. Preparation of Compound 46

[0342]

[0343] Compound 38 (210 mg, 0.5 mmol) was dissolved in acetonitrile (5 mL), and tert-butyl (2-(2-(2-iodoethoxy)ethoxy)ethoxy)ethyl)carbamate (305 mg, 0.75 mmol) and potassium carbonate (216 mg, 1.5 mmol) were added sequentially. The reaction mixture was heated to 65°C and reacted for 5 hours. After completion of the reaction, the mixture was filtered, and the residue was washed with acetonitrile. The filtrate was collected and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM:MeOH = 4:1) to obtain 218 mg of the desired product in a 62% yield. LC-MS (ESI) [M+H] + :693.5.

[0344] 23. Preparation of Compound 47

[0345]

[0346] Compound 46 (218 mg) was dissolved in a mixed solvent (DCM:TFA = 20:1, 10 mL) and the reaction mixture was incubated at 0°C for 1 hour. After the reaction was complete, the organic solvent was removed by concentration under reduced pressure to obtain the desired product. The product was used directly without further purification.

[0347] 24. Preparation of Compound 48

[0348]

[0349] 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 added sequentially. The reaction solution was left to react at room temperature for 3 hours. After the reaction was completed, 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 crude product was used directly without purification. LC-MS (ESI) [M+H] + :822.3.

[0350] 25. Preparation of Compound 49

[0351]

[0352] Compound 48 (100 mg, 0.12 mmol) was dissolved in a mixed solvent (DMSO:H2O = 1:1, 1 mL). 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 allowed to react at room temperature for 1 hour. After completion of the reaction, the mixture was filtered, and the filtrate was directly subjected to reverse phase chromatography to obtain 22 mg of the target compound in a 16% yield. LC-MS (ESI) [M+H] + :1140.4. 1 H NMR (400MHz, DMSO) δ9.43(t,J=5.8Hz,1H),8.21(t,J=5.6Hz,1H),7.91(t,1H),7.82(bs,2H),7.79(d,J=8.6Hz,1H),7 .68(d,J=8.6Hz,1H),7.44(d,J=8.1Hz,2H),7.36(d,J=8.1Hz,2H),7.01(s,1H),4.48(t,J=5.2Hz,2H),4.26(m,4H),3 .82–3.76(m,4H),3.67(t,J=6.4Hz,1H),3.62–3.55(m,6H),3.55–3.43(m,22H),3.39(t,J=5.0Hz,4H),3.33(t,J=6.3 Hz,2H),3.20(m,7H),2.31(t,J=6.4Hz,4H),2.06(t,J=11.3Hz,2H),1.67(m,8H),1.34(m,6H),0.91(t,J=7.3Hz,3H).

[0353] 26. Preparation of Compound 50

[0354]

[0355] Compound 50 was prepared by referring to the synthetic method of compound 38.

[0356] 27. Preparation of Compound 51

[0357]

[0358] Compound 51 was prepared by referring to the synthetic method of compound 45 with a yield of 56%. LC-MS (ESI) [M+H] + :599.4.

[0359] 28. Preparation of Compound 52

[0360]

[0361] Compound 52 was prepared by referring to the synthetic method of compound 38.

[0362] 29. Preparation of Compound 53

[0363]

[0364] Compound 53 was prepared by referring to the synthesis method of compound 45, except that 6-(maleimido)hexanoic acid succinimidyl ester was replaced with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester. The yield was 42%. LC-MS (ESI) [M+H] + :655.3.

[0365] 30. Preparation of Compound 54

[0366]

[0367] Tert-butyl methyl (2-(methylamino)ethyl)carbamate (1 g, 5.3 mmol) was dissolved in dichloromethane (10 mL). The reaction solution was placed at 0°C, and chloromethyl chloroformate (0.95 mL) and pyridine (0.86 mL) were added sequentially. The reaction was continued at 0°C for 2 hours. After the reaction was completed, dichloromethane and water were added, and the organic phase was washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The organic layers were combined and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 5:1) to obtain the desired product as a colorless oil. LC-MS (ESI) [M+H] + :281.3.

[0368] 31. Preparation of Compound 55

[0369]

[0370] Take N 4 Compound 54 (1080 mg, 3.9 mmol) and TBAI (1420 mg, 3.9 mmol) were added to 1-butyl-6-(4-((dimethylamino)methyl)benzyl)pyrido[3,2-d]pyrimidine-2,4-diamine (400 mg, 1.1 mmol) in chloroform (10 mL). The reaction mixture was heated to 60°C for 6 hours. After the reaction was complete, the mixture was concentrated and directly purified by column chromatography (DCM:MeOH = 4:1) to obtain 480 mg of the title compound in a 72% yield. LC-MS (ESI) [M] + :609.3.

[0371] 32. Preparation of Compound 56

[0372]

[0373] Compound 55 (480 mg) was dissolved in a mixed solvent (DCM:TFA = 10:1). The reaction mixture was incubated at 0°C for 1 hour. After completion of the reaction, the organic solvent was removed by concentration under reduced pressure to obtain the trifluoroacetate salt of the desired product. Diethyl 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 desired product. LC-MS (ESI) [M] + :509.3.

[0374] 33. Preparation of Compound 57

[0375]

[0376] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-3-methylbutanamide)-5-uridine pentylamide)benzyl (4-nitrobenzene) carbonate (MC-VC-PAB-NPC) (81.0 mg, 0.11 mmol) was dissolved in DMF (0.5 mL). 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 monitored by LCMS. After completion of the reaction, 30 mg of the desired product was obtained directly by RP-HPLC with a yield of 24%. LC-MS (ESI) [M] + :1107.4. 1 H NMR (400MHz, DMSO) δ10.16 (s, 1H), 8.38 (bs, 2H), 8.24 (bs, 1H), 7.90 (d, J = 7.7Hz, 1 H),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).

[0377] 34. Preparation of Compound 58

[0378]

[0379] Compound 58 was prepared by referring to the synthetic method of compound 57, except that MC-VC-PAB-NPC was replaced with MC-AAN-PAB-NPC. LC-MS (ESI) [M] + :1107.4. 1 H NMR (400MHz, DMSO) δ9.75(s,1H),8.33(bs,2H),8.24(s,1H),8.08(d,1H),7.76(t,1H),7.63(d,J=7.6H z,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=6.3Hz,2H), 2.09(t,J=7.5Hz,3H),1.67–1.55(m,2H),1.53–1.27(m,6H),1.24-1.18(m,10H),0.93(t,J=7.3Hz,3H).

[0380] 35. Preparation of Compound 59

[0381]

[0382] Compound 59 was prepared by referring to the synthetic method of compound 57, except that MC-VC-PAB-NPC was replaced by compound 41. LC-MS (ESI) [M] + :1183.5.

[0383] 36. Preparation of Compound 60

[0384]

[0385] Compound 59 (35 mg, 0.03 mmol) was dissolved in a mixed solvent (THF:H2O = 2:1), and lithium hydroxide monohydrate (16 mg, 0.3 mmol) was added. After stirring at room temperature for 30 minutes, acetic acid was added to adjust the pH to 7. The organic solvent was removed by concentration under reduced pressure, and methanol was added for dissolution. Reverse-phase chromatography was performed to obtain 5 mg of the desired product in a 15% yield. LC-MS (ESI) [M+H] + :1043.4.

[0386] Example 2: Preparation of anti-PD-L1 THIOMAB

[0387] 1. Gene Editing of Anti-PD-L1 THIOMAB

[0388] Avelumab (MSB0010718C, trade name Bavencio) is a fully humanized IgG1 monoclonal anti-PD-L1 antibody developed by Merck KGaA and Pfizer. It was approved by the FDA in 2017 for the treatment of Merkel cell carcinoma (Apolo et al., 2017; Shirley, 2018). The nucleotide sequences encoding its light and heavy chains are shown in SEQ ID NOs: 1 and 2, respectively, and the amino acid sequences of the light and heavy chains are shown in SEQ ID NOs: 3 and 4, respectively. In addition to binding to human PD-L1, avelumab also cross-reacts with murine PD-L1 (Deng et al., 2016). Therefore, using avelumab as a raw material to develop an anti-PD-L1 ADC drug can ensure that the antibody itself has sufficiently excellent biological activity and drugability, while also making the resulting ADC drug suitable for early mouse model testing and subsequent human clinical trials during development. The expression plasmid for avelumab consists of two parts: the light chain 159-pFuse-αPDL1-LC plasmid and the heavy chain 162-pFuse-αPDL1-hIgG-Fc2 plasmid, whose nucleotide sequences are shown in SEQ ID NOs: 5 and 6. These two plasmids are co-transfected into cells for expression, and after purification with protein A resin, the anti-PD-L1 antibody avelumab can be obtained.

[0389] The inventors constructed the THIOMAB expression plasmid using PCR site-directed mutagenesis. A pair of primers, perfectly complementary to the mutation site and oriented in opposite directions, were designed. Using the intact plasmid to be mutated as a template, a high-fidelity PCR enzyme was used to replicate and amplify the entire plasmid sequence containing the mutation site. Subsequently, the methylated template plasmid was removed using the DpnI restriction endonuclease, while retaining the unmethylated PCR product. Further plasmid transformation and culture were performed to amplify the intact plasmid containing the mutation site.

[0390] After the site-directed mutagenesis experiment was completed, the resulting plasmid was extracted and sequenced to determine whether the mutation was successful. The plasmid obtained after site-directed 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 sequencing comparison results are shown in Figure 1As shown. After comparison with the template plasmid, it was found that the sequence at the predetermined mutation site mutated from GTG to TGC, while the other bases did not change. Therefore, the 205th amino acid of the translated light chain mutated from valine to cysteine. These two artificially introduced cysteines will be used for the subsequent ADC drug conjugation reaction. The light chain encoding nucleotide sequence of the mutated antibody anti-PD-L1 THIOMAB is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain is shown in SEQ ID NO: 9.

[0391] 2. Expression of anti-PD-L1 antibodies and THIOMAB

[0392] After expression and purification, the anti-PD-L1 antibody was identified by reducing and non-reducing SDS PAGE analysis. Figure 2 The antibody was reduced to light chain (~25KD) and heavy chain (~50KD) in reducing SDS PAGE, but remained intact (~150KD) in non-reducing SDS PAGE.

[0393] After expression and purification, the anti-PD-L1 THIOMAB was obtained and identified by reducing and non-reducing SDS PAGE analysis. Figure 3 As shown, the SDS staining results of anti-PD-L1 THIOMAB (as shown Figure 3 As shown) with anti-PD-L1 antibodies (as shown) Figure 2 The results were consistent with those shown in Figure 2 (shown in Figure 2): the antibody was reduced to light chains (~25KD) and heavy chains (~50KD) in reducing SDS PAGE, while the intact antibody (~150KD) was retained in non-reducing SDS PAGE. Experiments have shown that the anti-PD-L1 THIOMAB obtained by gene editing basically retains the original structure of the antibody, which shows that the expression and self-assembly processes of THIOMAM are not affected by artificial mutations. At the same time, under the same conditions, the expression levels of the antibody and THIOMAB do not seem to differ much, which also shows that the expression and assembly processes of the antibody in the cell are not significantly affected.

[0394] To further identify the obtained THIOMAB, the inventors treated the intact THIOMAB according to the process of "TCEP reduction-purification-DHAA reoxidation-purification" to obtain THIOMAB with free thiol groups (THIOMAB-S2). The changes in its molecular weight were detected by Exactive Plus EMROrbitrap LC-MS. The results are as follows. Figure 4As shown. The molecular weight of the complete anti-PD-L1 THIOMAB is approximately 143791.75, while the molecular weight of THIOMAB-S2 with a free thiol group is approximately 143544.31, with a molecular weight difference of 247.44. Theoretically, the thiol group of the complete anti-PD-L1 THIOMAB is generally modified with a free cysteine. Therefore, compared with THIOMAB-S2, the molecular weight will be approximately 238 more than the molecular weight of the residue after the condensation of two cysteines and thiol groups. This error is acceptable for antibody macromolecules. Therefore, it can be judged that the molecular weight change of the obtained THIOMAB is in line with experimental expectations. This result proves that the inventors have successfully expressed a homogeneous anti-PD-L1 THIOMAB with two mutated cysteines through gene editing, which can be used for the subsequent construction of homogeneous ADC drugs. In subsequent experiments, the inventors also need to compare the results of their in vivo and in vitro experiments with natural anti-PD-L1 antibodies to study whether this artificial mutation affects the antibody's antigen binding ability and other activities.

[0395] like Figure 4 As shown in the figure, compared to the original anti-PD-L1 THIOMAB covered with cysteine, the reduced and dialyzed THIOMAB lost two cysteines, exposing two free sulfhydryl groups. This caused the expected molecular weight of THIOMAB to decrease by 238 to 143553.75, while the actual molecular weight of THIOMAB detected was 143544.31, a decrease of 247.44.

[0396] Example 3: Synthesis and identification of THIOMAB-based ADCs

[0397] 1. Synthesis and identification of ADC HE-S2

[0398] The synthesis diagram of ADC HE-S2 is as follows Figure 5 The inventors used anti-PD-L1 THIOMAB and compound 20 (prepared in Example 1) as raw materials to synthesize anti-PD-L1 antibodies with disulfide bond linkers and N 4 A conjugate of 6-butyl-6-(4-((dimethylamino)methyl)benzyl)pyridino[3,2-d]pyrimidine-2,4-diamine (hereinafter referred to as compound D18) was named ADCHE-S2. The attachment site on the antibody is cysteine ​​at position 226 of the amino acid sequence set forth in SEQ ID NO: 9 (light chain sequence).

[0399] The concentration and purity of the ADC HE-S2 obtained by the reaction were determined by Nonadrop, and the yield was estimated (about 30%-50%). A portion of the sample was sent to the National Protein Center for macromolecular MS to detect changes in the antibody molecular weight. The results were as follows: Figure 6 As shown in . Theoretically, modification of the thiol group in ADC HE-S2 with S2-D18 would increase the overall molecular weight of the ADC with a DAR of 2 by approximately 934 compared to THIOMAB. MS analysis revealed a molecular weight of 144,721.08 for the ADC, while that of THIOMAB was 143,791.75, a difference of 929.33. This error is acceptable for protein analysis at ~147 K. Therefore, it can be concluded that a homogeneous ADC drug with a DAR of 2, ADC HE-S2 (ADC1), has been successfully prepared, as shown below.

[0400]

[0401] The mass spectrometry results of ADC HE-S2 are as follows Figure 6 As shown, the molecular weight of ADC obtained by MS was 144721.08, while the molecular weight of THIOMAB was 143791.75.

[0402] 2. Synthesis and identification of other ADC drugs

[0403] 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, and 57 (prepared in Example 1) as starting materials) to synthesize two ADCs with different linkers: ADC VC (ADC2) and ADCs legumain (ADC3) and ADC4. The synthetic flow charts are shown in Figures 7 and 8, respectively.

[0404]

[0405] After MS identification (such as Figure 9 (As shown in the figure), based on the previously calculated analysis method, it can be calculated that the theoretical molecular weight of ADC VC should be approximately 1600 higher than that of THIOMAB. The MS results show that the difference between the two is 145399.23-143791.75 = 1607.48, which is very close to the theoretical value. Similarly, it can be verified that the theoretical molecular weight of ADC Legumain should be approximately 1600 higher than that of THIOMAB. The MS results show that the difference between the two is 145391.09-143791.75 = 1599.34, which is also very close to the theoretical calculation. Therefore, it can be concluded that two uniform ADCs, ADC VC and ADC Legumain, with a DAR of 2, have been obtained.

[0406] Example 4: Synthesis and identification of ADC based on wild-type PD-L1 antibody

[0407] 1. ADC Conjugation Steps and Methods

[0408] a. Preparation of reaction materials and reagents: The antibody (Avelumab described in Example 2) was prepared at a concentration of 5-10 mg / mL in PBS; a 10 mM aqueous TCEP solution was prepared; the conjugated small molecule (compounds 39, 40, 43, 44, 51, 45, 49, 53, 57, 58, 60, prepared in Example 1) was prepared at a concentration of 10 mM in DMSO; a 20 mM cysteine ​​solution was prepared in PBS;

[0409] b. Antibody reduction: Add 10 or 20 equivalents of TCEP (10 mM, shake at 37°C for 2-3 h, rotation speed 220 rpm / min;

[0410] c. Coupling reaction: After reducing the antibody, add 10-20 equivalents of the small molecule and shake at room temperature for 2-4 hours at 220 rpm / min;

[0411] d. Quenching reaction: Add cysteine ​​solution equivalent to the small molecule and incubate at 4 or 10°C for 30 minutes to terminate the reaction;

[0412] e. Desalting: After the reaction is terminated, desalt the solution using a 5 ml pre-packed desalting column from Cytiva.

[0413] 2. ADC DAR value determination

[0414] The DAR value calculation method is based on ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS):

[0415] a. Instrument: A Waters Acquity-Class ultra-high performance liquid chromatograph (UHPLC) was connected in series with a Waters Synapt G2-Si Q-TOF high-resolution mass spectrometer. High-purity nitrogen was used as the nebulizer gas, and high-purity argon was used as the collision gas.

[0416] b. Liquid chromatography conditions: The chromatographic column is ACQUITY UPLC Protein BEH SEC column ( The column was 2.1 mm × 150 mm, 1.7 μm. The column temperature was 25°C, and the injection volume was 15 μL. Isocratic elution was performed using 50 mM ammonium acetate as the mobile phase at a flow rate of 0.065 mL / min. The acquisition time was 10 min, and UV detection was performed at 280 nm.

[0417] Mass spectrometry conditions: the capillary voltage was set to 3.0 kV for ESI source positive ionization mode scanning; the cone voltage was 120 V, the source temperature and desolvation temperature were set to 120°C and 500°C, respectively; the cone gas and desolvation gas flow rates were set to 20 L / H and 600 L / H, respectively, and the primary mass spectrometry range was m / z 400 to m / z 8000.

[0418] c. Data Analysis: Data were analyzed using Waters Masslynx software, and mass spectrometry data were deconvoluted using the MaxEnt 1 plug-in. ADC DAR values ​​were determined by analyzing the deconvoluted mass spectra. First, peaks were integrated and peak area percentages were calculated, with the sum of peak area percentages being 100. The weighted average drug conjugate ratio of the ADC was calculated using the formula: DAR = Σweighted peak area / 100.

[0419] 3. Specific ADC product structure information

[0420]

[0421]

[0422] Example 5: Biological activity studies

[0423] 1. Antigen binding ability test of ADC HE-S2 (ADC1)

[0424] Sufficient and specific antigen affinity is the foundation of antibody biological activity and is crucial to the activity and efficacy of antibody drugs. Since the preparation of ADC drugs involves antibody modification, chemical treatment, and small molecule conjugation, these can destroy or affect the antibody's original antigen affinity and stability, resulting in impaired antigen-binding ability. Therefore, during the antibody processing process, the conditions used should be as mild as possible; the selection of modification and conjugation sites should also avoid the antigenic determinants of the original antibody to avoid damaging the antibody's antigen-binding ability. In addition, the antigen affinity of the resulting ADC drug should be tested and verified to ensure that the ADC drug retains sufficient targeting and specificity.

[0425] The antibody-antigen affinity of the ADC HE-S2 designed and prepared in this invention is a crucial foundation for achieving its anti-tumor activity. ADC HE-S2 requires targeted delivery of the immunomodulator D18 through specific binding of the antibody to PD-L1, which is highly expressed in tumors. Simultaneously, ADC HE-S2 can also bind to PD-L1 on the cell surface, blocking the tumor PD-1 / PD-L1 immunosuppressive signaling pathway and thereby inducing T cell anti-tumor responses. Therefore, after successfully preparing ADC HE-S2, the inventors first verified its ability to recognize and bind to the PD-L1 antigen in vitro.

[0426] Using PD-L1-positive MC38 cells as a platform, the inventors used fluorescence-activated cell sorting (FACS) to verify whether the ADC HE-S2 and the genetically modified anti-PD-L1 THIOMAB retained their original antibody-antigen binding ability. In the experiment, ADC HE-S2, the anti-PD-L1 antibody (i.e., avelumab described in Example 2), and the anti-PD-L1 THIOMAB (i.e., the mutant antibody prepared in Example 2) were incubated with MC38 cells as primary antibodies to allow sufficient contact and binding between the antibodies and cell surface PD-L1. After washing with PBS, the cells were treated with a FITC-labeled goat anti-human secondary antibody and incubated in the dark for 30 minutes to allow the secondary antibody to fully bind to the primary antibody. Two negative controls were set up at the same time. One was labeled as negative control (NC), in which only isotype control IgG was added to treat the cells without staining, indicating the fluorescence background of the cells themselves; the other was labeled as secondary antibody, which was stained with FITC-labeled secondary antibody after adding isotype control IgG together with other experimental groups to characterize the false positive fluorescence signal generated by nonspecific binding of secondary antibody to cells. TM The results were analyzed on a flow cytometer (TreeStar Inc.) to detect and compare the intensity of the FITC fluorescence signal on MC38 cells after treatment with different antibody drugs, in order to characterize the affinity of ADC, THIOMBA and antibody to PD-L1. All results were processed by FlowJo software (TreeStar Inc.) and the results were as follows: Figure 10 shown.

[0427] FACS results Figure 10 As shown, based on the blank control (NC) using an IgG isotype antibody, the control group stained with a fluorescent secondary antibody produced only a 1.7% false-positive signal offset. The mean fluorescence intensity (MFI) calculation showed that the MFI of the NC group was 142.00, while the mean fluorescence intensity detected by MC38 after secondary antibody staining was 166.75. While the difference was significant (p < 0.0001), it was small. This significant but slight difference is due to the false-positive signal generated by the nonspecific binding of the FITC secondary antibody used in the experiment to MC38 cells. The experiment demonstrated that this false-positive binding phenomenon is weak and does not affect the results. Therefore, the NC control group was used as a reference to evaluate the antibody-antigen binding ability of anti-PD-L1 antibody drugs.

[0428] The inventors observed similar FITC-positive signals on MC38 cells treated with anti-PD-L1 antibody, anti-PD-L1 THIOMAB, and ADC HE-S2. The fluorescence signal offset rates relative to the NC group were 35.7%, 36.6%, and 34.2%, respectively, significantly higher than the false-positive signal (1.7%) generated by nonspecific binding of the fluorescent secondary antibody. MFI calculations also showed that the evaluated fluorescence intensities of anti-PD-L1 antibody, anti-PD-L1 THIOMAB, and ADC HE-S2 were 445.3, 449.0, and 392.5, respectively, which were also significantly higher than the two control groups (p < 0.0001). This indicates that all three antibodies can specifically bind to PD-L1. At the same time, Student's t-test analysis demonstrated that the MFI detected for the gene-edited anti-PD-L1 THIOMAB and the further chemically modified ADCHE-S2 were not significantly different from those of the natural anti-PD-L1 antibody. This shows that the inventors' gene editing and chemical modification neither damaged the original antigen recognition affinity of the antibody nor caused the occurrence of non-specific off-target effects of the antibody.

[0429] Therefore, the inventors can confirm that ADC HE-S2 still maintains the antigen recognition affinity of the original anti-PD-L1 antibody and can be further applied in subsequent experiments and research and development.

[0430] 2. Internalization ability of ADC HE-S2 (ADC1)

[0431] The drug release of ADCs is widely believed to be dependent on the internalization ability of the corresponding antibody. The antibody-antigen complex formed after the ADC binds to the antigen protein on the cell surface needs to enter the target cell through the internalization process of the antigen protein, thereby releasing the coupled drug under the action of intracellular enzymes or other intracellular physiological environments. Therefore, the internalization ability of the complex formed after the selected antigen binds to the ADC is crucial for the realization of the ADC drug function. Since the ADC HE-S2 designed by the inventors should theoretically act simultaneously in cancer cells and DC cells, the inventors verified the ability of anti-PD-L1 antibody drugs to be internalized into tumor cells and DC cells through in vitro experiments.

[0432] Known pHrodo TM Red dye is a pH-sensitive fluorescent dye. When the surrounding environment becomes acidic, the fluorescence intensity of this molecule increases rapidly (Lehrman et al., 2018). When this dye is internalized into the cell along with the antigen-antibody complex, the pH value of the environment surrounding the dye will immediately drop to acidic, thereby generating an observable fluorescent signal. This property makes it possible to use pHrodo TM The red dye is used to monitor the internalization of the antibody. Live cell analysis system, inventors can pHrodo TM Image-based monitoring and quantitative analysis of the internalization of red dye-labeled anti-PD-L1 antibody. In 0.1 M sodium carbonate (pH 8.3) buffer, 1 mg / mL anti-PD-L1 THIOMAB and 3-5 equivalents of pHrodo TM The red dye was co-incubated for about 1 hour, and the residual small molecules were purified 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 future use; for long-term storage, it should be stored in a dark environment at -20°C to avoid repeated freezing and thawing. In order to characterize the internalization process of the PD-L1 / anti-PD-L1 antibody complex, the inventors chose to use pHrodo TM The anti-PD-L1 THIOMAB is labeled with a red dye. Previously, the inventors have verified that the anti-PD-L1 THIOMAB and ADC HE-S2 have the same antigen recognition ability. Therefore, the internalization ability of the anti-PD-L1 THIOMAB can also reflect the internalization ability of the ADC HE-S2.

[0433] (1) Anti-PD-L1 THIOMAB internalization experiment on cancer cells

[0434] Using MC38 and B16 PD-L1 positive tumor cells as materials, the internalization of PD-L1 / anti-PD-L1 antibody complex was verified. After adding fluorescently labeled anti-PD-L1 THIOMAB into the cells and incubating them in the dark for 24 hours, the cells were treated with The live cell analysis system captures the distribution of red fluorescent signals within cells. Figure 11 As shown in A.

[0435] It can be clearly observed that after the addition of labeled THIOMAB, a red signal was clearly generated in the cells, while the control group without antibody was almost not observed to have red fluorescence. From the synthesized image, it can be seen that there is almost no red fluorescence generated outside the cells, which proves the correlation between the fluorescence signal and the internalization of the antibody. Therefore, the inventors can The software analyzes the integrated intensity of the overall red fluorescent object to assess the internalization level of the fluorescently labeled THIOMAB, such as Figure 11 As shown in Figure B, after the addition of labeled THIOMAB, the overall red fluorescent signal intensity in MC38 cells reached 13701.63, significantly higher than the 84.88 in the control group. The signal intensity in B16 cells reached a high of 283613.60, also significantly higher than the 98.53 in the control group. The fluorescence signal in B16 cells was also stronger than that in MC38 cells, likely due to differences in PD-L1 internalization rates and intracellular pH.

[0436] (2) Internalization and selectivity experiments of anti-PD-L1 THIOMAB on DC cells

[0437] In order to verify the internalization of anti-PD-L1 THIOMAB on DC cells, the inventors obtained two types of DC cells, Cd274 knockout DC 2.4 cells and wild-type DC 2.4 cells, from Professor Tang Haidong of the School of Pharmacy of Tsinghua University. The Cd274 gene is the PD-L1 encoding gene. After knockout, DC 2.4 will not express PD-L1 protein, and thus is a PD-L1 negative DC cell; while wild-type DC 2.4 can express PD-L1, and is a PD-L1 positive DC cell (such as Figure 12 As shown in Figure 2 ). Based on this hypothesis, the inventors should be able to observe that anti-PD-L1 THIOMAB can be internalized into PD-L1-positive DC cells, but not into PD-L1-negative Cd274 knockout DC 2.4 cells. Therefore, by conducting anti-PD-L1 THIOMAB internalization experiments on these two cell types, the inventors can simultaneously verify the internalization ability of anti-PD-L1 THIOMAB on DC cells and the antibody's selectivity.

[0438] In the preliminary experiment, the inventors added 10% heat-inactivated fetal bovine serum (FBS) as a blocking agent to the culture medium of DC 2.4 cells according to conventional procedures. After incubation for 8 hours, the red fluorescent signal in the cells was detected by FACS. The results were as follows: Figure 13 As shown in . Unexpectedly, the FACS test results showed that the inventors observed the same proportion of red fluorescence-positive cells in both PD-L1-positive wild-type DC 2.4 cells and PD-L1-negative CD274 knockout DC 2.4 cells, which were 53.6% and 52.9%, respectively. Almost no red fluorescence-positive cells were observed in the negative control group without the addition of fluorescently labeled anti-PD-L1 THIOMAB (1.57% and 0.92%). Similar results were obtained after repeated use. These phenomena suggest that the fluorescent dye-labeled anti-PD-L1 THIOMAB can not only be successfully internalized 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 inventor's hypothesis.

[0439] The inventors conducted a literature review and found that the binding capacity of the Fc region of human IgG to mouse Fcγ receptors is very similar to that of its human orthologous Fcγ receptors (Dekkers et al., 2017). Therefore, the inventors speculated that the highly expressed and active mouse Fcγ receptors on the surface of DC2.4 cells can also bind to and internalize the humanized anti-PD-L1 antibody, thereby increasing the rate at which both cell types can internalize the anti-PD-L1 antibody.

[0440] To verify this hypothesis, the inventors used 10% mouse serum and 10% FBS as blocking agents and added them to the cell culture medium in groups. Compared with FBS, the mouse IgG rich in mouse serum can more effectively block the Fcγ receptors on the surface of DC2.4, thereby inhibiting the antibody internalization process mediated by the Fcγ receptors of DC cells. Before adding THIOMAB, DC cells should first be incubated at 4°C for 30 minutes to suspend the internalization process of cell surface receptors while allowing the IgG in mouse serum to fully bind to the Fcγ receptors on the surface of DC cells. Afterwards, fluorescently labeled anti-PD-L1 THIOMBA was added to each group of cells. Then, they were incubated in the dark at 4°C for 30 minutes to allow the antibody and antigen to fully contact and bind under the condition that cell internalization stopped.

[0441] The overall red fluorescence object integrated intensity-time change curve obtained from the internalization experiment of anti-PD-L1 THIOMAB is shown in Figure 2. Figure 14 As shown in A and B. Figure 14 The results in A show that after adding 10% FBS for blocking, very similar fluorescence intensity change curves can be observed in PD-L1-positive wild-type DC2.4 cells and PD-L1-negative CD274 knockout DC 2.4 cells; while no changes in fluorescence signals were observed in the negative control group without the addition of fluorescently labeled anti-PD-L1 THIOMAB.

[0442] like Figure 14 As shown in B, in the experimental group with 10% mouse serum added, the rate of increase in the integrated intensity of the overall red fluorescent object in the PD-L1 positive wild-type DC cells was significantly higher than that in the PD-L1 negative Cd274 knockout DC cells. Especially in the initial stage of the test (0-60min), the PD-L1 negative cells almost did not produce any fluorescent signal, and the change curve was similar to that of the negative control. After that, the fluorescent signal produced by the antibody began to appear in the PD-L1 negative DC cells, but the rate of signal enhancement (slope) still lagged behind that of the wild-type DC 2.4 cells. The final t-test analysis proved that there was a significant difference between the two (p < 0.0001). After 6 hours of culture, the cells were cultured with Live cell imaging of the experimental group blocked by mouse serum was performed to observe the distribution of red fluorescent signals in cells of each group. Figure 14 As shown in Figure A, the red fluorescence signal in wild-type DC 2.4 cells is significantly stronger than that in CD274-knockout DCs. This phenomenon can be explained by the fact that during the 30 minutes of incubation at 4°C before the assay, cell receptor internalization is nearly halted, while antigen-antibody binding is unaffected. This allows DC surface PD-L1 to fully bind to the anti-PD-L1 antibody, and Fcγ receptors are fully blocked by homologous IgG in mouse serum. When the assay begins, as the culture temperature gradually returns to 37°C, cell surface receptor internalization gradually resumes. The anti-PD-L1 THIOMAB, which previously bound strongly to PD-L1 on the wild-type DC surface, is rapidly internalized, while PD-L1-negative cells are unable to internalize the antibody. However, with prolonged incubation, PD-L1-negative DCs can internalize the anti-PD-L1 THIOMAB via recirculating Fcγ receptors, generating a fluorescent signal. However, this internalization is competitively inhibited by homologous mouse IgG in the culture medium. Therefore, the internalization rate of THIOMAB in Cd274 knockout DC cells still lags behind that of wild-type DC2.4 cells, which can internalize THIOMAB by binding to PD-L1.

[0443] This result validated the inventors' hypothesis and demonstrated the ability of anti-PD-L1 antibodies to internalize into cells by binding to cell surface PD-L1. Furthermore, after blocking with mouse serum, the difference in internalization speed of anti-PD-L1 THIOMAB in CD274 knockout and wild-type DC 2.4 cells also demonstrated the good selectivity of anti-PD-L1 antibodies.

[0444] Example 6: Antitumor Activity Study

[0445] 1. Study on the anti-tumor activity of ADC HE-S2 (ADC1)

[0446] To verify the anti-cancer activity of ADC HE-S2, the inventors first used MC38 to create a mouse model. After about a week of tumor loading, visible tumors could be observed on the surface of the mice. When the average tumor tissue volume of the mice reached 100 mm 3Afterwards, the mice were randomly divided into the following groups according to the final type of drug administration: IgG isotype control antibody group (abbreviated as IgG), anti-PD-L1 antibody group (anti-PD-L1), anti-PD-L1 THIOMAB group (Thiomab), D18 group (D18), D18 and anti-PD-L1 antibody combination treatment group (anti-PD-L1 & D18), and ADC HE-S2 group (ADC HE-S2), with 7-8 mice in each group. The IgG isotype control antibody group, anti-PD-L1 antibody (i.e., Avelumab described in Example 2) group, and anti-PD-L1 THIOMAB (i.e., the mutant antibody prepared in Example 2) group were administered twice a week, with each mouse receiving an intraperitoneal injection of 150 μg. The D18 group was administered twice a week, with each mouse receiving an intraperitoneal injection of 25 μg. In the D18 / anti-PD-L1 antibody combination treatment group, anti-PD-L1 antibody was administered twice a week at 150 μg per mouse each time; D18 was administered once at 25 μg per mouse each time; in the ADC HE-S2 group, ADC HE-S2 was administered once a week at 150 μg per mouse each time by intraperitoneal injection; and anti-PD-L1 antibody was administered once a week, staggered with ADC, at 150 μg per mouse each time by intraperitoneal injection.

[0447] The experimental results are as follows Figure 15 As shown in Figure A, the effects of individual treatments for MC38 tumors—anti-PD-L1 antibodies, anti-PD-L1 THIOMAB, and D18—were very similar: no significant difference in tumor growth inhibition was observed between the three agents, but all were slightly superior to the negative control IgG isotype antibody. This result suggests that both D18 and anti-PD-L1 antibodies possess some anticancer activity, and that anti-PD-L1 THIOMAB retains the biological function of the original antibody. The combination of D18 and anti-PD-L1 antibodies demonstrated significant tumor growth inhibition, exceeding that achieved by either agent alone. This result further validates the synergistic effect of D18 and PD-1 / PD-L1 blocking antibody therapy. ADC HE-S2 demonstrated impressive anticancer activity, significantly superior to the combination therapy group (P < 0.001). By day 24 of the experiment, tumor volume in the combination-treated mice began to increase significantly, while tumor size in the ADC-treated group remained at a low level.

[0448] In order to further study the survival rate of mice after long-term drug use, the inventors conducted a mouse survival experiment. The experiment lasted until the 70th day (as shown in Figure 2). Figure 15(As shown in Figure B) Six out of eight mice in the ADC HE-S2 group survived, and notably, the tumors in the six surviving mice were nearly undetectable. Four out of eight mice in the D18 and PD-L1 combination group survived, while one out of eight mice in the D18 group survived. All mice in the other two drug-alone groups and the negative control group died. This result demonstrates the potent anti-cancer activity of ADC HE-S2.

[0449] In addition, the inventors also used a B16 melanoma mouse model to further confirm the efficacy of ADC HE-S2 (e.g. Figure 15 C). The dosage and time of administration are also shown in Table 1. B16 melanoma is a tumor with low PD-L1 levels and a certain resistance to PD-1 / PD-L1 blockade therapy. As expected by the inventors, the inhibitory effects of various drugs on B16 melanoma were reduced compared with MC38 tumors. The efficacy of the three drugs, anti-PD-L1 antibody, anti-PD-L1 THIOMAB and D18, used alone was almost equivalent to that of the IgG isotype antibody. Only the combination treatment of anti-PD-L1 antibody + D18 and ADC HE-S2 still showed a certain tumor inhibitory effect. The growth rate of B16 tumors in mice treated with ADC HE-S2 was significantly slowed down compared with the combination treatment (P < 0.05). This result proves that in the B16 tumor model, the anti-tumor activity of ADC HE-S2 is also stronger than the combination treatment of anti-PD-L1 antibody and D18.

[0450] Table 1 Timetable for treatment of mice

[0451]

[0452]

[0453] Dosage: Antibody drugs, 150 μg per mouse per dose; D18, 25 μg per mouse per dose. - indicates no drug administration on that day. * Only the mice with the MC38 tumor model continued to the 24th day, while the mice with the B16 tumor model were no longer given the drug after the 21st day.

[0454] 2. ADC conjugation strategy expands the therapeutic window of D18

[0455] It is worth noting that since the DAR of ADC HE-S2 is 2 and the molecular weight is approximately 150K, it can be simply estimated that the D18 content in the ADC at each administration is 150μg / 150000×2×364=0.748μg, which is much lower than the dosage of the combination drug (25μg per mouse per administration). Therefore, the inventors hope to study the effect of varying the D18 dosage on the synergistic anticancer effect between D18 and anti-PD-L1 antibodies.

[0456] Therefore, the inventors used different doses of D18 (0.25 μg, 2.5 μg and 25 μg) in combination with 150 μg of anti-PD-L1 antibody to treat MC38 tumors (such as Figure 16 As shown). The mice were divided into 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)) according to the type of administration. When the average tumor volume of mice in each group reached 100 mm 3 The IgG isotype antibody and PD-L1 antibody were administered twice a week at 150 μg per mouse by intraperitoneal injection on D18. The mice were administered once a week at 25 μg, 2.5 μg, and 0.25 μg per mouse, respectively, on D18. The mice in the IgG isotype antibody group were intraperitoneally injected with the same dose of DMSO as on D18 as a control.

[0457] The anti-tumor results of gradient doses of D18 combined with anti-PD-L1 antibodies are as follows Figure 16 As shown. The experimental results show that D18 enhances the anti-tumor activity of anti-PD-L1 antibodies in a dose-dependent manner. When the dose of D18 per mouse was 25 μg, the combination therapy with anti-PD-L1 antibodies showed a more obvious tumor inhibitory activity. As the dose of D18 used decreased, the anti-tumor effect of the combination therapy of D18 and anti-PD-L1 antibodies continued to weaken. In fact, when the dose was reduced to 2.5 μg or 0.25 μg per mouse, its tumor inhibitory effect was no longer significantly different from that of the anti-PD-L1 antibody alone.

[0458] It is worth noting that the D18 dose (~0.75 μg) loaded into the ADC HE-S2 per administration is far less than 2.5 μg. Therefore, it can be concluded that through targeted ADC delivery, the inventors achieved a more significant tumor inhibition effect than the 25 μg D18 combined with anti-PD-L1 antibody therapy using a D18 dose (~0.75 μg) that is insufficient to be active in combination therapy.

[0459] Afterwards, the inventors tested the effects of each drug treatment on the body weight of mice in a mouse model to determine the toxic and side effects of the drugs used. The results are as follows: Figure 17 As shown. Healthy mice were divided into ADC group, anti-PD-L1 antibody + D18 group, anti-PD-L1 antibody group, D18 group, and IgG group according to the drug administration, with 5 mice in each group. The administration method and dosage of each group of mice were the same as those in the mouse tumor model experiment: the ADC group was administered with ADC HE-S2 once a week and an additional injection of anti-PD-L1 antibody, each of which was 150 μg; D18 was administered once a week, each of which was 25 μg; anti-PD-L1 antibody and IgG isotype antibody were administered twice a week, each of which was 150 μg.

[0460] The weight changes of mice in the anti-PD-L1 antibody group were almost the same as those in the blank control IgG group, demonstrating that the anti-PD-L1 antibody itself did not have significant toxic side effects on mice. After each injection of D18, the weight of mice in the anti-PD-L1 antibody + D18 group and the D18 group showed a certain degree of weight loss. Compared with D18 alone, the weight loss of mice in the anti-PD-L1 antibody + D18 group was significantly greater (p < 0.05). This result confirms the inventors' previous hypothesis that anti-PD-L1 antibodies, as immune checkpoint inhibitors, would amplify the toxic side effects of the TLR7 / 8 agonist D18. Although the ADC group also experienced a certain degree of weight loss after each injection of ADC HE-S2, the magnitude was significantly less than that of the D18 group (p < 0.05) and the combination drug group (p < 0.01). After administration, the weight of the mice quickly returned to normal, and ultimately, the weight change of mice in the ADC group was not significantly different from that in the control group. These results demonstrate that while the ADC HE-S2 also exhibits some toxicity, its toxicity is significantly lower than that of D18 alone or in combination, and it places less strain on the mice. The reduced amount of D18 actually loaded into the ADC is likely the primary reason for D18's reduced toxicity.

[0461] Ultimately, it can be concluded that ADC HE-S2, with its lower actual D18 dosage, exhibited superior tumor suppressive activity compared to combination therapy, while also significantly reducing the toxic side effects of the combination therapy. The inventors' proposed ADC strategy significantly reduced the minimum effective dose of D18 and expanded its therapeutic window.

[0462] 2. Anti-tumor experiments in mice with wild-type PD-L1 antibody-drug conjugate ADC10

[0463] MC38 cells (1x10 6 cells / mouse) were inoculated subcutaneously on the right side of the abdomen of mice. When the average tumor tissue volume of the mice reached 100 mm 3 Afterwards, the mice were randomly divided into groups according to the final type of drug administration: PBS control group and ADC 10 group, with 5-6 mice in each group. The drug was administered twice a week, with each mouse receiving an intraperitoneal injection of 10 mg / kg. The body weight of the mice was measured daily ( Figure 18 A), the tumor volume of mice was measured every 3 days ( Figure 18 B). Figure 18 As shown in B, the tumor inhibition rate of ADC was 70%.

[0464] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0465] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience, and preference of those skilled in the art.

[0466] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps. Sequence Listing <110> Tsinghua University Beijing Yufan Biotechnology Co., Ltd. <120> Antibody-drug conjugate and its preparation method and application <130> 1 <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 711 <212> DNA <213> Artificial Sequence <400> 1 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg tagtaccggt 60 caatccgcac tgactcaacc agccagcgtt agcggctccc ctggtcaatc tatcaccatc 120 agctgtaccg ggaccagctc agacgttggc ggttacaact acgtcagctg gtaccagcag 180 cacccgggta aagctccaaa gctgatgatt tatgatgtgt ctaatcgacc ttctggtgta 240 tctaaccgat tttcaggctc taaaagtgga aatactgctt ccctcacgat ctcagggctg 300 caagccgaag acgaagccga ttattattgt tctagctata catccagcag cacccgcgtg 360 tttggaacgg gaaccaaggt cacggttctg ggacagccca aagccaatcc taccgtcact 420 ctgttcccac ccagtagtga ggagctgcag gcaaataagg ctaccctggt ctgtcttata 480 tccgatttct atcccggggc agtcacagtc gcttggaagg cagatggctc tccagtgaag 540 gccggcgtcg aaacaactaa accttccaag cagtctaata acaagtacgc tgcttcttct 600 tacctttcac ttactcctga acaatggaag agccacagga gttactcttg tcaggtaacc 660 cacgaggggt ccactgtgga gaaaaccgtc gctcccacag agtgttcttg a 711 <210> 2 <211> 1413 <212> DNA <213> Artificial Sequence <400> 2 atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60 gaagtacagc tgcttgagag tggaggaggt ttggtacagc ccggcggatc cctccgcctg 120 tcctgtgcgg ctagtggctt tacattctca tcctatatca tgatgtgggt aagacaggcc 180 ccaggaaagg gcctggagtg ggttagttct atctacccct caggcgggat taccttctac 240 gcagatactg tgaagggcag gtttaccata tcccgagaca acagtaagaa taccctttac 300 cttcaaatga actcccttcg ggccgaggac actgcggtgt actattgcgc tcgcattaag 360 cttggcaccg tgacaaccgt gaactattgg ggtcaaggca cgctggtgac tgtctcttcc 420 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 480 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600 ggactctact ccctcagcag cgtggtgact gtgccctcta gcagcttggg cacccagacc 660 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgaaccc 720 aaatcttgcg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 780 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1140 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1320 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380 cagaagagcc tctccctgtc tccgggtaaa tga 1413 <210> 3 <211> 236 <212> PRT <213> Artificial Sequence <400> 3 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly 20 25 30 Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp 35 40 45 Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys 50 55 60 Ala Pro Lys Leu Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val 65 70 75 80 Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr 85 90 95 Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser 100 105 110 Tyr Thr Ser Ser Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr 115 120 125 Val Leu Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro 130 135 140 Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile 145 150 155 160 Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly 165 170 175 Ser Pro Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser 180 185 190 Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln 195 200 205 Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser 210 215 220 Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 235 <210> 4 <211> 470 <212> PRT <213> Artificial Sequence <400> 4 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 35 40 45 Phe Ser Ser Tyr Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Val Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr 65 70 75 80 Ala Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 85 90 95 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asn 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu[[ID=2,3]] 450 455 460 Ser Leu Ser Pro Gly Lys 4,65 470 <210> 5 <211> 4143 <212> DNA <213> Unknown <400> 5 ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg 60 agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa 120 Please note that some of the formatting might be a bit off due to the nature of the original text structure, but this is the closest translation while adhering to the rules.actgggaaag tgatgtcgtg tactggctcc gccttttttcc cgagggtggg ggagaaccgt 180 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac 240 agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 300 gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg 360 cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc 420 cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac 480 tctacgtctt tgtttcgttt tctgttctgc gccgttacag atccaagctg tgaccggcgc 540 ctacctgaga tcaccggcga aggagggcca ccatggagac agacacactc ctgctatggg 660. tactgctgct ctgggttcca ggtagtaccg gtcaatccgc actgactcaa ccagccagcg ttagcggctc ccctggtcaa tctatcacca tcagctgtac cgggaccagc tcagacgttg 720 gcggttacaa ctacgtcagc tggtaccagc agcaccggg taaagctcca aagctgatga 840. tttatgatgt gtctaatcga ccttctggtg tatctaaccg attttcaggc tctaaaagtg gaaatactgc ttccctcacg atctcagggc tgcaagccga agacgaagcc gattattatt 900 gttctagcta tacatccagc agcacccgcg tgtttggaac gggaaccaag gtcacggttc 960 tgggacagcc caaagccaat cctaccgtca ctctgttccc acccagtagt gaggagctgc 1020 aggcaaataa ggctaccctg gtctgtctta tatccgattt ctatcccggg gcagtcacag 1080 tcgcttggaa ggcagatggc tctccagtga aggccggcgt cgaaacaact aaaccttcca 1140 agcagtctaa taacaagtac gctgcttctt cttacctttc acttactcct gaacaatgga 1200 agagccacag gagttactct tgtcaggtaa cccacgaggg gtccactgtg gagaaaaccg 1260 tcgctcccac agagtgttct tgataagtgc tagctggcca gacatgataa gatacattga 1320 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg 1380 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa 1440 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta 1500 aaacctctac aaatgtggta tggaattaat tctaaaatac agcatagcaa aactttaacc 1560 tccaaatcaa gcctctactt gaatcctttt ctgagggatg aataaggcat aggcatcagg 1620 ggctgttgcc aatgtgcatt agctgtttgc agcctcacct tctttcatgg agtttaagat 1680 atagtgtatt ttcccaaggt ttgaactagc tcttcatttc tttatgtttt aaatgcactg 1740 acctcccaca ttcccttttt agtaaaatat tcagaaataa tttaaataca tcattgcaat 1800 gaaaataaat gttttttatt aggcagaatc cagatgctca aggcccttca taatatcccc 1860 cagtttagta gttggactta gggaacaaag gaacctttaa tagaaattgg acagcaagaa 1920 agcgagcttc tagcttatcc tcagtcctgc tcctctgcca caaagtgcac gcagttgccg 1980 gccgggtcgc gcagggcgaa ctcccgcccc cacggctgct cgccgatctc ggtcatggcc 2040 ggcccggagg cgtcccggaa gttcgtggac acgacctccg accactcggc gtacagctcg 2100 tccaggccgc gcacccacac ccaggccagg gtgttgtccg gcaccacctg gtcctggacc 2160 gcgctgatga acagggtcac gtcgtcccgg accacaccgg cgaagtcgtc ctccacgaag 2220 tcccgggaga acccgagccg gtcggtccag aactcgaccg ctccggcgac gtcgcgcgcg 2280 gtgagcaccg gaacggcact ggtcaacttg gccatgatgg ctcctcctgt caggagagga 2340 aagagaagaa ggttagtaca attgctatag tgagttgtat tatactatgc agatatacta 2400 tgccaatgat taattgtcaa actagggctg cagggttcat agtgccactt ttcctgcact 2460 gccccatctc ctgcccacccc tttcccaggc atagacagtc agtgacttac caaactcaca 2520 ggagggagaa ggcagaagct tgagacagac ccgcgggacc gccgaactgc gaggggacgt 2580 ggctagggcg gcttcttta tggtgcgccg gccctcggag gcagggcgct cggggaggcc 2640 tagcggccaa tctgcggtgg caggaggcgg ggccgaaggc cgtgcctgac caatccggag 2700 cacataggag tctcagcccc ccgccccaaa gcaaggggaa gtcacgcgcc tgtagcgcca 2760 gcgtgttgtg aaatgggggc ttgggggggt tggggccctg actagtcaaa acaaactccc 2820 attgacgtca atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc 2880 attgatgtac tgccaaaacc gcatcatcat ggtaatagcg atgactaata cgtagatgta 2940 ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccatttt 3000 accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa 3060 gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt 3120 tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc 3180 aggcgggcca tttaccgtaa gttatgtaac gcctgcaggt taattaagaa catgtgagca 3240 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 3300 ctccgccccc ctgacgagca tcaaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 3360 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 3420 ccgaccctgc cgcttaccgg atacctgtcc gccttctctcc cttcggggaag cgtggcgctt 3480 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 3540 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 3600 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 3660 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 3720 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 3780 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 3840 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 3900 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catggctagt 3960 taattaacat ttaaatcagc ggccgcaata aaatatcttt attttcatta catctgtgtg 4020 ttggtttttt gtgtgaatcg taactaacat acgctctcca tcaaaacaaa acgaaacaaa 4080[[ID=​​​​​​​​​​​​​​​​​​​​atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac 240 agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 300 gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg 360 cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc 420 cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac 480 tctacgtctt tgtttcgttt tctgttctgc gccgttacag atccaagctg tgaccggcgc 540 ctacctgaga tcaccggcga aggagggcca ccatgtacag gatgcaactc ctgtcttgca ttgcactaag tcttgcactt gtcacgaatt cggaagtaca gctgcttgag agtggaggag 660 gtttggtaca gcccggcgga tccctccgcc tgtcctgtgc ggctagtggc tttacattct 720 catcctatat catgatgtgg gtaagacagg ccccaggaaa gggcctggag tgggttagtt 780 ctatctaccc ctcaggcggg attaccttct acgcagatac tgtgaagggc aggtttacca 840 900. 900. 900. 900. 900. 900. 900. 900. 900 acactgcggt gtactattgc gctcgcatta agcttggcac cgtgacaacc gtgaactatt 960 ggggtcaagg cacgctggtg actgtctctt ccgcctccac caagggccca tcggtcttcc 1020 ccctggcacc ctcctccaag agcacctctg ggggcacagc ggccctgggc tgcctggtca 1080 aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg accagcggcg 1140 tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc agcgtggtga 1200 ctgtgccctc tagcagcttg ggcacccaga cctacatctg caacgtgaat cacaagccca 1260 gcaacaccaa ggtggacaag aaagttgaac ccaaatcttg cgacaaaact cacacatgcc 1320 caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc cccccaaaac 1380 ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga 1440 gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag gtgcataatg 1500 ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc agcgtcctca 1560 ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag 1620 ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc cgagaaccac 1680 aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc agcctgacct 1740 gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc aatgggcagc 1800 cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc ttcttctctct 1860 acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg 1920 tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg tctccgggta 1980 aatgataagt gctagctggc cagacatgat aagatacatt gatgagtttg gacaaaccac 2040 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 2100 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt 2160 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg 2220 tatggaatta attctaaaat acagcatagc aaaactttaa cctccaaatc aagcctctac 2280 ttgaatcctt ttctgaggga tgaataaggc ataggcatca ggggctgttg ccaatgtgca 2340 ttagctgttt gcagcctcac cttctttcat ggagtttaag atatagtgta ttttcccaag 2400 gtttgaacta gctcttcatt tctttatgtt ttaaatgcac tgacctccca cattcccttt 2460 ttagtaaaat attcagaaat aatttaaata catcattgca atgaaaataa atgtttttta 2520 ttaggcagaa tccagatgct caaggccctt cataatatcc cccagtttag tagttggact 2580 tagggaacaa aggaaccttt aatagaaatt ggacagcaag aaagcgagct tctagcttat 2640 cctcagtcct gctcctctgc cacaaagtgc acgcagttgc cggccgggtc gcgcagggcg 2700 aactcccgcc cccacggctg ctcgccgatc tcggtcatgg ccggcccgga ggcgtcccgg 2760 aagttcgtgg acacgacctc cgaccactcg gcgtacagct cgtccaggcc gcgcacccac 2820 acccaggcca gggtgttgtc cggcaccacc tggtcctgga ccgcgctgat gaacagggtc 2880 acgtcgtccc ggaccacacc ggcgaagtcg tcctccacga agtcccggga gaacccgagc 2940 cggtcggtcc agaactcgac cgctccggcg acgtcgcgcg cggtgagcac cggaacggca 3000 ctggtcaact tggccatgat ggctcctcct gtcaggagag gaaagagaag aaggttagta 3060 caattgctat agtgagttgt attatactat gcagatatac tatgccaatg attaattgtc 3120 aaactagggc tgcagggttc atagtgccac ttttcctgca ctgccccatc tcctgcccac 3180 cctttcccag gcatagacag tcagtgactt accaaactca caggagggag aaggcagaag 3240 cttgagacag acccgcggga ccgccgaact gcgaggggac gtggctaggg cggcttcttt 3300 tatggtgcgc cggccctcgg aggcagggcg ctcggggagg cctagcggcc aatctgcggt 3360 ggcaggaggc ggggccgaag gccgtgcctg accaatccgg agcacatagg agtctcagcc 3420 ccccgcccca aagcaagggg aagtcacgcg cctgtagcgc cagcgtgttg tgaaatgggg 3480 gcttgggggg gttggggccc tgactagtca aaacaaactc ccattgacgt caatggggtg 3540 gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa 3600 ccgcatcatc atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc 3660 cataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat 3720 agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa 3780 atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt 3840 cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt 3900 aagttatgta acgcctgcag gttaattaag aacatgtgag caaaaggcca gcaaaaggcc 3960 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 4020 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 4080 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 4140 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 4200 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 4260 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 4320 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 4380 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 4440 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 4500 tccggcaaac aaaccaccgc tggtagcggt ggttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatcttttt ctacggggtc tgacgctcag 4620 tggaacgaaa actcacgtta agggattttg gtcatggcta gttaattaac atttaaatca gcggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaat cgtaactaac atacgctctc catcaaaaca aaacgaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca ggtgccaga catttctcta tcgaa <210> 7 <211> 4143 <212> DNA <213> Description(Unknown) <400> 7 ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gccttttttcc cgagggtggg ggagaaccgt 180 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac 240 agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 300 gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg 360 cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc 420 cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac 480 tctacgtctt tgtttcgttt tctgttctgc gccgttacag atccaagctg tgaccggcgc 540 ctacctgaga tcaccggcga aggagggcca ccatggagac agacacactc ctgctatggg 660. tactgctgct ctgggttcca ggtagtaccg gtcaatccgc actgactcaa ccagccagcg ttagcggctc ccctggtcaa tctatcacca tcagctgtac cgggaccagc tcagacgttg 720 gcggttacaa ctacgtcagc tggtaccagc agcaccggg taaagctcca aagctgatga 840. tttatgatgt gtctaatcga ccttctggtg tatctaaccg attttcaggc tctaaaagtg gaaatactgc ttccctcacg atctcagggc tgcaagccga agacgaagcc gattattatt gttctagcta tacatccagc agcacccgcg tgtttggac gggaaccag gtcacggttc tgggacagcc caaagccaat cctaccgtca ctctgttccc acccagtagt gaggagctgc aggcaaataa ggctaccctg gtctgtctta tatccgattt ctatcccggg gcagtcacag 1080 tcgcttggaa ggcagatggc tctccagtga aggccggcgt cgaaacaact aaaccttcca 1140 agcagtctaa taacaagtac gctgcttctt cttacctttc acttactcct gaacaatgga 1200 agagccacag gagttactct tgtcaggtaa cccacgaggg gtccacttgc gagaaaaccg 1260 tcgctcccac agagtgttct tgataagtgc tagctggcca gacatgataa gatacattga 1320 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg 1380 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa 1440 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta 1500 aaacctctac aaatgtggta tggaattaat tctaaaatac agcatagcaa aactttaacc 1560 tccaaatcaa gcctctactt gaatcctttt ctgagggatg aataaggcat aggcatcagg 1620 ggctgttgcc aatgtgcatt agctgtttgc agcctcacct tctttcatgg agtttaagat 1680 atagtgtatt ttcccaaggt ttgaactagc tcttcatttc tttatgtttt aaatgcactg 1740 acctcccaca ttcccttttt agtaaaatat tcagaaataa tttaaataca tcattgcaat 1800 gaaaataaat gttttttatt aggcagaatc cagatgctca aggcccttca tatatcccc 1860 cagtttagta gttggactta gggaacaaag gaacctttaa tagaaattgg acagcaagaa 1920 agcgagcttc tagcttatcc tcagtcctgc tcctctgcca caaagtgcac gcagttgccg 1980 gccgggtcgc gcagggcgaa ctcccgcccc cacggctgct cgccgatctc ggtcatggcc 2040 ggcccggagg cgtcccggaa gttcgtggac acgacctccg accactcggc gtacagctcg 2100 tccaggccgc gcacccacac ccaggccagg gtgttgtccg gcaccacctg gtcctggacc 2160 gcgctgatga acagggtcac gtcgtcccgg accacaccgg cgaagtcgtc ctccacgaag 2220 tcccgggaga acccgagccg gtcggtccag aactcgaccg ctccggcgac gtcgcgcg 2280 gtgagcaccg gaacggcact ggtcaacttg gccatgatgg ctcctcctgt caggagagga 2340 aagagaagaa ggttagtaca attgctatag tgagttgtat tatactatgc agatatacta 2400 tgccaatgat taattgtcaa actagggctg cagggttcat agtgccactt ttcctgcact 2460 gccccatctc ctgcccacccc tttcccaggc atagacagtc agtgacttac caaactcaca 2520 ggagggagaa ggcagaagct tgagacagac ccgcgggacc gccgaactgc gaggggacgt 2580 ggctagggcg gcttcttta tggtgcgccg gccctcggag gcagggcgct cggggaggcc 2640 tagcggccaa tctgcggtgg caggaggcgg ggccgaaggc cgtgcctgac caatccggag 2700 cacataggag tctcagcccc ccgccccaaa gcaaggggaa gtcacgcgcc tgtagcgcca 2760 gcgtgttgtg aaatgggggc ttgggggggt tggggccctg actagtcaaa acaaactccc 2820 attgacgtca atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc 2880 attgatgtac tgccaaaacc gcatcatcat ggtaatagcg atgactaata cgtagatgta 2940 ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccatttt 3000 accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa 3060 gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt 3120 tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc 3180 aggcgggcca tttaccgtaa gttatgtaac gcctgcaggt taattaagaa catgtgagca 3240 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 3300 ctccgccccc ctgacgagca tcaaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 3360 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 3420 ccgaccctgc cgcttaccgg atacctgtcc gccttctctcc cttcggggaag cgtggcgctt 3480 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 3540 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 3600 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 3660 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 3720 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 3780 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg ttttttgtt 3840 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatcctttt gatctttct 3900 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catggctagt 3960 taattaacat ttaaatcagc ggccgcaata aaatatcttt attttcatta catctgtgtg 4020 ttggtttttt gtgtgaatcg taactaacat acgctctcca tcaaaacaaa acgaaacaaa 4080 acaaactagc aaaataggct gtccccagtg caagtgcagg tgccagaaca tttctctatc 4140 gaa 4143 <210> 8 <211> 711 <212> DNA <213> Artificial Sequence <400> 8 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg tagtaccggt 60 caatccgcac tgactcaacc agccagcgtt agcggctccc ctggtcaatc tatcaccatc 120 agctgtaccg ggaccagctc agacgttggc ggttacaact acgtcagctg gtaccagcag 180 cacccgggta aagctccaaa gctgatgatt tatgatgtgt ctaatcgacc ttctggtgta 240 tctaaccgat tttcaggctc taaaagtgga aatactgctt ccctcacgat ctcagggctg 300 caagccgaag acgaagccga ttattattgt tctagctata catccagcag cacccgcgtg 360 tttggaacgg gaaccaaggt cacggttctg ggacagccca aagccaatcc taccgtcact 420 ctgttcccac ccagtagtga ggagctgcag gcaaataagg ctaccctggt ctgtcttata 480 tccgatttct atcccggggc agtcacagtc gcttggaagg cagatggctc tccagtgaag 540 gccggcgtcg aaacaactaa accttccaag cagtctaata acaagtacgc tgcttcttct 600 tacctttcac ttactcctga acaatggaag agccacagga gttactcttg tcaggtaacc 660 cacgaggggt ccacttgcga gaaaaccgtc gctcccacag agtgttcttg a 711 <210> 9 <211> 236 <212> PRT <213> Artificial Sequence <400> 9 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly 20 25 30 0]Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp 35 40 45 Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys 50 55 60 Ala Pro Lys Leu Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val 65 70 75 80 Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr 85 90 95 Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser 100 105 110 Tyr Thr Ser Ser Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr 115 120 125 Val Leu Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro 130 135 140 Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile 145 150 155 160 Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly 165 170 175 Ser Pro Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser 180 185 190 Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln 195 200 205 Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser 210 215 220 Thr Cys Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 235

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has the following structure: （Ⅲ） in, Ab is an antibody or an antigen-binding fragment thereof; The antibody has a heavy chain sequence as shown in SEQ ID NO: 4 and a light chain sequence as shown in SEQ ID NO: 9; n is an integer from 1 to 10; The L part has the following structure: （Ⅴ） Wherein, Y is -S-; R L1 for ; R L2 -(CH2) i OCO-, i is an integer from 0 to 6; R a and R b Independently selected from: H, C1-C3 alkyl; R1 is selected from: a single bond, a C1-C6 alkylene group, a C1-C6 alkyleneoxy group; R4 and R5 are independently selected from C1-C6 alkyl; R2 is selected from: -NHR6, -OR6, -SR6, wherein R6 is C1-C6 alkyl or C1-C6 alkoxyalkyl; R3 is selected from: H, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxyalkyl; m is 0 or 1.

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 In the antibody-drug conjugate, the connection site between the antibody and L is the free thiol group on the cysteine ​​residue at position 226 of the amino acid sequence shown in SEQ ID NO:

9.

5. The antibody-drug conjugate according to claim 1, wherein R a and R b Independently selected from: H, methyl.

6. The antibody-drug conjugate according to claim 1, wherein R1 is -CH2-; or, R1 is -CH2O-, -CH2CH2O- or -CH2CH2CH2O-.

7. The antibody-drug conjugate according to claim 1, wherein R3 is selected from the group consisting of: H, methyl, methoxy.

8. The antibody-drug conjugate according to claim 1, wherein R4 and R5 are methyl groups.

9. The antibody-drug conjugate according to claim 1, wherein R2 is selected from: 、 、 、 .

10. The antibody-drug conjugate according to claim 1, wherein The D portion in the general formula I is selected from the following structures: 。 11. The antibody-drug conjugate according to claim 1, wherein The D portion in the general formula I is selected from the following structures: 。 12. The antibody-drug conjugate according to claim 1, wherein The D portion in the general formula I has the following structure: 。 13. The antibody-drug conjugate according to any one of claims 1 to 12, wherein: The L part has the following structure: 。 14. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that: The antibody drug conjugate has the following structure: in, Ab is an antibody or an antigen-binding fragment thereof, wherein the antibody has a heavy chain sequence as shown in SEQ ID NO: 4 and a light chain sequence as shown in SEQ ID NO: 9; n is 1-10.

15. The antibody-drug conjugate according to claim 14, wherein n is 2 or 4.

16. A modified anti-PD-L1 antibody, said antibody having a heavy chain sequence as shown in SEQ ID NO: 4 and a light chain sequence as shown in SEQ ID NO:

9.

17. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 15 and a pharmaceutically acceptable excipient.

18. Use of the antibody-drug conjugate according to any one of claims 1 to 15, or the antibody according to claim 16, in the preparation of a medicament for preventing and / or treating a disease; The disease is a tumor.

19. The use according to claim 18, characterized in that The tumor is selected from the group consisting of: lymphoma, blastoma, medulloblastoma, retinoblastoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumor, carcinoid tumor, gastrinoma, 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 tumors, head and neck cancer, and hematological malignancies.

20. The use according to claim 19, characterized in that The lung cancer is selected from the group consisting of small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung cancer, and squamous cell lung cancer.

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