WO2022237884A1 - 一种抗体药物偶联物及其制备方法和应用 - Google Patents

一种抗体药物偶联物及其制备方法和应用 Download PDF

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Publication number
WO2022237884A1
WO2022237884A1 PCT/CN2022/092556 CN2022092556W WO2022237884A1 WO 2022237884 A1 WO2022237884 A1 WO 2022237884A1 CN 2022092556 W CN2022092556 W CN 2022092556W WO 2022237884 A1 WO2022237884 A1 WO 2022237884A1
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Prior art keywords
antibody
alkyl
substituted
drug conjugate
cancer
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Ceased
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PCT/CN2022/092556
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English (en)
French (fr)
Inventor
廖学斌
王志松
贺磊
张岩
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Beijing Yufan Biotechnologies Co Ltd
Tsinghua University
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Beijing Yufan Biotechnologies Co Ltd
Tsinghua University
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Application filed by Beijing Yufan Biotechnologies Co Ltd, Tsinghua University filed Critical Beijing Yufan Biotechnologies Co Ltd
Priority to JP2023570204A priority Critical patent/JP7703050B2/ja
Priority to EP22806846.6A priority patent/EP4331616A4/en
Priority to US18/560,288 priority patent/US20240252670A1/en
Publication of WO2022237884A1 publication Critical patent/WO2022237884A1/zh
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Definitions

  • the present invention relates to the technical field of biochemical medicine, in particular to an antibody-drug conjugate (ADC), especially a conjugate of an antibody and an immunomodulator for immunotherapy, such as an anti-PD-L1 antibody and TLR7 And/or the conjugate of TLR8 agonist, and its pharmaceutical composition, preparation method and application.
  • ADC antibody-drug conjugate
  • Toll-like receptors are a relatively conservative receptor family in evolution, including at least 13 members, and 10 species (TLR1-10) are found in humans.
  • TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 are expressed on the cell surface and rapidly Recognize the products of bacterial metabolism.
  • TLR3, TLR7, TLR8, and TLR9 are expressed in cells, mainly for the monitoring and recognition of viral nucleic acids.
  • TLR3 recognizes double-stranded RNA
  • TLR7 and TLR8 recognize single-stranded RNA
  • TLR9 recognizes unmethylated CG coenzyme I, regulates the response of bacterial DNA and some viruses.
  • TLRs can specifically recognize pathogen-associated molecular patterns (PAMPs), play an important role in both innate immunity and acquired immunity, and serve as a bridge connecting innate immunity and acquired immunity.
  • PAMPs pathogen-associated molecular patterns
  • TLR7 after TLR7 recognizes the single-stranded RNA bound to the virus or the artificially synthesized small-molecule purine compound, it will recruit a specific adapter protein, activate a series of signal cascade reactions, initiate a high-level systemic adaptive immune response, and kill the infected virus cells, thereby completely eradicating the virus.
  • TLR7 agonists have been used to treat chronic viral infectious diseases, such as hepatitis B and hepatitis C.
  • TLR7 agonists can induce more rapid and effective immune protection as influenza vaccine adjuvants.
  • TLR7 agonists can not only directly stimulate pDC to secrete IFN- ⁇ , but also enhance the co-stimulation and antigen presentation ability of pDC, the activated pDC promotes the proliferation of CD4+ T cells, further activates CD8+ T cells, and kills Tumor cells, so the role of TLR7 agonists as immune adjuvants in the process of body recognition and killing tumors is also gradually getting attention.
  • ADC antibody-drug conjugate
  • an immunomodulator for immunotherapy such as a A conjugate of an anti-PD-L1 antibody and a TLR7 and/or TLR8 agonist
  • the above-mentioned antibody-drug conjugate has the following structure:
  • Ab is an antibody or an antigen-binding fragment thereof
  • D is a small molecule drug
  • L is the connecting unit connecting Ab and D;
  • n is an integer of 1-100.
  • the above-mentioned antibodies are monoclonal antibodies.
  • the forms of the above-mentioned antibodies may be, for example, chimeric antibodies, humanized antibodies, fully human antibodies and the like.
  • the above-mentioned antigen-binding fragments include Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, single-chain Fv (scFv), disulfide-bonded Fv (sdFv), fragments containing CDRs or Isolated CDRs, etc.
  • the above-mentioned antibodies are reactive to antigens or epitopes related to tumors, infectious microorganisms, or autoimmune diseases; specifically, the antigens targeted by the above-mentioned 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
  • MIF macrophage migration inhibitory factor
  • the above-mentioned antibody is an anti-PD-L1 antibody.
  • the above-mentioned antibodies comprise reactive groups (e.g., sulfhydryl, amino, carboxyl, amide, halogen, ester, acid halide, anhydride, epoxy, maleimide, aminooxy, azide base, alkynyl,
  • 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 cycloalkyl Alkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl), which can carry these reactive groups itself, or through mutation (such as site-directed mutagenesis), the known antibody
  • One or more amino acid residues are mutated to amino acids (including natural amino acids and unnatural amino acids, etc.) containing the same or different desired reactive groups.
  • the above-mentioned antibody is a modified anti-PD-L1 antibody, which is a modified anti-PD-L1 antibody (for example, atezolizumab (Atezolizumab), durvalumab) through genetic engineering.
  • a modified anti-PD-L1 antibody for example, atezolizumab (Atezolizumab), durvalumab
  • the above-mentioned antibody comprises a heavy chain and a light chain
  • amino acid sequence of the heavy chain is as shown in SEQ ID NO: 4, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences; and/or,
  • amino acid sequence of the light chain is as shown in SEQ ID NO: 9, or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO: 9 , 97%, 98%, 99% identical amino acid sequences.
  • the above-mentioned antibody has a light chain sequence as shown in SEQ ID NO:9.
  • the above 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 i.e. drug/antibody ratio (DAR)
  • DAR drug/antibody ratio
  • the linking site between the antibody and L is the free sulfhydryl group on the cysteine residue in the amino acid sequence of the antibody.
  • the antibody in the above-mentioned antibody-drug conjugate, 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 , especially 1-6.
  • the above-mentioned small molecule drug can be an immunomodulator, for example, a Toll-like receptor agonist (Toll-like receptors, TLR), specifically, a TLR7 and/or TLR8 agonist, such as in the patent application publication number WO2019/095455A1
  • TLR Toll-like receptors
  • TLR7 and/or TLR8 agonist such as in the patent application publication number WO2019/095455A1
  • TLR7 and/or TLR8 agonist such as in the patent application publication number WO2019/095455A1
  • WO2019/095455A1 The pyridopyrimidine derivatives and salts thereof.
  • part D in general formula I has the following structure:
  • L' is a linking group, which is selected from: single bond or C 1 -C 6 alkylene, C 1 -C 6 alkenylene, C 3 -C 6 cycloalkylene, wherein the groups can be any optionally substituted by C 1 -C 4 alkyl;
  • R 1 is selected from: single bond, C 1 -C 6 alkylene, C 1 -C 6 alkenylene, C 1 -C 6 alkyleneoxy, wherein said groups can be optionally replaced by C 1 - C 4 alkyl substitution;
  • X is selected from: -NR 4 -, -O-, -S-, single bond, -OCO-, -COO-, -NR 4 -(C 1 -C 6 alkylene)-NR 5 -, heterocyclylene (especially Nitrogen-containing heterocyclic group), wherein, R 4 and R 5 are independently selected from: H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, amino acid residue, oligopeptide residue, haloalkyl , carboxyl substituted alkyl, ester substituted alkyl, Wherein, a is an integer of 1-10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), R 8 and R 9 are independently selected from: H, C 1 -C 6 alkane group, or R8 and R9 together with the atoms in between form Or R 4 and R 5 form a heterocyclic group together with the nitrogen atom to which they are connected;
  • R 2 is selected from: -NR 6 R 7 , -OR 6 , -SR 6 , wherein R 6 and R 7 are independently selected from: H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkoxyalkyl, or R 6 and R 7 form a heterocyclic group together with the nitrogen atom to which they are attached, or R 6 forms a heterocyclic group with the oxygen atom to which they are attached, or R 6 forms a heterocyclic group with the oxygen atom to which they are attached The attached sulfur atoms together form a heterocyclic group;
  • R 3 is one or more independent substituents on the benzene ring, which are selected from: H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkoxyalkyl;
  • n is an integer of 0-4 (for example, 0, 1, 2, 3, 4).
  • formula II has the following structure:
  • the aforementioned L' is a C 1 -C 6 alkylene group, such as a C 1 -C 3 alkylene group.
  • the above-mentioned L' has the following structure:
  • R a and R b are independently selected from: H, C 1 -C 3 alkyl (such as methyl, ethyl, n-propyl, isopropyl), or, R a and R b are connected to the carbon
  • the atoms together form a C 3 -C 6 cycloalkylene (eg cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene).
  • L' can be selected from: -CH 2 -,
  • R 1 is C 1 -C 6 alkylene, such as C 1 -C 3 alkylene, such as -CH 2 -.
  • the above-mentioned R 1 is a single bond.
  • R 1 is C 1 -C 6 alkyleneoxy, such as C 1 -C 3 alkyleneoxy, such as -CH 2 O-, -CH 2 CH 2 O-, - CH2CH2CH2O- . _
  • R 3 is one or more independent substituents on the benzene ring, which are selected from: H, methyl, and methoxy.
  • m is 0 or 1.
  • a is an integer of 1-5, such as 1, 2, 3, 4, 5.
  • R 8 and R 9 are independently selected from: H, methyl, ethyl, n-propyl, isopropyl.
  • R4 and R5 are independently selected from: H, methyl, ethyl, n - propyl, isopropyl, n - butyl, tert-butyl, amino acid residues, oligopeptide residues, -CF3 , -CH 2 CF 3 , Or R 4 and R 5 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocyclic group.
  • the above-mentioned substituted or unsubstituted heterocyclic groups can be selected from:
  • the above-mentioned amino acid residues and amino acid residues in the oligopeptide are independently selected from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine one or more of the residues.
  • the above-mentioned amino acid residues are leucine residues.
  • the above oligopeptide residues are -asparagine-alanine-alanine-leucine-.
  • X is R 4 and R 5 are independently selected from C 1 -C 6 alkyl groups, for example, both R 4 and R 5 are methyl groups.
  • X is -NR 4 -, R 4 is selected from: H, C 1 -C 6 alkyl, for example, R 4 is H or methyl.
  • X is -NR 4 -(C 1 -C 6 alkylene)-NR 5 -, R 4 and R 5 are independently selected from C 1 -C 6 alkyl, for example, R 4 and R 5 are both methyl.
  • X is a heterocyclic group, especially a nitrogen-containing heterocyclic group, such as Ring A is a nitrogen-containing heterocyclic ring with 4 to 10 members, and ring A can be monocyclic, bicyclic or tricyclic (including fused ring, spiro ring, bridged ring); specifically, X can be
  • R 2 is selected from: -NHR 6 , -OR 6 , -SR 6 , wherein R 6 is C 1 -C 6 alkyl or C 1 -C 6 alkoxyalkyl, For example C 1 -C 4 alkyl, for example butyl, especially n-butyl, for example C 1 -C 4 alkoxyalkyl, for example methoxyethyl.
  • R 2 is selected from:
  • R 2 is selected from: -NR 6 R 7 , wherein, R 6 and R 7 form a heterocyclic group together with the nitrogen atom to which they are connected, for example,
  • part D in general formula I can be selected from the following structures:
  • part D in general formula I has the following structure:
  • the above-mentioned conjugate has the following structure:
  • the linking unit in the above-mentioned conjugate can be a chemically unstable linking unit (such as comprising a hydrazone, a disulfide group), an enzyme-catalyzed linking unit (such as comprising a peptide residue, a carbonate residue unstable to esterase ) and other forms.
  • a chemically unstable linking unit such as comprising a hydrazone, a disulfide group
  • an enzyme-catalyzed linking unit such as comprising a peptide residue, a carbonate residue unstable to esterase
  • the linking unit L linking Ab and D comprises a linking group Y, such as a single bond, -S-, -CO-, -NH-, -CONH-, (aminooxy);
  • Y is -S- or
  • L also contains a group Q
  • Q is a divalent saturated or unsaturated linear or branched C1-50 hydrocarbon chain, wherein 0-6 methylene units are independently substituted by the following: -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-, Amino acid residues, oligopeptide residues, wherein, k is selected from an integer between 1-10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), and each -Cy- is independently R is an optionally substituted divalent ring selected from
  • each -Cy- is independently an optionally substituted divalent ring selected from the group consisting of phenylene, bicyclic arylene, monocyclic cycloalkylene, bicyclic cycloalkylene, monocyclic Heteroaryl, bicyclic heteroarylene, monocyclic heterocycloalkylene, bicyclic heterocycloalkylene; especially, phenylene, monocyclic cycloalkylene, monocyclic heteroarylene, monocyclic heterocyclylene Cycloalkyl.
  • each -Cy- can be independently selected from the following:
  • R 11 is one or more independent substituents on the ring, and is selected from: H, halogen, -CN, -NO 2 , -CF 3 , -OCF 3 , -NH 2 , -OH, C 1 -C 6 alkyl, -O(C 1 -C 6 alkyl), -NH(C 1 -C 6 alkyl), -N(C 1 -C 6 alkyl)(C 1 -C 6 alkyl), mono
  • R 12 is selected from: H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkyl, Substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted heterocyclyl, substituted or
  • the monosaccharide or its derivatives can be selected from: arabinose, xylose, ribose, glucose, mannose, galactose, fructose, glucuronic acid, galacturonic acid.
  • the residues of monosaccharides or derivatives thereof are galacturonic acid residues, such as
  • each -Cy- is independently selected from the following:
  • R 10 may be selected from: H, C 1 -C 6 alkyl; in some embodiments of the present invention, R 10 is H or methyl.
  • amino acid residues and amino acid residues in oligopeptide residues are independently selected from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid Aminoamide, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, One or more of tyrosine, valine, citrulline, ornithine residues, especially selected from: valine, citrulline, alanine, asparagine, aspartic acid , glutamic acid, proline, glycine residues; in particular, the amino acid residues and the amino acid residues in the oligopeptide residues are L-type amino acid residues.
  • the oligopeptide residues consist of 2-10 amino acid residues, especially 2-6 amino acid residues, such as 2, 3, 4 or 5 amino acid residues.
  • the oligopeptide residues are selected from the group consisting of: valine-citrulline residues, alanine-alanine-asparagine residues, aspartic acid-valine residues , glutamic acid-valine residues, glycine-proline residues, etc.
  • part L in general formula I has the following structure:
  • Y is the linking group with the active group of Ab (such as sulfhydryl, amino, carboxyl, etc., especially sulfhydryl);
  • a combination of one or more of cycloalkylene and arylene, j is an integer of 0-10;
  • R L2 is selected from: single bond, -(CH 2 ) i OCO-, -(CH 2 ) i OCOO-, -(CH 2 ) i NH-COO-, amino acid residue, oligopeptide residue, wherein i is 0 Integer of -10;
  • R L5 , R L6 , R L7 and R L8 are 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 , wherein t is 0, 1 or 2;
  • Each R L9 and R L10 are independently selected from: H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted Substituted cycloalkylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl.
  • Y is selected from: a single bond, -S-, -CO-, -NH-, -CONH-, (aminooxy). In some embodiments of the present invention, Y is -S- or
  • amino acid residues and amino acid residues in oligopeptide residues are independently selected from: alanine, arginine, asparagine, aspartic acid, cysteine, Glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan , tyrosine, valine, citrulline, ornithine residues in one or more.
  • amino acid residues and amino acid residues in oligopeptide residues are L-type amino acid residues.
  • the oligopeptide residue consists of 2-10 amino acid residues, especially 2-6 amino acid residues, for example, 2, 3, 4 or 5 amino acid residues .
  • the oligopeptide residues can be selected from: valine-citrulline residues, alanine-alanine-asparagine residues, aspartic acid-valine residues residues, glutamate-valine residues, glycine-proline residues, etc.
  • R L2 is -(CH 2 ) i OCO-, i is an integer of 0-6, especially i is 1.
  • R L2 is -(CH 2 ) i , i is an integer of 1-10, especially i is 2.
  • R L2 is an oligopeptide residue selected from: valine-citrulline residues, alanine-alanine-asparagine residues, aspartic acid-valine residues amino acid residues, glutamic acid-valine residues, etc., especially for
  • R L1 , R L3 and R L4 are independently selected from: -(CH 2 ) j -, -(CH 2 ) j O-, -(CH 2 ) j NH-, -(CH 2 ) j CO- , -(CH 2 ) j OCOO-, -(CH 2 ) j OCONH-, -(CH 2 ) j NHCONH-, -(CH 2 ) j NHCO-, -O(CH 2 ) j COO-, -(CH 2 )
  • a combination of one or more of j COO- and -(CH 2 ) j CONH-, j is an integer of 0-10.
  • each j mentioned above can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 independently.
  • R L1 is
  • R L1 is -(CH 2 ) j CO-, j is an integer of 0-6, especially j is 5.
  • R L3 is -(CH 2 ) j NH-, j is an integer of 0-6, especially j is 0.
  • R L4 is -(CH 2 ) j -, j is an integer of 0-5, especially j is 1.
  • R L5 , R L6 , R L7 and R L8 are independently selected from: H, C1-6 alkyl, C1-6 haloalkyl, halogen, nitro, cyano, -OR L9 ;
  • R L8 is selected from: H, C1-6 alkyl; more specifically, R L5 , R L6 , R L7 and R L8 are independently selected from: H, halogen.
  • R L5 is H.
  • RL6 is H.
  • RL7 is H.
  • RL8 is H.
  • the above-mentioned part L has the following structure:
  • Y, R L1 and R L2 have the above-mentioned corresponding definitions in the present invention.
  • the above-mentioned part L may have the following structure:
  • the above-mentioned part L may also have the following structure:
  • R L2 is an oligopeptide residue, which has the above-mentioned definition of the present invention.
  • the above-mentioned part L has the following structure:
  • part L has the following structure:
  • k 1 and k 2 are independently selected from integers between 1-10.
  • the above-mentioned conjugate has the following structure:
  • the above-mentioned conjugate has the following structure:
  • the present invention also provides a stereoisomer (shown above) or a mixture thereof of the above-mentioned conjugate.
  • the present invention also provides a pharmaceutically acceptable salt, solvate and prodrug of the above conjugate.
  • the above-mentioned pharmaceutically acceptable salts may include organic or inorganic salts, for example, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetic acid Salt, pantothenate, succinate, citrate, tartrate, fumarate, maleate, gluconate, glucuronate, sugar, benzoate, lactic acid Salt, mesylate, ethanesulfonate, besylate, p-toluenesulfonate, arginate, aspartate, glutamate, pantothenate and ascorbate or Various.
  • organic or inorganic salts for example, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetic acid Salt, pantothenate, succinate, citrate, tartrate, fumarate, maleate, gluconate, glucuron
  • the present invention also provides a method for preparing the conjugate, which may include the step of coupling the conjugate (L-D) of the small molecule drug and the linking unit to the antibody.
  • the above preparation method further includes the steps of reducing and oxidizing the disulfide bond of the antibody.
  • the above-mentioned preparation method comprises the following steps:
  • step (2) incubating the antibody obtained in step (1) with an oxidizing agent
  • step (1) may also include removing excess disulfide bond reducing agent (eg, by ultrafiltration).
  • the disulfide bond reducing agent in step (1) may be tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), ⁇ -mercaptoethanol and the like.
  • the equivalent ratio of the antibody to the disulfide bond reducing agent is 1:8-15 (specifically such as 1:8, 1:9, 1:10, 1:11, 1:12).
  • the incubation temperature in step (1) is 35-40°C (specifically such as 35, 36, 37, 38, 39, 40°C); in one embodiment of the present invention, the incubation temperature is 37°C.
  • the incubation time in step (1) is 0.5-6 hours (specifically as 0.5, 1, 2, 3, 4, 5, 6 hours); in one embodiment of the present invention, the incubation time is 3 Hour.
  • step (2) may also include removing excess oxidizing agent (eg, by ultrafiltration).
  • the oxidizing agent in step (2) may be dehydroascorbic acid (DHAA), Cu(II) and the like.
  • the equivalent ratio of antibody to oxidant is 1:40-60 (specifically, such as 1:40, 1:45, 1:50, 1:55, 1:60).
  • the incubation temperature in step (2) is 20-30° C. (specifically such as 20, 22, 24, 25, 26, 28, 30° C.); in one embodiment of the present invention, the incubation temperature is room temperature .
  • the incubation time in step (2) is 1-6 hours (specifically such as 1, 2, 3, 4, 5, 6 hours); in one embodiment of the present invention, the incubation time is 3 hours.
  • the equivalent ratio of the antibody to the small molecule L-D is 1:10-20 (specifically such as 1:10, 1:12, 1:14, 1:15, 1:16, 1:18, 1:20).
  • the incubation temperature in step (3) is 20-30° C. (specifically such as 20, 22, 24, 25, 26, 28, 30° C.); in one embodiment of the present invention, the incubation temperature is room temperature .
  • the incubation time in step (3) is 6-48 hours (specifically such as 6, 12, 18, 20, 22, 24, 26, 28, 30, 36, 42, 48 hours); In one embodiment, the incubation time is 24 hours.
  • the above preparation method further includes the step of preparing the conjugate (L-D) of the small molecule drug and the linking unit.
  • the present invention also provides a modified anti-PD-L1 antibody, which combines known anti-PD-L1 antibodies (for example, Atezolizumab, Durvalumab) by genetic engineering means (such as Thiomab technology).
  • known anti-PD-L1 antibodies for example, Atezolizumab, Durvalumab
  • genetic engineering means such as Thiomab technology.
  • One or more amino acid residues of (Durvalumab, Avelumab, Cemiplimab, KN035, CS1001, BGB-A333, KL-A167, SHR-1316 and STI-A1014, etc.)
  • the bases are respectively mutated to cysteine, for example, amino acids in the heavy chain and/or light chain of avelumab are mutated to cysteine.
  • the above-mentioned antibody comprises a heavy chain and a light chain
  • amino acid sequence of the heavy chain is as shown in SEQ ID NO: 4, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences; and/or,
  • amino acid sequence of the light chain is as shown in SEQ ID NO: 9, or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO: 9 , 97%, 98%, 99% identical amino acid sequences.
  • the above-mentioned antibody has a light chain sequence as shown in SEQ ID NO:9.
  • the above 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.
  • the present invention also provides an application of the above-mentioned modified anti-PD-L1 antibody in the preparation of an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the present invention also provides a small molecular compound and pharmaceutically acceptable salts, stereoisomers, solvates, and prodrugs thereof.
  • the compound has the following structure:
  • Z is an active group.
  • the small molecular compound has the following structure:
  • m, X, L', R 1 , R 2 , R 3 , R L1 , R L2 , R L3 , R L4 , R L5 , R L6 , R L7 , and R L8 have the above-mentioned corresponding definitions of the present invention
  • Z is an active group.
  • Z is a reactive linking group with an amino acid reactive group (such as sulfhydryl, amino, carboxyl, etc., especially sulfhydryl); specifically, for example, the sulfhydryl reactive group can be maleimide, carboxyl, etc. , amide, halogen, disulfide, etc.; the amino reactive group can be ester group, acid halide, acid anhydride, carboxyl, epoxy, etc.; the carboxylamino reactive group can be hydroxyl, amino, halogen, acid halide basic
  • Z is
  • Z, R L1 , R L2 , R L3 , R L4 , R 1 , R 2 , R 3 , R 4 , R 5 , R a , R b , and m have the above-mentioned corresponding definitions in the present invention.
  • Z, R L1 , R L2 , R 1 , R 2 , R 3 , R 4 , R 5 , R a , R b , and m have the corresponding definitions above in the present invention.
  • Z, R L1 , and R L2 have the above-mentioned corresponding definitions of the present invention.
  • the compound represented by formula VII has the following structure:
  • the present invention also provides a compound and its pharmaceutically acceptable salts and stereoisomers, which can be used as linking unit parts of antibody-drug conjugates, which have the following structure:
  • Z, R L1 , R L2 , R L3 , R L4 , R L5 , R L6 , R L7 , and R L8 have the above-mentioned corresponding definitions of the present invention, and Z' is a reactive group.
  • Z' may be halogen (such as chlorine), carboxyl, ester, hydroxyl, epoxy, amine, etc.
  • the compound shown in formula IX has the following structure:
  • Z, R L1 , R L2 , and Z' have the above-mentioned corresponding definitions in the present invention.
  • the compound shown in formula IX has the following structure:
  • the aforementioned Z' is halogen (such as chlorine).
  • the above-mentioned Z' is a carbonate group (such as
  • the present invention also provides an application of the compound represented by the above formula IX in the preparation of an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the present invention also provides a pharmaceutical composition comprising the above-mentioned conjugate and pharmaceutically acceptable auxiliary materials.
  • the above-mentioned pharmaceutically acceptable auxiliary materials are conventional pharmaceutical auxiliary materials in the pharmaceutical field, for example: diluents, excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginate, Gelatin and polyvinylpyrrolidone, etc.; wetting agents such as glycerin, etc.; disintegrants such as agar, calcium carbonate and sodium bicarbonate, etc.; absorption enhancers such as quaternary ammonium compounds, etc.; surfactants such as cetyl alcohol, etc.; adsorption carriers such as kaolin and bentonite, etc.; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, etc.
  • other adjuvants such as flavoring agents, sweeteners, stabilizers, etc. can also be added to the pharmaceutical composition.
  • the above pharmaceutical composition will contain about 1 to about 99% by weight (specifically such as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%) of the above-mentioned conjugates of the present invention, and the rest are suitable auxiliary materials.
  • the above-mentioned pharmaceutical composition can be administered by any route of administration, which can be oral or parenteral administration, such as administration via lung, nose, rectum and/or intravenous injection.
  • routes of administration which can be oral or parenteral administration, such as administration via lung, nose, rectum and/or intravenous injection.
  • the formulations according to the invention may be suitable for local or systemic administration, in particular for dermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, pulmonary, buccal, sublingual, nasal, percutaneous, vaginal, oral or parenteral administration.
  • the preferred form for rectal administration is a suppository.
  • the dosage forms suitable for oral administration are tablets, pills, chewable gums, capsules, granules, drops or syrups and the like.
  • the dosage forms suitable for parenteral administration are solutions, suspensions, reconstitutable dry preparations or sprays and the like.
  • compositions of the present invention may be formulated for transdermal administration as deposits or patches in dissolved form.
  • Skin applications include ointments, gels, creams, lotions, suspensions or emulsions, and the like.
  • compositions of the present invention can be prepared according to conventional production methods in the field of pharmacy, for example, the active ingredient is mixed with one or more auxiliary materials, and then it is made into the required dosage form.
  • the above-mentioned conjugate of the present invention can be used as the only active ingredient, and can also be used in combination with one or more other active ingredients for the same indication, wherein the above-mentioned conjugate of the present invention It can be formulated for simultaneous, separate or sequential administration with the other active ingredient.
  • the present invention also provides a composition comprising the antibody and the small molecule drug of the present invention as active ingredients.
  • the antibody is an anti-PD-L1 antibody, for example, Atezolizumab, Durvalumab, Avelumab, Simiprimumab (Cemiplimab), KN035, CS1001, BGB-A333, KL-A167, SHR-1316 and STI-A1014, etc., and one or more known anti-PD-L1 antibodies (as described above) by genetic engineering means
  • Modified anti-PD-L1 antibodies obtained by mutating amino acid residues to cysteine, for example, mutating amino acids in the heavy chain and/or light chain of avelumab to cysteine.
  • the above-mentioned antibody comprises a heavy chain and a light chain
  • amino acid sequence of the heavy chain is as shown in SEQ ID NO: 4, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences; and/or,
  • amino acid sequence of the light chain is as shown in SEQ ID NO: 9, or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO: 9 , 97%, 98%, 99% identical amino acid sequences.
  • the above-mentioned antibody has a light chain sequence as shown in SEQ ID NO: 9
  • the above 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.
  • the small molecule drug can be an immunomodulator, for example, a Toll-like receptor agonist (Toll-like receptors, TLR), specifically, a TLR7 and/or TLR8 agonist, for example, Patent Application Publication No. Pyridopyrimidine derivatives and salts thereof described in WO2019/095455A1.
  • TLR Toll-like receptors
  • TLR7 and/or TLR8 agonist for example, Patent Application Publication No. Pyridopyrimidine derivatives and salts thereof described in WO2019/095455A1.
  • the small molecule drug in the above composition has the following structure:
  • R 1 , R 2 , R 3 , m, and L' have the above-mentioned corresponding definitions of the present invention
  • X' is selected from: H, halogen, -NR 4 R 5 , -N + R 4 R 5 , -OR 4 , -SR 4 , azido, phosphonic acid, phosphonic acid diethyl ester, haloalkyl, carboxyl , ester group, -CN; wherein, R 4 and R 5 are independently selected from: H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, amino acid residue, oligopeptide residue, haloalkyl, Or R 4 and R 5 form a heterocyclic group together with the nitrogen atom to which they are connected.
  • the small molecule drug in the above composition can be selected from the following structures:
  • the antibody in the above composition, is avelumab (Avelumab), and the small molecule drug has the following structure:
  • the present invention also provides 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 for preventing and/or treating Application in the medicine of disease.
  • the above-mentioned diseases may be diseases related to PD-L1 expression, or diseases related to TLR7 and/or TLR8 activity.
  • the aforementioned diseases may be one or more of respiratory diseases, immune diseases, viral diseases, and tumors.
  • the aforementioned respiratory diseases include, but are not limited to: asthma, chronic obstructive pulmonary disease, and adult respiratory distress syndrome.
  • the above immune diseases are autoimmune diseases, including but not limited to: systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, Sjogren's syndrome, polymyositis, vasculitis, Gerner's granulomatosis, sarcoidosis, ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis, Behcet's syndrome, etc.
  • autoimmune diseases including but not limited to: systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, Sjogren's syndrome, polymyositis, vasculitis, Gerner's granulomatosis, sarcoidosis, ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis, Behcet's syndrome, etc.
  • the pathogens of the above-mentioned viral diseases include but are not limited to: Adenoviridae (such as adenovirus), Herpesviridae (such as HSV1 (oral herpes), HSV2 (genital herpes), VZV (varicella), EBV (Estrogen- Bartholin virus), CMV (cytomegalovirus), poxviridae (eg, variola virus, vaccinia virus), papovavirus family (eg, papillomavirus), parvoviridae (eg, B19 virus), hepadnavirus Viridae (such as hepatitis B virus), polyomaviridae (such as polyomavirus), reoviridae (such as reovirus, rotavirus), picornaviridae (such as enterovirus, foot-and-mouth disease Viruses), Caliciviridae (such as Norwalk virus, Hepatitis E virus), Togaviridae
  • the above-mentioned tumors are malignant tumors, including but not limited to: lymphoma, blastoma, medulloblastoma, retinoblastoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumors, carcinoid tumors, gastric Melanoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancy, squamous cell carcinoma, epithelial squamous cell carcinoma, lung (small cell lung cancer) , non-small cell lung cancer, adenocarcinoma lung cancer, lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer, bowel cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder
  • the present invention also provides a disease prevention and/or treatment method, which comprises administering a therapeutically effective amount of the above-mentioned conjugate of the present invention or a pharmaceutically acceptable salt, solvate, prodrug, Stereoisomers, the above-mentioned antibody of the present invention, the compound represented by formula VII, and the steps of the composition.
  • the disease in the above-mentioned method has the above-mentioned definition of the present invention.
  • the present invention also provides a method for improving the body's positive immune response, which includes administering a therapeutically effective amount of the above-mentioned conjugate of the present invention or its pharmaceutically acceptable salt, solvate, prodrug, Stereoisomers, the above-mentioned antibody of the present invention, the compound represented by formula VII, and the steps of the composition.
  • the present invention also provides a method for enhancing the effect of chemotherapy, which comprises administering a therapeutically effective amount of the above-mentioned conjugate of the present invention or its pharmaceutically acceptable salt, solvate, prodrug, stereoisomer Body, the above-mentioned antibody of the present invention, the compound represented by formula VII, and the steps of composition.
  • the present invention also provides a method for improving the effect of immunotherapy, which comprises administering a therapeutically effective amount of the above-mentioned conjugate of the present invention or its pharmaceutically acceptable salt, solvate, prodrug, stereoisomeric compound to a system or individual in need.
  • a method for improving the effect of immunotherapy comprises administering a therapeutically effective amount of the above-mentioned conjugate of the present invention or its pharmaceutically acceptable salt, solvate, prodrug, stereoisomeric compound to a system or individual in need.
  • the above-mentioned therapeutically effective amount may vary according to factors such as administration route, patient's age, body weight, sex, type and severity of the disease to be treated, and the present invention is not specifically limited thereto.
  • 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 uses it for the construction of ADC. It was verified by experiments that the modified antibody and the resulting ADC maintain the antigen recognition affinity of the original anti-PD-L1 antibody, and can be internalized into cells by binding to cell surface PD-L1, with good selectivity. Through anti-tumor experiments, it was found that the obtained ADC has strong anti-tumor activity, can significantly improve the survival rate of tumor-bearing animals, and its toxicity is significantly lower than that of small molecule drugs administered alone and in combination with antibodies. The burden on the animal's body is less.
  • the ADC of 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 various diseases (such as tumors, viral diseases such as hepatitis B, etc.), and has good application prospects and value.
  • Figure 1 shows the sequencing results of plasmid ⁇ PD-L1 THIOMAB LC-V205C, in which, the sequence comparison of A. ⁇ PD-L1 THIOMAB LC-V205C sequencing sequence (top) and template light chain 159-pFuse- ⁇ PDL1-LC plasmid (bottom) Yes, the GTG site on the template plasmid is mutated to TGC; B.
  • the amino acid encoded at the mutation site of ⁇ PD-L1 THIOMAB LC-V205C is cysteine (Cys);
  • C The amino acid encoded at the mutation site of the template plasmid It is valine (Val).
  • Figure 2 shows the expression results of anti-PD-L1 antibodies identified by reducing and non-reducing SDS PAGE analysis.
  • Figure 3 shows the results of identifying the expression of anti-PD-L1 THIOMAB by reducing and non-reducing SDS PAGE analysis.
  • Figure 4 shows the mass spectrometry results of THIO-S2.
  • Figure 5 shows the coupling route of ADC HE-S2.
  • Figure 6 shows the mass spectrometry detection results of ADC HE-S2.
  • Figure 7 shows the coupling route of ADC VC.
  • Figure 8 shows the coupling route of ADC Legumain.
  • Figure 9 shows the mass spectrometric detection results of ADC VC and ADC Legumain.
  • Figure 10 shows the characterization results 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, they were stained with FITC-labeled goat anti-human secondary antibody, and the FITC signal intensity on the surface of MC38 was detected and analyzed by FACS technology.
  • A. The histograms of different colors represent the fluorescent signals measured by the negative control (NC), secondary antibody, anti-PD-L1 antibody, anti-PD-L1 THIOMAB and ADC HE-S2 from top to bottom .
  • B Mean fluorescence intensity (MFI) measured after treatment with different antibody drugs on MC38.
  • MFI Mean fluorescence intensity
  • NC group negative control with isotype antibody added only, without secondary antibody staining
  • secondary antibody group control group added with isotype antibody and stained with fluorescent secondary antibody.
  • the Student's t test was used to determine the significant difference between the groups, and **** indicates p ⁇ 0.0001.
  • Figure 11 shows the experimental results of the internalization of anti-PD-L1 THIOMAB into cancer cells.
  • MC38 and B16 cells were treated with pHrodo TM red dye-labeled anti-PD-L1 THIOMAB for 24 hours, A. used The living cell system collects high-definition phase images, red fluorescence images and combined images of cells; B. The histogram of the total red fluorescent object integrated intensity (total red object integrated intensity) obtained by software analysis, the ordinate data takes the logarithm of 10. Significant differences among groups were compared using Student's t test, **** indicates p ⁇ 0.0001.
  • Figure 12 shows the results of FACS detection 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.
  • Figure 13 shows the results of a preliminary experiment on the internalization of anti-PD-L1 THIOMAB in DC cells.
  • Figure 14 shows the experimental results of the internalization ability of the PD-L1/anti-PD-L1 antibody complex in DC cells.
  • Figure 15 shows the experimental results of verifying the anti-tumor activity of ADC HE-S2 through a mouse tumor model.
  • A Inhibitory activity of MC38 tumor growth in mice treated with ADC HE-S2, anti-PD-L1 antibody alone, D18 alone, anti-PD-L1 THIOMAB alone, and anti-PD-L1 antibody/D18 combination.
  • B Whole Animal Survival Studies. The vertical axis of the curve represents the percentage of tumor-bearing mice still alive after different days, and the horizontal axis represents the experimental days.
  • mice 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 MC38 tumor growth in mice.
  • the dosage and administration time of mice in each group are shown in Table 1. Significant differences among groups were compared using Student's t test, *** indicates P ⁇ 0.001.
  • Figure 16 shows the experimental results of the different tumor suppressive abilities of gradient doses of D18 combined with anti-PD-L1 antibodies.
  • D18 was administered once a week, and anti-PD-L1 antibody or IgG was administered twice a week.
  • 150 ⁇ g was administered to each mouse in each group.
  • the combination group was divided into groups, the anti-PD-L1+D18 (25 ⁇ g) group was injected with D18 25 ⁇ g per mouse each time; the anti-PD-L1+D18 (2.5 ⁇ g) group was injected with D18 2.5 ⁇ g per mouse each time; -PD-L1+D18 (0.25 ⁇ g) group was injected with D18 0.25 ⁇ g per mouse each time.
  • Significant differences among groups were compared using Student's t test, **** indicates P ⁇ 0.0001.
  • Figure 17 shows the effect of ADC HE-S2 on the body weight of mice.
  • 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 time of administration of each drug is marked by arrows.
  • FIG. 18 shows the results of the mouse anti-tumor experiment of ADC10.
  • Alkyl refers to a straight or branched hydrocarbon chain free radical without unsaturated bonds, and the hydrocarbon chain free radical is connected to the rest of the molecule by a single bond.
  • Typical alkyl groups contain from 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.
  • alkyl group is substituted by a cycloalkyl group, it corresponds to a "cycloalkylalkyl” radical, such as cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, etc. .
  • cycloalkylalkyl such as cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, etc.
  • aryl group it corresponds to an "aralkyl” radical, such as benzyl, benzhydryl or phenethyl.
  • heterocyclylalkyl it corresponds to a "heterocyclylalkyl" radical.
  • alkenyl refers to a straight or branched hydrocarbon chain radical containing at least two carbon atoms and at least one unsaturated bond, and the hydrocarbon chain radical is connected to other parts of the molecule by a single bond.
  • Typical alkenyl groups contain from 1 to 12 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, such as ethenyl, 1-methyl-ethylene group, 1-propenyl, 2-propenyl or butenyl, etc.
  • Alkoxy refers to a substituent formed after the hydrogen in a hydroxyl group is replaced by an alkyl group, and a typical alkoxy group contains 1 to 12 (such as 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12) carbon atoms, such as methoxy, ethoxy, propoxy, butoxy, etc.
  • Aryl refers to monocyclic or polycyclic radicals, including polycyclic radicals containing single aryl groups and/or fused aryl groups. Typical aryl groups contain 1 to 3 single 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.
  • Heterocyclyl includes heteroaromatic and heteroalicyclic groups containing 1 to 3 monocyclic and/or fused rings and 3 to about 18 ring atoms. Preferred heteroaromatic and heteroalicyclic groups contain 5 to about 10 ring atoms.
  • Suitable heteroaryl groups in the compounds of the invention contain 1, 2 or 3 heteroatoms selected from N, O or S atoms and include, for example, coumarins (including 8- Coumarin), quinolinyl (including 8-quinolyl, isoquinolyl), pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazole base, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolyl, phthalazinyl, pteridinyl, purinyl, Oxadiazolyl, thiadiazolyl, furacryl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuryl,
  • Suitable heteroalicyclic groups in the compounds of the present invention contain 1, 2 or 3 heteroatoms selected from N, O or S atoms
  • said heteroalicyclic groups include, for example, pyrrolidinyl , tetrahydrofuryl, dihydrofuran, tetrahydrothiophenyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, oxathiolanyl, piperazinyl, azetidine group, oxetanyl, thietanyl, homopiperidinyl, oxiranyl, thiiranyl, azepinyl, oxazepanyl, diazepanyl , triazepinyl, 1,2,3,6-tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, Dio
  • Halogen or "halo" means bromo, chloro, iodo or fluoro.
  • the compounds of the invention also include isotopically labeled forms, ie compounds that differ only in the presence of one or more isotopically enriched atoms.
  • isotopically labeled forms ie compounds that differ only in the presence of one or more isotopically enriched atoms.
  • Compounds are included within the scope of the present invention.
  • PD-L1 includes isoforms, mammalian, eg human PD-L1, species homologues of human PD-L1, and analogs comprising 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.
  • anti-PD-L1 antibody refers to an antibody that can specifically bind to PD-L1.
  • antibody means an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds (i.e., a "full antibody molecule"), and multimers thereof (eg, IgM) or antigen-binding fragments thereof.
  • Each heavy chain is comprised of a heavy chain variable region ("HCVR” or “VH”) and a heavy chain constant region (comprising domains CH1, CH2 and CH3).
  • Each light chain is comprised of a light chain variable region ("LCVR” or "VL”) and a light chain constant region (CL).
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the terms "antigen-binding portion" of an antibody, “antibody fragment” and the like include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex .
  • the term “antigen-binding portion” or “antibody fragment” of a PD-L1 antibody refers to one or more fragments of an antibody that retain the ability to specifically bind 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' ) fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; (vii) minimal identifying units (eg, isolated complementarity determining regions (CDRs)) consisting of amino acid residues that mimic hypervariable regions of antibodies.
  • CDRs complementarity determining regions
  • Antigen-binding fragments of antibodies will typically comprise at least one variable domain.
  • terapéuticaally effective amount refers to a dose that can achieve treatment, prevention, alleviation and/or alleviation of the diseases or conditions described in the present invention in a subject.
  • patient refers to any animal or cells thereof, whether in vitro or in situ, subject to the methods described herein, in some non-limiting embodiments Among them, it is a mammal, such as human, monkey, dog, rabbit, mouse and the like.
  • treating includes eradicating, removing, reversing, alleviating, altering or controlling a disease and/or condition after its onset.
  • prevention refers to the ability, by treatment, to avoid, minimize or make the onset or development of a disease and/or condition difficult before the onset of the disease and/or condition occurs.
  • disease refers to a physical state of the subject that is associated with the disease of the present invention.
  • compound 25 (308 mg, 1.0 mmol) was dissolved in dry DCM (5 mL), and a mixture of (4-aminophenyl)methanol (148 mg, 1.2 mmol) and pyridine (79 mg, 1.0 mmol) in DCM was slowly added dropwise solution (5 mL). The reaction mixture was stirred at room temperature for 2 h under argon protection. After the reaction was complete, the resulting solution was transferred into saturated ammonium chloride solution and extracted with DCM (3 x 50 mL). The organic layer was washed with water and brine, then dried over anhydrous Na2SO4 , filtered and rotary evaporated under reduced pressure to obtain crude product.
  • Compound 50 was prepared according to the synthetic method of compound 38.
  • Compound 52 was prepared according to the synthetic method of compound 38.
  • Compound 53 refers to the synthesis method of compound 45, replacing 6-(maleimido) hexanoic acid succinimide ester with 4-(N-maleimidomethyl) cyclohexane-1-carboxylate Acid succinimide ester prepared in 42% yield.
  • Avelumab (MSB0010718C, trade name Bavencio) is a fully humanized IgG1 monoclonal anti-PD-L1 antibody drug developed by Merck KGaA and Pfizer, which has been approved by the FDA in 2017 Approved for the treatment of Merkel-cell carcinoma (Apolo et al., 2017; Shirley, 2018), the coding nucleotide sequences of its light chain and heavy chain are shown in SEQ ID NO: 1 and 2, respectively As shown, the amino acid sequences of the light chain and the heavy chain are shown in SEQ ID NO: 3 and 4, respectively.
  • avelumab can also cross-react with murine PD-L1 (Deng et al., 2016).
  • Avelumab as a raw material to develop anti-PD-L1 ADC drugs can ensure that the antibody itself has sufficient biological activity and druggability, and at the same time make the obtained ADC drugs suitable for early mouse model tests and subsequent development. human clinical trials.
  • the expression plasmid of Avelumab consists of two parts, the light chain 159-pFuse- ⁇ PDL1-LC plasmid and the heavy chain 162-pFuse- ⁇ PDL1-hIgG-Fc2 plasmid, and its nucleotide sequences are shown in SEQ ID NO:5 and 6.
  • the 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.
  • the inventor has completed the construction of THIOMAB expression plasmid by the method of PCR site-directed mutagenesis. Design a pair of completely complementary primers containing the mutation site in opposite directions, use the complete plasmid to be mutated as a template, and use high-fidelity PCR enzymes to replicate and amplify the entire plasmid sequence with the mutation site. Afterwards, the methylated template plasmid was removed with DpnI significant endonuclease, while the unmethylated PCR product was retained. Further transformation and cultivation of the plasmid can amplify the complete plasmid with the mutation site.
  • the plasmid obtained by site-directed mutation of the light chain 159-pFuse- ⁇ PDL1-LC plasmid was named ⁇ PD-L1 THIOMAB LC-V205C, and its nucleotide sequence is shown in SEQ ID NO:7.
  • the results of the sequencing comparison are shown in Figure 1. After comparison with the template plasmid, it was found that the sequence at the predetermined mutation site was mutated from GTG to TGC, while other bases did not change, so the 205th amino acid of the light chain after translation was mutated from valine to cysteine.
  • the two artificially introduced cysteines will be used in subsequent conjugation reactions of ADC drugs.
  • the nucleotide sequence encoding the light chain 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.
  • Anti-PD-L1 antibodies were obtained after expression and purification, and identified by reducing and non-reducing SDS PAGE analysis. as shown in picture 2.
  • the antibody was reduced to light chain ( ⁇ 25KD) and heavy chain ( ⁇ 50KD) in reducing SDS PAGE, and remained intact antibody ( ⁇ 150KD) in non-reducing SDS PAGE.
  • Anti-PD-L1 THIOMAB was obtained after expression and purification, and identified by reducing and non-reducing SDS PAGE analysis. As shown in Figure 3, the SDS staining results of anti-PD-L1 THIOMAB (as shown in Figure 3) are basically consistent with those of anti-PD-L1 antibodies (as shown in Figure 2): the antibody was reduced to light chains in reducing SDS PAGE ( ⁇ 25KD) and heavy chain ( ⁇ 50KD), in non-reducing SDS PAGE, the antibody still remains intact ( ⁇ 150KD).
  • the anti-PD-L1 THIOMAB obtained by gene editing basically retains the original structure of the antibody, which shows that the expression process and self-assembly process of THIOMAM are not affected by artificial mutations.
  • the expression levels of the antibody and THIOMAB did not seem to be significantly different, which also indicated that the expression and assembly process of the intracellular antibody was not significantly affected.
  • THIOMAB In order to further identify the obtained THIOMAB, the inventors processed the complete THIOMAB according to the process of "TCEP reduction-purification-DHAA re-oxidation-purification" to obtain THIOMAB with free sulfhydryl groups (THIOMAB-S2). The change of its molecular weight was detected by Exactive Plus EMROrbitrap LC-MS. The result is shown in Figure 4.
  • the molecular weight of the intact anti-PD-L1 THIOMAB is about 143791.75, while that of THIOMAB-S2 with free thiols is about 143544.31, a molecular weight difference of 247.44.
  • the complete anti-PD-L1 THIOMAB sulfhydryl group will generally be modified with a free cysteine, so compared with THIOMAB-S2, the molecular weight will be two more than the molecular weight of residues after condensation of cysteine and sulfhydryl group , about 238. 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 the experimental expectation.
  • This result proves that the inventors have successfully expressed a homogeneous anti-PD-L1 THIOMAB with two mutant cysteines through gene editing, which can be used for the construction of homogeneous ADC drugs.
  • the inventors also need to compare the results of in vivo and in vitro experiments with natural anti-PD-L1 antibodies to study whether this artificial mutation will affect the antigen-binding ability and other activities of the antibody.
  • Embodiment 3 Synthetic route and identification of ADC based on THIOMAB
  • a schematic diagram of the synthesis of ADC HE-S2 is shown in Figure 5.
  • the inventors used anti-PD-L1 THIOMAB and compound 20 (prepared in Example 1) as raw materials to synthesize an anti-PD-L1 antibody with a disulfide bond linker and N 4 -butyl-6-(4-((di The conjugate of methylamino)methyl)benzyl)pyridin[3,2-d]pyrimidine-2,4-diamine (hereinafter referred to as compound D18) is named ADC HE-S2.
  • the linking site on the antibody is the cysteine at position 226 of the amino acid sequence (light chain sequence) shown in SEQ ID NO:9.
  • the mass spectrometry results of ADC HE-S2 are shown in Figure 6.
  • the molecular weight of ADC obtained by MS is 144721.08, while that of THIOMAB is 143791.75.
  • Example 4 Synthetic route and identification of ADC based on wild-type PD-L1 antibody
  • reaction raw materials and reagent preparation the concentration of the antibody (Avelumab described in Example 2) is prepared as a PBS solution of 5-10 mg/mL; the TCEP aqueous solution with a concentration of 10 mM is prepared; the coupling small molecule (compound 39, 40, 43, 44, 51, 45, 49, 53, 57, 58, 60, prepared in Example 1) were prepared as a DMSO solution with a concentration of 10mM; a cysteine PBS solution with a concentration of 20mM was prepared;
  • Antibody reduction add 10 or 20 equivalents of TCEP (10mM, shake at 37°C for 2-3h, and rotate at 220rpm/min;
  • Liquid chromatography conditions the chromatographic column is ACQUITY UPLC Protein BEH SEC chromatographic column ( 2.1mm ⁇ 150mm, 1.7 ⁇ m), the column temperature is 25°C, and the injection volume is 15 ⁇ L.
  • Isocratic elution 50mM ammonium acetate as mobile phase, flow rate 0.065mL/min, acquisition time 10min, UV detection wavelength 280nm.
  • Mass spectrometry conditions the capillary voltage was set to 3.0kV for ESI source positive ionization mode scanning; the cone voltage was 120V, the source temperature and desolvation temperature were set to 120°C and 500°C, respectively; the flow rates of cone gas and desolvation gas were set to 20L/H and 600L/H, the primary mass spectrum ranges from m/z 400 to m/z 8000.
  • Embodiment 5 biological activity research
  • ADC1 ADC HE-S2
  • Sufficient specific antigen affinity is the basis of antibody biological activity and is crucial to the activity and efficacy of antibody drugs. Since the preparation of ADC drugs involves the modification of antibodies, chemical treatment, and small molecule coupling, all of these may destroy or affect the original antigen affinity and stability of antibodies, resulting in impaired antigen binding ability of antibodies. Therefore, in the process of processing antibodies, the conditions used need to be as mild as possible; the selection of modification and conjugation sites should also avoid the antigenic determinants of the original antibody, so as not to damage the antigen-binding ability of the antibody. In addition, the antigen affinity ability of the obtained ADC drug should also be verified to ensure that the ADC drug retains sufficient targeting and specificity.
  • ADC HE-S2 designed and prepared in the present invention is an important basis for realizing its anti-tumor activity.
  • ADC HE-S2 requires the specific binding of the antibody to PD-L1 highly expressed in the tumor to achieve the targeted delivery of the immune modulator D18.
  • ADC HE-S2 can also block the tumor PD-1/PD-L1 immunosuppressive signaling pathway through the binding of PD-L1 on the cell surface, thereby inducing T cell anti-tumor response. Therefore, after successfully preparing ADC HE-S2, the inventor first verified its ability to recognize and bind PD-L1 antigen in vitro.
  • the inventors verified whether the ADC HE-S2 and the genetically modified anti-PD-L1 THIOMAB retained the original antibody antigen-binding ability through fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • ADC HE-S2, anti-PD-L1 antibody (i.e. Avelumab described in Example 2) and anti-PD-L1 THIOMAB (i.e. the mutant antibody prepared in Example 2) were incubated with MC38 cells as primary antibodies, so that The antibody can fully contact and bind to PD-L1 on the cell surface.
  • the cells were treated with FITC-labeled goat anti-human secondary antibody, and incubated in the dark for 30 minutes to fully combine the secondary antibody with the primary antibody.
  • Negative control NC
  • the control IgG was stained with FITC-labeled secondary antibody together with other experimental groups to characterize the false positive fluorescent signal generated by the non-specific binding of the secondary antibody to the cells.
  • the stained MC38 cells were analyzed on a BD FACSAria TM III flow cytometer to detect and compare the intensity of FITC fluorescence signals on MC38 cells treated with different antibody drugs, so as to characterize ADC, THIOMBA and antibodies against PD- L1 affinity. All the results were processed by FlowJo software (TreeStar Inc.), and the results are shown in FIG. 10 .
  • the FACS results are shown in Figure 10. Based on the blank control group (NC) using IgG isotype antibodies, the false positive signal offset rate of the control group stained with fluorescent secondary antibodies is only 1.7%; the mean fluorescence intensity (MFI) calculation results It was shown that the MFI of the NC group was 142.00, while the average fluorescence intensity detected by MC38 after secondary antibody staining was 166.75, although the difference was significant (p ⁇ 0.0001), but the difference was small. This significant but weak difference is the false positive signal generated by the non-specific binding of the FITC secondary antibody used in the experiment to MC38 cells. Experiments have shown that the phenomenon of this false positive combination is weak and will not affect the results. Therefore, the NC control group was used as a reference basis to evaluate the antibody antigen binding ability of anti-PD-L1 antibody drugs.
  • MFI mean fluorescence intensity
  • the inventors observed similar positive FITC signals on MC38 cells treated with anti-PD-L1 antibody, anti-PD-L1 THIOMAB and ADC HE-S2, and the shift rates of fluorescence signals relative to the NC group were 35.7%, 36.6 % and 34.2%, much higher than the false positive signal (1.7%) produced by the non-specific binding of the fluorescent secondary antibody.
  • MFI calculation 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 those of the two control groups (p ⁇ 0.0001). It shows that all three antibodies can specifically bind to PD-L1.
  • ADC HE-S2 still maintains the antigen recognition affinity of the original anti-PD-L1 antibody, which can be further applied to subsequent experiments and research and development.
  • Drug release from ADCs is widely considered to be dependent on the internalization ability of the corresponding antibody.
  • the antibody-antigen complex formed after the ADC binds to the antigenic protein on the cell surface needs to enter the target cell with the internalization of the antigenic protein, thereby releasing the conjugated drug under the action of intracellular enzymes or other intracellular physiological environments . Therefore, the internalization ability of the complex formed after the combination of the selected antigen and ADC is crucial for the realization of the drug function of the ADC. Since the ADC HE-S2 designed by the inventor should theoretically function in both cancer cells and DC cells, the inventors verified the ability of anti-PD-L1 antibody drugs to internalize into tumor cells and DC cells through in vitro experiments .
  • pHrodo TM red dye is a pH-sensitive fluorescent dye, and the fluorescence intensity of this molecule will increase rapidly when the surrounding environment becomes acidic (Lehrman et al., 2018). When the dye is internalized into the cell along with the antigen-antibody complex, the pH value of the surrounding environment of the dye will immediately drop to acidic, thereby producing an observable fluorescent signal. This property allows monitoring of antibody internalization using pHrodo TM red dye. pass With the live cell analysis system, the inventors can perform image-based monitoring and quantitative analysis of the internalization process of pHrodo TM red dye-labeled anti-PD-L1 antibody.
  • anti-PD-L1 THIOMAB has the same antigen recognition ability as ADC HE-S2, so the internalization ability of anti-PD-L1 THIOMAB can also reflect the internalization ability of ADC HE-S2.
  • the integrated intensity of red fluorescent objects in MC38 was 13701.63, which was significantly higher than 84.88 in the control group; the signal intensity in B16 was as high as 283613.60, which was also significantly higher than 98.53 in the control group.
  • the fluorescent signal in B16 cells was also stronger than that in MC38 cells, which may be caused by the differences in the internalization rate of PD-L1 in different cells and the difference in intracellular pH.
  • DC 2.4 In order to verify the internalization of anti-PD-L1 THIOMAB on DC cells, the inventors obtained two kinds of DC cells, Cd274 knockout DC 2.4 cells and wild-type DC 2.4 cells, from Mr. Tang Haidong, School of Pharmacy, Tsinghua University.
  • the Cd274 gene is the gene encoding PD-L1.
  • DC 2.4 After knockout, DC 2.4 will not be able to express PD-L1 protein, so it is a PD-L1-negative DC cell; while wild-type DC 2.4 can express PD-L1, which is a PD-L1-positive DC cells (as shown in Figure 12).
  • 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, through the anti-PD-L1 THIOMAB internalization experiments of these two types of cells, the inventors can simultaneously verify the internalization ability of anti-PD-L1 THIOMAB on DC cells and the selectivity of the antibody.
  • the inventor added 10% heat-inactivated fetal bovine serum (FBS) as a blocking agent to the culture medium of DC 2.4 cells according to the routine operation, and after incubation for 8 hours, the red fluorescent signal in the cells was detected by FACS.
  • FBS heat-inactivated fetal bovine serum
  • Figure 13 the results of FACS detection showed that the inventors observed the same proportion of red fluorescent positive cells in PD-L1-positive wild-type DC 2.4 cells or in PD-L1-negative CD274 knockout DC 2.4 cells, The ratios are 53.6% and 52.9%, respectively.
  • the inventors used 10% mouse serum and 10% FBS as blocking agents and added them to the cell culture medium in groups.
  • the rich mouse IgG in mouse serum can more effectively block the Fc ⁇ receptor on the surface of DC 2.4, thereby inhibiting the antibody internalization process mediated by DC cell Fc ⁇ receptor.
  • DC cells should first be incubated at 4°C for 30 minutes to suspend the internalization process of cell surface receptors and allow IgG in mouse serum to fully bind to Fc ⁇ receptors on the surface of DC cells.
  • fluorescently labeled anti-PD-L1 THIOMBA was added to each group of cells. Then incubate at 4°C in the dark for 30 minutes, so that the antibody and antigen can fully contact and combine under the condition that the internalization of the cells stops.
  • the overall red fluorescent object integration intensity-time curves obtained from the internalization experiments of anti-PD-L1 THIOMAB are shown in Figure 14A and B.
  • the results in Figure 14A show that after adding 10% FBS to block, very similar fluorescence intensity curves can be observed in PD-L1-positive wild-type DC 2.4 cells and PD-L1-negative CD274 knockout DC 2.4 cells ; No change in fluorescence signal was observed in the negative control group without fluorescently labeled anti-PD-L1 THIOMAB.
  • the red fluorescent signal in the wild-type DC 2.4 cells was significantly stronger than that in the Cd274-knockout DC cells. This phenomenon can be explained as the PD-L1 on the surface of DC cells can resist PD-L1 because the receptor internalization process of the cells is almost stopped and the antigen-antibody binding is not affected during the incubation at 4°C for 30 min before the detection. Antibody binding is sufficient, and Fc ⁇ receptors are also sufficiently blocked by cognate IgG in mouse serum.
  • Embodiment 6 antitumor activity research
  • mice In order to verify the anticancer activity of ADC HE-S2, the inventors first created a mouse model with MC38. After tumor-bearing, visible tumors can be observed on the surface of the mice in about a week. When the average tumor tissue volume of the mice reached 100 mm, the mice were randomly divided into groups according to the type of final administration, followed by: IgG isotype control antibody group (IgG for short), anti-PD-L1 antibody group (anti-PD- L1), anti-PD-L1 THIOMAB group (Thiomab), D18 group (D18), D18 combined with anti-PD-L1 antibody therapy group (anti-PD-L1&D18) and ADC HE-S2 group (ADC HE-S2), each Group 7-8 mice.
  • IgG isotype control antibody group IgG for short
  • anti-PD-L1 antibody group anti-PD- L1
  • anti-PD-L1 THIOMAB group Thiomab
  • D18 group D18 combined
  • IgG isotype control antibody group anti-PD-L1 antibody (i.e. Avelumab described in Example 2) group, anti-PD-L1 THIOMAB (i.e. the mutant antibody prepared in Example 2) group were administered twice a week, each time Each mouse was injected intraperitoneally with 150 ⁇ g. The D18 group was administered twice a week, and each mouse was intraperitoneally injected with 25 ⁇ g each time.
  • anti-PD-L1 antibody i.e. Avelumab described in Example 2
  • anti-PD-L1 THIOMAB i.e. the mutant antibody prepared in Example 2
  • the anti-PD-L1 antibody was administered twice a week, 150 ⁇ g each time; D18 was administered once, 25 ⁇ g each time; ADC HE-S2 group was administered weekly ADC HE -S2 was administered once, with 150 ⁇ g intraperitoneally injected per mouse each time; anti-PD-L1 antibody was administered once a week, at a time staggered from ADC, with 150 ⁇ g intraperitoneally injected per mouse each time.
  • Fig. 15A The experimental results are shown in Fig. 15A.
  • the effects of anti-PD-L1 antibody, anti-PD-L1 THIOMAB and D18 alone are very similar: there is no significant difference in the inhibitory activity of tumor growth among each other, but all are slightly better than negative ones.
  • Efficacy of control IgG isotype antibodies This result shows that both D18 and anti-PD-L1 antibodies have certain anti-cancer activity, and anti-PD-L1 THIOMAB also retains the biological function of the original antibody.
  • the combination therapy of D18 and anti-PD-L1 antibody showed very good tumor growth inhibitory activity, and its effect was far better than that of either group alone.
  • ADC HE-S2 showed a very surprising anticancer activity, and the tumor growth inhibitory activity was significantly better than that of the combined treatment group (P ⁇ 0.001).
  • the tumor volume of the mice in the combined treatment group began to increase significantly; while in the ADC treatment group, the tumor size of the mice was maintained at a very low level.
  • mice in the ADC HE-S2 group were 6/8, and it is worth emphasizing that the tumors of the last six surviving mice were almost undetectable.
  • the survival rate of the mice in the D18 and PD-L1 combination group was 4/8; the survival rate of the mice in the D18 group was 1/8; while the mice in the other two single drug groups and the negative control group all died. This result also proved the strong anticancer activity of ADC HE-S2.
  • B16 melanoma is considered to be a tumor with low PD-L1 level and resistance to PD-1/PD-L1 blocking therapy. Results As expected by the inventors, compared with MC38 tumors, the inhibitory effects of various drugs on B16 melanoma were all decreased. The efficacy of anti-PD-L1 antibody, anti-PD-L1 THIOMAB and D18 three-drug monotherapy group was almost comparable to the results of IgG isotype antibody.
  • mice were divided into IgG isotype antibody control group (IgG for short), anti-PD-L1 antibody group (anti-PD-L1), anti-PD-L1 antibody + D18 (25 ⁇ g) group (anti-PD-L1) according to the type of administration.
  • IgG isotype antibody control group
  • anti-PD-L1 antibody group anti-PD-L1 antibody + D18 (25 ⁇ g) group
  • anti-PD-L1 anti-PD-L1
  • Anti-IgG isotype antibody and PD-L1 antibody were administered twice a week, intraperitoneally injected 150 ⁇ g/monkey for each administration; D18 was administered once a week, and each administration was injected intraperitoneally with 25 ⁇ g/monkey and 2.5 ⁇ g respectively according to the group /mouse and 0.25 ⁇ g/mouse; mice in the IgG isotype antibody group were intraperitoneally injected weekly with the same dose of DMSO as the administration on D18 as a control.
  • the anti-tumor results of gradient doses of D18 combined with anti-PD-L1 antibody are shown in Figure 16.
  • the experimental results proved that the function of D18 to enhance the anti-tumor activity of anti-PD-L1 antibody showed a dose-dependent characteristic.
  • the combination therapy with anti-PD-L1 antibody showed a more obvious tumor suppressive activity.
  • the anti-tumor effect of the combination therapy of D18 and anti-PD-L1 antibody also continued to weaken. In fact, when the dose was reduced to 2.5 ⁇ g or 0.25 ⁇ g per mouse, the tumor inhibitory effect was no longer significantly different from that of anti-PD-L1 antibody alone.
  • the loaded D18 dose ( ⁇ 0.75 ⁇ g) of ADC HE-S2 per administration was much less than 2.5 ⁇ g. Therefore, it can be judged that through ADC targeted drug delivery, the inventors have achieved a more significant effect than the combined treatment of 25 ⁇ g of D18 and anti-PD-L1 antibody by using a dose of D18 ( ⁇ 0.75 ⁇ g) that is not enough to exert activity in the combination drug. Tumor suppressor effect.
  • mice 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 administration, with 5 mice in each group.
  • mice in each group were the same as those in the mouse tumor model experiment: ADC HE-S2 was administered once a week in the ADC group, and anti-PD-L1 antibody was injected once, each time 150 ⁇ g; D18 weekly Administer once, 25 ⁇ g each time; anti-PD-L1 antibody and IgG isotype antibody are administered twice a week, 150 ⁇ g each time.
  • the weight change of the mice in the anti-PD-L1 antibody group was almost the same as that in the blank control IgG group, which proved that the anti-PD-L1 antibody itself had little toxic side effects on the mice.
  • the body weight of the mice showed a certain degree of decline.
  • the weight loss of the mice in the anti-PD-L1 antibody + D18 group was particularly significant (p ⁇ 0.05). This result confirmed the inventor's previous hypothesis that anti-PD as an immune checkpoint inhibitor -L1 antibody will increase the toxic side effects of TLR7/8 agonist D18.
  • ADC HE-S2 exhibits better tumor suppressive activity than that of combination therapy through lower actual D18 usage, and at the same time significantly reduces the toxic side effects of combination therapy.
  • the minimum effective dose of D18 is greatly reduced, and the therapeutic window of D18 is expanded.
  • mice MC38 cells (1x10 6 cells/mouse) were subcutaneously inoculated into the right abdomen of the mice.
  • the mice were randomly divided into groups according to the type of final administration, in order: PBS control group and ADC 10 group, with 5-6 mice in each group.
  • the drug was administered twice a week, and the intraperitoneal injection dose of each mouse was 10 mg/kg.
  • the weight change of the mice was weighed every day ( FIG. 18A ), and the tumor volume of the mice was measured every 3 days ( FIG. 18B ). As shown in Figure 18B, the tumor inhibition rate of ADC was 70%.

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Abstract

本发明公开了一种抗体药物偶联物及其制备方法和应用，特别是一种抗PD-L1抗体与TLR7和/或TLR8激动剂的偶联物及其药物组合物、制备方法和应用。本发明通过基因编辑获得带有突变半胱氨酸的修饰的抗PD-L1抗体，其基本上保留了原抗体的结构，可用于抗体药物偶联物的构建。通过抗肿瘤实验发现，所得抗体药物偶联物具有较佳的活性，例如强大的抗肿瘤活性，可明显提高荷瘤动物的生存率，且毒性显著降低，对实验动物身体的负担较小，大大降低了其中小分子药物单独使用时的最低有效剂量，扩大了其治疗窗，有望用于多种疾病(例如肿瘤、病毒性疾病如乙肝等)治疗药物的开发，具有良好的应用前景和价值。

Description

一种抗体药物偶联物及其制备方法和应用 技术领域
本发明涉及生化医药技术领域，具体涉及一种抗体药物偶联物(ADC)，特别是一种用于免疫治疗的抗体与免疫调节剂的偶联物，例如一种抗PD-L1抗体与TLR7和/或TLR8激动剂的偶联物，及其药物组合物、制备方法和应用。
背景技术
Toll样受体是进化中比较保守的一个受体家族，至少包括13个成员，在人中发现10种(TLR1-10)，TLR1、TLR2、TLR4、TLR5、TLR6、TLR10表达于细胞表面，快速识别细菌代谢的产物，TLR3、TLR7、TLR8、TLR9表达在细胞内，主要是对病毒核酸的监测和识别，TLR3识别双链RNA，而TLR7和TLR8识别单链RNA，TLR9识别未甲基化的CG辅酶Ⅰ，调节细菌DNA和某些病毒的反应。
TLR能特异识别病原相关分子模式(PAMP)，在天然免疫和获得性免疫中都发挥着重要的作用，是连接天然免疫和获得性免疫的桥梁。其中，TLR7在识别结合病毒的单链RNA或人工合成的小分子嘌呤类化合物后，会招募特异接头蛋白，激活一系列信号级联反应，启动高水平的系统性适应性免疫应答，杀伤感染病毒的细胞，从而彻底清除病毒。临床上己经开始利用TLR7激动剂治疗慢性病毒性感染疾病，如乙型肝炎、丙型肝炎等。此外，TLR7激动剂作为流感疫苗佐剂能诱导更加迅速有效的免疫保护。在抗肿瘤方面，TLR7激动剂不仅可以直接刺激pDC分泌IFN-α，还会增强pDC的共刺激和抗原递呈能力，激活的pDC促进CD4+T细胞的增殖，进一步激活CD8+T细胞，杀伤肿瘤细胞，故而TLR7激动剂作为免疫佐剂在机体识别和杀伤肿瘤过程中的作用也在逐渐得到重视。
发明内容
本发明的发明人在其之前的研发成果基础上，继续深入研发出一种抗体药物偶联物(ADC)(特别是一种用于免疫治疗的抗体与免疫调节剂的偶联物，例如一种抗PD-L1抗体与TLR7和/或TLR8激动剂的偶联物)，其具有更佳的治疗效果，显著降低了毒副作用，扩大了免疫调节剂(如TLR7和/或TLR8激动剂)的治疗窗。
具体地，上述抗体药物偶联物具有如下结构：
Figure PCTCN2022092556-appb-000001
其中，
Ab为抗体或其抗原结合片段；
D为小分子药物；
L为连接Ab和D的连接单元；
n为1-100的整数。
具体地，上述抗体为单克隆抗体。
具体地，上述抗体的形式可以为，例如，嵌合抗体、人源化抗体、全人源抗体等。
具体地，上述抗原结合片段包括Fab片段、F(ab')2片段、Fd片段、Fv片段、dAb片段、单链Fv(scFv)、二硫键连接的Fv(sdFv)、含有CDR的片段或分离的CDR等。
具体地，上述抗体对肿瘤、感染性微生物或自身免疫性疾病等相关的抗原或其表位是反应性的；具体地，上述抗体靶向的抗原例如：Claudin18.2、GPC3、HER-2/neu、碳酸酐酶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、甲胎蛋白(AFP)、VEGF、ED-B纤连蛋白、EGP-1、EGP-2、EGF受体(ErbB1)、ErbB2、ErbB3、因子H、FHL-1、Flt-3、叶酸受体、Ga 733、GROB、HMGB-1、缺氧诱导因子(HIF)、HM1.24、胰岛素样生长因子(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、神经节糖苷、HCG、HLA-DR、CD66a-d、MAGE、mCRP、MCP-1、MIP-1A、MIP-1B、巨噬细胞移动抑制因子(MIF)、MUC1、MUC2、MUC3、MUC4、MUC5、PD-1、PD-L1、胎盘生长因子(PIGF)、PSA、PSMA、PSMA二聚物、PAM4抗原、NCA-95、NCA-90、A3、A33、Ep-CAM、KS-1、Le(y)、间皮素、S100、腱生蛋白、TAC、Tn抗原、Thomas-Friedenreich抗原、肿瘤坏死抗原、肿瘤血管生成抗原、TNF-α、TRAIL受体(R1和R2)、VEGFR、RANTES、T101、癌干细胞抗原、补体因子C3、C3a、C3b、C5a、C5和致癌基因产物等。
在本发明的一个实施方式中，上述抗体为抗PD-L1抗体。
具体地，上述抗体包含反应活性基团(如，巯基、氨基、羧基、酰胺基、卤素、酯基、酰卤素、酸酐、环氧基、马来酰亚胺基、胺基氧基、叠氮基、炔基、
Figure PCTCN2022092556-appb-000002
其中R可选自：H、取代或未取代的烷基、取代或未取代的芳基、取代或未取代的芳烷基、取代或未取代的环烷基、取代或未取代的环烷基烷基、取代或未取代的杂环基、取代或未取代的杂环基烷基)，其可以本身自带这些反应活性基团，也可通过突变(如定点突变)，将已知抗体的一个或多个氨基酸残基突变为含有相同或不同的所需反应活性基团的氨基酸(包括天然氨基酸和非天然氨基酸等)而获得。在本发明的一个实施方式中，上述抗体通过突变(例如通过Thiomab技术)在抗体的氨基酸序列的特定位置插入/取代为半胱氨酸残基。
在本发明的一个实施例中，上述抗体为修饰的抗PD-L1抗体，其通过基因工程手段将已知的抗PD-L1抗体(例如，阿特珠单抗(Atezolizumab)、德瓦鲁单抗(Durvalumab)、阿维单抗(Avelumab)、西米普利单抗(Cemiplimab)、KN035、CS1001、BGB-A333、KL-A167、SHR-1316和STI-A1014等)的一个或多个氨基酸残基分别突变为半胱氨酸获得。
具体地，上述抗体包含重链和轻链；
其中，重链的氨基酸序列如SEQ ID NO：4所示，或为与SEQ ID NO：4具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；和/或，
轻链的氨基酸序列如SEQ ID NO：9所示，或为与SEQ ID NO：9具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列。
更具体地，上述抗体具有如SEQ ID NO：9所示的轻链序列。
在本发明的一个实施例中，上述抗体具有如SEQ ID NO：4所示的重链序列和如SEQ ID NO：9所示的轻链序列。
具体地，上述n(即药物/抗体比率(DAR))可以为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；例如，1-50，1-40，1-20，1-10，1-6，1-4；在本发明的一些实施例中，n＝2或4。
在本发明的一个实施方式中，上述抗体药物偶联物中，抗体与L的连接位点为抗体的氨基酸序列中半胱氨酸残基上的游离巯基。
在本发明的一个实施例中，上述抗体药物偶联物中，抗体具有如SEQ ID NO：4所示的重链序列和如SEQ ID NO：9所示的轻链序列，抗体与L的连接位点为SEQ ID NO：9所示氨基酸序列的第226位的半胱氨酸残基上的游离巯基；优选地，n＝2。
在本发明另一个实施例中，上述抗体药物偶联物中，抗体具有如SEQ ID NO：4所示的重链序列和如SEQ ID NO：3所示的轻链序列，n为1-10，特别是1-6。
具体地，上述小分子药物可以为免疫调节剂，例如，Toll样受体激动剂(Toll-like receptors，TLR)，具体如，TLR7和/或TLR8激动剂，例如专利申请公开号WO2019/095455A1中所述吡啶并嘧啶衍生物及其盐。
在本发明的一个实施方式中，通式Ⅰ中D部分具有如下结构：
Figure PCTCN2022092556-appb-000003
其中，
L'为连接基团，其选自：单键或C 1-C 6亚烷基、C 1-C 6亚烯基、C 3-C 6亚环烷基，其中所述基团均可任选地被C 1-C 4烷基取代；
R 1选自：单键、C 1-C 6亚烷基、C 1-C 6亚烯基、C 1-C 6亚烷氧基，其中所述基团均可任选地被C 1-C 4烷基取代；
X选自：
Figure PCTCN2022092556-appb-000004
-NR 4-、-O-、-S-、单键、-OCO-、-COO-、-NR 4-(C 1-C 6亚烷基)-NR 5-、亚杂环基(特别是亚含氮杂环基)，其中，R 4和R 5独立地选自：H、C 1-C 6烷基、C 1-C 6烷氧基、氨基酸残基、寡肽残基、卤代烷基、羧基取代烷基、酯基取代烷基、
Figure PCTCN2022092556-appb-000005
其中，a为1-10的整数(例如1、2、3、4、5、6、7、8、9、10)，R 8和R 9独立地选自：H、C 1-C 6烷基，或R 8和R 9与其中间的原子一起形成
Figure PCTCN2022092556-appb-000006
或者R 4和R 5与二者所连接的氮原子一起形成杂环基；
R 2选自：-NR 6R 7、-OR 6、-SR 6，其中，R 6和R 7独立地选自：H、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氧基烷基，或者R 6和R 7与二者所连接的氮原子一起形成杂环基，或R 6与其所连接的氧原子一起形成杂环基，或R 6与其所连接的硫原子一起形成杂环基；
R 3为苯环上一个或多个独立的取代基，其选自：H、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氧基烷基；
m为0-4的整数(例如0、1、2、3、4)。
具体地，式Ⅱ具有如下结构：
Figure PCTCN2022092556-appb-000007
在本发明的一个实施方式中，上述L'为C 1-C 6亚烷基，例如C 1-C 3亚烷基。
在本发明的一个实施方式中，上述L'具有如下结构：
Figure PCTCN2022092556-appb-000008
其中，R a和R b独立地选自：H、C 1-C 3烷基(例如甲基、乙基、正丙基、异丙基)，或，R a和R b与其所连接的碳原子一起形成C 3-C 6亚环烷基(例如亚环丙基、亚环丁基、亚环戊基、亚环己基)。
具体地，L'可选自：-CH 2-、
Figure PCTCN2022092556-appb-000009
在本发明的一个实施方式中，上述R 1为C 1-C 6亚烷基，例如C 1-C 3亚烷基，例如-CH 2-。
在本发明另一个实施方式中，上述R 1为单键。
在本发明的一个实施方式中，上述R 1为C 1-C 6亚烷氧基，例如C 1-C 3亚烷氧基，例如-CH 2O-、-CH 2CH 2O-、-CH 2CH 2CH 2O-。
具体地，R 3为苯环上一个或多个独立的取代基，其选自：H、甲基、甲氧基。
在本发明的实施例中，m为0或1。
具体地，a为1-5的整数，例如1、2、3、4、5。
具体地，R 8和R 9独立地选自：H、甲基、乙基、正丙基、异丙基。
具体地，上述R 4和R 5独立地选自：H、甲基、乙基、正丙基、异丙基、正丁基、叔丁基、氨基酸残基、寡肽残基、-CF 3、-CH 2CF 3、
Figure PCTCN2022092556-appb-000010
Figure PCTCN2022092556-appb-000011
或者R 4和R 5与二者所连接的氮原子一起形成取代或未取代的杂环基。
具体地，上述取代或未取代的杂环基可以选自：
Figure PCTCN2022092556-appb-000012
Figure PCTCN2022092556-appb-000013
具体地，上述氨基酸残基以及寡肽中的氨基酸残基独立地选自：丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸残基中的一种或多种。在本发明的一个实施例中，上述氨基酸残基为亮氨酸残基。在本发明的一个实施例中，上述寡肽残基为-天冬酰胺-丙氨酸-丙氨酸-亮氨酸-。
在本发明的一些实施例中，X为
Figure PCTCN2022092556-appb-000014
R 4和R 5独立地选自C 1-C 6烷基，例如，R 4和R 5均为甲基。
在本发明另一些实施例中，X为-NR 4-，R 4选自：H、C 1-C 6烷基，例如R 4为H或甲基。
在本发明另一些实施例中，X为-NR 4-(C 1-C 6亚烷基)-NR 5-，R 4和R 5独立地选自C 1-C 6烷基，例如，R 4和R 5均为甲基。
在本发明另一些实施例中，X为亚杂环基，特别是亚含氮杂环基，例如
Figure PCTCN2022092556-appb-000015
A环为4至10元含氮杂环，A环可以为单环、双环或三环(包括稠环、螺环、桥环)；具体地，X可以为
Figure PCTCN2022092556-appb-000016
Figure PCTCN2022092556-appb-000017
在本发明的一个实施方式中，R 2选自：-NHR 6、-OR 6、-SR 6，其中，R 6为C 1-C 6烷基或C 1-C 6烷氧基烷基，例如C 1-C 4烷基，例如丁基，特别是正丁基，例如C 1-C 4烷氧基烷基，例如甲氧基乙基。本发明的一些实施例中，R 2选自：
Figure PCTCN2022092556-appb-000018
在本发明另一个实施方式中，R 2选自：-NR 6R 7，其中，R 6和R 7与二者所连接的氮原子一起形成杂环基，例如，
Figure PCTCN2022092556-appb-000019
具体地，通式Ⅰ中D部分可以选自如下结构：
Figure PCTCN2022092556-appb-000020
Figure PCTCN2022092556-appb-000021
Figure PCTCN2022092556-appb-000022
在本发明的一个实施例中，通式Ⅰ中D部分具有如下结构：
Figure PCTCN2022092556-appb-000023
在本发明的一个实施方式中，上述偶联物具有如下结构：
Figure PCTCN2022092556-appb-000024
其中，Ab、n、m、R 1、R 2、R 3、R 4、R 5、R a、R b具有本发明上述定义。
具体地，上述偶联物中的连接单元可以为化学不稳定连接单元(如包含腙、二硫基)、酶催化连接单元(如包含肽残基、对酯酶不稳定的碳酸盐残基)等形式。
具体地，连接Ab和D的连接单元L包含与Ab的活性基团(如巯基、氨基、羧基等，特别是巯基)的连接基团Y，例如单键、
Figure PCTCN2022092556-appb-000025
-S-、-CO-、-NH-、-CONH-、
Figure PCTCN2022092556-appb-000026
(胺基氧基)；在本发明的一些实施例中，Y为-S-或
Figure PCTCN2022092556-appb-000027
进一步地，L还包含基团Q，Q为二价的饱和或不饱和的直链或支链的C1-50烃链，其中0-6个亚甲基单元独立地被以下取代：-Cy-、-O-、-NR 10-、-S-、-OC(O)-、-C(O)O-、-C(O)-、-S(O)-、-S(O) 2 -、-NR 10S(O) 2-、-S(O) 2-NR 10-、-NR 10-C(O)-、-C(O)NR 10-、-OC(O)NR 10-、-NR 10-C(O)O-、
Figure PCTCN2022092556-appb-000028
Figure PCTCN2022092556-appb-000029
氨基酸残基、寡肽残基，其中，k选自1-10之间的整数(例如1、2、3、4、5、6、7、8、9、10)，每个-Cy-独立地为选自以下的任选地经取代的二价环：亚芳基、亚环烷基、亚杂环基；R 10选自：H、-OH、C 1-C 6烷基、C 3-C 6环烷基、杂环烷基。
具体地，每个-Cy-独立地为选自以下的任选地经取代的二价环：亚苯基、双环亚芳基、单环亚环烷基、双环亚环烷基、单环亚杂芳基、双环亚杂芳基、单环亚杂环烷基、双环亚杂环烷基；特别是，亚苯基、单环亚环烷基、单环亚杂芳基、单环亚杂环烷基。
更具体地，每个-Cy-可以独立地选自以下：
Figure PCTCN2022092556-appb-000030
Figure PCTCN2022092556-appb-000031
Figure PCTCN2022092556-appb-000032
其中，R 11为环上一个或多个独立的取代基，并选自：H、卤素、-CN、-NO 2、-CF 3、-OCF 3、-NH 2、-OH、C 1-C 6烷基、-O(C 1-C 6烷基)、-NH(C 1-C 6烷基)、-N(C 1-C 6烷基)(C 1-C 6烷基)、单糖或其衍生物的残基，R 12选自：H、取代或未取 代的烷基、取代或未取代的芳基、取代或未取代的芳烷基、取代或未取代的环烷基、取代或未取代的环烷基烷基、取代或未取代的杂环基、取代或未取代的杂环基烷基。
具体地，单糖或其衍生物可以选自：阿拉伯糖、木糖、核糖、葡萄糖、甘露糖、半乳糖、果糖、葡萄糖醛酸、半乳糖醛酸。在本发明的一些实施例中，单糖或其衍生物的残基为半乳糖醛酸残基，例如
Figure PCTCN2022092556-appb-000033
在本发明的一些实施例中，每个-Cy-独立地选自以下：
Figure PCTCN2022092556-appb-000034
Figure PCTCN2022092556-appb-000035
具体地，R 10可以选自：H、C 1-C 6烷基；在本发明的一些实施例中，R 10为H或甲基。
具体地，上述Q的定义中，氨基酸残基以及寡肽残基中的氨基酸残基独立地选自：丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、瓜氨酸、鸟氨酸残基中的一种或多种，特别是选自：缬氨酸、瓜氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、脯氨酸、甘氨酸残基中的一种或多种；特别是，氨基酸残基以及寡肽残基中的氨基酸残基为L型氨基酸的残基。
具体地，上述Q的定义中，寡肽残基由2-10个氨基酸残基组成，特别是由2-6个氨基酸残基组成，例如由2、3、4或5个氨基酸残基组成。
具体地，上述Q的定义中，寡肽残基选自：缬氨酸-瓜氨酸残基、丙氨酸-丙氨酸-天冬酰胺残基、天冬氨酸-缬氨酸残基、谷氨酸-缬氨酸残基、甘氨酸-脯氨酸残基等。
在本发明的一些实施例中，寡肽残基为
Figure PCTCN2022092556-appb-000036
Figure PCTCN2022092556-appb-000037
在本发明的一个实施方式中，通式Ⅰ中L部分具有如下结构：
Figure PCTCN2022092556-appb-000038
其中，
Y为与Ab的活性基团(如巯基、氨基、羧基等，特别是巯基)的连接基团；
R L1、R L3和R L4独立地选自：
Figure PCTCN2022092556-appb-000039
-(CH 2) jO-、-(CH 2) jN(R L9)-、-(CH 2) jCO-、-(CH 2) jOCO-、-(CH 2) jOCON(R L9)-、-(CH 2) jN(R L9)CON(R L10)-、-(CH 2) jN(R L9)CO-、-O(CH 2) jCOO-、-(CH 2) jCOO-、-(CH 2) jCON(R L9)-、-(CH 2) jS-S-、-(CH 2) jN(R L9)-NH＝、
Figure PCTCN2022092556-appb-000040
Figure PCTCN2022092556-appb-000041
Figure PCTCN2022092556-appb-000042
亚环烷基和亚芳基中一种或多种的组合，j为0-10的整数；
R L2选自：单键、-(CH 2) iOCO-、-(CH 2) iOCOO-、-(CH 2) iNH-COO-、氨基酸残基、寡肽残基，其中i为0-10的整数；
R L5、R L6、R L7和R L8独立地选自：H、取代或未取代的烷基、卤素、硝基、氰基、-OR L9、-NR L9R L10、-S(O) tR L9、-C(O)OR L9、-C(O)R L9和-C(O)NR L9R L10，其中，t为0、1或2；
各R L9和R L10独立地选自：H、取代或未取代的烷基、取代或未取代的芳基、取代或未取代的芳烷基、取代或未取代的环烷基、取代或未取代的环烷基烷基、取代或未取代的杂环基、取代或未取代的杂环基烷基。
具体地，Y选自：单键、
Figure PCTCN2022092556-appb-000043
-S-、-CO-、-NH-、-CONH-、
Figure PCTCN2022092556-appb-000044
(胺基氧基)。在本发明的一些实施例中，Y为-S-或
Figure PCTCN2022092556-appb-000045
具体地，上述R L2的定义中，氨基酸残基以及寡肽残基中的氨基酸残基独立地选自：丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、 甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、瓜氨酸、鸟氨酸残基中的一种或多种。特别是，氨基酸残基以及寡肽残基中的氨基酸残基为L型氨基酸的残基。
具体地，上述R L2的定义中，寡肽残基由2-10个氨基酸残基组成，特别是由2-6个氨基酸残基组成，例如由2、3、4或5个氨基酸残基组成。
具体地，上述R L2的定义中，寡肽残基可以选自：缬氨酸-瓜氨酸残基、丙氨酸-丙氨酸-天冬酰胺残基、天冬氨酸-缬氨酸残基、谷氨酸-缬氨酸残基、甘氨酸-脯氨酸残基等。
在本发明的一个实施例中，R L2为-(CH 2) iOCO-，i为0-6的整数，特别是i为1。
在本发明的一个实施例中，R L2为-(CH 2) i，i为1-10的整数，特别是i为2。
在本发明另一个实施例中，R L2为寡肽残基，选自：缬氨酸-瓜氨酸残基、丙氨酸-丙氨酸-天冬酰胺残基、天冬氨酸-缬氨酸残基、谷氨酸-缬氨酸残基等，特别是为
Figure PCTCN2022092556-appb-000046
Figure PCTCN2022092556-appb-000047
具体地，R L1、R L3和R L4独立地选自：-(CH 2) j-、-(CH 2) jO-、-(CH 2) jNH-、-(CH 2) jCO-、-(CH 2) jOCOO-、-(CH 2) jOCONH-、-(CH 2) jNHCONH-、-(CH 2) jNHCO-、-O(CH 2) jCOO-、-(CH 2) jCOO-和-(CH 2) jCONH-中一种或多种的组合，j为0-10的整数。
具体地，上述各j可以独立地为0、1、2、3、4、5、6、7、8、9、10。
在本发明的一个实施例中，R L1为
Figure PCTCN2022092556-appb-000048
在本发明另一个实施例中，R L1为-(CH 2) jCO-，j为0-6的整数，特别是j为5。
在本发明的一个实施例中，R L3为-(CH 2) jNH-，j为0-6的整数，特别是j为0。
在本发明的一个实施例中，R L4为-(CH 2) j-，j为0-5的整数，特别是j为1。
具体地，R L5、R L6、R L7和R L8独立地选自：H、C1-6烷基、C1-6卤代烷基、卤素、硝基、氰基、-OR L9；R L8选自：H、C1-6烷基；更具体地，R L5、R L6、R L7和R L8独立地选自：H、卤素。
在本发明的一个实施例中，R L5为H。
在本发明的一个实施例中，R L6为H。
在本发明的一个实施例中，R L7为H。
在本发明的一个实施例中，R L8为H。
在本发明的一个实施方式中，上述L部分具有如下结构：
Figure PCTCN2022092556-appb-000049
其中，Y、R L1、R L2具有本发明上述相应定义。
在本发明的一个实施例中，上述L部分可以具有如下结构：
Figure PCTCN2022092556-appb-000050
在本发明另一个实施例中，上述L部分还可以具有如下结构：
Figure PCTCN2022092556-appb-000051
其中，R L2为寡肽残基，具有本发明上述定义。
具体地，上述L部分具有如下结构：
Figure PCTCN2022092556-appb-000052
Figure PCTCN2022092556-appb-000053
Figure PCTCN2022092556-appb-000054
在本发明另一些实施例中，L部分具有如下结构：
Figure PCTCN2022092556-appb-000055
Figure PCTCN2022092556-appb-000056
k 1和k 2独立地选自1-10之间的整数。
在本发明的一个实施方式中，上述偶联物具有如下结构：
Figure PCTCN2022092556-appb-000057
其中，Ab、n和D具有本发明上述相应定义。
在本发明的一些实施例中，上述偶联物具有如下结构：
Figure PCTCN2022092556-appb-000058
Figure PCTCN2022092556-appb-000059
Figure PCTCN2022092556-appb-000060
Figure PCTCN2022092556-appb-000061
其中，Ab和n具有本发明上述相应定义。
本发明还提供一种上述偶联物的立体异构体(如上所示)或其混合物。
本发明还提供一种上述偶联物的药学上可接受的盐、溶剂化物和前药。
具体地，上述药学上可接受的盐可包括有机盐或无机盐，例如，盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐、甲酸盐、乙酸盐、三氟乙酸盐、泛酸盐、琥珀酸盐、枸橼酸盐、酒石酸盐、富马酸盐、马来酸盐、葡糖酸盐、葡糖醛酸盐、糖酸盐、苯甲酸盐、乳酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐、精氨酸盐、天冬氨酸盐、谷氨酸盐、泛酸盐和抗坏血酸盐中的一种或多种。
本发明还提供一种上述偶联物的制备方法，其可以包括将小分子药物与连接单元的结合物(L-D)与抗体进行偶联的步骤。
具体地，在偶联步骤前，上述制备方法还包括将抗体的二硫键还原和氧化的步骤。
具体地，上述制备方法包括如下步骤：
(1)将抗体与二硫键还原剂孵育；
(2)将步骤(1)所得抗体与氧化剂孵育；
(3)将步骤(2)所得抗体与上述结合物L-D孵育。
具体地，步骤(1)还可包括(例如，通过超滤)除去多余的二硫键还原剂。
具体地，步骤(1)中所述二硫键还原剂可以为三(2-羧乙基)膦(TCEP)、二硫苏糖醇(DTT)、β-巯基乙醇等。
具体地，步骤(1)中，抗体与二硫键还原剂的当量比例为1:8-15(具体如1:8、1:9、1:10、1:11、1:12)。
具体地，步骤(1)中的孵育的温度为35-40℃(具体如35、36、37、38、39、40℃)；在本发明的一个实施例中，该孵育温度为37℃。
具体地，步骤(1)中的孵育的时间为0.5-6小时(具体如0.5、1、2、3、4、5、6小时)；在本发明的一个实施例中，该孵育时间为3小时。
具体地，步骤(2)还可包括(例如，通过超滤)除去多余的氧化剂。
具体地，步骤(2)中的氧化剂可以为脱氢抗坏血酸(DHAA)、Cu(II)等。
具体地，步骤(2)中，抗体与氧化剂的当量比例为1:40-60(具体如1:40、1:45、1:50、1:55、1:60)。
具体地，步骤(2)中的孵育的温度为20-30℃(具体如20、22、24、25、26、28、30℃)；在本发明的一个实施例中，该孵育温度为室温。
具体地，步骤(2)中的孵育的时间为1-6小时(具体如1、2、3、4、5、6小时)；在本发明的一个实施例中，该孵育时间为3小时。
具体地，步骤(3)中，抗体与小分子L-D的当量比例为1:10-20(具体如1:10、1:12、1:14、1:15、1:16、1:18、1:20)。
具体地，步骤(3)中的孵育的温度为20-30℃(具体如20、22、24、25、26、28、30℃)；在本发明的一个实施例中，该孵育温度为室温。
具体地，步骤(3)中的孵育的时间为6-48小时(具体如6、12、18、20、22、24、26、28、30、36、42、48小时)；在本发明的一个实施例中，该孵育时间为24小时。
在本发明的一个实施方式中，上述制备方法还包括小分子药物与连接单元的结合物(L-D)的制备步骤。
本发明还提供一种修饰的抗PD-L1抗体，其通过基因工程手段(例如Thiomab技术)将已知的抗PD-L1抗体(例如，阿特珠单抗(Atezolizumab)、德瓦鲁单抗(Durvalumab)、阿维单抗(Avelumab)、西米普利单抗(Cemiplimab)、KN035、CS1001、BGB-A333、KL-A167、SHR-1316和STI-A1014等)的一个或多个氨基酸残基分别突变为半胱氨酸获得，例如将阿维单抗(Avelumab)的重链和/或轻链中的氨基酸突变为半胱氨酸。
具体地，上述抗体包含重链和轻链；
其中，重链的氨基酸序列如SEQ ID NO：4所示，或为与SEQ ID NO：4具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；和/或，
轻链的氨基酸序列如SEQ ID NO：9所示，或为与SEQ ID NO：9具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列。
具体地，上述抗体具有如SEQ ID NO：9所示的轻链序列。
在本发明的一个实施例中，上述抗体具有如SEQ ID NO：4所示的重链序列和如SEQ ID NO：9所示的轻链序列。
本发明还提供一种上述修饰的抗PD-L1抗体在制备抗体药物偶联物(ADC)中的应用。
本发明还提供一种小分子化合物及其药学上可接受的盐、立体异构体、溶剂化物、前药，该化合物具有如下结构：
Figure PCTCN2022092556-appb-000062
其中，m、X、L'、R 1、R 2、R 3、Q具有本发明上述相应定义，
Z为活性基团。
在本发明的一个实施方式中，该小分子化合物具有如下结构：
Figure PCTCN2022092556-appb-000063
其中，m、X、L'、R 1、R 2、R 3、R L1、R L2、R L3、R L4、R L5、R L6、R L7、R L8具有本发明上述相应定义，
Z为活性基团。
具体地，Z为与氨基酸的活性基团(如巯基、氨基、羧基等，特别是巯基)的反应连接基团；具体地，例如，巯基反应活性基团可以为马来酰亚胺基、羧基、酰胺基、卤素、二硫基等；氨基反应性基团可以为酯基、酰卤基、酸酐、羧基、环氧基等；羧基氨基反应性基团可以为羟基、氨基、卤素、酰卤基等
在本发明的一些实施例中，Z为
Figure PCTCN2022092556-appb-000064
具体地，式Ⅶ所示化合物具有如下结构：
Figure PCTCN2022092556-appb-000065
其中，Z、R L1、R L2、R L3、R L4、R 1、R 2、R 3、R 4、R 5、R a、R b、m具有本发明上述相应定义。
更具体地，式Ⅶ所示化合物具有如下结构：
Figure PCTCN2022092556-appb-000066
其中，Z、R L1、R L2、R 1、R 2、R 3、R 4、R 5、R a、R b、m具有本发明上述相应定义。
进一步具体地，式Ⅶ所示化合物具有如下结构：
Figure PCTCN2022092556-appb-000067
其中，Z、R L1、R L2具有本发明上述相应定义。
在本发明的一些实施例中，式Ⅶ所示化合物具有如下结构：
Figure PCTCN2022092556-appb-000068
Figure PCTCN2022092556-appb-000069
Figure PCTCN2022092556-appb-000070
Figure PCTCN2022092556-appb-000071
本发明还提供一种化合物及其药学上可接受的盐、立体异构体，其可用作抗体药物偶联物的连接单元部分，其具有如下结构：
Figure PCTCN2022092556-appb-000072
其中，Z、R L1、R L2、R L3、R L4、R L5、R L6、R L7、R L8具有本发明上述相应定义，Z'为反应活性基团。
具体地，Z'可以为卤素(例如氯)、羧基、酯基、羟基、环氧基、胺基等。具体地，式Ⅸ所示化合物具有如下结构：
Figure PCTCN2022092556-appb-000073
其中，Z、R L1、R L2、Z'具有本发明上述相应定义。
在本发明的一些实施例中，式Ⅸ所示化合物具有如下结构：
Figure PCTCN2022092556-appb-000074
Figure PCTCN2022092556-appb-000075
在本发明的一些实施例中，上述Z'为卤素(例如氯)。
在本发明另一些实施例中，上述Z'为碳酸酯基(例如
Figure PCTCN2022092556-appb-000076
本发明还提供一种上述式Ⅸ所示化合物在制备抗体药物偶联物(ADC)中的应用。
本发明还提供一种包含上述偶联物和药学上可接受的辅料的药物组合物。
具体地，上述药学上可接受的辅料为药学领域常规的药物辅料，例如：稀释剂、赋形剂如水等，填充剂如淀粉、蔗糖等；粘合剂如纤维素衍生物、藻酸盐、明胶和聚乙烯吡咯烷酮等；湿润剂如甘油等；崩解剂如琼脂、碳酸钙和碳酸氢钠等；吸收促进剂如季铵化合物等；表面活性剂如十六烷醇等；吸附载体如高岭土和皂粘土等；润滑剂如滑石粉、硬脂酸钙和镁、聚乙二醇等。另外，还可以在药物组合物中加入其它辅剂如香味剂、甜味剂、稳定剂等。
具体地，根据所需给药方式，上述药物组合物将包含约1至约99重量％(具体如1％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、99％)的本发明上述偶联物、其余为适宜的辅料。
具体地，上述药物组合物可用于以任何给药途径给药，可以是口服或肠道外给药，如经肺、鼻、直肠和/或静脉注射给药。因此，根据本发明的制剂可适用于局部或全身给药，特别是用于皮肤、皮下、肌肉、关节内、腹膜内、肺部、口腔、舌下、鼻、经皮穿刺、阴道、口服的或肠道外给药。直肠给药优选形式为栓剂。
适用于口服给药的剂型为片剂、丸剂、咀嚼胶姆剂、胶囊剂、颗粒剂、滴剂或糖浆剂等。适用于肠道外给药的剂型为溶液、悬浮液、可复水的干剂或喷雾剂等。
本发明的组合物可配制成溶解形式的沉积物或贴剂用于经皮给药。皮肤应用包括软膏剂、凝胶剂、霜剂、洗剂、悬浮液或乳液等。
本发明的药物组合物的各种剂型可以按照药学领域的常规生产方法制备，例如，使活性成分与一种 或多种辅料混合，然后将其制成所需的剂型。
具体地，上述药物组合物中，本发明上述偶联物可以作为唯一的活性成分，也可与一种或多种其它用于相同适应症的活性成分联用，其中，本发明上述偶联物与该其他活性成分可配制用于同时、单独或顺序给药(simultaneous,separate or sequential administration)。
本发明还提供一种组合物，其包含本发明所述抗体和小分子药物作为活性成分。
具体地，上述组合物中，抗体为抗PD-L1抗体，例如，阿特珠单抗(Atezolizumab)、德瓦鲁单抗(Durvalumab)、阿维单抗(Avelumab)、西米普利单抗(Cemiplimab)、KN035、CS1001、BGB-A333、KL-A167、SHR-1316和STI-A1014等，以及通过基因工程手段将已知的抗PD-L1抗体(如前所述)的一个或多个氨基酸残基分别突变为半胱氨酸获得的修饰的抗PD-L1抗体，例如将阿维单抗(Avelumab)的重链和/或轻链中的氨基酸突变为半胱氨酸。
具体地，上述抗体包含重链和轻链；
其中，重链的氨基酸序列如SEQ ID NO：4所示，或为与SEQ ID NO：4具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；和/或，
轻链的氨基酸序列如SEQ ID NO：9所示，或为与SEQ ID NO：9具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列。
具体地，上述抗体具有如SEQ ID NO：9所示的轻链序列
在本发明的一个实施例中，上述抗体具有如SEQ ID NO：4所示的重链序列和如SEQ ID NO：9所示的轻链序列。
具体地，上述组合物中，小分子药物可以为免疫调节剂，例如，Toll样受体激动剂(Toll-like receptors，TLR)，具体如，TLR7和/或TLR8激动剂，例如专利申请公开号WO2019/095455A1中所述吡啶并嘧啶衍生物及其盐。
具体地，上述组合物中的小分子药物具有如下结构：
Figure PCTCN2022092556-appb-000077
其中，R 1、R 2、R 3、m、L'具有本发明上述相应定义；
X'选自：H、卤素、-NR 4R 5、-N +R 4R 5、-OR 4、-SR 4、叠氮基、膦酸基、膦酸二乙酯基、卤代烷基、羧基、酯基、-CN；其中，R 4和R 5独立地选自：H、C 1-C 6烷基、C 1-C 6烷氧基、氨基酸残基、寡肽残基、卤代烷基，或者R 4和R 5与二者所连接的氮原子一起形成杂环基。
更具体地，上述组合物中的小分子药物可以选自如下结构：
Figure PCTCN2022092556-appb-000078
Figure PCTCN2022092556-appb-000079
Figure PCTCN2022092556-appb-000080
Figure PCTCN2022092556-appb-000081
在本发明的一个实施例中，上述组合物中，抗体为阿维单抗(Avelumab)，小分子药物具有如 下结构：
Figure PCTCN2022092556-appb-000082
本发明还提供一种上述偶联物或其药学上可接受的盐、溶剂化物、前药、立体异构体、本发明上述抗体、式Ⅶ所示化合物、组合物在制备预防和/或治疗疾病的药物中的应用。
具体地，上述疾病可以为PD-L1表达相关的疾病，也可以为TLR7和/或TLR8活性相关疾病。
具体地，上述疾病可以为呼吸系统疾病、免疫性疾病、病毒性疾病、肿瘤中的一种或多种。
具体地，上述呼吸系统疾病包括但不限于：哮喘、慢性阻塞性肺病、成人呼吸窘迫综合征。
具体地，上述免疫性疾病为自身免疫病，包括但不限于：系统性红斑狼疮、类风湿性关节炎、炎性肠病、斯耶格伦氏综合症、多肌炎、脉管炎、韦格纳肉芽肿、结节病、强直性脊柱炎、赖特综合征、牛皮癣性关节炎、贝赫切特综合征等。
具体地，上述病毒性疾病的病原体包括但不限于：腺病毒科(如腺病毒)、疱疹病毒科(如HSV1(口腔疱疹)、HSV2(外生殖器疱疹)、VZV(水痘)、EBV(埃-巴二氏病毒)、CMV(巨细胞病毒))、痘病毒科(如天花病毒、牛痘病毒)、乳多泡病毒科(如乳头瘤病毒)、细小病毒科(如B19病毒)、嗜肝DNA病毒科(如乙型肝炎病毒)、多瘤病毒科(如多瘤病毒)、呼肠孤病毒科(如呼肠弧病毒、轮状病毒)、小核糖核酸病毒科(如肠道病毒、口蹄疫病毒)、嵌杯样病毒科(如诺沃克病毒、戊型肝炎病毒)、披膜病毒科(如风疹病毒)、沙粒病毒科(如淋巴细胞性脉络丛脑膜炎病毒)、逆转录病毒科(HIV-1、HIV-2、HTLV-1)、黄病毒科(如登革热病毒、寨卡病毒、乙型脑炎病毒、基孔肯亚病毒、黄热病病毒、丙型肝炎病毒、西尼罗病毒等)、正粘病毒科(如流感病毒(如甲型流感病毒、乙型流感病毒、丙型流感病毒等))、副粘病毒科(如1型人副流感病毒(HPV)、2型HPV、3型HPV、4型HPV、仙台病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞病毒、新城疫病毒等)、布尼亚病毒科(如加利福尼亚脑炎病毒、汉坦病毒)、弹状病毒科(如狂犬病毒)、丝状病毒科(如埃博拉病毒、马尔堡病毒)、冠状病毒科(如HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV、SARS-CoV-2等)、星状病毒科(如星状病毒)、博尔纳病毒科(如博尔纳病毒)；更具体地，上述病毒性疾病可以为流感、SARS、COVID-19、病毒性肝炎(如甲型肝炎、乙型肝炎、丙型肝炎、丁型肝炎等)、AIDS、狂犬病、登革热、埃博拉病毒病等。
具体地，上述肿瘤为恶性肿瘤，其包括但不限于：淋巴瘤、母细胞瘤、髓母细胞瘤、视网膜母细胞瘤、脂肪肉瘤、滑膜细胞肉瘤、神经内分泌肿瘤、类癌肿瘤、胃泌素瘤、胰岛细胞癌、间皮瘤、神经鞘瘤、听神经瘤、脑膜瘤、腺癌、黑色素瘤、白血病或淋巴样恶性肿瘤、鳞状细胞癌、上皮鳞状细胞癌、肺癌(小细胞肺癌、非小细胞肺癌、腺癌肺癌、肺鳞癌)、腹膜癌、肝细胞癌、胃癌、肠癌、胰腺癌、成胶质细胞瘤、子宫颈癌、卵巢癌、肝癌、膀胱癌、肝癌、乳腺癌、转移性乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫癌、唾液腺癌、肾癌、前列腺癌、外阴癌、甲状腺癌、肝癌、肛门癌、阴茎癌、梅克尔细胞癌、食管癌、胆道肿瘤、头颈部癌和血液恶性肿瘤；特别是结肠癌、膀胱癌、黑色素瘤、脑膜瘤、肺癌、胰腺癌。
本发明还提供一种疾病的预防和/或治疗方法，其包括向有需要的系统或个体给予治疗有效量的本发明上述偶联物或其药学上可接受的盐、溶剂化物、前药、立体异构体、本发明上述抗体、式Ⅶ所示化合物、组合物的步骤。
具体地，上述方法中所述疾病具有本发明上述定义。
本发明还提供一种提高机体正向免疫应答的方法，其包括向有需要的系统或个体给予治疗有效量的本发明上述偶联物或其药学上可接受的盐、溶剂化物、前药、立体异构体、本发明上述抗体、式Ⅶ所示化合物、组合物的步骤。
本发明还提供一种增强化疗效果的方法，其包括向有需要的系统或个体给予治疗有效量的本发明上述偶联物或其药学上可接受的盐、溶剂化物、前药、立体异构体、本发明上述抗体、式Ⅶ所示化合物、组合物的步骤。
本发明还提供一种提高免疫治疗效果的方法，其包括向有需要的系统或个体给予治疗有效量的本发明上述偶联物或其药学上可接受的盐、溶剂化物、前药、立体异构体、本发明上述抗体、式Ⅶ所示化合物、组合物的步骤。
具体地，上述治疗有效量可根据给药途径、患者的年龄、体重、性别、所治疗的疾病的类型和严重程度等等因素而变化，本发明对此不作具体限定。
本发明通过基因编辑获得带有两个突变半胱氨酸的修饰的抗PD-L1抗体，其基本上保留了原抗体的结构，将其用于ADC的构建。通过实验验证，该修饰抗体和所得ADC保持着原抗PD-L1抗体的抗原识别亲和能力，可通过结合细胞表面PD-L1而内化到胞内，具有良好的选择性。通过抗肿瘤实验发现，所得ADC具有强大的抗肿瘤活性，可明显提高荷瘤动物的生存率，且毒性显著低于其中小分子药物单独给药及其与抗体的联合给药的毒性，对实验动物身体的负担较小。本发明所述ADC大大降低了其中小分子药物单独使用时的最低有效剂量，扩大了其治疗窗，有望用于多种疾病(例如肿瘤、病毒性疾病如乙肝等)治疗药物的开发，具有良好的应用前景和价值。
附图说明
图1所示为质粒αPD-L1 THIOMAB LC-V205C测序结果，其中，A.αPD-L1 THIOMAB LC-V205C测序序列(上)与模板轻链159-pFuse-αPDL1-LC质粒(下)的序列比对，模板质粒上的GTG位点被突变为TGC；B.αPD-L1 THIOMAB LC-V205C突变位点处编码的氨基酸为半胱氨酸(Cys)；C.模板质粒突变位点处编码的氨基酸为缬氨酸(Val)。
图2所示为通过还原和非还原SDS PAGE分析鉴定抗PD-L1抗体的表达结果。
图3所示为通过还原和非还原SDS PAGE分析鉴定抗PD-L1 THIOMAB的表达结果。
图4所示为THIO-S2的质谱分析结果。
图5所示为ADC HE-S2的偶联路线。
图6所示为ADC HE-S2的质谱检测结果。
图7所示为ADC VC的偶联路线。
图8所示为ADC Legumain的偶联路线。
图9所示为ADC VC和ADC Legumain的质谱检测结果。
图10所示为ADC HE-S2和抗PD-L1 THIOMAB的抗体抗原亲和能力的表征结果。MC38细胞用抗PD-L1抗体、抗PD-L1 THIOMAB和ADC HE-S2处理后，用FITC标记的山羊抗人二级抗体染色并通过FACS技术检测分析MC38表面的FITC信号强度。A.不同颜色的直方图从上而下分别代表阴性对照(NC)、二抗(secondary antibody)、抗PD-L1抗体，抗PD-L1 THIOMAB和ADC HE-S2各组所测得的荧光信号。B.MC38上不同抗体类药物处理后所测的平均荧光强度(MFI)。NC组：只加入同型抗体的阴性对照，不加二抗染色；二抗组：加入同型抗体并用荧光二抗染色的对照组。使用Student’s t test确定比较各组之间的显著性差异，并用 ****表示p＜0.0001。
图11所示为抗PD-L1 THIOMAB内化到癌细胞内部的实验结果。MC38和B16细胞用pHrodo TM红色染料标记的抗PD-L1 THIOMAB处理24h后，A.使用
Figure PCTCN2022092556-appb-000083
活细胞系统采集得到细胞的高清相位图像，红色荧光图像和结合图像；B.
Figure PCTCN2022092556-appb-000084
软件分析得到的总体红色荧光对象集成强度(total red object integrated intensity)直方图，纵坐标数据取10的对数。使用Student’s t test比较各组之间的显著性差异， ****表示p＜0.0001。
图12所示为FACS检测Cd274敲除型和野生型DC 2.4细胞表面的PD-L1水平的结果。KO:Cd274敲除型DC 2.4细胞,WT:野生型DC 2.4细胞。使用Student’s t test比较各组之间的显著性差异， **表示P＜0.01。
图13所示为抗PD-L1 THIOMAB在DC细胞内内化的预实验结果。在10％FBS培养基中培养PD-L1阳性的野生型DC 2.4细胞和PD-L1阴性的Cd274敲除DC 2.4细胞，待细胞状态稳定后内加入pHrodo TM红色染料标记的抗PD-L1 THIOMAB，孵育过夜。之后通过FACS分析细胞内红色荧光的强度，以表征THIOMAB的内化水平。
图14所示为DC细胞中PD-L1/抗PD-L1抗体复合物的内化能力的实验结果。pHrodo TM红色染料标记的抗PD-L1 THIOMAB在野生型DC 2.4细胞系和Cd274敲除DC 2.4细胞系内的总体红色荧光对象集成强度-时间变化曲线。在4℃下孵育细胞30分钟以暂时抑制细胞表明受体的内化过程。加入标记的抗PD-L1抗体后，在4℃下培养30分钟以使抗原和抗体完全结合。然后通过
Figure PCTCN2022092556-appb-000085
活细胞系统分析抗体的内化。A.将细胞在含10％FBS的培养基中培养。B.将细胞在含10％小鼠血清的培养基中培养。C.6小时后，用
Figure PCTCN2022092556-appb-000086
活细胞系统采集到的DC 2.4细胞的高清相位图像，红色荧光图像和结合图像。使用Student’s t test比较各组之间的显著性差异， ****表示P＜0.0001。
图15所示为通过小鼠肿瘤模型验证ADC HE-S2的抗肿瘤活性的实验结果。A.用ADC HE-S2治疗、抗PD-L1抗体单独用药、D18单独用药、抗PD-L1 THIOMAB单独用药和抗PD-L1抗体/D18联合治疗对小鼠MC38肿瘤生长的抑制活性。B.整体动物生存研究。曲线纵坐标表示不同天数后仍 然存活的荷瘤小鼠的百分比，横坐标表示实验天数。C.ADC HE-S2治疗、抗PD-L1抗体单独用药、D18单独用药、抗PD-L1 THIOMAB单独用药和抗PD-L1抗体/D18联合治疗对小鼠MC38肿瘤生长的抑制活性。各组小鼠给药剂量与给药时间如表1中所示。使用Student’s t test比较各组之间的显著性差异， ***表示P＜0.001。
图16所示为梯度剂量的D18联合抗PD-L1抗体具有不同的肿瘤抑制能力的实验结果。D18每周给药一次，抗PD-L1抗体或IgG每周给药两次。抗体类药物给药时，每组每只老鼠给药150μg。联用组按照组别，anti-PD-L1+D18(25μg)组每次每只老鼠注射D18 25μg；anti-PD-L1+D18(2.5μg)组每次每只老鼠注射D18 2.5μg；anti-PD-L1+D18(0.25μg)组每次每只老鼠注射D18 0.25μg。使用Student’s t test比较各组之间的显著性差异， ****表示P＜0.0001。
图17所示为ADC HE-S2对小鼠体重的影响结果。用ADC HE-S2、抗PD-L1抗体+D18联用、抗PD-L1抗体单独用药、D18单独用药和同型IgG处理小鼠，记录其体重变化。各种药物的给药时间用箭头标注。使用Student’s t test比较各组之间的显著性差异， *表示P＜0.05， **表示P＜0.01，ns表示无显著性差异。
图18所示为ADC10的小鼠抗肿瘤实验结果。
具体实施方式
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
在本发明的内容中，以下术语具有如下详述的含义。
“烷基”指的是直链或支链的且不含不饱和键的烃链自由基，且该烃链自由基以单键与分子其它部分连接。典型的烷基基团含有1至12(例如1、2、3、4、5、6、7、8、9、10、11、12)个碳原子，如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、新戊基、叔戊基、正己基、异己基等。如果烷基被环烷基取代，其相应为“环烷基烷基”自由基，如环丙基甲基、环丙基乙基、环丁基甲基、环戊基甲基、环己基甲基等。如果烷基被芳基取代，那么其相应为“芳烷基”自由基，如苄基、二苯甲基或苯乙基。如果烷基被杂环基取代，那么其相应为“杂环基烷基”自由基。
“烯基”指的是至少含两个碳原子、至少一个不饱和键的直链或支链的烃链自由基，且该烃链自由基以单键与分子其它部分连接。典型的烯基基团含有1至12(例如1、2、3、4、5、6、7、8、9、10、11、12)个碳原子，如乙烯基、1-甲基-乙烯基、1-丙烯基、2-丙烯基或丁烯基等。
“烷氧基”指的是羟基中的氢被烷基取代后形成的取代基，典型的烷氧基基团含有1至12(例如1、2、3、4、5、6、7、8、9、10、11、12)个碳原子，如甲氧基、乙氧基、丙氧基、丁氧基等。
“芳基”指的是单环或多环自由基，包括含单芳基基团和/或稠芳基基团的多环自由基。典型的芳基基团包含1至3个单环或稠环及6至约18个碳环原子，优选6至约14个碳环原子，如苯基、萘基、联苯基、茚基、菲基或蒽基自由基。
“杂环基”包括含1至3个单环和/或稠环及3至约18个环原子的杂芳香族基团和杂脂环基团。优选的杂芳香族基团和杂脂环基团含5至约10个环原子。本发明的化合物中的合适的杂芳基含1、2或3种杂原子，所述杂原子选自N、O或S原子，所述杂芳基包括，例如，香豆素(包括8-香豆素)、喹啉基(包括8-喹啉基、异喹啉基)、吡啶基、吡嗪基、吡唑基、嘧啶基、呋喃基、吡咯基、噻吩基、噻唑基、异噻唑基、三唑基、四唑基、异恶唑基、恶唑基、咪唑基、吲哚基、异吲哚基、吲唑基、吲嗪基、酞嗪基、蝶啶基、嘌呤基、恶二唑基、噻二唑基、呋吖基、哒嗪基、三嗪基、噌啉基、苯并咪唑基、苯并呋喃基、苯并呋吖基、苯并噻吩基、苯并噻唑基、苯并恶唑基、喹唑啉基、喹喔啉基、萘啶基和呋喃并吡啶基，等等。本发明的化合物中的合适的杂脂环基团含1、2或3种杂原子，所述杂原子选自N、O或S原子，所述杂脂环基团包括，如，吡咯烷基、四氢呋喃基、二氢呋喃、四氢噻吩基、四氢噻喃基、哌啶基、吗啉基、硫代吗啉基、氧硫杂环己烷基、哌嗪基、氮杂环丁烷基、氧杂环丁烷基、硫杂环丁烷基、高哌啶基、氧杂环丙烷基、硫杂环丙烷基、吖庚因基、氧氮杂环庚基、二吖庚因基、三吖庚因基、1,2,3,6-四氢吡啶基、2-吡咯啉基、3-吡咯啉基，二氢吲哚基、2H-吡喃基、4H-吡喃基、二氧杂环己烷基、1,3-二氧戊环基、吡唑啉基、二噻烷基、二硫戊环基、二氢吡喃基、二氢噻吩基、吡唑烷基、咪唑啉基、咪唑烷基、3-氮杂双环[3.1.0]己基、3-氮杂双环[4.1.0]庚基、3H-吲哚基和喹嗪基，等等。
“卤素”或“卤基”是指溴代、氯代、碘代或氟代。
除非另有说明，本发明的化合物还包括同位素标记的形式，即区别仅在于存在一种或多种富含 同位素的原子的化合物。例如，具有仅用氘或氚来替代至少一个氢原子、或者使用富含 13C或 14C的碳来替代至少一个碳、或者使用富含 15N的氮来替代至少一个氮的现有结构的化合物均包含在本发明范围内。
术语“PD-L1”包括同种型，哺乳动物的，例如人PD-L1，人PD-L1的物种同源物，以及包含至少一个与PD-L1的共同表位的类似物。PD-L1的氨基酸序列，例如人PD-L1，是本领域已知的。本发明中，“抗PD-L1抗体”是指可与PD-L1特异性结合的抗体。
术语“抗体”意指包含四个多肽链，即通过二硫键互连的两个重(H)链和两个轻(L)链(即，“全抗体分子”)的免疫球蛋白分子，以及其多聚体(例如，IgM)或其抗原结合片段。每个重链包含重链可变区(“HCVR”或“VH”)和重链恒定区(包含域CH1、CH2和CH3)。每个轻链包含轻链可变区(“LCVR”或“VL”)和轻链恒定区(CL)。VH和VL区可进一步细分为高变区，称为互补决定区(CDR)，其间散布着较保守的区域，称为框架区(FR)。各VH和VL由三个CDR和四个FR构成，以下列顺序由氨基端排列至羧基端：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
如本文中所用，术语抗体的“抗原结合部分”、“抗体片段”等包括特异性地结合抗原以形成复合物的任何天然存在、可以酶促方法获得、合成或基因工程改造的多肽或糖蛋白。如本文所用，术语PD-L1抗体的“抗原结合部分”或“抗体片段”指的是保持与PD-L1特异性结合的能力的抗体的一个或多个片段。抗体片段可包括Fab片段、F(ab') 2片段、Fv片段、dAb片段、含有CDR的片段或分离CDR。抗原结合片段的非限制性实例包括：(i)Fab片段；(ii)F(ab') 2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；(vi)dAb片段；(vii)由模拟抗体高变区的氨基酸残基组成的最小鉴别单位(例如，分离的互补决定区(CDR))。如本文中所用的表述“抗原结合片段”内还涵盖其它工程改造分子，如双功能抗体、三功能抗体、四功能抗体和微抗体等。抗体的抗原结合片段将典型地包含至少一个可变结构域。
术语“治疗有效量”是指可在受试者中实现治疗、预防、减轻和/或缓解本发明所述疾病或病症的剂量。
术语“患者”、“对象”、“个体”等等在本文中可交换使用，并指的是服从本文描述方法的任何动物或其细胞，不论是体外或原位，在一些非限制性实施方式中，其为哺乳动物，例如人、猴、狗、兔、鼠等。
术语“治疗”包括在疾病和/或病症发作之后，根除、移除、逆转、缓解、改变或控制该疾病和/或病症。
术语“预防”指在疾病和/或病症发作之前，通过治疗以避免、最小化或令该疾病和/或病症难于发作或发展的能力。
术语“疾病”是指所述受试者的一种身体状态，该身体状态与本发明所述疾病有关。
本文所引用的各种出版物、专利和公开的专利说明书，其公开内容通过引用整体并入本文。
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：小分子荷载的制备
1、(R)-2-((5-5-硝基吡啶-2-基)二硫代环己基)丙-1-醇(化合物24)的制备
Figure PCTCN2022092556-appb-000087
在0℃，氩气保护下，向5-硝基吡啶-2-硫醇(560mg，3.59mmol)的无水DCM(8mL)溶液中滴加SOCl 2(316μL，3.94mmol)。将所得混合物在室温搅拌2h，反应完成后，将反应混合物在减压下浓缩以提供呈黄色粉末的中间体24。向中间体4在干燥的DCM(10mL)中的溶液中逐滴加入甲基(R)-2-巯基丙-1-醇(根据WO2013055987中描述的程序制备，360mg，3.88mmol)在干燥的DCM中的溶液(5mL)。将反应混合物在氩气保护下于室温搅拌过夜。反应完成后，将反应混合物过滤并将滤液减压浓缩，得到黄色固体。将残余物悬浮在水中，并用碳酸氢钠溶液碱化，然后用DCM(3×100mL)萃取。有机相经无水Na 2SO 4干燥，过滤并减压浓缩。将残余物通过硅胶快速色谱纯化(PE∶EA＝10∶3)，得到化合物24。产率：76％。1H NMR(400MHz，氯仿-d)δ9.23(dd，J＝2.7，0.8Hz，1H)，8.33(dd，J＝8.9，2.6Hz，1H)，7.71(dd，J＝8.8，0.7Hz，1H)，4.48(t，J＝6.7Hz，1H)，3.62(ddd，J＝11.8、7.1、4.3Hz，1H)， 3.38(ddd，J＝11.9、7.3、4.4Hz，1H)，3.11(pd，J＝7.0，4.4Hz，1H)，1.28(d，J＝6.9Hz，3H)。13C NMR(101MHz，氯仿-d)δ167.95，145.12，142.45，131.48，120.85，64.00，49.68，16.73。C 8H 11N 2O 3S 2MS(ESI)m/z[M+H] +计算值为247.0，实测值为247.2。
2、(R)-2-((5-硝基吡啶-2-基)二硫环己基)丙基(4-(羟甲基)苯基)氨基甲酸酯(化合物26)的制备
Figure PCTCN2022092556-appb-000088
将化合物24(246mg，1mmol)和吡啶(79mg，1mmol)溶于干燥的DCM中(5mL)，于0℃搅拌。氩气保护下，向0℃的混合物中逐滴加入加三光气(145mg，0.5mmol)的无水DCM(5mL)溶液，继续搅拌1h。反应结束后，将所得反应液减压旋蒸，得到(R)-2-((5-硝基吡啶-2-基)二硫烷基)丙基碳氯化物(化合物25)，无需进一步纯化即可直接使用。随后，将化合物25(308mg，1.0mmol)溶解在干燥的DCM(5mL)中，并缓慢滴加(4-氨基苯基)甲醇(148mg，1.2mmol)和吡啶(79mg，1.0mmol)的DCM混合溶液(5mL)。将反应混合物在氩气保护下于室温搅拌2h。反应完成后，将所得溶液转移至饱和氯化铵溶液中，并用DCM(3×50mL)萃取。有机层用水和盐水洗涤，然后用无水Na 2SO 4干燥，过滤并在减压旋蒸，得到粗产物。粗产物通过硅胶快速色谱纯化(PE∶EA＝2∶1)，得到化合物26，为白色固体。产率：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)δ168.63，152.87，145.03，142.06，136.88，136.37，131.61，128.04，119.47，118.73，67.26，64.86，45.57，17.10。Mass(ESI)：C 16H 18N 3O 5S 2m/z[M+H] +计算值为396.1，实测值为396.2。
3、(R)-2-((5-硝基吡啶-2-基)二硫环己基)丙基(4-(苄氯)苯基)氨基甲酸酯(化合物19)的制备
Figure PCTCN2022092556-appb-000089
0℃下，化合物26(100mg，0.253mmol)的无水DCM(5mL)溶液中滴加亚硫酰氯(22μL，0.304mmol)。将反应混合物从0℃中取出，室温下搅拌30分钟。反应结束后，减压旋蒸除去过量的亚硫酰氯，得到期望的产物，化合物19，为白色固体。产率：96％。1H NMR(400MHz，氯仿-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，4H)，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(101MHz，氯仿-d)δ168.59，152.71，145.06，142.11，137.56，132.88，131.60，129.59，119.47，118.71，67.34，45.90，45.51，17.07。C 16H1 7ClN 3O 4S 2MS(ESI)：m/z[M+H] +计算值为414.0，实测值为414.2。
4、化合物20的制备
Figure PCTCN2022092556-appb-000090
向化合物N 4-丁基-6-(4-((二甲基氨基)甲基)苄基)吡啶[3,2-d]嘧啶-2,4-二胺(40mg，0.11mmol)的DMF(0.5mL)溶液中加入化合物19(50mg，0.12mmol)，TBAB(8mg，0.022mmol)和DIEA(18μL，0.11mmol)。将反应混合物在室温搅拌并通过LC-MS进行监测。当反应完成时，减压旋蒸除去DMF。将残余物重新溶解在10％的甲醇溶液中，并在室温下搅拌2h。将得到的溶液旋蒸浓缩之后通过相制备高效液相色谱法(RPHPLC)纯化，得到化合物20，为甲酸盐。产率：68.0％。 1H NMR(400MHz，甲醇-d4)δ9.09(d，J＝2.6Hz，1H)，8.32(s，1H)，8.31(dd，J＝8.7，2.6Hz，1H)，8.09(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). 13C NMR(101MHz，甲醇-d4)δ168.46，165.88，160.00，157.48，155.18，153.27，144.32，142.10，141.94，141.18，133.68，133.63，133.31，131.74，129.62，129.31，126.30，125.85，125.76，121.22，120.11，118.01，68.23，67.23，67.2043.08，40.56，30.66，19.77，15.98，12.78。C 37H 44N 9O 4S 2MS(ESI)：m/z[M] +,计算值为742.3，实测值为742.3。HRMS(ESI)[M] +：计算值为742.2958，实测值为742.2968。
5、MC-Val-Cit-PAB-Cl(化合物29)的制备
Figure PCTCN2022092556-appb-000091
0℃下，向MC-Val-Cit-PAB-OH(57mg，0.1mmol)的无水DCM(5mL)溶液中滴加亚硫酰氯(8.7μL，0.304mmol)。将反应混合物从0℃中取出，室温下搅拌12小时。反应结束后，减压旋蒸除去过量的亚硫酰氯，得到所需的产物为棕色固体29，产物未经纯化直接使用。
6、化合物30的制备
Figure PCTCN2022092556-appb-000092
向化合物N 4-丁基-6-(4-((二甲基氨基)甲基)苄基)吡啶[3,2-d]嘧啶-2,4-二胺(40mg，0.11mmol)的DMF(1mL)溶液中加入化合物29(71mg，0.12mmol)，TBAB(8mg，0.022mmol)和DIEA(18μL，0.11mmol)。将反应混合物在室温搅拌并通过LC-MS进行监测。当反应完成时，减压旋蒸除去DMF。将残余物重新溶解在10％的甲醇溶液中，并在室温下搅拌2h。将得到的溶液旋蒸浓缩之后通过相制备高效液相色谱法(RPHPLC)纯化，得到化合物30，为甲酸盐。产率：55.0％。 1H 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,6H),1.74–1.53(m,4H),1.60–1.38(m,2H),1.41–1.19(m,6H),1.14–0.80(m,9H).MS(ESI)[M] +：计算值为920.15，实测值为920.30。
7、(S)-N 1-(4-(氯甲基)苯基)-2-((R)-2-((S)-2-(6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺)丙酰胺)丙酰胺)琥珀酰胺(化合物31)的制备
Figure PCTCN2022092556-appb-000093
0℃下，向(S)-2-((R)-2-((S)-2-(6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺)丙酰胺)丙酰胺)-N 1-(4-(羟甲基)苯基)琥珀酰胺(100mg，0.174mmol)的无水DCM(5mL)溶液中滴加亚硫酰氯(15μL，0.210mmol)。将反应混合物从0℃中取出，室温下搅拌30分钟。反应结束后，减压旋蒸除去过量的亚硫酰氯，得到期望的产物，为棕色固体。产物未经纯化直接使用。
8、化合物32的制备
Figure PCTCN2022092556-appb-000094
向化合物N 4-丁基-6-(4-((二甲基氨基)甲基)苄基)吡啶[3,2-d]嘧啶-2,4-二胺(40mg，0.11mmol)的DMF(1mL)溶液中加入化合物31(71mg，0.12mmol)，TBAB(8mg，0.022mmol)和DIEA(18μL，0.11mmol)。将反应混合物在室温搅拌并通过LC-MS进行监测。当反应完成时，减压旋蒸除去DMF。将残余物重新溶解在10％的甲醇溶液中，并在室温下搅拌2h。将得到的溶液旋蒸浓缩之后通过相制备高效液相色谱法(RPHPLC)纯化，得到化合物甲酸盐32。产率：48.0％。LC-MS(ESI)[M] +：计算值为920.10，实测值为920.25。
9、化合物33的制备
Figure PCTCN2022092556-appb-000095
取商业可得的化合物(2S,3R,4S,5S,6S)-2-(2-氨基-4-(羟甲基)苯氧基)-6-(甲氧羰基)四氢-2H-吡喃-3,4,5-三醇三醋酸酯(455mg，1mmol)溶于DMF(5mL)，依次加入6-(马来酰亚胺基)己酸琥珀酰亚胺酯(340mg，1.1mmol)、1-羟基苯并三唑(135mg,1mmol)和N,N-二异丙基乙胺(210μL，1.2mmol)，室温搅拌2小时。将反应液用EA/H 2O萃取，有机相浓缩得粗产物。粗产物经硅胶柱层析(DCM:MeOH＝10:1)纯化得到目标产物278mg。LC-MS(ESI)[M] +：649.3。
10、化合物34的制备
Figure PCTCN2022092556-appb-000096
0℃下，向(2S,3R,4S,5S,6S)-2-(2-(6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺)-4-(羟甲基)苯氧基)-6-(甲氧基羰基)四氢-2H-吡喃-3,4,5-三酰三醋酸酯(化合物33)(65mg，0.1mmol)的无水DCM(2mL)溶液中滴加亚硫酰氯(8μL，0.12mmol)。将反应混合物0℃搅拌30分钟。反应结束后，减压旋蒸除去有机溶剂得到目标产物。产物未经纯化直接使用。
11、化合物35的制备
Figure PCTCN2022092556-appb-000097
向化合物(2S,3R,4S,5S,6S)-2-(4-(氯甲基)-2-(6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺)苯氧 基)-6-(甲氧基羰基)四氢-2H-吡喃-3,4,5-三酰三醋酸酯(67mg，0.1mmol)(化合物34)的DMF(1mL)溶液中加入N 4-丁基-6-(4-((二甲基氨基)甲基)苄基)吡啶[3,2-d]嘧啶-2,4-二胺(40mg，0.11mmol)，TBAB(8mg，0.022mmol)和DIEA(18μL，0.11mmol)。将反应混合物在室温搅拌并通过LC-MS进行监测。当反应完成时，将反应液过滤之后通过相制备高效液相色谱法(RPHPLC)纯化，得到目标产物30mg，产率为28％。LC-MS(ESI)[M] +：995.4。
12、化合物36的制备
Figure PCTCN2022092556-appb-000098
取化合物35(30mg，0.03mmol)溶于混合溶剂(THF:H 2O＝2:1)，加入一水氢氧化锂(15mg，0.3mmol)，室温搅拌30min后，加入醋酸调节pH至7。减压浓缩除去有机溶剂，再加入甲醇溶解，反相色谱制备得目标产物12mg，产率46％。LC-MS(ESI)[M] +：855.4。
13、化合物37的制备
Figure PCTCN2022092556-appb-000099
取N 4-丁基-6-(4-(氯甲基)苄基)吡啶[3,2-d]嘧啶-2,4-二胺(356mg，1mmol，合成方法参照中国专利CN 108069963 B实施例中化合物10的制备方法)溶于DMF中，加入2-Boc-2,6-二氮杂螺[3.3]庚烷半草酸盐(980mg，0.2mmol)和碳酸钾(420mg，0.3mmol)，35℃搅拌过夜。反应结束后，反应液用乙酸乙酯和饱和食盐水萃取，用水洗涤3次。有机相经无水硫酸钠干燥，旋干得到粗产物。粗产物经过硅胶柱层析(DCM-MeOH体系)纯化得目标产物340mg，产率为65％。LC-MS(ESI)[M+H] +：518.3。
14、化合物38的制备
Figure PCTCN2022092556-appb-000100
取6-(4-((2-氨基-4-(丁基氨基)吡啶[3,2-d]嘧啶-6-基)甲基)苄基)-2,6-二氮杂螺[3.3]庚烷-2-羧酸叔丁酯(化合物37)(260mg，0.5mmol)溶于混合溶剂(DCM:TFA＝10:1)。将反应液置于0℃搅拌1小时。减压浓缩除去有机溶剂得粗产物。往粗产物中加入乙醚超声，过滤得到目标产物。LC-MS(ESI)[M+H] +：418.3。
15、化合物39的制备
Figure PCTCN2022092556-appb-000101
取4-((S)-2-((S)-2-(6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺)-3-甲基丁酰胺)-5-尿苷戊酰胺)苄基(4-硝基苯)碳酸酯(81.0mg，0.11mmol)溶于DMF(0.5mL)中，加入6-(4-((2,6-二氮杂螺[3.3]庚烷-2-基)甲基)苄基)-N 4-丁基吡啶[3,2-d]嘧啶-2,4-二胺(化合物38)(42.0mg，0.10mmol)和N,N-二异丙基乙胺(21μL，0.12mmol)，室温搅拌，LCMS检测反应。反应结束后，直接RP-HPLC制备得目标产物20mg，产率为20％。LC-MS(ESI)[M+H] +：1016.4。 1H 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).
16、化合物40的制备
Figure PCTCN2022092556-appb-000102
取商业可得的4-((S)-4-氨基-2-((S)-2-(6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺基)丙酰胺基)-4-氧代丁酰胺基)苄基(4-硝基苯)碳酸酯(81.0mg，0.11mmol)溶于DMF(0.5mL)中，加入6-(4-((2,6-二氮杂螺[3.3]庚烷-2-基)甲基)苄基)-N 4-丁基吡啶[3,2-d]嘧啶-2,4-二胺(化合物38)(42.0mg，0.10mmol)和N,N-二异丙基乙胺(21μL，0.12mmol)，室温搅拌，LCMS检测反应。反应结束后，直接RP-HPLC制备得目标产物15mg，产率为16％。LC-MS(ESI)[M+H] +：1016.4。
17、化合物41的制备
Figure PCTCN2022092556-appb-000103
取(2S,3R,4S,5S,6S)-2-(2-(6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺)-4-(羟甲基)苯氧基)-6-(甲氧基羰基)四氢-2H-吡喃-3,4,5-三酰三醋酸酯(260mg，0.4mmol)溶于DMF(2mL)，依次加入二(对硝基苯)碳酸酯(182mg，0.6mmol)和N,N-二异丙基乙胺(105μL，0.6mmol)，反应6小时。反应液经EA/H 2O萃取三次，合并有机相，无水硫酸钠干燥，减压浓缩得粗产物。粗产物经硅胶柱层析(DCM:MeOH＝15:1)纯化得到目标产物250mg(产率为78％)。LC-MS(ESI)[M+H] +：814.4。
18、化合物42的制备
Figure PCTCN2022092556-appb-000104
取(2S,3R,4S,5S,6S)-2-(2-(6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺)-4-((((4-硝基苯氧基)羰基)甲基)苯氧基)-6-(甲氧基羰基)四氢-2H-吡喃-3,4,5-三基三醋酸酯(化合物41)(82mg，0.1mmol)溶于DMF(1mL)，加入6-(4-((2,6-二氮杂螺[3.3]庚烷-2-基)甲基)苄基)-N 4-丁基吡啶[3,2-d]嘧啶-2,4-二胺(化合物38)(42.0mg，0.10mmol)和N,N-二异丙基乙胺(21μL，0.12mmol)，室温搅拌，LCMS检测反应。反应结束后，直接RP-HPLC制备得目标产物30mg，产率为27％。LC-MS(ESI)[M+H] +：1092.4。
19、化合物43的制备
Figure PCTCN2022092556-appb-000105
取化合物42(30mg，0.027mmol)溶于混合溶剂(THF:H 2O＝2:1)，加入一水氢氧化锂(15mg， 0.27mmol)，室温搅拌30min后，加入醋酸调节pH至7。减压浓缩除去有机溶剂，再加入甲醇溶解，反相色谱制备得目标产物15mg，产率51％。LC-MS(ESI)[M+H] +：952.3。 1H NMR(400MHz,CD 3OD)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).
20、化合物44的制备
Figure PCTCN2022092556-appb-000106
取化合物38(41.8mg，1.0mmol)溶于DMF(2mL)，加入6-(马来酰亚胺基)己酸琥珀酰亚胺酯(34mg，0.11mmol)，室温反应3小时。将反应液用乙酸乙酯/饱和食盐水萃取，有机相用水洗涤3次，无水硫酸钠干燥，减压浓缩除去有机相得粗产物。粗产物经过硅胶柱层析(DCM:MeOH＝5:1)得到目标产物28mg，产率为48％。LC-MS(ESI)[M+H] +：611.4。 1H 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).
21、化合物45的制备
Figure PCTCN2022092556-appb-000107
取化合物38(21mg，0.5mmol)溶于甲醇(1mL)，加入2-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)乙醛(28mg，2mmol)，室温搅拌30min后分批加入氰基硼氢化钠(8mg，13mmol)，继续反应1小时。反应液过滤，直接反相色谱制备6mg，产率为23％。LC-MS(ESI)[M+H] +：541.3。 1H NMR(400MHz,CDCl 3):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).
22、化合物46的制备
Figure PCTCN2022092556-appb-000108
取化合物38(210mg，0.5mmol)溶于乙腈(5mL)，依次加入(2-(2-(2-碘乙氧基)乙氧基)乙氧基)乙基)氨基甲酸叔丁酯(305mg，0.75mmol)和碳酸钾(216mg，1.5mmol)，将反应液升温到65℃反应5小时。反应结束后，过滤，滤渣经乙腈洗涤。收集滤液，减压浓缩得粗产物。粗产物经硅胶柱层析(DCM:MeOH＝4:1)纯化得目标产物218mg，产率为62％。LC-MS(ESI)[M+H] +：693.5。
23、化合物47的制备
Figure PCTCN2022092556-appb-000109
取化合物46(218mg)溶于混合溶剂(DCM:TFA＝20:1，10mL)中，将反应液置于0℃反应1小时。待反应结束后，减压浓缩除去有机溶剂得目标产物。产物不经纯化直接使用。
24、化合物48的制备
Figure PCTCN2022092556-appb-000110
取化合物47(184mg，0.3mmol)溶于DMF(1mL)中，依次加入2,5-二氧代吡咯烷-1-基3-(2-(2-叠氮乙氧基)乙氧基)乙氧基)丙酸酯(106mg，0.3mmol)和三乙胺(135μL，0.9mmol)。将反应液置于室温反应3小时。反应结束后，加入二氯甲烷和水萃取，有机相经饱和氯化铵水溶液和水洗涤后，用无水硫酸钠干燥，减压浓缩得粗产物。所得粗产物不经纯化直接使用。LC-MS(ESI)[M+H] +：822.3。
25、化合物49的制备
Figure PCTCN2022092556-appb-000111
取化合物48(100mg，0.12mmol)溶于混合溶剂(DMSO:H 2O＝1:1,1mL)中，加入4-((2,5-二氧代-2,5-二氢-1H-吡咯-1-基)甲基)-N-(丙-2-炔-1-基)环己烷-1-甲酰胺(66mg，0.24mmol)和溴化亚铜(51mg，0.36mmol)。将反应液置于室温反应1小时。反应结束后，过滤，滤液直接反相色谱制备得到目标化合物22mg，产率为16％。LC-MS(ESI)[M+H] +：1140.4。 1H 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＝6.3Hz,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).
26、化合物50的制备
Figure PCTCN2022092556-appb-000112
化合物50参照化合物38的合成方法制备。
27、化合物51的制备
Figure PCTCN2022092556-appb-000113
化合物51参照化合物45的合成方法制备，产率为56％。LC-MS(ESI)[M+H] +：599.4。
28、化合物52的制备
Figure PCTCN2022092556-appb-000114
化合物52参照化合物38的合成方法制备。
29、化合物53的制备
Figure PCTCN2022092556-appb-000115
化合物53参照化合物45的合成方法，将6-(马来酰亚胺基)己酸琥珀酰亚胺酯替换为4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯制备而得，产率为42％。LC-MS(ESI)[M+H] +：655.3。
30、化合物54的制备
Figure PCTCN2022092556-appb-000116
取甲基(2-(甲基氨基)乙基)氨基甲酸叔丁酯(1g，5.3mmol)溶于二氯甲烷(10mL)中，将反应液置于0摄氏度，依次加入氯甲酸氯甲酯(0.95mL)和吡啶(0.86mL)，0℃继续反应2小时。反应结束后，加入二氯甲烷和水分层，有机相用饱和氯化钠水溶液洗涤，再用无水硫酸钠干燥，合并有机层，浓缩得粗产物。粗产物经硅胶柱层析(PE:EA＝5:1)纯化得目标产物为无色油状物。LC-MS(ESI)[M+H] +：281.3。
31、化合物55的制备
Figure PCTCN2022092556-appb-000117
取N 4-丁基-6-(4-((二甲基氨基)甲基)苄基)吡啶[3,2-d]嘧啶-2,4-二胺(400mg，1.1mmol)的氯仿(10mL)中，加入化合物54(1080mg，3.9mmol)和TBAI(1420mg，3.9mmol)，将反应液升温至60℃反应6小时。反应液结束，将反应液浓缩后直接柱层析纯化(DCM:MeOH＝4:1)得目标化合物480mg，产率为72％。LC-MS(ESI)[M] +：609.3。
32、化合物56的制备
Figure PCTCN2022092556-appb-000118
取化合物55(480mg)溶于混合溶剂(DCM:TFA＝10:1)中，将反应液置于0℃反应1小时，反应结束，减压浓缩除去有机溶剂得到目标产物的三氟乙酸盐。往黄色油状物中加入乙醚超声，析出固体，过滤。滤渣为目标产物的三氟乙酸盐。LC-MS(ESI)[M] +：509.3。
33、化合物57的制备
Figure PCTCN2022092556-appb-000119
取4-((S)-2-((S)-2-(6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺)-3-甲基丁酰胺)-5-尿苷戊酰胺)苄基(4-硝基苯)碳酸酯(MC-VC-PAB-NPC)(81.0mg，0.11mmol)溶于DMF(0.5mL)中，加入化合物56(51mg，0.10mmol)、HOBT(13.5mg，0.10mmol)和N,N-二异丙基乙胺(53μL，0.3mmol)，室温搅拌，LCMS检测反应。反应结束后，直接RP-HPLC制备得目标产物30mg，产率为24％。LC-MS(ESI)[M] +：1107.4。 1H NMR(400MHz,DMSO)δ10.16(s,1H),8.38(bs,2H),8.24(bs,1H),7.90(d,J＝7.7Hz,1H),7.75(t,1H),7.66–7.37(m,7H),7.26(m,2H),7.00(s,2H),6.24(s,2H),5.50(s,2H),5.26–5.06(m,2H),5.04–4.87(m,2H),4.61–4.12(m,8H),3.60–3.27(m,8H),2.89(m,12H),2.25–2.05(m,2H),1.97(m,1H),1.79–1.09(m,13H),1.00–0.71(m,8H).
34、化合物58的制备
Figure PCTCN2022092556-appb-000120
化合物58参照化合物57的合成方法，将MC-VC-PAB-NPC替换为MC-AAN-PAB-NPC制备而得。LC-MS(ESI)[M] +：1107.4。 1H 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＝7.6Hz,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).
35、化合物59的制备
Figure PCTCN2022092556-appb-000121
化合物59参照化合物57的合成方法，将MC-VC-PAB-NPC替换为化合物41制备而得。LC-MS(ESI)[M] +：1183.5。
36、化合物60的制备
Figure PCTCN2022092556-appb-000122
取化合物59(35mg,0.03mmol)溶于混合溶剂(THF:H 2O＝2:1)，加入一水氢氧化锂(16mg，0.3mmol)，室温搅拌30min后，加入醋酸调节pH至7。减压浓缩除去有机溶剂，再加入甲醇溶解，反相色谱制备得目标产物5mg，产率15％。LC-MS(ESI)[M+H] +：1043.4。
实施例2：抗PD-L1 THIOMAB的制备
1、抗PD-L1 THIOMAB的基因编辑
Avelumab(MSB0010718C，商品名Bavencio)是由默克雪兰诺(Merck KGaA)和辉瑞(Pfizer)开发的一种完全人源化的IgG1型单克隆抗PD-L1抗体药物，已经于2017年被FDA批准用于默克尔细胞癌(Merkel-cell carcinoma)的治疗(Apolo et al.,2017；Shirley,2018)，其轻链和重链的编码核苷酸序列分别如SEQ ID NO:1和2所示，轻链和重链的氨基酸序列分别如SEQ ID NO:3和4所示。Avelumab除了可以跟人PD-L1结合之外，还可以与鼠源的PD-L1发生交叉反应(Deng et al.,2016)。因此以Avelumab为原料，进行抗PD-L1的ADC药物的开发可以保证抗体本身具有足够优良的生物活性和成药性，同时也使所得到的ADC药物适用于前期的小鼠模型试验，以及后续开发时的人类临床试验。Avelumab的表达质粒由轻链159-pFuse-αPDL1-LC质粒和重链162-pFuse-αPDL1-hIgG-Fc2质粒两部分组成，其核苷酸序列如SEQ ID NO:5和6所示。将这两种质粒共转染到细胞内表达，经过蛋白质A树脂纯化后，即可得到抗PD-L1抗体Avelumab。
发明人通过PCR定点突变的方法，完成了THIOMAB表达质粒的构建。设计一对包含突变位点的完全互补、方向相反的引物，以待突变的完整质粒为模板，利用高保真PCR酶复制扩增出带有突变位点的整个质粒序列。之后，用DpnI显著性内切酶去除甲基化的模板质粒，而保留未被甲基化的PCR产物。进一步通过质粒转化与培养，即可扩增出带有突变位点的完整质粒。
在定点突变实验完成后，提取所得质粒测序，以鉴定突变是否成功。由轻链159-pFuse-αPDL1-LC质粒定点突变后得到的质粒被命名为αPD-L1 THIOMAB LC-V205C，其核苷酸序列如SEQ ID NO:7所示。测序对比结果如图1所示。与模板质粒比对后发现预定突变位点处序列从GTG突变为了TGC，而其他碱基没有发生变化，因此翻译后的轻链第205位氨基酸从缬氨酸突变为了半胱氨酸。这两个人工引入的半胱氨酸将被用于后续的ADC药物的偶联反应。突变所得抗体抗PD-L1 THIOMAB的轻链编码核苷酸序列如SEQ ID NO:8所示，轻链的氨基酸序列如SEQ ID NO:9所示。
2、抗PD-L1抗体和THIOMAB的表达
表达纯化后得抗PD-L1抗体，通过还原和非还原SDS PAGE分析进行鉴定。如图2所示。该抗体在还原性SDS PAGE中被还原为轻链(～25KD)和重链(～50KD)，在非还原SDS PAGE中则仍 然保持完整的抗体(～150KD)。
表达纯化后得到抗PD-L1 THIOMAB，通过还原和非还原SDS PAGE分析进行鉴定。如图3所示，抗PD-L1 THIOMAB的SDS染色结果(如图3所示)跟抗PD-L1抗体基本(如图2所示)一致：抗体在还原性SDS PAGE中被还原为轻链(～25KD)和重链(～50KD)，在非还原SDS PAGE中则仍然保持完整的抗体(～150KD)。实验证明，基因编辑所得到的抗PD-L1 THIOMAB基本保留了抗体原先的结构，这说明了THIOMAM表达过程和自我组装过程并没有受到人工突变的影响。同时，相同条件下，抗体与THIOMAB的表达量上似乎差别也不大，也说明了细胞内抗体表达和组装过程未受明显影响。
为了进一步鉴定所得的THIOMAB，发明人将完整的THIOMAB按照“TCEP还原-纯化-DHAA重新氧化-纯化”的流程进行了处理，得到了有游离巯基的THIOMAB(THIOMAB-S2)。通过Exactive Plus EMROrbitrap LC-MS检测了其分子量的变化。结果如图4所示。完整的抗PD-L1 THIOMAB分子量约为143791.75，而具有游离巯基的THIOMAB-S2的分子量约为143544.31，分子量相差247.44。理论上，完整的抗PD-L1 THIOMAB巯基上一般会被修饰上一个游离的半胱氨酸，因此相对于THIOMAB-S2，分子量会多出两个半胱氨酸与巯基缩合后残基的分子量，约为238。这个误差对于抗体大分子而言是可以接受的。因此，可以判断所得到的THIOMAB的分子量变化，符合实验预期。这一结果证明，发明人通过基因编辑，成功表达出了均质的、带有两个突变半胱氨酸的抗PD-L1THIOMAB，可用于之后的均质ADC药物的构建。在后续实验中，发明人还需要通过对其体内体外实验的结果与天然的抗PD-L1抗体相对比，研究这种人工突变是否会影响抗体的抗原结合能力和其他活性。
如图4所示，与半胱氨酸覆盖的原始抗PD-L1 THIOMAB相比，还原并透析后的THIOMAB失去了两个半胱氨酸，暴露出两个自由的巯基来。这使得此时的THIOMAB的预期分子量减少了238，变成了143553.75，而实际上检测到的THIOMAB分子量为143544.31，减少了247.44。
实施例3：基于THIOMAB的ADC的合成路线与鉴定
1、ADC HE-S2的合成与鉴定
ADC HE-S2的合成示意图如图5所示。发明人以抗PD-L1 THIOMAB和化合物20(实施例1制备)为原料，合成了带有二硫键连接子的抗PD-L1抗体和N 4-丁基-6-(4-((二甲基氨基)甲基)苄基)吡啶[3,2-d]嘧啶-2,4-二胺(以下简称为化合物D18)的偶联物，命名为ADC HE-S2。其中，抗体上的连接位点为SEQ ID NO：9所示氨基酸序列(轻链序列)的第226位的半胱氨酸。
将反应得到的ADC HE-S2通过Nonadrop测定浓度和纯度后，估算产率(约为30％-50％)。取部分样品送至国家蛋白质中心用大分子MS检测抗体分子量的变化，结果如图6中所示。理论上，ADC HE-S2中巯基被S2-D18修饰后，DAR为2的ADC整体分子量变化量相对于THIOMAB而言会增大～934。MS检测所得ADC的分子量为144721.08，而THIOMAB分子量则为143791.75，分子量相差929.33。这个误差对于～147K的蛋白质谱分析而言，是可以接受的。因此可以判断，已成功制备了均一的、DAR为2的ADC药物，ADC HE-S2(ADC1)，如下所示。
Figure PCTCN2022092556-appb-000123
ADC HE-S2的质谱检测结果如图6所示，MS所得ADC的分子量为144721.08，而THIOMAB分子量则为143791.75。
2、其他ADC药物的合成与鉴定
在合成ADC HE-S2的实验方案成熟之后，发明人又利用这种偶联策略(以抗PD-L1 THIOMAB和化合物30、化合物32、化合物57(实施例1制备)为原料)合成了具有不同连接子的两种ADC：ADC VC(ADC2)与ADC Legumain(ADC3)、ADC4。合成流程图分别如7和8中所示。
Figure PCTCN2022092556-appb-000124
经过MS鉴定(如图9所示)，根据之前的计算分析方法，可以计算验证ADC VC理论上的分子量应该比THIOMAB大约1600。MS结果可知两者相差为145399.23-143791.75＝1607.48，与理论值非常接近。同理还可以验证ADC Legumain理论上的分子量应该比THIOMAB大约增加1600。MS结果可知两者相差为145391.09-143791.75＝1599.34，同样非常接近于理论计算。因此可以得出结论，得到了均一的、DAR为2的两种ADC，ADC VC与ADC Legumain。
实施例4：基于野生型PD-L1抗体的ADC的合成路线与鉴定
1.ADC偶联步骤和方法
a.反应原料和试剂配制:将抗体(实施例2中所述Avelumab)浓度配制为5-10mg/mL的PBS溶液；配置浓度为10mM的TCEP水溶液；将偶联小分子(化合物39、40、43、44、51、45、49、53、57、58、60，实施例1制备)配制为浓度10mM的DMSO溶液；配制浓度为20mM的半胱氨酸PBS溶液；
b.抗体还原：加入10或20当量的TCEP(10mM，37℃震荡2-3h，转速为220rpm/min；
c.偶联反应：抗体还原后，加入10-20当量的小分子，室温震荡2-4h，转速为220rpm/min；
d.淬灭反应：加入与小分子当量的半胱氨酸溶液，4或10℃孵育30min以终止反应；
e、脱盐：反应终止后，利用Cytiva的5ml预装脱盐柱脱盐。
2.ADC DAR值测定
采用超高效液相色谱仪(UHPLC)串联质谱分析(MS)DAR值计算方法：
a.仪器：采用Waters Acquity-Class超高效液相色谱仪(UHPLC)串联Waters Synapt G2-Si Q-TOF高分辨质谱系统，采用高纯氮气作为雾化气，高纯氩气作为碰撞气。
b.液相色谱条件：色谱柱为ACQUITY UPLC Protein BEH SEC色谱柱(
Figure PCTCN2022092556-appb-000125
2.1mm×150mm,1.7μm)，色谱柱温度为25℃，进样量为15μL。等度洗脱：50mM醋酸铵作为流动相，流速0.065mL/min，采集时间为10min，紫外检测波长为280nm。
质谱条件：毛细管电压设置为3.0kV进行ESI源正电离模式扫描；锥孔电压为120V，源温度和脱溶温度分别设定为120℃和500℃；锥孔气和脱溶剂气流量分别设置为20L/H和600L/H，一级质谱范围m/z 400～m/z8000。
c.数据分析：用沃特世Masslynx软件对数据进行分析，使用MaxEnt 1插件对质谱数据进行解卷积处理。通过对质谱解卷积图谱进行分析测定ADC DAR值。首先，对峰进行积分，计算峰面积百分比，峰面积百分比和为100。根据公式：DAR＝Σ加权峰面积/100计算ADC的加权平均药物偶联比。
3.具体ADC产物结构信息
Figure PCTCN2022092556-appb-000126
Figure PCTCN2022092556-appb-000127
实施例5：生物活性研究
1、ADC HE-S2(ADC1)的抗原结合能力检测
足够的特异性的抗原亲和能力是抗体生物活性的基础，对于抗体类药物的活性和疗效至关重要。由于ADC药物制备过程中涉及到对抗体的改造、化学处理以及小分子偶联，这些都有可能破坏或影响抗体的原有的抗原亲和能力和稳定性，造成抗体抗原结合能力受损。因此，在处理抗体的过程中，所用条件需要尽可能温和；改造和偶联位点的选择也应该避开原抗体的抗原决定簇，以免损伤抗体的抗原结合能力。此外，还应当检验验证所得ADC药物的抗原亲和能力，确保ADC药物保留有足够的靶向性和特异性。
本发明中设计并制备ADC HE-S2的抗体抗原亲和能力是实现其抗肿瘤活性的重要基础。ADC HE-S2需要通过抗体与肿瘤中高表达的PD-L1的特异性结合来实现免疫调节剂D18的靶向运输。同时，ADC HE-S2还可以通过细胞表面的PD-L1结合，阻断肿瘤PD-1/PD-L1免疫抑制信号通路，从而诱导T细胞抗肿瘤反应。因此成功制备了ADC HE-S2之后，发明人首先在体外验证了其识别并 结合PD-L1抗原的能力。
以PD-L1阳性的MC38为平台，发明人通过了荧光激活细胞分选技术(FACS)验证了ADC HE-S2和基因修饰后的抗PD-L1 THIOMAB是否保留了原有的抗体抗原结合能力。实验中，把ADC HE-S2、抗PD-L1抗体(即实施例2中所述Avelumab)和抗PD-L1 THIOMAB(即实施例2制备的突变抗体)作为一级抗体与MC38细胞孵育，使抗体与细胞表面PD-L1可以充分接触并结合。PBS清洗后再用FITC标记的山羊抗人二级抗体处理细胞，避光孵育30分钟使二抗与一抗充分结合。同时设置两组阴性对照，其一标记为阴性对照(NC)，只加入同型对照IgG处理细胞，不做染色，表示细胞本身的荧光背景；其二标记为二抗(secondary antibody)，在加入同型对照IgG后与其他实验组一起用FITC标记的二抗染色，以表征二级抗体与细胞非特异性结合产生的假阳性荧光信号。染色后的MC38细胞在BD FACSAria TM III流式细胞仪上进行分析，检测并比较不同抗体类药物处理后MC38细胞上的FITC荧光信号的强弱，以此来表征ADC、THIOMBA和抗体对PD-L1的亲和能力。所有结果均通过FlowJo软件(TreeStarInc.)处理，所得如图10所示。
FACS结果如图10所示，以使用IgG同型抗体的空白对照组(NC)为基础，使用荧光二抗染色的对照组产生假阳性信号偏移率只有1.7％；平均荧光强度(MFI)计算结果显示，NC组的MFI为142.00而二抗染色后MC38检测到的平均荧光强度则为166.75，虽然差异显著(p＜0.0001)但是差值较小。这种显著而微弱的差异就是实验所用FITC二抗与MC38细胞非特异性结合所产生的假阳性信号。实验证明，这种假阳性结合的现象较弱，不会对结果产生影响。因此以NC对照组作为参照基础来评估抗PD-L1抗体类药物的抗体抗原结合能力。
在抗PD-L1抗体、抗PD-L1 THIOMAB和ADC HE-S2处理后的MC38细胞上发明人观测到了相近的FITC阳性信号，相对于NC组产生的荧光信号偏移率分别为35.7％、36.6％和34.2％，远远高于荧光二抗非特异性结合所产生的假阳性信号(1.7％)。MFI计算也表明，抗PD-L1抗体、抗PD-L1 THIOMAB和ADC HE-S2的评价荧光强度分别为445.3，449.0和392.5，同样显著高于两个对照组(p＜0.0001)。说明三种抗体都可以与PD-L1产生特异性结合。同时，Student’s t法分析证明，经历过基因编辑的抗PD-L1THIOMAB以及进一步化学修饰的ADC HE-S2与天然的抗PD-L1抗体相比，所检测到的MFI无显著性差异。这说明发明人的基因编辑和化学修饰既没有造成抗体原有的抗原识别亲和能力的损伤，也没有引起抗体的非特异性脱靶现象的出现。
因此，发明人可以确认ADC HE-S2依然保持着原先抗PD-L1抗体的抗原识别亲和能力，可以进一步应用于之后的实验和研发。
2、ADC HE-S2(ADC1)的内化能力
ADC的药物释放被广泛认为是依赖于对应抗体的内化能力。ADC结合细胞表面的抗原蛋白后形成的抗体-抗原复合物，需要随着抗原蛋白的内化过程进入靶向细胞，从而在胞内酶或其他胞内生理环境的作用下释放出偶联的药物。因此，所选抗原与ADC结合后所形成复合物的内化能力对于ADC药物功能的实现至关重要。由于发明人设计的ADC HE-S2理论上应该在癌细胞内和DC细胞内同时发挥作用，因此发明人通过体外实验，验证了抗PD-L1抗体类药物内化到肿瘤细胞和DC细胞的能力。
已知pHrodo TM红色染料是一种pH敏感型荧光染料，当周围环境变酸时，这种分子荧光强度会迅速提高(Lehrman et al.,2018)。当这种染料随着抗原抗体复合物内化到细胞内部后，染料周围环境的pH值会随即降为酸性，从而产生可观察的荧光信号。这种性质使得可以利用pHrodo TM红色染料来监测到抗体的内化过程。通过
Figure PCTCN2022092556-appb-000128
活细胞分析系统，发明人可以对pHrodo TM红色染料标记的抗PD-L1抗体的内化过程进行基于图像的监测与定量分析。在0.1M碳酸钠(pH8.3)缓冲液中，1mg/mL的抗PD-L1 THIOMAB和3-5当量的pHrodo TM红色染料共同孵育大约1h，透析纯化去除残留的小分子从而得到荧光染料标记的抗PD-L1 THIOMAB。标记后的抗体应该避光储存在4℃中备用；长期储存时应避光保存在-20℃环境中，避免反复冻融。为了表征PD-L1/抗PD-L1抗体复合物的内化过程，发明人选择使用pHrodo TM红色染料来标记抗PD-L1 THIOMAB。在前文中，发明人已经验证了抗PD-L1 THIOMAB与ADC HE-S2具有相同的抗原识别能力，因此抗PD-L1 THIOMAB的内化能力，也可以反应出ADC HE-S2的内化能力。
(1)抗PD-L1 THIOMAB在癌细胞上的内化实验
以MC38和B16两种PD-L1阳性的肿瘤细胞为材料，验证了表征PD-L1/抗PD-L1抗体复合物的内化。在细胞内加入荧光标记的抗PD-L1 THIOMAB后，避光孵育24h后，用
Figure PCTCN2022092556-appb-000129
活细胞分析系统拍摄细胞内红色荧光信号的分布，结果如图11A所示。
可以明显观察到在加入了标记的THIOMAB后，细胞内明显产生了红色信号，而不加抗体的对 照组则几乎观察不到红色荧光。从合成的图像中可以看到，细胞外几乎没有红色荧光的产生，这证明了荧光信号与抗体内化的相关性。因此发明人可以通过
Figure PCTCN2022092556-appb-000130
软件分析得到的总体红色荧光对象集成强度来评估荧光标记的THIOMAB的内化水平，如图11B所示。加入标记的THIOMAB之后，MC38内的总体红色荧光对象集成强度为13701.63，显著高于对照组的84.88；B16内信号强度更是高达283613.60，同样显著高于对照组的98.53。B16细胞内的荧光信号同样强于MC38细胞，这可能是不同细胞PD-L1内化速率以及细胞内pH值的差异等原因导致的。
(2)抗PD-L1 THIOMAB在DC细胞上的内化和选择性实验
为了验证抗PD-L1 THIOMAB在DC细胞上的内化，发明人从清华大学药学院的唐海东老师处得到了Cd274敲除型DC 2.4细胞和野生型DC 2.4细胞两种DC细胞。Cd274基因为PD-L1编码基因，敲除后的DC 2.4将不能表达PD-L1蛋白，因此为PD-L1阴性DC细胞；而野生型DC 2.4则可以表达PD-L1，为PD-L1阳性DC细胞(如图12所示)。根据假设，发明人应该可以观察到抗PD-L1THIOMAB可以内化到PD-L1阳性的DC细胞内，而无法内化到PD-L1阴性的Cd274敲除型DC 2.4细胞内部。因此通过这两种细胞的抗PD-L1 THIOMAB内化实验，发明人可以同时验证抗PD-L1THIOMAB在DC细胞上的内化能力以及抗体的选择性。
预实验中，发明人依照常规操作，在DC 2.4细胞的培养基中加入了10％的热灭活胎牛血清(FBS)作为封闭剂，孵育8h后用FACS检测细胞内的红色荧光信号，结果如图13中所示。意外的是，FACS检测结果表明，在PD-L1阳性的野生型DC 2.4细胞中还是在PD-L1阴性的CD274敲除DC 2.4细胞中，发明人都观测到了相同的比例的红色荧光阳性细胞，比例分别为53.6％和52.9％。未加入荧光标记的抗PD-L1THIOMAB的阴性对照组中几乎没有观测到红色荧光阳性的细胞(1.57％和0.92％)。多次重复使用都得到了类似的结果，这些现象暗示着：被荧光染料标记的抗PD-L1 THIOMAB不但可以顺利内化到PD-L1阳性的DC 2.4细胞内，也可以内化到PD-L1阴性的Cd274敲除型DC 2.4细胞中。这一出乎意料的结果与发明人的假设相悖。
发明人通过文献调查发现，人类IgG的Fc段与小鼠Fcγ受体的结合能力和其与人类直系同源Fcγ受体的结合能力非常类似(Dekkers et al.,2017)。因此发明人推测DC 2.4细胞表面高表达、高活性的鼠源Fcγ受体同样可以结合并内化人源化抗PD-L1抗体，从而使得两种细胞都可以内化抗PD-L1抗体的速率。
为了验证这一猜想，发明人使用10％的小鼠血清和10％的FBS作为封闭剂分别按组加入到了细胞培养基中。相对于FBS，小鼠血清内富含的鼠源IgG可以更加有效地封闭DC 2.4表面的Fcγ受体，从而抑制DC细胞Fcγ受体所介导的抗体内化过程。在加入THIOMAB前，DC细胞首先应该先在4℃下孵育30分钟，暂停细胞表面受体的内化过程的同时，也使得小鼠血清中的IgG与DC细胞表面的Fcγ受体能充分结合。之后，将荧光标记的抗PD-L1 THIOMBA加入到各组细胞内。接着再在4℃下避光培养30min，使抗体与抗原在细胞内化停止的条件下，充分接触并结合。
抗PD-L1 THIOMAB的内化实验得到的总体红色荧光对象集成强度-时间变化曲线如图14A和B中所示。图14A中的结果表明，加入了10％FBS封闭后，在PD-L1阳性的野生型DC 2.4细胞和PD-L1阴性的CD274敲除DC 2.4细胞中，可以观测到非常相似的荧光强度变化曲线；而未加入荧光标记的抗PD-L1THIOMAB的阴性对照组中都没有观测到荧光信号的变化。
如图14B所示，在加入了10％小鼠血清的实验组中，在PD-L1阳性的野生型DC细胞中，总体红色荧光对象集成强度增强的速度明显高于PD-L1阴性的Cd274敲除型DC细胞。尤其是在检测的开始阶段(0-60min)，PD-L1阴性细胞则几乎没有荧光信号产生，变化曲线与阴性对照相似，之后PD-L1阴性的DC细胞内开始出现抗体产生的荧光信号，但是信号增强的速率(斜率)依然落后于野生型DC 2.4细胞，最终t检验分析证明两者之间存在显著性差异(p＜0.0001)。培养6小时后，用
Figure PCTCN2022092556-appb-000131
活细胞拍摄小鼠血清阻断的实验组，观察各组细胞内红色荧光信号的分布。结果如图14A所示，野生型DC 2.4细胞内的红色荧光信号要显著强于Cd274敲除型DC细胞。这一现象可以解释为，由于在检测前30min的4℃孵育中，细胞的受体内化过程几乎停止而抗原-抗体的结合不受影响，所以DC细胞表面的PD-L1可以抗PD-L1抗体充分结合，Fcγ受体也会被小鼠血清中的同源IgG充分封闭。当检测开始时，随着培养温度逐渐恢复到37℃，细胞表面受体内化过程也逐渐恢复，之前与野生型DC细胞表面的PD-L1充分结合的抗PD-L1 THIOMAB也得以随着迅速内化，而PD-L1阴性的细胞则不能内化。而随着孵育时间的延长，PD-L1阴性的DC细胞可以通过重新循环出来的Fcγ受体来内化抗PD-L1 THIOMAB，从而产生荧光信号。但是在培养基内小鼠同源IgG的作用下，这种内化过程被竞争性抑制。因此在Cd274敲除型DC细胞中THIOMAB的内化速度依然落后于可以通过与PD-L1结合而内化THIOMAB的野生型DC2.4细胞。
这一结果验证了发明人的猜想并且证明了抗PD-L1抗体可以通过结合细胞表面PD-L1而内化到胞内的能力。同时，在用小鼠血清封闭后，抗PD-L1 THIOMAB在Cd274敲除型和野生型DC 2.4细胞中的内化速度的表现出的差异，也证明抗PD-L1抗体具有良好的选择性。
实施例6：抗肿瘤活性研究
1、ADC HE-S2(ADC1)的抗肿瘤活性研究
为了验证ADC HE-S2的抗癌活性，发明人首先用MC38制造小鼠模型。荷瘤之后，大约一周左右可以在小鼠体表观察到可见的肿瘤。当小鼠平均肿瘤组织体积达到100mm 3后，根据最终给药种类将小鼠随机分组，依次为：IgG同种型对照抗体组(简称为IgG)、抗PD-L1抗体组(anti-PD-L1)、抗PD-L1 THIOMAB组(Thiomab)、D18组(D18)、D18与抗PD-L1抗体联合治疗组(anti-PD-L1&D18)和ADC HE-S2组(ADC HE-S2)，每组7-8只小鼠。IgG同种型对照抗体组、抗PD-L1抗体(即实施例2中所述Avelumab)组、抗PD-L1 THIOMAB(即实施例2制备的突变抗体)组每周给药两次，每次每只小鼠腹膜内注射150μg。D18组每周给药两次，每次每只小鼠腹膜内注射25μg。D18/抗PD-L1抗体组合治疗组中，每周抗PD-L1抗体给药两次，每次每只150μg；D18给药一次，每次每只25μg；ADC HE-S2组每周ADC HE-S2给药一次，每次每只小鼠腹膜内注射150μg；抗PD-L1抗体每周给药一次，时间与ADC错开，每次每只小鼠腹膜内注射150μg。
实验结果如图15A所示。对于MC38肿瘤，抗PD-L1抗体、抗PD-L1 THIOMAB和D18这三种药物单独治疗的效果都非常类似：彼此之间对于肿瘤生长的抑制活性没有显著性差异，但是都略优于作为阴性对照的IgG同型抗体的疗效。这一结果说明，D18和抗PD-L1抗体都具有一定抗癌活性，而抗PD-L1 THIOMAB同样保留了原先抗体的生物功能。而D18与抗PD-L1抗体的组合治疗则表现出了非常好的肿瘤生长抑制活性，其效果远远好于任何一组单独用药的治疗。这一结果再次验证了D18与PD-1/PD-L1阻断抗体治疗之间的协同作用。ADC HE-S2表现出来了非常令人惊喜的抗癌活性，肿瘤生长抑制活性明显要要优于联合治疗组(P＜0.001)。当实验进行到第24天，联合治疗组的小鼠肿瘤体积开始有了较为明显的上升；而在ADC治疗组中，小鼠的肿瘤大小一直被维持在了很低的水平。
为了进一步研究小鼠长期用药后的生存率，发明人又进行了小鼠生存实验。实验最终持续到了第70天(如图15B所示)。ADC HE-S2组的小鼠生存率为6/8，值得强调的是，最后存活的六只老鼠的肿瘤已经几乎检测不到了。D18和PD-L1联合用药组中小鼠生存率为4/8；D18组中小鼠生存率则为1/8；而其他两种单独用药组和阴性对照组中的老鼠则全部死亡。这一结果同样证明了ADC HE-S2强大的抗癌活性。
此外，发明人还使用B16黑色素瘤小鼠模型以进一步证实ADC HE-S2的疗效(如图15C所示)。给药剂量和时间同样如表1所示。B16黑色素瘤被是一种PD-L1水平较低的、对PD-1/PD-L1阻断疗法有一定耐药性的肿瘤。结果如发明人所预料，相对于MC38肿瘤，各种药物对B16黑色素瘤的抑制效果均有所下降。抗PD-L1抗体、抗PD-L1 THIOMAB和D18三种药物的单独用药组的疗效几乎与IgG同型抗体的结果相当。只有抗PD-L1抗体+D18的组合治疗以及ADC HE-S2依然表现出一定的肿瘤抑制效果。而ADC HE-S2治疗后的小鼠与联合治疗相比，其身上的B16肿瘤生长速度明显减慢(P＜0.05)，这一结果证明在B16肿瘤模型上，ADC HE-S2的抗肿瘤活性同样强于抗PD-L1抗体与D18两种药物的联合治疗。
表1小鼠治疗用药时间表
Figure PCTCN2022092556-appb-000132
Figure PCTCN2022092556-appb-000133
给药剂量：抗体类药物，每只小鼠每次给药150μg；D18，每只小鼠每次给药25μg。-表示当天不给药。 *只有MC38肿瘤模型的小鼠进行到了第24天，B16肿瘤模型的小鼠在第21天后不再给药。
2、ADC偶联策略扩大了D18的治疗窗
值得注意的是，由于ADC HE-S2的DAR为2，分子量约为150K，可以简单估算出每次给药时ADC中D18的含量为150μg/150000×2×364＝0.748μg，远远小于联合用药时的给药剂量(每次每只小鼠的给药剂量为25μg)。因此，发明人希望研究D18剂量变化对于D18与抗PD-L1抗体之间的协同抗癌作用的影响。
因此，发明人将不同剂量的D18(0.25μg、2.5μg和25μg)与150μg的抗PD-L1抗体联合使用以治疗MC38肿瘤(如图16所示)。将小鼠按照给药类型分组为IgG同型抗体对照组(简称为IgG)、抗PD-L1抗体组(anti-PD-L1)、抗PD-L1抗体+D18(25μg)组(anti-PD-L1+D18(25μg))、抗PD-L1抗体+D18(2.5μg)组(anti-PD-L1+D18(2.5μg))和抗PD-L1抗体+D18(0.25μg)组(anti-PD-L1+D18(0.25μg))。在每组小鼠肿瘤平均体积达到100mm 3后开始给药。IgG同型抗体抗和PD-L1抗体每周给药两次，每次给药腹腔注射150μg/每只；D18每周给药一次，每次给药按照组别分别腹腔注射25μg/只、2.5μg/只和0.25μg/只；IgG同型抗体组的小鼠每周腹腔注射与D18给药时相同剂量的DMSO作为对照。
梯度剂量的D18联合抗PD-L1抗体的抗肿瘤结果如图16所示。实验结果证明，D18增强抗PD-L1抗体的抗肿瘤活性的功能，表现出剂量依赖型的特点。当D18每次每只小鼠的给药剂量为25μg时，与抗PD-L1抗体的联合治疗表现出较为明显的肿瘤抑制活性。而随着所用D18剂量的减少，D18与抗PD-L1抗体联合治疗的抗肿瘤作用也不断减弱。事实上，当所用剂量下降为每只老鼠每次2.5μg或0.25μg时，其肿瘤抑制效果已经与抗PD-L1抗体单独用药无显著性差异了。
值得注意的是，ADC HE-S2每次给药时装载的D18剂量(～0.75μg)是远远小于2.5μg的。因此可以判断，通过ADC靶向给药，发明人利用不足以在联合用药中发挥活性的D18剂量(～0.75μg)，取得了比25μg的D18与抗PD-L1抗体联合治疗时更为显著的肿瘤抑制效果。
之后，发明人在小鼠模型中检测了各组药物治疗对小鼠体重的影响，从而判断所用药物的毒副作用，结果如图17所示。将健康的小鼠按照给药分为ADC组、抗PD-L1抗体+D18组、抗PD-L1抗体组、D18组和IgG组，每组5只小鼠。各组小鼠的给药方式和剂量与小鼠肿瘤模型试验中相同：ADC组每周ADC HE-S2给药一次，另外在注射一次抗PD-L1抗体，每次均为150μg；D18每周给药一次，每次25μg；抗PD-L1抗体和IgG同型抗体每周给药两次，每次150μg。
抗PD-L1抗体组中小鼠的体重变化几乎与空白对照的IgG组一致，证明抗PD-L1抗体本身对小鼠的毒副作用并不大。抗PD-L1抗体+D18组和D18组每次注射完D18后，小鼠体重都会表现出一定程度的下降。相对于D18单独用药，抗PD-L1抗体+D18组小鼠的体重下降程度要格外显著(p＜0.05)，这一结果印证了发明人之前的假设，即作为免疫检查点抑制剂的抗PD-L1抗体，会加大TLR7/8激动剂D18的毒副作用。ADC组在每次注射ADC HE-S2之后，虽然也会造成一定程度的体重下降，但是幅度显著小于D18组(p＜0.05)和联合用药组(p＜0.01)。在给药之后，小鼠的体重也迅速回归了正常，最终ADC组的小鼠体重变化与对照组无显著性差异。这些结果证明，ADC HE-S2虽然同样也表现出了一定的毒性，但是毒性显著低于D18单独用药和联合用药，对小鼠身体的负担较小。ADC中实际所荷载的D18较少应该是D18毒性降低的主要原因。
最终可以得出结论，ADC HE-S2通过更低的实际D18使用量，表现出了比联合用药时更好的肿瘤抑制活性，同时还显著降低了联合治疗的毒副作用。通过发明人所提出的ADC策略，大大降低了D18使用时的最低有效剂量，扩增了D18的治疗窗。
2.基于野生型PD-L1抗体的抗体偶联药物ADC10的小鼠抗肿瘤实验
将MC38细胞(1x10 6cells/只)接种到小鼠右侧腹部皮下。当小鼠平均肿瘤组织体积达到100mm 3后，根据最终给药种类将小鼠随机分组，依次为：PBS对照组和ADC 10组，每组5-6只小鼠。每周给药两次，每次每只小鼠腹膜内注射剂量为10mg/kg。每天称量小鼠体重变化(图18A)，每3天测量一次小鼠肿瘤体积(图18B)。如图18B所示，ADC的抑瘤率为70％。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。
本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。

Claims (52)

  1. 一种抗体药物偶联物或其药学上可接受的盐、溶剂化物、前药、立体异构体，所述抗体药物偶联物具有如下结构：
    Figure PCTCN2022092556-appb-100001
    其中，
    Ab为抗体或其抗原结合片段；
    D为小分子药物；
    L为连接Ab和D的连接单元；
    n为1-100的整数。
  2. 如权利要求1所述的抗体药物偶联物，其特征在于，所述抗体靶向的抗原选自：Claudin18.2、GPC3、HER-2/neu、碳酸酐酶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、甲胎蛋白(AFP)、VEGF、ED-B纤连蛋白、EGP-1、EGP-2、EGF受体(ErbB1)、ErbB2、ErbB3、因子H、FHL-1、Flt-3、叶酸受体、Ga 733、GROB、HMGB-1、缺氧诱导因子(HIF)、HM1.24、胰岛素样生长因子(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、神经节糖苷、HCG、HLA-DR、CD66a-d、MAGE、mCRP、MCP-1、MIP-1A、MIP-1B、巨噬细胞移动抑制因子(MIF)、MUC1、MUC2、MUC3、MUC4、MUC5、PD-1、PD-L1、胎盘生长因子(PIGF)、PSA、PSMA、PSMA二聚物、PAM4抗原、NCA-95、NCA-90、A3、A33、Ep-CAM、KS-1、Le(y)、间皮素、S100、腱生蛋白、TAC、Tn抗原、Thomas-Friedenreich抗原、肿瘤坏死抗原、肿瘤血管生成抗原、TNF-α、TRAIL受体(R1和R2)、VEGFR、RANTES、T101、癌干细胞抗原、补体因子C3、C3a、C3b、C5a、C5和致癌基因产物。
  3. 如权利要求1所述的抗体药物偶联物，其特征在于，所述抗体为抗PD-L1抗体。
  4. 如权利要求1所述的抗体药物偶联物，其特征在于，所述抗体包含重链和轻链；
    其中，所述轻链的氨基酸序列如SEQ ID NO：9所示，或为与SEQ ID NO：9具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    优选地，所述抗体具有如SEQ ID NO：9所示的轻链序列；
    更优选地，所述抗体具有如SEQ ID NO：4所示的重链序列和如SEQ ID NO：9所示的轻链序列；或，所述抗体具有如SEQ ID NO：4所示的重链序列和如SEQ ID NO：3所示的轻链序列。
  5. 如权利要求1-4任一项所述的抗体药物偶联物，其特征在于，n为1-10的整数，优选为1-6的整数；优选地，n＝2或4。
  6. 如权利要求4所述的抗体药物偶联物，其特征在于，所述抗体药物偶联物中，所述抗体与L的连接位点为SEQ ID NO：9所示氨基酸序列的第226位的半胱氨酸残基上的游离巯基。
  7. 如权利要求1-6任一项所述的抗体药物偶联物，其特征在于，通式Ⅰ中D部分具有如下结构：
    Figure PCTCN2022092556-appb-100002
    其中，
    L'为连接基团，其选自：单键、C 1-C 6亚烷基、C 1-C 6亚烯基、C 3-C 6亚环烷基，其中所述基团均可任选地被C 1-C 4烷基取代；
    R 1选自：单键、C 1-C 6亚烷基、C 1-C 6亚烯基、C 1-C 6亚烷氧基，其中所述基团均可任选地被C 1-C 4烷基取代；
    X选自：
    Figure PCTCN2022092556-appb-100003
    -NR 4-、-O-、-S-、单键、-OCO-、-COO-、-NR 4-(C 1-C 6亚烷基)-NR 5-、亚杂环基，其中，R 4和R 5独立地选自：H、C 1-C 6烷基、C 1-C 6烷氧基、氨基酸残基、寡肽残基、卤代烷基、羧基取代烷基、酯基取代烷基、
    Figure PCTCN2022092556-appb-100004
    其中，a为1-10的整数，R 8和R 9独立地选自：H、C 1-C 6烷基，或R 8和R 9与其中间的原子一起形成
    Figure PCTCN2022092556-appb-100005
    或者R 4和R 5与二者所连接的氮原子一起形成杂环基；
    R 2选自：-NR 6R 7、-OR 6、-SR 6，其中，R 6和R 7独立地选自：H、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氧基烷基，或者R 6和R 7与二者所连接的氮原子一起形成杂环基，或R 6与其所连接的氧原子一起形成杂环基，或R 6与其所连接的硫原子一起形成杂环基；
    R 3为苯环上一个或多个独立的取代基，其选自：H、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氧基烷基；
    m为0-4的整数。
  8. 如权利要求7所述的抗体药物偶联物，其特征在于，L'具有如下结构：
    Figure PCTCN2022092556-appb-100006
    其中，R a和R b独立地选自：H、C 1-C 3烷基，或，R a和R b与其所连接的碳原子一起形成C 3-C 6亚环烷基；
    优选地，L'选自：-CH 2-、
    Figure PCTCN2022092556-appb-100007
  9. 如权利要求7所述的抗体药物偶联物，其特征在于，R 1为C 1-C 6亚烷基，例如C 1-C 3亚烷基，例如-CH 2-；或，
    R 1为单键；或，
    R 1为C 1-C 6亚烷氧基，例如C 1-C 3亚烷氧基，例如-CH 2O-、-CH 2CH 2O-、-CH 2CH 2CH 2O-。
  10. 如权利要求7所述的抗体药物偶联物，其特征在于，R 3为苯环上一个或多个独立的取代基，其独立地选自：H、甲基、甲氧基。
  11. 如权利要求7所述的抗体药物偶联物，其特征在于，m为0或1。
  12. 如权利要求7所述的抗体药物偶联物，其特征在于，R 8和R 9独立地选自：H、甲基、乙基、正丙基、异丙基。
  13. 如权利要求7所述的抗体药物偶联物，其特征在于，R 4和R 5独立地选自：H、甲基、乙基、正丙基、异丙基、正丁基、叔丁基、氨基酸残基、寡肽残基、-CF 3、-CH 2CF 3、
    Figure PCTCN2022092556-appb-100008
    Figure PCTCN2022092556-appb-100009
    Figure PCTCN2022092556-appb-100010
    或者R 4和R 5与二者所连接的氮原子一起形成取代或未取代的杂环基；
    优选地，所述取代或未取代的杂环基选自：
    Figure PCTCN2022092556-appb-100011
    Figure PCTCN2022092556-appb-100012
    优选地，所述氨基酸残基选自：丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸残基；
    优选地，所述寡肽残基中的氨基酸残基独立地选自：丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸残基中的一种或多种；
    更优选地，X为
    Figure PCTCN2022092556-appb-100013
    R 4和R 5独立地选自C 1-C 6烷基，例如甲基；
    更优选地，X为-NR 4-，R 4选自：H、C 1-C 6烷基，例如R 4为H或甲基；
    更优选地，X为-NR 4-(C 1-C 6亚烷基)-NR 5-，R 4和R 5独立地选自C 1-C 6烷基，例如，R 4和R 5均为甲基；
    更优选地，X为亚杂环基，特别是亚含氮杂环基，例如
    Figure PCTCN2022092556-appb-100014
    A环为4至10元含氮杂环，A环可以为单环、双环或三环(包括稠环、螺环、桥环)；进一步优选地，X为
    Figure PCTCN2022092556-appb-100015
    Figure PCTCN2022092556-appb-100016
    Figure PCTCN2022092556-appb-100017
  14. 如权利要求7所述的抗体药物偶联物，其特征在于，R 2选自：-NHR 6、-OR 6、-SR 6，其中，R 6为C 1-C 6烷基或C 1-C 6烷氧基烷基；或，
    R 2为-NR 6R 7，其中，R 6和R 7与二者所连接的氮原子一起形成杂环基；
    优选地，所述杂环基选自：
    Figure PCTCN2022092556-appb-100018
    Figure PCTCN2022092556-appb-100019
  15. 如权利要求7所述的抗体药物偶联物，其特征在于，R 2选自：
    Figure PCTCN2022092556-appb-100020
    Figure PCTCN2022092556-appb-100021
  16. 如权利要求7所述的抗体药物偶联物，其特征在于，通式Ⅰ中D部分选自如下结构：
    Figure PCTCN2022092556-appb-100022
    Figure PCTCN2022092556-appb-100023
    Figure PCTCN2022092556-appb-100024
  17. 如权利要求7所述的抗体药物偶联物，其特征在于，通式Ⅰ中D部分具有如下结构：
    Figure PCTCN2022092556-appb-100025
  18. 如权利要求1-17任一项所述的抗体药物偶联物，其特征在于，L包含与Ab的活性基团的连接基团Y，例如单键、
    Figure PCTCN2022092556-appb-100026
    -S-、-CO-、-NH-、-CONH-、
    Figure PCTCN2022092556-appb-100027
    优选地，L还包含基团Q，Q为二价的饱和或不饱和的直链或支链的C1-50烃链，其中0-6个亚甲基单元独立地被以下取代：-Cy-、-O-、-NR 10-、-S-、-OC(O)-、-C(O)O-、-C(O)-、-S(O)-、-S(O) 2-、-NR 10S(O) 2-、-S(O) 2-NR 10-、-NR 10-C(O)-、-C(O)NR 10-、-OC(O)NR 10-、-NR 10-C(O)O-、
    Figure PCTCN2022092556-appb-100028
    Figure PCTCN2022092556-appb-100029
    氨基酸残基、寡肽残基，其中，k选自1-10之间的整数，每个-Cy-独立地为选自以下的任选地经取代的二价环：亚芳基、亚环烷基、亚杂环基；R 10选自：H、-OH、C 1-C 6烷基、C 3-C 6环烷基、杂环烷基；
    优选地，每个-Cy-可以独立地选自以下：
    Figure PCTCN2022092556-appb-100030
    Figure PCTCN2022092556-appb-100031
    Figure PCTCN2022092556-appb-100032
    Figure PCTCN2022092556-appb-100033
    其中，R 11为环上一个或多个独立的取代基，并选自：H、卤素、-CN、-NO 2、-CF 3、-OCF 3、-NH 2、-OH、C 1-C 6烷基、-O(C 1-C 6烷基)、-NH(C 1-C 6烷基)、-N(C 1-C 6烷基)(C 1-C 6烷基)、单糖或其衍生物的残基，R 12选自：H、取代或未取代的烷基、取代或未取代的芳基、取代或未取代的芳烷基、取代或未取代的环烷基、取代或未取代的环烷基烷基、取代或未取代的杂环基、取代或未取代的杂环基烷基；
    更优选地，每个-Cy-独立地选自以下：
    Figure PCTCN2022092556-appb-100034
    Figure PCTCN2022092556-appb-100035
  19. 如权利要求1-17任一项所述的抗体药物偶联物，其特征在于，通式Ⅰ中L部分具有如下结构：
    Figure PCTCN2022092556-appb-100036
    其中，
    Y为与Ab的活性基团的连接基团；
    R L1、R L3和R L4独立地选自：
    Figure PCTCN2022092556-appb-100037
    -(CH 2) jO-、-(CH 2) jN(R L9)-、-(CH 2) jCO-、-(CH 2) jOCOO-、-(CH 2) jOCON(R L9)-、-(CH 2) jN(R L9)CON(R L10)-、-(CH 2) jN(R L9)CO-、-O(CH 2) jCOO-、-(CH 2) jCOO-、-(CH 2) jCON(R L9)-、-(CH 2) jS-S-、-(CH 2) jN(R L9)-NH＝、
    Figure PCTCN2022092556-appb-100038
    Figure PCTCN2022092556-appb-100039
    Figure PCTCN2022092556-appb-100040
    环烷基和芳基中一种或多种的组合，j为0-10的整数；
    R L2选自：单键、-(CH 2) iOCO-、-(CH 2) iOCOO-、-(CH 2) iNH-COO-、氨基酸残基、寡肽残基，其中i为0-10的整数；
    R L5、R L6、R L7和R L8独立地选自：H、取代或未取代的烷基、卤素、硝基、氰基、-OR L9、-NR L9R L10、-S(O) tR L9、-C(O)OR L9、-C(O)R L9和-C(O)NR L9R L10，其中，t为0、1或2；
    各R L9和R L10独立地选自：H、取代或未取代的烷基、取代或未取代的芳基、取代或未取代的芳烷基、取代或未取代的环烷基、取代或未取代的环烷基烷基、取代或未取代的杂环基、取代或未取代的杂环基烷基。
  20. 如权利要求19所述的抗体药物偶联物，其特征在于，所述L部分具有如下结构：
    Figure PCTCN2022092556-appb-100041
  21. 如权利要求19或20所述的抗体药物偶联物，其特征在于，Y选自：单键、
    Figure PCTCN2022092556-appb-100042
    -S-、-CO-、-NH-、-CONH-、
    Figure PCTCN2022092556-appb-100043
  22. 如权利要求19或20所述的抗体药物偶联物，其特征在于，R L1为
    Figure PCTCN2022092556-appb-100044
    或，R L1为-(CH 2) jCO-，j为0-6的整数。
  23. 如权利要求19或20所述的抗体药物偶联物，其特征在于，R L2为-(CH 2) iOCO-，i为0-6的整数，或，R L2为寡肽残基，所述寡肽残基选自：缬氨酸-瓜氨酸残基、丙氨酸-丙氨酸-天冬酰胺残基、天冬氨酸-缬氨酸残基、谷氨酸-缬氨酸残基。
  24. 如权利要求23所述的抗体药物偶联物，其特征在于，所述L部分具有如下结构：
    Figure PCTCN2022092556-appb-100045
  25. 如权利要求1-17任一项所述的抗体药物偶联物，其特征在于，所述L部分具有如下结构：
    Figure PCTCN2022092556-appb-100046
    Figure PCTCN2022092556-appb-100047
    Figure PCTCN2022092556-appb-100048
    Figure PCTCN2022092556-appb-100049
    Figure PCTCN2022092556-appb-100050
  26. 如权利要求1-6任一项所述的抗体药物偶联物，其特征在于，所述抗体药物偶联物具有如下结构：
    Figure PCTCN2022092556-appb-100051
    Figure PCTCN2022092556-appb-100052
    Figure PCTCN2022092556-appb-100053
    Figure PCTCN2022092556-appb-100054
  27. 一种权利要求1-26任一项所述的抗体药物偶联物的制备方法，其包括将小分子药物与连接单元的结合物与抗体进行偶联的步骤。
  28. 一种修饰的抗PD-L1抗体，其包含重链和轻链，
    其中，所述轻链的氨基酸序列如SEQ ID NO：9所示，或为与SEQ ID NO：9具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性的氨基酸序列；
    优选地，所述抗体具有如SEQ ID NO：9所示的轻链序列；
    更优选地，所述抗体具有如SEQ ID NO：4所示的重链序列和如SEQ ID NO：9所示的轻链序列。
  29. 一种化合物或其药学上可接受的盐、溶剂化物、前药、立体异构体，其具有如下结构：
    Figure PCTCN2022092556-appb-100055
    其中，
    Z为活性基团；
    L'为连接基团，其选自：单键、C 1-C 6亚烷基、C 1-C 6亚烯基、C 3-C 6亚环烷基，其中所述基团均可任选地被C 1-C 4烷基取代；
    R 1选自：单键、C 1-C 6亚烷基、C 1-C 6亚烯基、C 1-C 6亚烷氧基，其中所述基团均可任选地被C 1-C 4烷基取代；
    X选自：
    Figure PCTCN2022092556-appb-100056
    -NR 4-、-O-、-S-、单键、-OCO-、-COO-，其中，R 4和R 5独立地选自：H、C 1-C 6烷基、C 1-C 6烷氧基、氨基酸残基、寡肽残基、卤代烷基、羧基取代烷基、酯基取代烷基、
    Figure PCTCN2022092556-appb-100057
    其中，a为1-10的整数，R 8和R 9独立地选自：H、C 1-C 6烷基，或R 8和R 9与其中间的原子一起形成
    Figure PCTCN2022092556-appb-100058
    或者R 4和R 5与二者所连接的氮原子一起形成杂环基；
    R 2选自：-NR 6R 7、-OR 6、-SR 6，其中，R 6和R 7独立地选自：H、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氧基烷基，或者R 6和R 7与二者所连接的氮原子一起形成杂环基，或R 6与其所连接的氧原子一起形成杂环基，或R 6与其所连接的硫原子一起形成杂环基；
    R 3为苯环上一个或多个独立的取代基，其选自：H、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氧基烷基；
    m为0-4的整数；
    Q为二价的饱和或不饱和的直链或支链的C1-50烃链，其中0-6个亚甲基单元独立地被以下取代：-Cy-、-O-、-NR 10-、-S-、-OC(O)-、-C(O)O-、-C(O)-、-S(O)-、-S(O) 2-、-NR 10S(O) 2-、-S(O) 2-NR 10-、-NR 10-C(O)-、-C(O)NR 10-、-OC(O)NR 10-、-NR 10-C(O)O-、
    Figure PCTCN2022092556-appb-100059
    Figure PCTCN2022092556-appb-100060
    氨基酸残基、寡肽残基，其中，k选自1-10之间的整数，每个-Cy-独立地为选自以下的任选地经取代的二价环：亚芳基、亚环烷基、亚杂环基；R 10选自：H、-OH、C 1-C 6烷基、C 3-C 6环烷基、杂环烷基；
    优选地，每个-Cy-可以独立地选自以下：
    Figure PCTCN2022092556-appb-100061
    Figure PCTCN2022092556-appb-100062
    Figure PCTCN2022092556-appb-100063
    其中，R 11为环上一个或多个独立的取代基，并选自：H、卤素、-CN、-NO 2、-CF 3、-OCF 3、-NH 2、-OH、C 1-C 6烷基、-O(C 1-C 6烷基)、-NH(C 1-C 6烷基)、-N(C 1-C 6烷基)(C 1-C 6烷基)、单糖或其衍生物的残基，R 12选自：H、取代或未取代的烷基、取代或未取代的芳基、取代或未取代的芳烷基、取代或未取代的环烷基、取代或未取代的环烷基烷基、取代或未取代的杂环基、取代或未取代的杂环基烷基；
    更优选地，每个-Cy-独立地选自以下：
    Figure PCTCN2022092556-appb-100064
    Figure PCTCN2022092556-appb-100065
  30. 如权利要求29所述的化合物，其特征在于，所述化合物具有如下结构：
    Figure PCTCN2022092556-appb-100066
    其中，
    R L1、R L3和R L4独立地选自：
    Figure PCTCN2022092556-appb-100067
    -(CH 2) jO-、-(CH 2) jN(R L9)-、-(CH 2) jCO-、-(CH 2) jOCOO-、-(CH 2) jOCON(R L9)-、-(CH 2) jN(R L9)CON(R L10)-、-(CH 2) jN(R L9)CO-、-O(CH 2) jCOO-、-(CH 2) jCOO-、-(CH 2) jCON(R L9)-、-(CH 2) jS-S-、-(CH 2) jN(R L9)-NH＝、
    Figure PCTCN2022092556-appb-100068
    Figure PCTCN2022092556-appb-100069
    Figure PCTCN2022092556-appb-100070
    环烷基和芳基中一种或多种的组合，j为0-10的整数；
    R L2选自：单键、-(CH 2) iOCO-、-(CH 2) iOCOO-、-(CH 2) iNH-COO-、氨基酸残基、寡肽残基，其中i为0-10的整数；
    R L5、R L6、R L7和R L8独立地选自：H、取代或未取代的烷基、卤素、硝基、氰基、-OR L9、-NR L9R L10、-S(O) tR L9、-C(O)OR L9、-C(O)R L9和-C(O)NR L9R L10，其中，t为0、1或2；
    各R L9和R L10独立地选自：H、取代或未取代的烷基、取代或未取代的芳基、取代或未取代的芳烷基、取代或未取代的环烷基、取代或未取代的环烷基烷基、取代或未取代的杂环基、取代或未 取代的杂环基烷基。
  31. 如权利要求30所述的化合物，其特征在于，所述化合物具有如下结构：
    Figure PCTCN2022092556-appb-100071
    其中，R a和R b独立地选自：H、C 1-C 3烷基，或，R a和R b与其所连接的碳原子一起形成C 3-C 6亚环烷基。
  32. 如权利要求29所述的化合物，其特征在于，L'选自：-CH 2-、
    Figure PCTCN2022092556-appb-100072
    Figure PCTCN2022092556-appb-100073
  33. 如权利要求29或30所述的化合物，其特征在于，R 1为C 1-C 6亚烷基，例如C 1-C 3亚烷基，例如-CH 2-；或，
    R 1为单键；或，
    R 1为C 1-C 6亚烷氧基，例如C 1-C 3亚烷氧基，例如-CH 2O-、-CH 2CH 2O-、-CH 2CH 2CH 2O-。
  34. 如权利要求29或30所述的化合物，其特征在于，R 3为苯环上一个或多个独立的取代基，其选自：H、甲基、甲氧基。
  35. 如权利要求29或30所述的化合物，其特征在于，m为0或1。
  36. 如权利要求29或30所述的化合物，其特征在于，R 8和R 9独立地选自：H、甲基、乙基、正丙基、异丙基。
  37. 如权利要求29或30所述的化合物，其特征在于，R 4和R 5独立地选自：H、甲基、乙基、正丙基、异丙基、正丁基、叔丁基、氨基酸残基、寡肽残基、-CF 3、-CH 2CF 3、
    Figure PCTCN2022092556-appb-100074
    Figure PCTCN2022092556-appb-100075
    Figure PCTCN2022092556-appb-100076
    或者R 4和R 5与二者所连接的氮原子一起形成取代或未取代的杂环基；
    优选地，所述取代或未取代的杂环基选自：
    Figure PCTCN2022092556-appb-100077
    Figure PCTCN2022092556-appb-100078
    优选地，所述氨基酸残基选自：丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸残基；
    优选地，所述寡肽残基中的氨基酸残基独立地选自：丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸残基中的一种或多种。
  38. 如权利要求29或30所述的化合物，其特征在于，R 2选自：-NHR 6、-OR 6、-SR 6，其中，R 6为C 1-C 6烷基或C 1-C 6烷氧基烷基；或，
    R 2为-NR 6R 7，其中，R 6和R 7与二者所连接的氮原子一起形成杂环基；
    优选地，所述杂环基选自：
    Figure PCTCN2022092556-appb-100079
    Figure PCTCN2022092556-appb-100080
  39. 如权利要求29或30所述的化合物，其特征在于，R 2选自：
    Figure PCTCN2022092556-appb-100081
    Figure PCTCN2022092556-appb-100082
  40. 如权利要求30所述的化合物，其特征在于，R L1为
    Figure PCTCN2022092556-appb-100083
    或，R L1为-(CH 2) jCO-，j为0-6的整数。
  41. 如权利要求30所述的化合物，其特征在于，R L2为-(CH 2) iOCO-，i为0-6的整数，或，R L2为寡肽残基，所述寡肽残基选自：缬氨酸-瓜氨酸残基、丙氨酸-丙氨酸-天冬酰胺残基、天冬氨酸-缬氨酸残基、谷氨酸-缬氨酸残基。
  42. 如权利要求30所述的化合物，其特征在于，R L3为-(CH 2) jNH-，j为0-6的整数。
  43. 如权利要求30所述的化合物，其特征在于，R L4为-(CH 2) j-，j为0-5的整数。
  44. 如权利要求29-43任一项所述的化合物，其特征在于，Z选自：马来酰亚胺基、羧基、酰胺基、卤素、二硫基、酯基、酰卤基、酸酐、环氧基、羟基；
    优选地，Z为
    Figure PCTCN2022092556-appb-100084
  45. 如权利要求29所述的化合物，其特征在于，所述化合物具有如下结构：
    Figure PCTCN2022092556-appb-100085
    Figure PCTCN2022092556-appb-100086
    Figure PCTCN2022092556-appb-100087
    Figure PCTCN2022092556-appb-100088
    Figure PCTCN2022092556-appb-100089
  46. 一种药物组合物，其包含权利要求1-26任一项所述的抗体药物偶联物和药学上可接受的辅料。
  47. 一种组合物，其包含抗PD-L1抗体和小分子药物作为活性成分，其中，所述小分子药物为Toll样受体激动剂。
  48. 如权利要求47所述的组合物，其特征在于，所述小分子药物具有如下结构：
    Figure PCTCN2022092556-appb-100090
    其中，L'为连接基团，其选自：单键、C 1-C 6亚烷基、C 1-C 6亚烯基、C 3-C 6亚环烷基，其中所述基团均可任选地被C 1-C 4烷基取代；
    R 1选自：单键、C 1-C 6亚烷基、C 1-C 6亚烯基、C 1-C 6亚烷氧基，其中所述基团均可任选地被C 1-C 4烷基取代；
    X选自：
    Figure PCTCN2022092556-appb-100091
    -NR 4-、-O-、-S-、单键、-OCO-、-COO-，其中，R 4和R 5独立地选自：H、C 1-C 6烷基、C 1-C 6烷氧基、氨基酸残基、寡肽残基、卤代烷基、羧基取代烷基、酯基取代烷基、
    Figure PCTCN2022092556-appb-100092
    其中，a为1-10的整数，R 8和R 9独立地选自：H、C 1-C 6烷基，或R 8和R 9与其中间的原子一起形成
    Figure PCTCN2022092556-appb-100093
    或者R 4和R 5与二者所连接的氮原子一起形成杂环基；
    R 2选自：-NR 6R 7、-OR 6、-SR 6，其中，R 6和R 7独立地选自：H、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氧基烷基，或者R 6和R 6与二者所连接的氮原子一起形成杂环基，或R 6与其所连接的氧原子一起形成杂环基，或R 6与其所连接的硫原子一起形成杂环基；
    R 3为苯环上一个或多个独立的取代基，其选自：H、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氧基烷基；
    m为0-4的整数；
    X'选自：H、卤素、-NR 4R 5、-N +R 4R 5、-OR 4、-SR 4、叠氮基、膦酸基、膦酸二乙酯基、卤代烷基、羧基、酯基、-CN；其中，R 4和R 5独立地选自：H、C 1-C 6烷基、C 1-C 6烷氧基、氨基酸残基、寡肽残基、卤代烷基，或者R 4和R 5与二者所连接的氮原子一起形成杂环基。
  49. 如权利要求47所述的组合物，其特征在于，所述小分子药物选自如下结构：
    Figure PCTCN2022092556-appb-100094
    Figure PCTCN2022092556-appb-100095
    Figure PCTCN2022092556-appb-100096
    Figure PCTCN2022092556-appb-100097
  50. 如权利要求47-49任一项所述的组合物，其特征在于，所述抗PD-L1抗体选自：Atezolizumab、Durvalumab、Avelumab、Cemiplimab、KN035、CS1001、BGB-A333、KL-A167、SHR-1316和STI-A1014；或，
    所述抗体具有如SEQ ID NO：9所示的轻链序列；优选地，所述抗体具有如SEQ ID NO：4所示的重链序列和如SEQ ID NO：9所示的轻链序列。
  51. 一种权利要求1-26任一项所述的抗体药物偶联物、权利要求28所述的抗体、权利要求29-45任一项所述的化合物、权利要求47-50任一项所述的组合物在制备预防和/或治疗疾病的药物中的应用。
  52. 如权利要求51所述的应用，其特征在于，所述疾病选自：呼吸系统疾病、免疫性疾病、病毒性疾病、肿瘤；
    优选地，所述呼吸系统疾病选自：哮喘、慢性阻塞性肺病、成人呼吸窘迫综合征；
    优选地，所述免疫性疾病为自身免疫病，选自：系统性红斑狼疮、类风湿性关节炎、炎性肠病、斯耶格伦氏综合症、多肌炎、脉管炎、韦格纳肉芽肿、结节病、强直性脊柱炎、赖特综合征、牛皮癣性关节炎、贝赫切特综合征；
    优选地，所述病毒性疾病选自：流感、SARS、COVID-19、甲型肝炎、乙型肝炎、丙型肝炎、丁型肝炎、AIDS、狂犬病、登革热、埃博拉病毒病；
    优选地，所述肿瘤选自：淋巴瘤、母细胞瘤、髓母细胞瘤、视网膜母细胞瘤、脂肪肉瘤、滑膜细胞肉瘤、神经内分泌肿瘤、类癌肿瘤、胃泌素瘤、胰岛细胞癌、间皮瘤、神经鞘瘤、听神经瘤、脑膜瘤、腺癌、黑色素瘤、白血病或淋巴样恶性肿瘤、鳞状细胞癌、上皮鳞状细胞癌、肺癌(小细胞肺癌、非小细胞肺癌、腺癌肺癌、肺鳞癌)、腹膜癌、肝细胞癌、胃癌、肠癌、胰腺癌、成胶质细胞瘤、子宫颈癌、卵巢癌、肝癌、膀胱癌、肝癌、乳腺癌、转移性乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫癌、唾液腺癌、肾癌、前列腺癌、外阴癌、甲状腺癌、肝癌、肛门癌、阴茎癌、梅克尔细胞癌、食管癌、胆道肿瘤、头颈部癌和血液恶性肿瘤。
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WO2024231224A2 (en) 2023-05-05 2024-11-14 BioNTech SE Antibody-drug conjugates employing novel linker-payload systems for enhanced targeting of cancer-associated antigens

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