PEGylated antibody hydroxyl-containing drug conjugates

Abstract

In order to provide homogeneous conjugates with high potency and low toxicity, antibody drug conjugates (ADCs), particularly PEGylated mono- or bispecific antibody hydroxyl-containing drug conjugates prepared by site-specific conjugation, are provided herein. The present disclosure also relates to methods for preparing the antibody hydroxyl-containing drug conjugates, compositions comprising the antibody hydroxyl-containing drug conjugates, and their use in treating diseases.

Description

[Technical field]

[0001] This international patent application claims priority to International Patent Application No. PCT / CN2022 / 076012, filed February 11, 2022, the entire contents of which are incorporated by reference for all purposes.

[0002] The present invention relates to antibody hydroxyl-containing drug conjugates, particularly multispecific antibody hydroxyl-containing drug conjugates in which the hydroxyl groups of the payload are used to attach the drug to the antibody. Specifically, the present invention relates to long-acting PEGylated single or bispecific single chain antibody drug conjugates prepared by site-specific conjugation of PEGylated drug conjugates (in which the hydroxyl groups of the payload are coupled to polyethylene glycol (PEG) moieties) to mono- or bispecific antibodies. [Background technology]

[0003] Chemotherapy is one of the main therapeutic options in cancer treatment and is widely used in clinical practice, but serious obstacles such as drug resistance, systemic toxicity, and a narrow therapeutic window still remain.

[0004] Antibody-drug conjugates (ADCs) are a new type of targeted drug. By 2021, 12 ADCs (excluding one fusion ADC) have been approved by the FDA, and more than 100 ADC candidates are being tested in nearly 300 clinical trials (Beacon Targeted Therapies, Hanson Wade, UK research report). The biggest challenge in ADC development today is the need to administer them at doses very close to the maximum tolerated dose (MTD) to demonstrate therapeutic activity, resulting in a very narrow therapeutic window (Beck, A. et al., Nat. Rev. Drug Discov., 2017, 16, 315-337; Vankemmelbeke, M. et al., Ther. Deliv., 2016, 7, 141-144; Tolcher, AW et al., Ann. Oncol., 2016, 27, 2168-2172). The toxicity profile of ADCs is typically comparable to that of standard-of-care chemotherapeutic agents, and they are known to have dose-limiting toxicities associated with their cytotoxic warheads (Coats, S. et al. Clin. Cancer Res., 2019, 25, 5441-5448). As a result, 9 of the 12 FDA-approved ADCs require black box warning labels due to serious side effects, limiting their use in various cancer indications, and over 80 ADCs have been discontinued midway through clinical trials.

[0005] In addition, there are some genotoxicities that are directly related to the design and structure of ADCs. For example, ADC toxicity can result from off-target / off-tumor binding to Fc receptors (FcγR) or lectin receptors (such as mannose receptor) on normal cells (Donaghy, H. et al. MAbs, 2016, 8, 659-671), which releases the cytotoxic payload inside the cell and kills FcγR- or mannose-expressing cells (Gorovits, B. et al. Cancer Immunol Immunother, 2013, 62, 217-223). Another type of Fc-dependent toxicity results from ADC aggregates, which activate Fcγ receptors on immune cells, are internalized via FcγR, and ultimately kill such target-negative cells (Aoyama, M. et al. Pharmaceutical Research, 2022, 39, 89-103).

[0006] A large number of natural or synthetic cytotoxic compounds can be used as payloads for ADCs. The majority of ADCs in development or approved utilize an amino-containing payload to form a stable carbamate bond with a self-immolating spacer, which in turn is attached to a trigger molecule. The carbamate bond that is formed allows the payload to remain attached to the antibody during blood circulation. However, there is another large class of cytotoxic compounds in which the only functional group available for attachment to the antibody is a hydroxyl group. This class of hydroxyl-containing compounds has not received as much research and development in the development of ADCs as has been the case with cytotoxic amino-containing compounds. For compounds that have a hydroxyl group as the only functional group available for coupling, the same strategy of attaching the amino-containing compound to the antibody via a self-immolating moiety (such as 4-aminophenyl alcohol (PAB)) to form a stable carbamate bond cannot be pursued; instead of the carbamate bond, an unstable carbonate bond is formed, which can result in premature release of the payload.

[0007] Antibody drugs, including ADCs, face several obstacles that affect intratumoral distribution. The main mode of intratumoral transport of antibodies is based on diffusion, which is influenced by the size of the antibody, binding affinity, tumor microenvironment, angiogenesis, and the proximity of the target antigen (Xenaki, KT et al. Front Immunol, 2017, 8, 1287). Antibodies or ADCs with a large size, such as 150 kd molecular weight, have difficulty leaking out of the blood vessels and penetrating deep into tumor tissue, while antibody fragments with a small size showed a significant increase in tumor biodistribution (Li, Z. et al. MAbs, 2016, 8, 113-119). Binding site barrier (BSB) is another obstacle to antibody penetration into tumors (Miao, L. et al. ACS Nano, 2016, 10, 9243-9258). Since the high affinity of antibodies for cellular targets is the main cause of the binding site barrier, studies have adopted a strategy of co-administering unconjugated competing antibodies with ADCs. This strategy has been found to reduce the impact of the binding site barrier on ADCs and allow ADCs to be distributed more uniformly within solid tumors (Evans, R. et al. Sci Rep., 2022, 12, 7677). However, this approach requires two antibody-related products, dramatically increasing the cost of treatment.

[0008] Therefore, there is a need for new ADC technologies that address the above-mentioned issues. Summary of the Invention

[0009] The present invention addresses an unmet need by providing non-immunogenic polymer-modified antibody hydroxyl-containing drug conjugates prepared by site-specific conjugation of polymer-modified (e.g., PEGylated) hydroxyl-containing drug conjugates to either monospecific or multispecific antibody fragments. The antibody fragments can be monovalent or multivalent with respect to the antigen.

[0010] In one embodiment, the present invention provides a compound of formula Ia [ka] The polymer-antibody-drug conjugate moiety is provided as follows: P can be a non-immunogenic polymer; T can be a multifunctional (e.g., trifunctional) small molecule linker moiety and can have at least one functional group that allows for site-specific conjugation to a monospecific or multispecific antibody or protein; A can be any monospecific or multispecific antibody or protein; D can be any hydroxyl-containing cytotoxic molecule (n≧1), and each D can be the same or different.

[0011] In particular, an embodiment of the present invention is a conjugate of formula Ib: [ka] [In the formula, P can be a non-immunogenic polymer; M is H or C 1-50 an end-capping group selected from alkyl and aryl, where one or more carbons of said alkyl may be optionally replaced with a heteroatom; y may be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; T may be a multifunctional linker having two or more functional groups, including, but not limited to, trifunctional or tetrafunctional or other cyclic or acyclic multifunctional moieties (e.g., lysine), where T and (L 1 ) a and T and (L 2 ) b The bonds with may be the same or different; L 1 and L 2 may each independently be a bifunctional linker; a and b can each be an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; B may be a branched linker, where each branch may include an extended spacer (optional), a trigger unit, one or more self-immolative spacers, or any combination thereof, where the trigger unit may be an amino acid sequence, an enzymatically cleavable disulfide bond or a pH-sensitive linker, or any cleavable bond that may release the hydroxyl-containing drug D by some cleavage mechanism; A can be any monospecific or multispecific antibody, or antibody fragment, single chain antibody, nanobody (single domain antibody), or antigen binding protein, including any antigen-binding fragment, which can be monovalent or multivalent with respect to the antigen; D can be any cytotoxic hydroxyl-containing small molecule or derivative thereof; n may be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. to provide.

[0012] Another embodiment of the present invention is a conjugate of formula Ic: [ka] wherein each variable is as defined for formula Ib. to provide.

[0013] In some embodiments, each branch of B comprises an extended spacer (optional), a trigger moiety (e.g., an amino acid sequence or a disulfide moiety or a carbohydrate moiety such as a β-glucoronide or β-galactoside), which is linked to a hydroxyl-containing drug D via one or more self-immolative spacers cleavable by, for example, cathepsin B, plasmin, matrix metalloproteinases (MMPs), glutathione, thioredoxin family members (WCGH / PCK), thioreductase (Arunachalam, B. et. al. Proc. Natl. Acad. Sci. USA, 2000, 97, 745-750). Examples of one or two self-immolative spacers include, but are not limited to, the following: [ka] wherein n is 1 or 2; Y is a carbohydrate moiety; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 is H or C 1-10 Alkyl or -(CH2CH2-O) 1-10 -CH3 or any combination thereof; X=O, S, or N. In such embodiments, D can be any small molecule or peptide or derivative thereof that contains an active -OH functional group.

[0014] In some embodiments, A is a monospecific antibody that is monovalent or bivalent with respect to the antigen, for example a monospecific single chain antibody that is monovalent or bivalent with respect to the antigen.

[0015] In some embodiments, A is a multispecific antibody, such as a bispecific single chain antibody.

[0016] In some embodiments, the two binding domains of a bispecific antibody bind to two of the same tumor-associated antigen (TAA) molecules at two different epitopes or bind to two different TAA molecules.

[0017] In a further embodiment, A is a single chain anti-PDL1xanti-CD47 antibody that binds to PDL1 and CD47 expressed on cancer cells.

[0018] In another embodiment, A is a single chain anti-HER2(1)xanti-HER2(2) antibody that binds to HER2 expressed on cancer cells.

[0019] In yet another embodiment, A is a single chain anti-cMet(1)xanti-cMet(2) antibody that binds to cMet expressed on cancer cells.

[0020] In some embodiments, the antibody has the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0021] In some embodiments, the two binding domains of a single chain antibody are linked via a linker, wherein the linker is 1 The antibody may include moieties such as cysteine ​​or unnatural amino acid residues for site-specific conjugation to the antibody.

[0022] In some embodiments, D may be selected from any hydroxyl-containing DNA crosslinking agent, microtubule inhibitor, DNA alkylating agent, topoisomerase inhibitor, protein degrading agent, STING agonist, or combinations thereof.

[0023] In some embodiments, D is selected from the group consisting of Dxd, SN38, calicheamicin, pyrrolobenzodiazepines, sibiromycin, tomaymycin, duocarmycin, neothramycin, DC-81, psymberin, vinca alkaloids, laulimalide, taxanes, tubulysin, rhizoxin, discodermolide, taccalonolide, and the like. olide A or B or AF or AJ, taccalonolide AI-epoxide, epothilone A and B, paclitaxel, docetaxel, doxorubicin, camptothecin, tafuramycin A, PNU-159682, uncialamycin, β-amanitin, amatoxin, thailanstatin, or any hydroxyl-containing cytotoxic compound, or analogs / derivatives thereof, or combinations thereof.

[0024] In some embodiments, D is SN38 or Dxd (potent topoisomerase I inhibitors), or duocarmycin (a DNA alkylating agent), or an analog / derivative thereof, or a combination thereof.

[0025] In some embodiments, D is SN38, and -NR 1 -(CH2) n CH2-NR 2 -(EDA) to a self-immolative spacer such as 4-aminobenzyl alcohol (PAB), where n=1 or 2, and R 1 or R 2 is H, low molecular weight alkyl, or -(CH2CH2O) 1-10 -CH3 (1-10 PEG units), and the PAB is linked to a trigger moiety such as valine-citrulline.

[0026] In other embodiments, D is Dxd and is linked to a self-immolative spacer such as 4-aminobenzyl alcohol (PAB) via -NR1-(CH2)nCH2-NR2-(EDA), where n=1 or 2, R1 or R2 can be H, a low molecular weight alkyl, or -(CH2CHO)1-10-CH3 (1-10 PEG units), and the PAB is linked to a trigger moiety such as valine-citrulline.

[0027] In a further embodiment, D is duocarmycin DM, -NR 1 -(CH2) n CH2-NR 2 -(EDA) to a self-immolative spacer such as 4-aminobenzyl alcohol (PAB), where n=1 or 2, and R 1 or R 2 is H, low molecular weight alkyl, or -(CH2CH2O) 1-10 -CH3 (1-10 PEG units), and the PAB is linked to a trigger moiety such as valine-citrulline.

[0028] In any of the above aspects and embodiments, the non-immunogenic polymer may be selected from the group consisting of polyethylene glycol (PEG), dextran, carbohydrate polymer, polyalkylene oxide, polyvinyl alcohol, hydroxypropyl methacrylamide (HPMA), and copolymers thereof. Preferably, the non-immunogenic polymer is a PEG, such as a branched PEG or a linear PEG, where the PEG may be attached to the multifunctional moiety T via either a permanent or cleavable bond. The total molecular weight of the PEG may be 3000-100,000 daltons, e.g., 5000-80,000, 10,000-60,000, 10000-30000, or 20,000-40,000 daltons, e.g., about 10000, 20000, 30000, or 40000 daltons.

[0029] (L 1 ) aThe functional group for site-specific conjugation that forms a bond between and Protein A may be selected from the group consisting of thiol, maleimide, 2-pyridyldithio variants, aromatic or vinyl sulfone, acrylate, bromo or iodoacetamide, azide, alkyne, dibenzocyclooctyl (DBCO), carbonyl, 2-amino-benzaldehyde or 2-amino-acetophenone group, hydrazide, oxime, potassium acyltrifluoroborate, O-carbamoylhydroxylamine, transcyclooctene, tetrazine, triarylphosphine, boronic acid, iodine, and the like.

[0030] In some embodiments, (L 1 ) a may include a bond formed from an azide and an alkyne, or from a maleimide and a thiol. In some embodiments, the alkyne may be dibenzocyclooctyl (DBCO).

[0031] In some embodiments, T can be lysine, aspartic acid, glutamic acid, serine, tyrosine, or any other molecule with a trifunctional group, P can be PEG, y can be 1, while the alkyne can be dibenzocyclooctyl (DBCO).

[0032] In some embodiments, A may be derived from an azide-labeled mono- or multispecific antibody or antibody fragment, a single chain antibody, a nanobody, or an antigen-binding protein, including any antigen-binding fragment thereof, or a combination thereof, where the azide is the corresponding (L 1 ) a In other embodiments, A may be derived from a thiol-labeled mono- or multispecific antibody or antibody fragment, a single chain antibody, a nanobody, or an antigen-binding protein, including any antigen-binding fragment thereof, or a combination thereof, where the thiol is conjugated to the corresponding (L 1 ) a The compound can be conjugated to a maleimide in

[0033] The above antibody-drug conjugates can be prepared according to a method comprising: (i) preparing a non-immunogenic polymer-drug conjugate having a terminal functional group capable of site-specifically conjugating to an antibody or protein or modified form thereof; and (ii) site-specifically conjugating the non-immunogenic polymer-drug conjugate to an antibody or protein or modified structure thereof to form a compound of formula Ia, Ib, or Ic. In some instances, the antibody or protein can be modified with a small molecule linker prior to conjugation.

[0034] The present invention also provides pharmaceutical formulations comprising the above-described antibody-hydroxyl-containing drug conjugates, e.g., PEGylated single- or bispecific single chain antibody-hydroxyl-containing drug conjugates which are mono- or multivalent to an antigen, and a pharma- ceutically acceptable carrier.

[0035] The present invention further provides a method for treating a disease in a subject in need thereof, comprising administering an effective amount of the above-described antibody-hydroxyl-containing drug conjugate, e.g., a PEGylated single or bispecific single chain antibody drug conjugate that is monovalent or multivalent with respect to an antigen.

[0036] The details of one or more embodiments of the invention are set forth below. Other features, objects, and advantages of the invention will be apparent from the drawings, the description, and the claims.

[0037] The present disclosure further provides the following embodiments.

[0038] Embodiment 1. A compound of formula (Ib): [ka] [In the formula, P is a non-immunogenic polymer; M is H or C 1-50 an end-capping group selected from alkyl and aryl, where one or more carbons of said alkyl may be optionally replaced with a heteroatom; y is an integer selected from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is an antibody or antigen-binding fragment thereof; T is a multifunctional small molecule linker moiety; L 1 and L 2 are each independently a hetero- or homobifunctional linker; a and b are each an integer selected from 0 to 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; B is a branched linker, where each branch has an amino acid sequence or a disulfide bond or a carbohydrate moiety or a cleavable bond attached to one or more self-immolative spacers, where cleavage of said amino acid sequence or disulfide bond or carbohydrate moiety or cleavable bond by an enzyme triggers a self-immolative mechanism to release a hydroxyl-containing D or derivative thereof; D is, independently, a cytotoxic hydroxyl-containing small molecule or peptide; n is an integer selected from 1 to 25, e.g., 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25].

[0039] Embodiment 2. T is a trifunctional linker derived from a molecule having three functional groups independently selected from hydroxyl, amino, hydrazinyl, azide, alkene, alkyne, carboxyl (aldehyde, ketone, ester, carboxylic acid, anhydride, acid halide), thiol, disulfide, nitrile, epoxide, imine, nitro, and halide, where T and (L 1 ) a and T and (L 2 ) b The compound of embodiment 1, wherein the bonds to are the same or different.

[0040] Embodiment 3. The compound of embodiment 2, wherein T is lysine, aspartic acid, glutamic acid, serine, tyrosine, or any other cyclic or acyclic molecule having a trifunctional group, or a derivative thereof.

[0041] Embodiment 4.(L 1 ) a The compound according to any one of the preceding embodiments, wherein one of the functional groups at the linker terminus of is capable of site-specific conjugation with A and is selected from the group consisting of thiol, maleimide, 2-pyridyldithio variants, aromatic or vinyl sulfone, acrylate, bromo or iodoacetamide, azide, alkyne, dibenzocyclooctyl (DBCO), carbonyl, 2-amino-benzaldehyde or 2-amino-acetophenone group, hydrazide, oxime, potassium acyltrifluoroborate, O-carbamoylhydroxylamine, transcyclooctene, tetrazine, triarylphosphine, boronic acid, and iodine.

[0042] Embodiment 5. The compound according to any one of embodiments 1 to 4, wherein the antibody is a monospecific or multispecific full-length antibody, a single-chain antibody, a nanobody (single domain antibody), or an antigen-binding domain thereof.

[0043] Embodiment 6. The compound of any one of embodiments 1 to 5, wherein the antibody is a monospecific single chain antibody.

[0044] Embodiment 7. The compound of embodiment 6, wherein the monospecific single chain antibody binds to a tumor-associated antigen (TAA), such as Her2, cMet, PDL1, or CD47.

[0045] Embodiment 8. The compound of embodiment 7, wherein the monospecific single chain antibody has two binding domains that bind to Her2.

[0046] Embodiment 9. The compound of embodiment 8, wherein the monospecific single chain antibody has the amino acid sequence shown in SEQ ID NO:3.

[0047] Embodiment 10. The compound of any one of embodiments 1 to 5, wherein the antibody is a bispecific antibody, such as a bispecific single chain antibody.

[0048] Embodiment 11. The compound of embodiment 10, wherein the two binding domains of the bispecific antibody bind to the same tumor-associated antigen (TAA), to two different TAAs, or to a TAA and an antigen expressed on T cells or NK cells (e.g. a component of the T cell receptor).

[0049] Embodiment 12. The compound of embodiment 11, wherein the antibody is an anti-PDL1 x anti-CD47 single chain bispecific antibody or an anti-HER2(1) x anti-HER2(2) single chain bispecific antibody or an anti-cMet(1) x anti-cMet(2) single chain bispecific antibody.

[0050] Embodiment 13. The compound of embodiment 12, wherein the antibody has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:6.

[0051] Embodiment 14. The two binding domains of a monospecific single chain antibody are linked via a linker, wherein the linker is 1 ) a 10. The compound of any one of embodiments 6 to 9, comprising a moiety such as a cysteine ​​or an unnatural amino acid residue for site-specific conjugation to.

[0052] Embodiment 15. The two binding domains of a bispecific single chain antibody are linked via a linker, wherein the linker is 1 ) a 14. The compound of any one of embodiments 10-13, comprising a moiety such as a cysteine ​​or an unnatural amino acid residue for site-specific conjugation to.

[0053] Embodiment 16. The non-natural amino acid residue is selected from the group consisting of the genetically encoded alkene lysine (such as N6-(hex-5-enoyl)-L-lysine), 2-amino-8-oxononanoic acid, m- or p-acetylphenylalanine, amino acids containing β-diketone side chains (such as 2-amino-3-(4-(3-oxobutanoyl)phenyl)propanoic acid), (S)-2-amino-6-(((1R,2R)-2-azidocyclopentyloxy)carbonylamino)hexanoic acid, azidohomoalanine, the pyrrolidine analog N6-((prop-2-yn-1-yloxy)carbonyl)-L-lysine, (S)-2-amino-6-pent-4-ynamidohexanoic acid, (S)-2-amino-6-((prop-2-yn ... 16. The compound according to any one of embodiments 14-15, wherein the compound is selected from the group consisting of Nε-acryloyl-l-lysine, Nε-5-norbornen-2-yloxycarbonyl-l-lysine, N-ε-(cyclooct-2-yn-1-yloxy)carbonyl)-L-lysine, N-ε-(2-(cyclooct-2-yn-1-yloxy)ethyl)carbonyl-L-lysine, and genetically encoded tetrazine amino acids such as 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine.

[0054] Embodiment 17. The compound of any one of embodiments 1-16, wherein D is selected from a DNA crosslinking agent, a microtubule inhibitor, a DNA alkylating agent, a topoisomerase inhibitor, a protein degrading agent, a STING agonist, or a combination thereof.

[0055] Embodiment 18.D is selected from the group consisting of Dxd, SN38, calicheamicin, pyrrolobenzodiazepines, sibiromycin, tomaymycin, duocarmycin, neothramycin, DC-81, psymberin, vinca alkaloids, laulimalide, taxanes, tubulysin, rhizoxin, discodermolide, taccalonolide A or B or AF or taccalonolide B or AF. or AJ, taccalonolide AI-epoxide, epothilone A and B, paclitaxel, docetaxel, doxorubicin, camptothecin, tafuramycin A, PNU-159682, uncialamycin, β-amanitin, amatoxin, thailanstatin, or any hydroxyl-containing cytotoxic compound, or analogs / derivatives thereof, or combinations thereof.

[0056] Embodiment 19. A compound according to any one of embodiments 1 to 18, wherein the non-immunogenic polymer is polyethylene glycol (PEG).

[0057] Embodiment 20. The compound of embodiment 19, wherein the PEG is a linear PEG or a branched PEG.

[0058] Embodiment 21. The compound of any one of embodiments 19-20, wherein at least one end of the polyethylene glycol is capped with methyl or a low molecular weight alkyl.

[0059] Embodiment 22. A compound according to any one of embodiments 19 to 21, wherein the total molecular weight of the PEG is between 3,000 and 100,000 daltons.

[0060] Embodiment 23. A compound according to any one of embodiments 19 to 22, wherein PEG is linked to a tri- or tetra-functional or any other cyclic or acyclic polyfunctional moiety T (e.g. lysine) via a permanent or cleavable bond.

[0061] Embodiment 24. A compound of formula (Ic): [ka] [In the formula, P is a linear PEG; A is an antibody or antigen-binding fragment thereof; L 1 and L 2 are each independently a bifunctional linker; a and b are each an integer selected from 0 to 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; B is a branched linker, where each branch has an amino acid sequence or a disulfide bond or a carbohydrate moiety or a cleavable bond attached to one or more self-immolative spacers, where cleavage of said amino acid sequence or disulfide bond or carbohydrate moiety by an enzyme, e.g., cathepsin B, plasmin, matrix metalloproteinase (MMP), glutathione, thioredoxin family member (WCGH / PCK), thioreductase, triggers a self-immolative mechanism to release D or a derivative thereof; D is, independently, a cytotoxic hydroxyl-containing small molecule or peptide; n is an integer selected from 1 to 25, e.g., 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25].

[0062] Embodiment 25.(L 1 ) a25. The compound of embodiment 24, wherein one of the functional groups at the linker terminus of is capable of site-specific conjugation with A and is selected from the group consisting of thiol, maleimide, 2-pyridyldithio variants, aromatic or vinyl sulfone, acrylate, bromo or iodoacetamide, azide, alkyne, dibenzocyclooctyl (DBCO), carbonyl, 2-amino-benzaldehyde or 2-amino-acetophenone group, hydrazide, oxime, potassium acyltrifluoroborate, O-carbamoylhydroxylamine, transcyclooctene, tetrazine, triarylphosphine, boronic acid, and iodine.

[0063] Embodiment 26. The compound according to any one of embodiments 24 to 25, wherein the antibody is a monospecific or multispecific full-length antibody, a single-chain antibody, a nanobody (single domain antibody), or an antigen-binding domain thereof.

[0064] Embodiment 27. The compound of embodiment 26, wherein the antibody is a monospecific single chain antibody, optionally wherein said monospecific single chain antibody binds to a tumor associated antigen (TAA), such as Her2, cMet, PDL1, or CD47.

[0065] Embodiment 28. The compound of embodiment 27, wherein the monospecific single chain antibody has two binding domains that bind to Her2.

[0066] Embodiment 29. The compound of embodiment 28, wherein the monospecific single chain antibody has the amino acid sequence shown in SEQ ID NO:3.

[0067] Embodiment 30. The compound of embodiment 26, wherein the antibody is a bispecific antibody, such as a bispecific single chain antibody.

[0068] Embodiment 31. The compound of embodiment 30, wherein the two binding domains of the bispecific antibody bind to the same tumor-associated antigen (TAA), to two different TAAs, or to a TAA and an antigen expressed on T cells or NK cells (e.g. a component of the T cell receptor).

[0069] Embodiment 32. The compound of embodiment 30, wherein the antibody is an anti-PDL1 x anti-CD47 single chain bispecific antibody or an anti-HER2(1) x anti-HER2(2) single chain bispecific antibody or an anti-cMet(1) x anti-cMet(2) single chain bispecific antibody.

[0070] Embodiment 33. The compound of embodiment 32, wherein the antibody has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:6.

[0071] Embodiment 34. The two binding domains of a monospecific single chain antibody are linked via a linker, wherein the linker is 1 ) a 30. The compound of any one of embodiments 27-29, comprising a moiety such as a cysteine ​​or an unnatural amino acid residue for site-specific conjugation to.

[0072] Embodiment 35. The two binding domains of a bispecific single chain antibody are linked via a linker, wherein the linker is 1 ) a 34. The compound of any one of embodiments 30-33, comprising a moiety such as a cysteine ​​or an unnatural amino acid residue for site-specific conjugation to.

[0073] Embodiment 36. The non-natural amino acid residue is selected from the group consisting of non-genetically encoded alkene lysine (such as N6-(hex-5-enoyl)-L-lysine), 2-amino-8-oxononanoic acid, m- or p-acetylphenylalanine, amino acids containing β-diketone side chains (such as 2-amino-3-(4-(3-oxobutanoyl)phenyl)propanoic acid), (S)-2-amino-6-(((1R,2R)-2-azidocyclopentyloxy)carbonylamino)hexanoic acid, azidohomoalanine, pyrrolidine analog N6-((prop-2-yn-1-yloxy)carbonyl)-L-lysine, (S)-2-amino-6-pent-4-ynamidohexanoic acid, (S)-2-amino-6-((prop-2-ynyl) 36. The compound according to any one of embodiments 34-35, wherein the compound is selected from the group consisting of Nε-acryloyl-l-lysine, Nε-5-norbornen-2-yloxycarbonyl-l-lysine, N-ε-(cyclooct-2-yn-1-yloxy)carbonyl)-L-lysine, N-ε-(2-(cyclooct-2-yn-1-yloxy)ethyl)carbonyl-L-lysine, and genetically encoded tetrazine amino acids such as 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine.

[0074] Embodiment 37. The compound of any one of embodiments 24-36, wherein D is selected from a DNA crosslinking agent, a microtubule inhibitor, a DNA alkylating agent, a topoisomerase inhibitor, a STING agonist, a protein degrading agent, or a combination thereof.

[0075] Embodiment 38. The hydroxyl-containing D is Dxd, SN38, calicheamicin, pyrrolobenzodiazepine, sibiromycin, tomaymycin, duocarmycin, neothramycin, DC-81, psymberin, vinca alkaloid, laulimalide, taxane, tubulysin, rhizoxin, discodermolide, taccalonolide A or B or The compound of embodiment 37, wherein is selected from AF or AJ, taccalonolide AI-epoxide, epothilone A and B, paclitaxel, docetaxel, doxorubicin, camptothecin, tafuramycin A, PNU-159682, uncialamycin, β-amanitin, amatoxin, thailanstatin, or any hydroxyl-containing cytotoxic compound, or an analog / derivative thereof, or a combination thereof.

[0076] Embodiment 39. A compound according to any one of embodiments 24 to 38, wherein the total molecular weight of the PEG is between 3,000 and 100,000 daltons.

[0077] Embodiment 40.L 1 and L 2 However, each independently, -(CH2) a XY(CH2) b -, -X(CH2) a O(CH2CH2O) c (CH2) b Y-, -(CH2) a Heterocyclyl-, -(CH2) a X-, -X(CH2) a Y-, -W1-(CH2) a C(O)NR1(CH2) b O(CH2CH2O) c (CH2) dC(O)-, -C(O)(CH2) a O(CH2CH2O) b (CH2) c W2C(O)(CH2) d NR1-, and -W3-(CH2) a C(O)NR1(CH2) b O(CH2CH2O) c (CH2) d W2C(O)(CH2) e C(O)- is selected from the group consisting of In the formula, a, b, c, d, and e are each independently an integer selected from 0 to 25, for example, 0 to 20, 0 to 15, 0 to 10, 0 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; X and Y are each independently C(=O), NR2, S, O, N3, CR3R 4、 R1, R2, R3, and R4 are each independently selected from hydrogen, C 1-10 Alkyl, or (CH2) 1-10 C(=O); W1 and / or W3 are derived from a maleimide-based moiety and W2 represents a triazolyl- or tetrazolyl-containing group; and the heterocyclyl group is selected from a maleimide-derived moiety or a tetrazolyl- or triazolyl-based moiety.

[0078] Embodiment 41.L 1 and L 2 However, each independently, [ka] is selected from 40. The compound of any one of embodiments 1-39, wherein n and m are each independently an integer selected from 0 to 20, e.g., 0 to 15, 0 to 10, 0 to 5, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0079] Embodiment 42. The compound according to any one of embodiments 1 to 41, wherein the branched linker B comprises an extended spacer (optional), a trigger unit, one or more self-immolative spacers, or any combination thereof, and optionally, said trigger unit is an amino acid sequence or a disulfide bond or a β-glucoronide or β-galactoside trigger moiety cleavable by an enzyme (such as, for example, cathepsin B, plasmin, matrix metalloproteinase (MMP), β-glucuronidase, β-galactosidase, glutathione, thioredoxin family member (WCGH / PCK), or thioreductase).

[0080] Embodiment 43. The branched linker B is: [ka] [In the formula, a, b, c, d, e, and f are each independently an integer selected from 1 to 25, e.g., 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25; n is an integer selected from 1 to 10; (A) tis a trigger unit of the amino acid sequence Val-Cit, Val-Ala, Val-Lys, Phe-Lys, Phe-Cit, Phe-Arg, Phe-Ala, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, D-Phe-Phe-Lys, Phe-Phe-Lys, Gly-Phe-Lys, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, or Ala-Leu-Ala-Leu; PAB is para-aminobenzyl alcohol; EDA-NR 1 (CH2) m NR 2 where m is 2 or 3, and R 1 and R 2 are each independently H, a low molecular weight alkyl, or -(CH2CH2O) l -CH3, where l is an integer selected from 1 to 10; Each of the Ex's are as follows: -NR 1 (CH2) x O(CH2CH2O) y (CH2) z C(O)-, -C(O)(CH2) x NR 1 -, -NR 1 (CH2) x O(CH2CH2O) y (CH2) z NR 2 -, -NR 1 (CH2) x NR 2 -, -NR 1 (CH2) x O(CH2CH2O) y (CH2) z O-, -O(CH2) x NR 1 -, -C(O)(CH2) x O-, -O(CH2) x O(CH2CH2O)y (CH2) z C(O)-, -C(O)(CH2) x O(CH2CH2O) y (CH2) z C(O)-, -C(O)(CH2) x C(O)-, Or nothing (wherein x, y, and z are each independently an integer selected from 0 to 25, e.g., 0 to 20, 0 to 15, 0 to 10, 0 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25; R 1 and R 2 are each independently hydrogen or C 1-10 represents an alkyl group) is an extended spacer comprising a linker chain independently selected from The compound of embodiment 42, selected from:

[0081] Embodiment 44. The branched linker B is: [ka] [wherein n=1 or 2; b is an integer selected from 1 to 10; R 1 and R 2 are each independently H, a low molecular weight alkyl, or -(CH2CH2O) m -CH3, where m=1-10.

[0082] Embodiment 45. The following: [ka] [ka] [ka] [wherein SCA1 and SCA2 are anti-PDL1 and anti-CD47 single chain antibodies or anti-HER2(1) and anti-HER2(2) single chain antibodies or anti-cMet(1) and anti-cMet(2) single chain antibodies, preferably having the amino acids shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 6; mPEG has a total molecular weight of 3,000 to 100,000 daltons, for example 10,000 to 40,000 daltons; [ka] is] or a pharma- ceutically acceptable salt thereof.

[0083] Embodiment 46. The following: [ka] [In the formula, SCA1 and SCA2 are anti-PDL1 and anti-CD47 single chain antibodies or anti-HER2(1) and anti-HER2(2) single chain antibodies or anti-cMet(1) and anti-cMet(2) single chain antibodies, preferably having the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:6; n is an integer selected from 110 to 1800, preferably n is an integer selected from 220 to 910, or preferably the total molecular weight of PEG is 10000 to 40000 daltons, for example about 10000, 20000, 30000, or 40000 daltons; [ka] is] or a pharma- ceutically acceptable salt thereof.

[0084] Embodiment 47. The method according to the present invention comprises the steps of: a) preparing a non-immunogenic polymer-modified (e.g., PEGylated) hydroxyl-containing drug conjugate having free functional groups for site-specific conjugation; b) site-specifically conjugating said non-immunogenic polymer-modified (e.g., PEGylated) hydroxyl-containing drug conjugate to an antibody to provide a compound of formula (Ib) or (Ic). A method for preparing a compound according to any one of embodiments 1 to 46, comprising:

[0085] Embodiment 48. A pharmaceutical formulation comprising an effective amount of a compound according to any one of embodiments 1 to 46 and a pharma- ceutically acceptable salt, carrier, or excipient.

[0086] Embodiment 49. A compound according to any one of embodiments 1 to 46 for use in the treatment of a cancer selected from the group consisting of non-Hodgkin's lymphoma, B-cell acute and chronic lymphocytic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemia, T-cell lymphoma and leukemia, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, renal cancer, bladder cancer, gastric cancer, colon cancer, colorectal cancer, salivary gland cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, head and neck cancer, and endometrial cancer.

[0087] Embodiment 50. A compound according to any one of embodiments 1-46 for use in combination with an effective amount of another anti-cancer agent or an immunosuppressant in the treatment of a cancer selected from the group consisting of non-Hodgkin's lymphoma, B-cell acute and chronic lymphocytic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemia, T-cell lymphoma and leukemia, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, renal cancer, bladder cancer, gastric cancer, colon cancer, colorectal cancer, salivary gland cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, head and neck cancer, and endometrial cancer.

[0088] Embodiment 51. A method for treating cancer in a subject, comprising administering to the subject an effective amount of a compound according to any one of embodiments 1 to 46, wherein the cancer is selected from the group consisting of non-Hodgkin's lymphoma, B-cell acute and chronic lymphocytic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemia, T-cell lymphoma and leukemia, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, renal cancer, bladder cancer, gastric cancer, colon cancer, colorectal cancer, salivary gland cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, head and neck cancer, and endometrial cancer.

[0089] Embodiment 52. The method of embodiment 51, further comprising administering to the subject an effective amount of another anti-cancer or immunosuppressant agent.

[0090] Embodiment 53. The use of a compound according to any one of embodiments 1 to 46 in the manufacture of a medicament for treating cancer in a subject, wherein the cancer is selected from the group consisting of non-Hodgkin's lymphoma, B-cell acute and chronic lymphocytic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemia, T-cell lymphoma and leukemia, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, renal cancer, bladder cancer, gastric cancer, colon cancer, colorectal cancer, salivary gland cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, head and neck cancer, and endometrial cancer.

[0091] Embodiment 54. The use according to embodiment 53, wherein the compound is combined with another anticancer or immunosuppressant agent.

[0092] Embodiment 55. The use of a compound of any one of embodiments 1-46 and another anticancer or immunosuppressant agent in the manufacture of a medicament for treating cancer in a subject, wherein the cancer is selected from the group consisting of non-Hodgkin's lymphoma, B-cell acute and chronic lymphocytic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemia, T-cell lymphoma and leukemia, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, renal cancer, bladder cancer, gastric cancer, colon cancer, colorectal cancer, salivary gland cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, head and neck cancer, and endometrial cancer. [Brief description of the drawings]

[0093] [Figure 1] FIG. 1 illustrates diagrammatically the reaction scheme for preparing the compound Fmoc-Val-Cit-PAB-PNP (5) described in Example 1. [Diagram 2] FIG. 2 illustrates diagrammatically the reaction scheme for preparing compound Val-Cit-PAB-DEA-SN38 (10) described in Example 1. [Diagram 3] FIG. 3 illustrates diagrammatically an alternative reaction scheme for preparing compound Val-Cit-PAB-DEA-SN38 (10) described in Example 1. [Figure 4] FIG. 4 illustrates a reaction scheme for preparing branched intermediate compound 16 described in Example 1. [Diagram 5] FIG. 5 illustrates diagrammatically the reaction scheme for preparing the compound 30 kmPEG-Lys(Mal)-3(Val-Cit-PAB-DEA-SN38) (22) described in Example 1. [Figure 6] FIG. 6 illustrates diagrammatically the reaction scheme for preparing the compound 30 kmPEG-Lys(Mal)-6(Val-Cit-PAB-DEA-SN38) (28) of Example 2. [Figure 7] FIG. 7 illustrates diagrammatically the reaction scheme for preparing the compound Val-Cit-PAB-DEA-Duo-DM (33) of Example 3. [Figure 8] FIG. 8 illustrates a reaction scheme for preparing the branched intermediate compound (40) of Example 3. [Figure 9] FIG. 9 illustrates diagrammatically an alternative reaction scheme for preparing the branched intermediate compound (40) of Example 3. [Figure 10] FIG. 10 diagrammatically illustrates the reaction scheme for preparing the compound 20 kmPEG-Glu(Mal)-4(Val-Cit-PAB-DEA-Duo-DM) (46) of Example 3. [Figure 11] FIG. 11 illustrates a reaction scheme for preparing the branched intermediate compound (48) of Example 4. [Figure 12] FIG. 12 illustrates diagrammatically the reaction scheme for preparing the compound Mal-20kPEG-3(Val-Cit-PAB-DEA-Duo-DM) (51) of Example 4. [Figure 13] FIG. 13 illustrates diagrammatically the reaction scheme for preparing the compound 30 kmPEG-(SCAPDL1xSCACD47)-3(Val-Cit-PAB-DEA-SN38) (53) of Example 6. [Figure 14] FIG. 14 diagrammatically illustrates the reaction scheme for preparing the compound 30 kmPEG-(SCAPDL1xSCACD47)-6(Val-Cit-PAB-DEA-SN38) (54) of Example 7. [Figure 15] FIG. 15 diagrammatically illustrates the reaction scheme for preparing the compound 20 kmPEG-(SCAPDL1xSCACD47)-4(Val-Cit-PAB-DEA-Duo-DM) (55) of Example 8. [Figure 16] FIG. 16 diagrammatically illustrates the reaction scheme for preparing the compound SCAPDL1xSCACD47-20kPEG-3(Val-Cit-PAB-DEA-Duo-DM) (56) of Example 9. [Figure 17] FIG. 17 illustrates a reaction scheme for preparing the branched intermediate compound (58) of Example 10. [Figure 18]FIG. 18 illustrates diagrammatically the reaction scheme for preparing the branched intermediate compound N3-PEG6-3(Val-Cit-PAB-DEA-SN38) (62) of Example 11. [Figure 19] FIG. 19 illustrates diagrammatically the reaction scheme for preparing the branched intermediate compounds N3-PEG6-2 (Val-Cit-PAB-DEA-SN38) (65) and N3-PEG6-4 (Val-Cit-PAB-DEA-SN38) (66) of Example 12. [Figure 20] FIG. 20 diagrammatically illustrates the reaction scheme for preparing 30 kmPEG-Lys(PEG2-Mal)-DBCO (68) of Example 13. [Figure 21] FIG. 21 diagrammatically illustrates the reaction scheme for preparing 30 kmPEG-Lys(PEG2-Mal)-2(Val-Cit-PAB-DEA-SN38) (69) of Example 14. [Figure 22] FIG. 22 illustrates diagrammatically the reaction scheme for preparing 30 km PEG-Lys(PEG2-Mal)-3(Val-Cit-PAB-DEA-SN38) (70) of Example 15. [Figure 23] FIG. 23 illustrates diagrammatically the reaction scheme for preparing 30 km PEG-Lys(PEG2-Mal)-4(Val-Cit-PAB-DEA-SN38) (71) of Example 16. [Figure 24] FIG. 24 diagrammatically illustrates the reaction scheme for preparing 20 km PEG-Lys(PEG-Mal)-2(Val-Cit-PAB-DEA-SN38) (73) of Example 17. [Diagram 25] FIG. 25 illustrates diagrammatically the reaction scheme for preparing Mal-PEG2-20kPEG-2(Val-Cit-PAB-DEA-SN38) (76) of Example 18. [Figure 26] FIG. 26 illustrates diagrammatically the reaction scheme for preparing Val-Cit-PAB-DEA-Dxd (81) of Example 19. [Figure 27]FIG. 27 diagrammatically illustrates the reaction scheme for preparing 20 kmPEG-Lys(PEG-Mal)-2(Val-Cit-PAB-DEA-Dxd) (83) of Example 20. [Figure 28] FIG. 28 diagrammatically illustrates the reaction scheme for preparing 30 kmPEG(SCAHer2xSCAHer2)-2(Val-Cit-PAB-DEA-SN38) (86) of Example 23. [Figure 29] FIG. 29 diagrammatically illustrates the reaction scheme for preparing 30 kmPEG(SCAPDL1xSCACD47)-2(Val-Cit-PAB-DEA-SN38) (87) of Example 24. [Diagram 30] FIG. 30 diagrammatically illustrates the reaction scheme for preparing 30 kmPEG(SCAHer2xSCAHer2)-3(Val-Cit-PAB-DEA-SN38) (88) of Example 25. [Diagram 31] FIG. 31 diagrammatically illustrates the reaction scheme for preparing 30 kmPEG(SCAPDL1xSCACD47)-3(Val-Cit-PAB-DEA-SN38) (89) of Example 26. [Diagram 32] FIG. 32 diagrammatically illustrates the reaction scheme for preparing 30 kmPEG(SCAPDL1xSCACD47)-4(Val-Cit-PAB-DEA-SN38) (90) of Example 27. [Diagram 33] FIG. 33 diagrammatically illustrates the reaction scheme for preparing 20 kmPEG(SCAPDL1xSCACD47)-2(Val-Cit-PAB-DEA-SN38) (91) of Example 28. [Diagram 34] FIG. 34 diagrammatically illustrates the reaction scheme for preparing SCAPDL1xSCACD47-20kPEG-2(Val-Cit-PAB-DEA-SN38) (92) of Example 29. [Diagram 35] FIG. 35 diagrammatically illustrates the reaction scheme for preparing 30 kmPEG(SCAPDL1xSCACD47)-2(Val-Cit-PAB-DEA-Dxd) (93) of Example 30. [Diagram 36]FIG. 36 illustrates diagrammatically the reaction scheme for preparing 30 kmPEG(SCAc-MetxSCAc-Met)-2(Val-Cit-PAB-DEA-SN38) (94) of Example 31. [Figure 37] FIG. 37 illustrates the in vitro cytotoxicity of Compound 86 and Compound 88 of Example 32 against tumor cell lines. [Figure 38] FIG. 38 illustrates the in vitro cytotoxicity of compounds 87, 89, 90, 91, and 92 of Example 33 against tumor cell lines. [Figure 39] FIG. 39 illustrates the in vitro cytotoxicity of compound 93 of Example 34 against tumor cell lines. [Diagram 40] FIG. 40 illustrates the in vitro cytotoxicity of compound 94 of Example 35 against tumor cell lines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] In the present invention, PEGylated mono- or multispecific antibody-hydroxyl-containing drug conjugates are provided, in which the hydroxyl groups of the payload react to bind the payload to the antibody. Using the present invention, it is possible to produce ADCs with hydroxyl-containing cytotoxic payloads that are stable in the blood circulation until they reach the target, and can be internalized and released into the target cells, killing the target cells.

[0095] Furthermore, the present invention provides a novel antibody structural form of PEGylated single or bispecific single chain antibody hydroxyl-containing drug conjugates, which not only does not exhibit the toxicity to megakaryocytes or other normal cells mediated by the Fc portion of IgG-based antibodies, but also increases the therapeutic window, enhances the antitumor effect of the conjugates, and increases tumor penetration, internalization, and lysosomal trafficking. Thus, the present invention extends current ADC technology to allow a vast number of cytotoxic hydroxyl-containing compounds to be used as ADC payloads, improving current cancer therapies for the treatment of solid tumors.

[0096] I. Complex In one embodiment of the invention, a compound of formula (Ia): [ka] is provided.

[0097] In the compound, P can be a non-immunogenic polymer. T can be a multifunctional moiety, such as a trifunctional small molecule linker moiety, and can have at least one functional group capable of site-specific conjugation to an antibody or protein. A can be any monospecific or multispecific antibody or protein, such as a full-length antibody, single-chain antibody, nanobody, or any antigen-binding fragment thereof, or a combination thereof. D can be any hydroxy-containing cytotoxic small molecule or peptide (n=1-25), and each D can be the same or different.

[0098] In particular, an embodiment of the present invention is a conjugate of formula Ib or Ic: [ka] [In the formula, P can be a non-immunogenic polymer, such as PEG; M is H or C 1-50 an end-capping group selected from alkyl and aryl, where one or more carbons of said alkyl may be optionally replaced with a heteroatom; y may be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; T may be a moiety having two or more functional groups, where T and (L 1 ) a and T and (L 2 ) b The bonds with may be the same or different; L 1 and L 2 may each independently be a bifunctional linker; a and b can each be an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; B may be a branched linker, where each branch may include an extended spacer (optional), a trigger unit, one or more self-immolative spacers, or any combination thereof, where the trigger unit may be an amino acid sequence or a β-glucoronide or β-galactoside trigger moiety cleavable by an enzyme such as cathepsin B, plasmin, matrix metalloproteinase (MMP), β-glucuronidase, or β-galactosidase; a pH-sensitive linker that may trigger the release of the hydroxyl-containing drug D or its derivative under acidic pH conditions, or a disulfide bond linker that may trigger the release of the hydroxyl-containing drug D or its derivative by glutathione, a thioredoxin family member (WCGH / PCK), or a thioreductase; A can be any monospecific or multispecific antibody, or antibody fragment, single chain antibody, nanobody (single domain antibody), or antigen binding protein, including any antigen-binding fragment, which is monovalent or multivalent towards the antigen; D may be any hydroxyl-containing cytotoxic small molecule or peptide or derivative thereof, which can be released from B by either enzymatic cleavage and / or a self-immolative mechanism or pH-induced hydrolysis; each hydroxyl-containing D may be the same or different; n may be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25. to provide.

[0099] In some embodiments, each branch of B contains a trigger moiety (e.g., an amino acid sequence or a disulfide moiety or a β-glucoronide or β-galactoside) and is linked to the hydroxyl-containing drug D via one or more self-immolative spacers. Examples of self-immolative spacers include, but are not limited to, the following: [ka] [Wherein, X=O or NH or S, n=1 or 2, and R 1 , R 2 , R 3 , R 4 is H, C 1-10 Alkyl or -(CH2CH2O) m -CH3, where m is an integer from 1 to 10. Examples include:

[0100] In some embodiments, each branch of B may contain a disulfide bond linker that may be cleaved by enzymatic cleavage (e.g., by glutathione, a thioredoxin family member (WCGH / PCK), or a thioreductase) followed by release of the hydroxyl-containing drug D or a derivative thereof at the tumor site and / or inside the tumor cell by one or more self-immolative mechanisms.

[0101] In some embodiments, A is a single chain bispecific antibody capable of binding to two different antigens, such as PDL1 and CD47 (SCAPDL1xSCACD47).

[0102] In some embodiments, the amino acid sequence of SCAPDL1xSCACD47 is DIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKGGSGGSGGSGSG GQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSGCGGSSGG SDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTHHHHHH (SEQ ID NO: 1) It could be.

[0103] In some embodiments, A is a single chain bispecific antibody capable of binding to two different epitopes on two Her2 antigens, such as SCAHer2(1)xSCAHer2(2).

[0104] In some embodiments, the amino acid sequence of SCAHer2(1)xSCAHer2(2) is DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTGGSGGSGGSGGSGGEVQLV ESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGCGSGGSGGSGGSG GEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGSGGSGGSGGSGGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTHHHHHH (SEQ ID NO: 2) It could be.

[0105] In some embodiments, A is a single chain monospecific antibody capable of binding to two identical epitopes on two Her2 antigens, such as SCAHer2(1)xSCAHer2(1).

[0106] In some embodiments, the amino acid sequence of SCAHer2(1)xSCAHer2(1) is DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGGSGGSGGSGGSGGEVQLV ESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGCGSGGSGGSGGSG GDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGGSGGSGGSGGSGGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSHHHHHH (SEQ ID NO: 3) It could be.

[0107] In some embodiments, A is a single chain bispecific antibody capable of binding to two different antigens, such as Her2 and Her3 (SCAHer2xSCAHer3).

[0108] In some embodiments, the amino acid sequence of SCAHer2IVxSCAHer3 is DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGGSGGSGGSGGSGGEVQLV ESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGCGSGGSGGSGGSG GQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNTNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSSGGSGGSGGSGGSGGDIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKHHHHHH (SEQ ID NO: 4) It could be.

[0109] In some embodiments, A is a single chain bispecific antibody capable of binding to two different antigens, such as Met1 and Met2 (SCAc-Met1xSCAc-Met2).

[0110] In some embodiments, the amino acid sequence of SCAc-Met1xSCAc-Met2 is DIQMTQSPSSLSASVGDRVTITCSVSSSVSSIYLHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCIQYSGYPLTFGGGTKVEIKGGSGGSGGSGGSGGQVQLV QSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGRVNPNRGGTTYNQKFEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARTNWLDYWGQGTTVTVSGCGSGGSGGSGGSGGQVQ LVQSGAEVKKPGASVKVSCKASGYIFTAYTMHWVRQAPGQGLEWMGWIKPNNGLANYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSEITTEFDYWGQGTLVTVSSGGSGGSGGSGGSGGDIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFLHWYQQKPGQPPKLLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPLTFGGGTKVEIKRHHHHHH (SEQ ID NO: 5) It could be.

[0111] In some embodiments, the amino acid sequence of SCAc-Met(1)xSCAc-Met(2) is DIMMSQSPSSLTVSVGEKVTVSCKSSQSLLYTSSQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTITSVKADDLAVYYCQQYYAYPWTFGGGTKLEIKGGSGGSGGSGGSGGQVQL QQSGPELVRPGASVKMSCRASGYTFTSYWLHWVKQRPGQGLEWIGMIDPSNSDTRFNPNFKDKATLNVDRSSNTAYMLLSSLTSADSAVYYCATYGSYVSPLDYWGQGTSVTVSSGCGSGGSGGSGGQV QLVESGPGLVKPSQTLSLTCAVSGGSITTNYYYWSWIRQSPGKGLEWMGVIAYDGSTDYSPSLKSRTSISRDTSKNQFSLQLSSVTPEDTAVYYCARDVRVIATGWATANALDAWGQGTLVTVSSGGSGGSGGSGGSGGQSVLTQPPSVSGSPGKTVTISCAGTSSDVGYGNYVSWYQQLPGTAPKLLIFAVSYRASGIPDRFSGSKSGNTAFLTISGLQSEDEADYYCASYRSSNNAAVFGGGTHLTVLHHHHHH (sequence number 6). It could be.

[0112] In some embodiments, the hydroxyl-containing drug D can be released either at the tumor site or inside tumor cells by either enzyme-triggered or pH-induced hydrolysis followed by one or more self-immolative mechanisms.

[0113] In some embodiments, the hydroxyl-containing drug D may be selected from any DNA crosslinking agent, microtubule inhibitor, DNA alkylating agent, topoisomerase inhibitor, protein degrading drug, STING agonist, or combinations thereof.

[0114] In some embodiments, the hydroxyl-containing drug D is selected from the group consisting of Dxd, SN38, calicheamicin, pyrrolobenzodiazepines, sibiromycin, tomaymycin, duocarmycin, neothramycin, DC-81, psymberin, vinca alkaloids, laulimalide, taxanes, tubulysin, rhizoxin, discodermolide, taccalonolide, and the like. ide A or B or AF or AJ, taccalonolide AI-epoxide, epothilone A and B, paclitaxel, docetaxel, doxorubicin, camptothecin, tafuramycin A, PNU-159682, uncialamycin, β-amanitin, amatoxin, thailanstatin, or any hydroxyl-containing cytotoxic compound, or analogs / derivatives thereof, or combinations thereof.

[0115] In some embodiments, D is SN38 or Dxd (potent topoisomerase I inhibitors), or duocarmycin (a DNA alkylating agent), or an analog / derivative thereof, or a combination thereof.

[0116] In a further embodiment, the hydroxyl-containing drug D is linked to a double self-immolative spacer, such as ethylenediamine (EDA) or a derivative thereof, and 4-aminobenzyl alcohol (PAB), which is linked to a trigger moiety, such as valine-citrulline (Val-Cit-PAB-EDA-D).

[0117] In one embodiment of the invention, a method is provided for preparing a PEGylated hydroxyl-containing drug conjugate that can be site-specifically conjugated to a protein or antibody, such as an antibody fragment or a single chain mono- or multispecific antibody. In another embodiment of the invention, a method is provided for preparing a PEGylated single chain bispecific antibody hydroxyl-containing drug conjugate.

[0118] To synthesize a PEGylated single chain bispecific antibody hydroxyl-containing drug conjugate, the coding sequence of a mono- to pentavalent monospecific single chain antibody or single chain bispecific antibody or a vector carrying the coding sequence can be synthesized and introduced, for example, into a CHO expression system. The protein can be expressed and purified as previously described (WO2018075308).

[0119] For the synthesis of PEGylated hydroxyl-containing drug conjugates whose side chains have site-specific conjugation functional groups, the terminal functional groups of PEG, such as hydroxyl or carboxyl groups and the like, can be activated and conjugated with trifunctional small molecule moieties, such as Boc- or Fmoc-protected lysine, to form terminally branched heterobifunctional PEGs, followed by removal of the protecting groups. The PEGylated compound after deprotection can be coupled with a small molecule linker having a site-specific conjugation functional group, such as maleimide or DBCO, to form PEG-Lys(Mal)-OH or PEG-Lys(DBCO)-OH. The PEG-Lys(Mal)-OH or PEG-Lys(DBCO)-OH can then be coupled with a branching moiety, each of which is linked to a hydroxyl-containing drug D, e.g., SN38, via a trigger unit and a double self-immolative spacer, to form PEG-lys(Mal)-B(Val-Cit-PAB-EDA-SN38). n or PEG-lys(DBCO)-B(Val-Cit-PAB-EDA-SN38) n where n is an integer from 1 to 20, e.g., 4. The final step in the synthesis is site-specific conjugation of the PEGylated hydroxyl-containing drug conjugate to a thiol- or azide-labeled single-chain monospecific or bispecific antibody to form compounds of formula Ia and Ib. Alternatively, the PEGylated hydroxyl-containing drug conjugate Mal-PEG-B-(Val-Cit-PAB-EDA-SN38) n or DBCO-PEG-B-(Val-Cit-PAB-EDA-SN38) n(wherein n is an integer, e.g., 4) can be synthesized from commercially available heterobifunctional PEGs using a similar procedure, and the PEGylated hydroxyl-containing drug conjugates are site-specifically conjugated to thiol- or azide-labeled single chain bispecific antibodies to form compounds of formula Ic.

[0120] II. Polyethylene glycol (PEG) moiety In one embodiment of the invention, the linear PEG is [ka] where n can be an integer from 1 to about 2300, preferably to provide a polymer with a total molecular weight (if desired) of 3000 to 100,000 Daltons or more. M can be H, methyl, or other low molecular weight alkyl. Non-limiting examples of M include H, methyl, ethyl, isopropyl, propyl, butyl, or F1(CH2)qCH2, where F and F1 can independently be terminal functional groups such as hydroxyl, carboxyl, thiol, halide, amino groups, and the like, which can be functionalized, activated, and / or conjugated to small molecule spacers or linkers. Q and m can be any integer from 0 to 10.

[0121] In another embodiment of the invention, the method can also be carried out using alternative branched PEGs, such as those described below: [ka] where PEG is polyethylene glycol. M can be an integer from 2 to 10, preferably to provide a branched PEG with a total molecular weight (if desired) of 3,000 to 100,000 daltons or more. M can be methyl or other low molecular weight alkyl. L can be a functional linking moiety to which two or more PEGs are attached. Non-limiting examples of such linking moieties are any amino acid such as glycine, alanine, lysine, or 1,3-diamino-2-propanol, triethanolamine, any 5- or 6-membered aromatic or aliphatic ring to which two or more functional groups are attached. S is any non-cleavable spacer. F can be a terminal functional group such as hydroxyl, carboxyl, thiol, amino group, etc. i is 0 or 1. When i is equal to 0, the above formula becomes [ka] wherein each variable PEG, m, M, or L has the same definition as above.

[0122] The method of the present invention comprises the steps of: It may also be practiced with alternative polymers such as dextrans, carbohydrate polymers, polyalkylene oxides, polyvinyl alcohol, or other similar non-immunogenic polymers whose end groups can be functionalized or activated. The foregoing list is merely illustrative and is not intended to limit the types of non-antigenic polymers suitable for use herein.

[0123] III. Trifunctional Linker T T is P, (L 1 ) a , and (L 2 ) brepresents a trifunctional linker that bonds to T. T can be derived from a molecule having any combination of three functional groups, non-limiting examples of which include hydroxyl, amino, hydrazinyl, azide, alkene, alkyne, carboxyl (aldehyde, ketone, ester, carboxylic acid, anhydride, acid halide), thiol, disulfide, nitrile, epoxide, imine, nitro, and halide. The functional groups of the trifunctional linker can be the same or different. In some embodiments, one or two of the functional groups may be protected to achieve selective conjugation with other reaction partners. A variety of protecting groups are known in the art, including those shown in Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York). Functional groups may also be converted to other groups before or after the reaction of T with another reaction partner. For example, a hydroxyl group may be converted to a mesylate or tosylate group. A halide may be replaced with an azide group. The acid functionality of T may be converted to an alkyne functionality by coupling with an amino group containing a terminal alkyne.

[0124] In exemplary embodiments, T is derived from 1,3-diamino-2-propanol, triethanolamine, lysine, aspartic acid, glutamic acid, serine, or tyrosine. One or more of the functional groups on these molecules may be protected for selective reaction. In some embodiments, T is derived from a Boc-protected lysine.

[0125] IV. Bifunctional Linker L 1 and L 2 Both linkers L 1 and L 2 is the following: -(CH2) a XY(CH2) b -, -X(CH2) a O(CH2CH2O) c (CH2)b Y-, -(CH2) a -heterocyclyl-, -(CH2) a X-, -X(CH2) a Y-, -W1-(CH2) a C(O)NR1(CH2) b O(CH2CH2O) c (CH2) d X-, -X(CH2) a O(CH2CH2O) b (CH2) c W2C(O)(CH2) d Y-, and -W3-(CH2) a C(O)NR1(CH2) b O(CH2CH2O) c (CH2) d W2C(O)(CH2) e X- wherein a, b, c, d, and e are each independently an integer selected from 0 to 25, e.g., 0 to 20, 0 to 15, 0 to 10, 0 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25; X and Y are each independently selected from C(=O), NR2, S, O, CR3R 4、 or nothing; R1, R2, R3, and R4 are each independently selected from hydrogen, C 1-10 Alkyl, or (CH2) 1-10represents C(=O); W1 and / or W3 are derived from a maleimide-based moiety and W2 represents a triazolyl- or tetrazolyl-containing group; the heterocyclyl group is selected from a maleimide-derived moiety or a tetrazolyl- or triazolyl-based moiety. Non-limiting examples of maleimide-based moieties include succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-aminocaproate) (LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl)isocyanate (PMPI). Alternatively, the heterocyclyl linking group of the linker may be tetrazolyl or triazolyl formed by conjugation of different linker moieties such as DBCO and azide.

[0126] In some exemplary embodiments, (L 1 ) a and (L 2 ) b is the following: [ka] [wherein n and m are integers and independently selected from 0 to 20].

[0127] In some other non-limiting exemplary embodiments, each linker unit may also be derived from a haloacetyl-based moiety selected from N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), or N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[0128] V. Branched Linker B The branched linker B may comprise a branching unit, an extension spacer (optional), a trigger unit, one or more self-immolative spacers, or any combination thereof.

[0129] In some embodiments, the branching unit comprises: [ka] 1. X, Y, Z, or W = NR 1 , N.R. 2 , C(=O), O, N, or nothing, where R 1 and R 2 are independently hydrogen or C 1-10 Represents an alkyl group 2. a, b, and c are integers between 0 and 10 The present invention includes structures that may be independently selected from:

[0130] In another embodiment, the branching unit comprises: [ka] 1.X, Y, Z, U, V, W=C(=O), NR 1 , N.R. 2 , O, N, or nothing, where R 1 and R 2 are independently hydrogen or C 1-10 Represents an alkyl group 2. a, b, c, d, e = 0 to 10 The present invention includes structures that may be independently selected from:

[0131] In some embodiments, the extended spacer in each branch is -X(CH2) a O(CH2CH2O) b (CH2) c Y-, -X(CH2) a Y-, or any combination thereof, wherein a, b, and c are each an integer selected from 0 to 25, inclusive of all subunits, and X and Y are each an integer selected from NR 1 , N.R. 2 , C(O), O, or nothing, where R 1 and R 2 are independently hydrogen or C 1-10 Represents an alkyl group.

[0132] In some embodiments, a branch unit (e.g., having two branches) with or without an extended spacer may be joined by two or more branch units (e.g., having two branches) to form a branch unit with four branches.

[0133] In other embodiments, the trigger unit comprises any amino acid sequence or any carbohydrate moiety or disulfide bond or PH sensitive bond or any cleavable bond that can be enzymatically or chemically cleaved.

[0134] In some embodiments, the self-immolative spacer is: [ka] [wherein n is 1 or 2; R 1 , R 2 , R 3 and R 4 are independently hydrogen, C 1-10 Alkyl, or -(CH2CH2O) m represents CH3, where m=1-10; X and Y can be NH or O or S. The structure may be selected from:

[0135] In some embodiments, two self-immolative spacers can be linked to each other. For example, [ka]

[0136] In some embodiments, the branched linker B is: [ka] [ka] [ka] [In the formula, a, b, c, d, e, and f are each independently an integer selected from 1 to 25; n is an integer selected from 1 to 10; (A) n is a trigger unit of an amino acid sequence, each A is an independent amino acid, and n is any integer from 1 to 25; PAB is 4-aminobenzyl alcohol; EDA, HNR 1 CH2CH2NHR 2 or HNR 1 CH2CH2CH2NHR 2 and R 1 and R 2 are independently hydrogen, C 1-10 Alkyl group, or -(CH2CH2O) m CH3, where m is any integer from 1 to 10; Ex: -NR 1 (CH2) a O(CH2CH2O) b (CH2) c C(O)-, -C(O)(CH2) a NR 1 -, -NR 1 (CH2) a O(CH2CH2O) b (CH2) c NR 2 -, -NR 1 (CH2) a NR 2 -, -NR 1 (CH2) a O(CH2CH2O) b (CH2) c O-, -O(CH2) a NR 1 -, -C(O)(CH2) a O-, -O(CH2) a O(CH2CH2O) b (CH2) c C(O)-, -C(O)(CH2) a O(CH2CH2O) b (CH2) c C(O)-, -C(O)(CH2) a C(O)-, Or nothing (wherein a, b, and c are each an integer selected from 0 to 25, including all subunits; R 1 and R 2 are independently hydrogen or C 1-10 represents an alkyl group) and may be selected from:

[0137] In some other embodiments, the amino acid sequence trigger unit can be Val-Cit, Val-Ala, Val-Lys, Phe-Lys, Phe-Cit, Phe-Arg, Phe-Ala, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, D-Phe-Phe-Lys, Phe-Phe-Lys, Gly-Phe-Lys, Gly-Phe-Leu-Gly, or Ala-Leu-Ala-Leu, Gly-Gly-Phe-Gly.

[0138] For preferred embodiments, the amino acid sequence may be Val-Cit, Phe-Lys, or Val-Lys.

[0139] For some exemplary embodiments, the branched linker B is: [ka] [ka] [wherein n=1 or 2; b is an integer selected from 1 to 10; R 1 and R 2 are each independently H, a low molecular weight alkyl, or a low molecular weight PEG [-(CH2CH2O) m -CH3, m = 1 to 10] may be selected from:

[0140] VI. Linking group Different moieties of the conjugates of the invention can be linked via a variety of chemical bonds. Examples include, but are not limited to, amide, ester, disulfide, ether, amino, carbamate, hydrazine, thioether, and carbonate. For example, the terminal hydroxyl group of a PEG moiety (P) may be activated and subsequently coupled to a lysine (T) to provide a desired point of attachment between P and T of formula Ia or Ib. T and (L 1 ) a Or T and (L 2 ) b or (L 2 )b The linking group between and B is a linker (L 2 ) b and the carboxyl group of lysine (T) or (L 1 ) a The carboxyl group of T and the amino group of (L 2 ) b Depending on the desired properties of the conjugate, a suitable linking group may be attached to the antibody portion (A) and the adjacent linker L. 1 or between any two amino acids, or between an amino acid and paraaminobenzyl alcohol, or between paraaminobenzyl alcohol and N,N'-dimethylethylenediamine or a derivative thereof.

[0141] In some embodiments, the linking group between different parts of the conjugate may result from the coupling of a pair of functional groups that have a unique chemical affinity or selectivity for each other. These types of coupling or ring formation allow for site-specific conjugation for the introduction of protein or antibody moieties to PEGylated moieties. Non-limiting examples of these functional groups that cause site-specific conjugation include thiol, maleimide, 2'-pyridyldithio variants, aromatic or vinyl sulfones, acrylates, bromo or iodoacetamides, azides, alkynes, dibenzocyclooctyl (DBCO), carbonyl, 2-amino-benzaldehyde or 2-amino-acetophenone groups, hydrazides, oximes, potassium acyltrifluoroborates, O-carbamoylhydroxylamines, transcyclooctene, tetrazines, triarylphosphines, boronic acids, and alkynes.

[0142] VII. Cytotoxic Compound D In some embodiments, D can be any hydroxyl-containing compound, including but not limited to, vinca alkaloids, laulimalide, colchicine, tubulysin, cryptophycin, hemiasterin, cemadotin, rhizoxin, discodermolide, taccalonolide A or B or AF or AJ, taccalonolide AI-epoxide, CA-4, epothilones A and B, taxanes, paclitaxel, docetaxel, epothilones, iSGD-1882, centanamycin, amycin, PNU-159682, uncialamycin, indolinobenzodiazepine dimer, β-amanitin, amatoxin, thailanstatin, anthracycline, daunomycin, larotaxel, tesetaxel, ortataxel, CC-1065, Dxd, SN38, topotecan, CPT-11, camptothecin, rubitecan, bryostatin, kallistatin, biceresin, duocarmycin, eleutherobin, pancratic Statins, sarcodictyin, spongiostatin, estramustine, prednimustine, chlorozotocin, ranimustine, calicheamicin, dynemicin, esperamicin, neocarzinostatin chromophore, aclacinomycin, azithromycin, bleomycin, caminomycin, carzinophilin, chromomycin, daunorubicin, detorubicin, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin omycin), mycophenolic acid, nogalamycin, peplomycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, fludarabine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine (cytosine arabinoside, ara-C), gemcitabine, capecitabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calsterone, epithiostanol,Trilostane, elliptinium acetate, maytansinoids, ansamitocin, mitoxantrone, mopidamol, pentostatin, pirarubicin, etoposide, podophyllotoxin, rhizoxin, tenuazonic acid, T-2 mycotoxin, verracurin, roridin A, anguidine, vindesine, mannomustine, mitobronitol, mitolactol, vinblastine, mitoxantrone, vincristine, vinorelbine, teniposide, xeloda, raloxifene, 4-hydroxytamoxifen, estradiol, trioxyphene, keoxifene, LY117018, onapristone, bicalutamide, leuprolide, goserelin, or a pharma- ceutically acceptable salt, acid, or derivative thereof, or a combination thereof.

[0143] VIII. Antibodies and Targets A large number of therapeutic antibodies against cell surface molecules and / or their ligands are known. These antibodies can be used for the selection and construction of tailor-made specific recognition binding moieties in mono- or multispecific PEGylated antibody hydroxyl-containing drug conjugates. Examples include Monjuvi / tafasitamab (CD19), Rituxan / MabThera / rituximab (CD20), H7 / ocrelizumab (CD20), Zevalin / ibritumomab (CD20), Arzerra / ofatumumab (CD20), HLL2 / epratuzumab, inotuzumab (CD22), Zenapax / daclizumab, Simulect / basiliximab (CD25), Herceptin / trastuzumab, pertuzumab (H er2 / ERBB2), Mylotarg / gemtuzumab (CD33), Raptiva / efalizumab (Cd11a), Erbitux / cetuximab (EGFR, epidermal growth factor receptor), IMC-1121B (VEGF receptor 2), Tysabri / natalizumab (a4β1 and the α4 subunit of α4β7 integrin), ReoPro / abciximab (gpIIb-gpIIa and αvβ3-integrin), Orthoclone OKT3 / muromonab-CD3 (CD3), Benlysta / belimumab (BAFF), Tolerx / otelixizumab (CD3), Soliris / eculizumab (C5 complement protein), Actemra / tocilizumab (IL-6R), Panorex / edrecolomab (EpCAM, epithelial cell adhesion molecule), CEA-CAM5 / labetuzumab (CD66 / CEA, carcinoembryonic antigen), CT-11 (PD-1, programmed death 1 These include T cell inhibitory receptor, CD279), H224G11 (c-Met receptor), SAR3419 (CD19), IMC-A12 / cizutumumab (IGF-1R, insulin-like growth factor 1 receptor), MEDI-575 (PDGF-R, platelet-derived growth factor receptor), CP-675, 206 / tremelimumab (cytotoxic T lymphocyte antigen 4), RO5323441 (placental growth factor or PGF), HGS1012 / mapatuzumab (TRAIL-R1), SGN-70 (CD70), Vedotin (SGN-35) / brentuximab (CD30), and ARH460-16-2 (CD44).

[0144] Numerous cell surface markers and their ligands are known. For example, cancer cells may express, but are not limited to, carbonic anhydrase IX, alpha-fetoprotein, alpha-actinin-4, A3 (antigen specific for A33 antibody), ART-4, B7-1, B7-H1, Ba-733, BAGE, BrE3 antigen, CA125, CAMEL, CAP-1, CASP-8 / m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CDS, CD8, CD1-1A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, C D133, CD137, CD138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXC L12, HIF-1-α, colon-specific antigen p (CSAp), CEA (CEACAM5), CEACAM6, c-met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, GROB, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2 or 1a, IGF-1R, IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL- 18, IL-25, insulin-like growth factor 1 (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, M ART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1 / 2, M It has been reported that pancreatic cancers express at least one of the following: UM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, 5100, survivin, survivin 2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, bcl-6, Kras, c-Met, oncogene markers, and cell surface markers and / or ligands for oncogene products (Sensi, M. et al., Clin. Cancer Res., 2006, 12, 5023-5032;Parmiani, G. et al., J. Immunol., 2007, 178, 1975-1979;Castelli, C. et al., Cancer Immunol. Immunother., 2005, 54, 187-207). Thus, antibodies that recognize such specific cell surface receptors or their ligands can be used for specific and selective recognition binding moieties in the multispecific ADCs of the invention, which can target and bind multiple cell surface markers or ligands associated with diseases. Antibodies against the above antigens can be used as binding domains or moieties to prepare the mono- or multispecific PEGylated antibody hydroxyl-containing drug conjugates of the invention.

[0145] In some embodiments, mono- or multispecific PEGylated antibody hydroxyl-containing drug conjugates are used to target tumor-associated antigens (TAA) for the treatment of cancer / tumors, as reported in Herberman, "Immunodiagnosis of Cancer", Fleisher ed., "The Clinical Biochemistry of Cancer", page 347 (American Association of Clinical Chemists, 1979), as well as US4150149; US4361544; US4444744. Reports on tumor-associated antigens can also be found in Mizukami, Y. et al. Nature Med., 2005, 11, 992-997; Hatfield, KJ et al., Curr. Cancer Drug Tar., 2005, 5, 229-248; Vallbohmer, D. et al., J. Clin. Oncol. 2005, 23, 3536-3544; and Ren, Y. et al., Ann. Surg. 2005, 242, 55-63, each of which is incorporated herein by reference with respect to the identified TAAs. If the disease involves lymphoma, leukemia, or autoimmune disease, the target antigens may be CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD47, CD54, CD67, CD74, CD79a, CD80, CD126, CD138, CD154, CD273 (PD-L2), CD274 ( PD-L1), CXCR4, B7, MUC1 or 1a, HM1.24, HLA-DR, tenascin, VEGF, P1GF, ED-B fibronectin, oncogenes, oncogene products (e.g., c-Met or PLAGL2), CD66a-d, necrosis antigens, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4), and TRAIL-R2 (DR5).

[0146] A variety of bispecific PEGylated antibody hydroxyl-containing drug conjugates can be prepared against two different targets. Examples of antigen pairs include CD19 / CD3, BCMA / CD3, combinations of different antigens of the HER family (EGFR, HER2, HER3), IL17RA / IL7R, IL-6 / IL-23, IL-1-β / IL-8, IL-6 or IL-6R / IL-21 or IL-21R, ANG2 / VEGF, VEGF / PDGFR-β, VEGF combinations of antigens selected from the group consisting of FLT3, c-FMS / CSF1R, RET, c-Met, EGFR, Her2 / neu, HER3, HER4, IGFR, PDGFR, c-KIT, BCR, integrins, and MMPs having water-soluble ligands selected from the group consisting of VEGF, EGF, PIGF, PDGF, HGF, and angiopoietins, ERBB-3cC-Met, ERBB-2 / C-MET, EGF receptor 1 / CD3, EGFR / HER3, PSCA / CD3, C-MET / CD3, endosialin / CD3, EPCAM / CD3, IGF-1R / CD3, FAP alpha / CD3, EGFR / IGF-1R, IL 17A / F, EGF receptor 1 / CD3, and CD19 / CD16. Further examples of bispecific ADCs may have (i) a first specificity for a carbohydrate epitope of an antigen selected from the group consisting of Lewis x, Lewis b, and Lewis y structures, Globo H structures, KH1, Tn antigen, TF antigen, and carbohydrate structures of mucins, glycolipids and glycosphingolipids such as CD44, Gg3, Gb3, GD3, GD2, Gb5, Gm1, Gm2, and sialyltetraosylceramide, and (ii) a second specificity for an ErbB receptor tyrosine kinase selected from the group consisting of EGFR, HER2, HER3, and HER4. GD2 in combination with the second antigen binding site is associated with an immune cell selected from the group consisting of T lymphocytes, NK cells, B lymphocytes, dendritic cells, monocytes, macrophages, neutrophils, mesenchymal stem cells, neural stem cells.

[0147] A monospecific or bispecific antibody can be conjugated to another monospecific or bispecific antibody using the methods disclosed herein to prepare a multispecific PEGylated ADC, which may have additive / synergistic effects compared to a single-targeted ADC.

[0148] In some embodiments, multispecific PEGylated ADCs of the invention are prepared using pairs of antibodies that specifically interact with and exhibit measurable affinity for the following pairs of targets: [Table 1] [Table 2]

[0149] In some embodiments, the PEGylated BsADC comprises a bispecific single chain antibody, in which the two binding domains of the bispecific single chain antibody are linked via a peptide linker. In some embodiments, the peptide linker comprises a moiety such as a cysteine ​​or a non-natural amino acid residue that can be used to site-specifically conjugate the antibody to a non-immunogenic polymeric hydroxyl-containing drug conjugate (e.g., a PEGylated hydroxyl-containing drug conjugate). In some other embodiments, one or both of the two binding domains of the bispecific single chain antibody comprise a cysteine ​​or a non-natural amino acid residue that can be used to site-specifically conjugate the antibody to a non-immunogenic polymeric hydroxyl-containing drug conjugate (e.g., a PEGylated hydroxyl-containing drug conjugate).

[0150] In a preferred embodiment, the PEGylated bispecific hydroxyl-containing drug conjugate is a conjugate of two antibodies or antigen-binding fragments thereof (such as Fabs, scFvs, nanobodies, and the like) that specifically interact with two different epitopes of Her2 and exhibit measurable affinity.

[0151] IX.Synthesis Once the desired size and shape of the PEG is selected, the terminal functional groups of the PEG, such as hydroxyl, carboxyl, and the like, can be converted to terminally branched heterobifunctional groups using any art-recognized method (WO2018075308). In general terms, terminally branched heterobifunctional PEGs can be prepared by activating the terminal hydroxyl or carboxyl groups of PEG with N-hydroxysuccinimide in the presence of a base, such as 4-dimethylaminopyridine (DMAP), pyridine, and the like, in the case of terminal hydroxyl groups, using reagents such as di(N-succinimidyl)carbonate (DSC), triphosgene, and the like, and in the case of terminal carboxyl groups, using coupling agents such as N,N-diisopropylcarbodiimide (DIPC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and the like, to form an activated PEG.

[0152] The activated PEG can then be reacted with a multifunctional small molecule such as the lysine derivative H-Lys(Boc)-OH in the presence of a base such as diisopropylamine (DIPEA) to form a terminally branched heterobifunctional PEG with a free carboxyl group and a Boc-protected amino group PEG-Lys(Boc)-COOH. As will be appreciated by those skilled in the art, other terminal functional groups of PEG such as halide, amino, thiol groups, and the like, as well as other trifunctional small molecules containing any combination of three functional groups from the list of -NH2, -NHNH2, -COOH, -OH, -C(O)X (X=halide), -N=C=O, -SH, anhydride, halide, maleimide, C=C, C≡C, and the like, or protected forms thereof, can be used as alternatives for the same purpose, if desired.

[0153] Removal of the Boc with TFA followed by reaction with a maleimide-labeled spacer such as NHS-PEG2-maleimide forms PEG-Lys(Mal)-COOH.

[0154] Separately, a cytotoxic drug (e.g., SN38) linked to a trigger unit (e.g., val-cit) via two self-immolative spacers (e.g., PAB-EDA) is coupled to a branching unit with an extension by a coupling agent such as EDCI / HOBt to form a compound BD: e.g. [ka] Create a.

[0155] The target product can be formed by coupling PEG-Lys(Mal)-COOH with BD using a coupling agent such as DCC to form the PEGylated drug conjugate PEG-Lys(Mal)-4(Val-Cit-PAB-EDA-SN38).

[0156] Monospecific antibodies or bispecific antibodies such as SCAHer2IIxSCAHer2IV that are bivalent for an antigen can be prepared by genetic engineering of an expression system. For example, DNA encoding a bispecific scFv can be synthesized and introduced into an expression system (e.g., CHO cells). The protein of interest is then expressed and purified by chromatographic techniques.

[0157] To prepare PEGylated single chain antibody hydroxyl-containing drug conjugates, PEGylated hydroxyl-containing drug conjugates bearing the functional groups maleimide or DBCO can be site-specifically reacted with free thiol or azide functional groups of genetically inserted or protein-derivatized bifunctional antibodies [e.g., SCAPDL1xSCACD47 or SCAHer2(1)xSCAHer2(2) or SCAcMet(1)xcMet(2)] to form PEG-Lys(SCAPDL1xSCACD47)-4(Val-Cit-PAB-EDA-SN38) or PEG-Lys(SCAHer2(1)xSCAHer2(2))-4(Val-Cit-PAB-EDA-SN38) or PEG-Lys(SCAcMet(1)xSCAcMet(2))-4(Val-Cit-PAB-EDA-SN38).

[0158] PEGylated multispecific hydroxyl-containing antibody drug conjugates can be similarly prepared using multispecific antibodies instead of mono- or bispecific antibodies.

[0159] In addition to the thiol / maleimide or DBCO / azide site-specific conjugation group pairs enumerated in the present invention, as those skilled in the art will appreciate, other known site-specific conjugation group pairs, such as transcyclooctene / tetrazine pairs; carbonyl / hydrazide; carbonyl / oxime; Suzuki-Miyaura cross-coupling agent pairs; Sonogashira cross-coupling agent pairs; Staudinger ligation reagent pairs; Knoevenagel-Intra Michael addition reagent pairs; active amine / acrylate pairs, and the like, can also be similarly designed and used as alternatives for the same purpose, if desired. The above list of site-specific conjugation group pairs is merely illustrative and is not intended to limit the types of site-specific conjugation group pairs suitable for use herein.

[0160] X. Composition The present invention also provides compositions, such as pharmaceutical compositions, that contain the compounds of the present invention, which are formulated with pharma- ceutically acceptable carriers.For example, the pharmaceutical compositions of the present invention can contain compounds that bind to two different epitopes of Her2 receptor (e.g., PEGylated bispecific hydroxyl-containing antibody-drug conjugates).

[0161] The therapeutic formulations of the present invention can be prepared by mixing the mono- or polyspecific molecular drug conjugates having the desired purity with any physiologically acceptable carrier, excipient, or stabilizer, and can be in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are non-toxic at the dosages and concentrations used by the administering subject, and include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. sol, etc.); low molecular weight proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as Tween, Pluronics, or PEG.

[0162] The formulation may also contain one or more active compounds, preferably compounds with complementary activities and that do not adversely affect each other, as required for the particular indication being treated. For example, the formulation may further contain another antibody or multispecific antibody, a cytotoxic agent, a chemotherapeutic agent, or an ADC. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0163] The pharmaceutical compositions of the invention can be administered in combination therapy, i.e., in combination with other agents. Examples of therapeutic agents that can be used in combination therapy are described in more detail below.

[0164] The preparations used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane. Sterile injection can be prepared by mixing the required amount of active compound with one or more combinations of the above-listed components in a suitable solvent, and then optionally by sterile microfiltration. In general, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and the other required components listed above. In the case of sterile powder for preparing sterile injection, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which produces a powder of active ingredient and any other desired ingredients from its previously sterile-filtered solution.

[0165] XI. Dose The amount of active ingredient that can be combined with carrier materials to create a single dosage form varies depending on the subject being treated and the specific method of administration. The amount of active ingredient that can be combined with carrier materials to create a single dosage form is generally the amount of the composition that produces a therapeutic effect. Generally, out of 100 percent, this amount ranges from about 0.01% to about 99% of the active ingredient combined with a pharma- ceutically acceptable carrier, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 50%.

[0166] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, or multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased according to the exigencies of the therapeutic situation. For ease of administration and uniformity of dose, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as unitary doses for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specifications of the dosage unit form of the present invention are determined by and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the technology of combining such active compounds for the treatment of individual sensitivities.

[0167] For administration of the PEGylated mono- or multispecific hydroxyl-containing drug conjugates of the invention, the dosage is about 0.0001-100 mg / kg of host body weight, more typically 0.01-50 mg / kg of host body weight. For example, dosages can be 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, or 15 mg / kg of body weight, or within the range of 1-15 mg / kg. Exemplary treatment regimens entail administration daily, once every other day, twice weekly, once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every three months, or once every six months. Preferred dosing regimens for the PEGylated mono- or multispecific hydroxyl-containing drug conjugates of the invention include intravenous administration of 1 mg / kg body weight or 3 mg / kg body weight, where the mono- or multispecific drug conjugates are administered using one of the following dosing schedules: (i) six times every three weeks, then once a month; (ii) once every three weeks; (iii) once at 3 mg / kg body weight, then 1 mg / kg body weight every three weeks.

[0168] Dosage and frequency vary according to the half-life of the mono- or multispecific drug conjugate in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency can vary according to whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are administered relatively infrequently for a long period of time. Some patients continue to receive treatment until they die. In therapeutic applications, sometimes it is necessary to use relatively high doses at relatively short intervals until the progression of the disease is reduced or terminated, preferably until the patient shows partial or complete improvement of disease symptoms. The patient can then undergo a prophylactic regimen.

[0169] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied for a particular patient, composition, and method of administration to obtain an amount of the active ingredient effective to achieve a desired therapeutic response without toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, such as the activity of the particular composition of the present invention employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, medical condition, overall health, and medical history of the patient being treated, as well as other factors well known in the medical arts.

[0170] A "therapeutically effective dose" of the PEGylated mono- or multispecific hydroxyl-containing drug conjugates of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of injury or disability due to disease affliction. For example, for the treatment of tumors, a "therapeutically effective dose" preferably inhibits cell proliferation or tumor growth or metastasis by at least about 10%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, compared to a subject not receiving the treatment. The ability of an agent or compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of the composition can be evaluated by testing the ability of the compound to inhibit in vitro by assays known to those of skill in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size, reduce metastasis, or improve symptoms in a subject. One of skill in the art can determine such amounts based on factors such as the subject's physical type, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0171] XII. Administration The compositions of the present invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As one of skill in the art will appreciate, the route and / or method of administration will vary depending on the desired outcome. Preferred routes of administration of the PEGylated single or multispecific hydroxyl-containing drug conjugates of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as administration by injection or infusion. As used herein, the phrase "parenteral administration" refers to a method of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the mono- or multispecific drug conjugates of the invention may be administered via non-parenteral routes such as topical, epidermal, or mucosal routes of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical.

[0172] Active compound can be prepared with carriers that prevent the rapid release of compound, such as release controlled formulations, such as implants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Many methods for preparing such formulations are patented or known to those skilled in the art.See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0173] The therapeutic composition can be administered using medical devices known in the art.For example, the therapeutic composition of the present invention can be administered using needleless hypodermic injectors such as the devices disclosed in US5399163, US5383851, US5312335, US5064413, US4941880, US4790824 and US4596556.Examples of well-known implants and components that can be used in the present invention include those described in US4487603, US4486194, US4447233, US4447224, US4439196 and US4475196.These patents are incorporated herein by reference.Many other such implants, delivery systems and components are known to those skilled in the art.

[0174] XIII. Treatment The PEGylated mono- or multispecific hydroxyl-containing drug conjugates disclosed herein may be used in the preparation of drugs for the treatment of oncological, cardiovascular, infectious, inflammatory, autoimmune, metabolic (e.g., endocrine), or neurological (e.g., neurodegenerative) diseases. Illustrative, non-limiting examples of these diseases include Alzheimer's disease, non-Hodgkin's lymphoma, B-cell acute and chronic lymphocytic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemia, T-cell lymphoma and leukemia, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, cancer (e.g., oral cancer, gastrointestinal cancer, colon cancer, stomach cancer, airway cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, endometrial cancer, cervical cancer, bladder cancer, pancreatic cancer, bone cancer, liver cancer, gallbladder cancer, kidney cancer, skin cancer, and testicular cancer), melanoma, sarcoma, glioma, and skin cancer, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus Nephritis, rheumatic fever, polyglandular syndrome, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, poststreptococcal nephritis, erythema nodosum, Takayasu arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, obstructive Thromboangiitis, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis / dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, or fibrosing alveolitis.

[0175] In one aspect, the invention relates to the treatment of a subject in vivo with the above-described PEGylated mono- or multispecific hydroxyl-containing drug conjugates, whereby the growth and / or metastasis of cancerous tumors is inhibited. In one embodiment, the invention provides a method of inhibiting the growth and / or limiting the metastatic spread of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of a mono- or multispecific molecular drug conjugate.

[0176] Non-limiting examples of preferred cancers to be treated include chronic or acute leukemias such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphocytic lymphoma, breast cancer, ovarian cancer, melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate cancer), colon cancer, and lung cancer (e.g., non-small cell lung cancer). Additionally, the present invention encompasses refractory or recurrent malignant tumors whose growth may be inhibited using the antibodies of the present invention. Other examples of cancers that may be treated using the methods of the present invention include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers such as asbestos-induced cancer, and combinations of the above cancers.

[0177] The term "subject" as used herein is intended to include humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, with mammals being preferred, such as non-human primates, sheep, dogs, cats, cows, and horses. Preferred subjects include human patients who require enhanced immune response. The method is particularly suitable for human patients who have a disease that can be treated by enhancing immune response.

[0178] The above treatment may be combined with standard cancer treatment. For example, it may be effectively combined with chemotherapy regimen. In these cases, it may be possible to reduce the dose of the chemotherapy agent administered (Mokyr, M. et al. Cancer Res., 1998, 58, 5301-5304).

[0179] Other antibodies that may be used to activate host immune responses may be used with the PEGylated mono- or multispecific hydroxyl-containing drug conjugates of the present invention. These include molecules that target the surface of dendritic cells and activate DC function and antigen presentation. For example, anti-CD40 antibodies can effectively replace T cell helper activity (Ridge, J. et al., Nature, 1998, 393, 474-478) and may be used in combination with the mono- or multispecific drug conjugates of the present invention (Ito, N. et al., Immunobiology, 2000, 201, 527-540). Similarly, antibodies targeting T cell costimulatory molecules such as CTLA-4 (US5811097), CD28 (Haan, J. et al., Immunol. Lett., 2014, 162, 103-112), OX-40 (Weinberg, A. et al., J. Immunol., 2000, 164, 2160-2169), 4-1BB (Melero, I. et al., Nature Med., 1997, 3, 682-685), and ICOS (Hutloff, A. et al., Nature, 1999, 397, 262-266) or antibodies targeting PD-1 (US8008449) and PD-L1 (US7943743; US8168179) may provide increased levels of T cell activation. In another example, the mono- or multispecific drug conjugates of the invention may be used in combination with anti-tumor antibodies such as Rituxan (rituximab), Herceptin (trastuzumab), Bexxar (tositumomab), Zevalin (ibritumomab), Campath (alemtuzumab), Lymphocide (epratuzumab), Avastin (bevacizumab), and Tarceva (erlotinib), and the like.

[0180] Definition of Terms The term "alkyl" as used herein refers to a hydrocarbon chain, generally about 1 to 25 atoms in length. Such hydrocarbon chains are preferably, but not necessarily, saturated and may be branched or straight-chained, although straight-chained is generally preferred. 1-10 The term alkyl includes alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbons. 1-25 Alkyl includes all alkyls having 1 to 25 carbons. Exemplary alkyl groups include methyl, ethyl, isopropyl, n-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl, 3-methyl-3-pentyl, and the like. When three or more carbon atoms are referred to, "alkyl" as used herein includes cycloalkyl. Unless otherwise specified, alkyl can be substituted or unsubstituted.

[0181] As used herein, the term "functional group" refers to a group that may be used, under normal conditions of organic synthesis, to form a covalent bond between the entity to which it is attached and another entity that generally has another functional group. A "bifunctional linker" refers to a linker that has two functional groups capable of forming two bonds with other moieties of a complex.

[0182] The term "derivative" as used herein refers to a chemically modified compound having additional structural moieties to introduce new functional groups or adjust the properties of the original compound.

[0183] The term "protecting group" as used herein refers to a moiety that prevents or blocks the reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions.Various protecting groups are well known in the art and are described, for example, in TW Greene and GM Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and PJ Kocienski, Protecting Groups, Third Ed., Thieme Chemistry, 2003, and the references cited therein.

[0184] As used herein, the term "PEG" refers to polyethylene glycol. PEG for use in the present invention is generally represented by -(CH2CH2O) n - structures. PEGs may have a variety of molecular weights, structures, or geometries. The PEG group may include a capping group that does not readily undergo chemical transformation under typical synthetic reaction conditions. Examples of capping groups include -OC 1-25 Examples include -Oaryl and -Oalkyl.

[0185] As used herein, the term "PEGylation" refers to chemical modification with polyethylene glycol.

[0186] The term "linker" as used herein refers to an atom or a collection of atoms used to connect interconnecting moieties (such as an antibody and a cytotoxic drug). A linker may be cleavable or non-cleavable. Preparation of various linkers for conjugates is described in literature such as Goldmacher et al., Antibody-drug Conjugates and Immunotoxins: From Pre-clinical Development to Therapeutic Applications, Chapter 7, in Linker Technology and Impact of Linker Design on ADC properties, Edited by Phillips GL;Ed. Springer Science and Business Media, New York (2013). Cleavable linkers include groups or moieties that can be cleaved under some biological or chemical conditions. Examples include enzymatically cleavable valine-citrulline amino acid sequences, disulfide linkers, 1,4- or 1,6-benzyl removal, trimethyl lock cleavable systems, bicine-based self-cleaving systems, acid-labile silyl ether linkers, and photolabile linkers.

[0187] The term "linking group" or "bond" as used herein refers to a functional group or moiety that connects different parts of a compound or complex. Examples of linking groups include, but are not limited to, amides, esters, carbamates, ethers, thioethers, disulfides, hydrazones, oximes, and semicarbazides, carbodiimides, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. For example, the linker moiety and the polymer moiety may be bonded to each other via an amide or carbamate linking group.

[0188] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein to describe a sequence of amino acid residues in a polymer. Peptides, polypeptides, or proteins may be composed of the standard 20 naturally occurring amino acids, in addition to rare amino acids and synthetic amino acid analogs. They may be any amino acid chain, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0189] A "recombinant" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein made by recombinant DNA technology, i.e., made from a cell transformed with a foreign DNA construct encoding the desired peptide. A "synthetic" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein prepared by chemical synthesis. The term "recombinant", when used with respect to, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or by the modification of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell so modified. Fusion proteins containing one or more of the above sequences and heterologous sequences are included within the scope of the present invention. A heterologous polypeptide, nucleic acid, or gene is a polypeptide, nucleic acid, or gene from a foreign species, or if from the same species, is significantly modified from its original form. Two fusion domains or sequences are heterologous to each other if they are not adjacent to each other in the naturally occurring protein or nucleic acid.

[0190] An "isolated" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein that is separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide / protein may comprise at least 10% (i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%) of the dry weight of the purified product. Purity may be measured by any appropriate standard method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated polypeptide / protein according to the present invention may be purified from a natural source, produced by recombinant DNA technology, or produced by chemical methods.

[0191] "Antigen" refers to a substance that binds to a substance that elicits an immune response or the product of that response. The term "epitope" refers to the region of an antigen to which an antibody or T cell binds.

[0192] The term "antibody" as used herein includes intact antibodies and any antigen-binding fragments or single chains thereof. Intact antibodies are glycoproteins that contain at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (V H ) and the heavy chain constant region. The heavy chain constant region is made up of three domains, C H 1. C H 2, and C H Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ), and the light chain constant region consists of one domain. H and V L The regions can be further subdivided into highly variable regions called complementarity determining regions (CDRs) and highly conserved regions called framework regions (FRs). H and V LEach of the heavy chain variable regions consists of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. The CDRs and FRs of the heavy chain variable region are HFRl, HCDRl, HFR2, HCDR2, HFR3, HCDR3, HFR4. The CDRs and FRs of the light chain variable region are LFRl, LCDRl, LFR2, LCDR2, LFR3, LCDR3, LFR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.

[0193] As used herein, an "antibody fragment" may comprise a portion of a complete antibody, generally including the antigen-binding region and / or the variable region of the complete antibody and / or the Fc region of the antibody that retains FcR binding ability. Examples of antibody fragments include linear antibodies; single-chain antibody molecules; nanobodies; and multispecific antibodies formed from antibody fragments.

[0194] The term "antigen-binding fragment or portion" of an antibody (or simply "antibody fragment or portion") as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be carried out by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment or portion" of an antibody include (i) Fab fragments, V L , V H , C L , and C H (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fab' fragment, essentially a Fab but with part of the hinge region; (iv) Fd fragment, a V H and C H(v) Fv fragment, consisting of the V arm of an antibody; L and a VH domain; (vi) a dAb, consisting of a VH domain; (vii) an isolated complementarity determining region (CDR); and (viii) a nanobody, consisting of a heavy chain variable region comprising a single variable domain and two constant domains. In addition, the two domains of the Fv fragment, V L and V H Although the V and VD are encoded by separate genes, they can be joined by a synthetic linker that allows them to be prepared as a single protein chain using recombinant methods, where V L and V H The domains pair to form a monovalent molecule (known as a single chain Fv (scFv); see, e.g., Bird, RE et al., Science, 1988, 242, 423-426; and Huston, JS et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment or portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0195] As used herein, the term "Fc fragment" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain.

[0196] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population. That is, each antibody comprising such a population is identical except for possible minor natural mutations. Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which generally contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as requiring that the antibody be made by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by the hybridoma method originally described by Kohler and Milstein (Kohler, G. et al., Nature, 1975, 256, 495-497), which is incorporated herein by reference, or may be prepared by recombinant DNA methods (US4816567), which is incorporated herein by reference. Monoclonal antibodies may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson T. et al., Nature, 1991, 352, 624-628 and Marks JD et al., J Mol Biol, 1991, 222, 581-597, each of which is incorporated herein by reference.

[0197] As used herein, monoclonal antibodies specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity (see US4816567; Morrison, SL et al., Proc. Natl. Acad. Sci. USA, 1984, 81, 6851-6855; Neuberger, MS et al., Nature, 1984, 312, 604-608; Takeda, S. et al., Nature, 1985, 314, 452-454; PCT / GB8500392, each of which is incorporated herein by reference).

[0198] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (such as mouse, rat, rabbit, or non-human primate) (donor antibody) having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR residues are those of a human immunoglobulin sequence. Optionally, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. For further details, see Jones, PT et al., Nature, 1986, 321, 522-525; Riechmann, L. et al., Nature, 1988, 332, 323-329; Presta, LG Curr. Opion. Struct. Biol., 1992, 2, 593-596; US5225539, each of which is incorporated herein by reference.

[0199] "Human antibody" refers to any antibody having fully human sequences, such as antibodies obtainable from human hybridomas, human phage display libraries, or transgenic mice expressing human antibody sequences.

[0200] The term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier, making the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0201] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents, and the like. A "pharmaceutically acceptable carrier" does not cause undesirable physiological effects upon or after administration to a subject. A carrier in a pharmaceutical composition must also be "acceptable" in the sense that it is compatible with and can stabilize the active ingredient. One or more solubilizing agents may be utilized as pharmaceutical carriers to deliver active agents. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to obtain a composition that can be used as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Other suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). The therapeutic compound may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, Berge, SM et al., J. Pharm. Sci., 1997, 66, 1-19).

[0202] As used herein, "treatment" or "therapy" refers to the administration of a compound or agent to a subject having a disease or at risk of developing a disease for the purpose of curing, alleviating, ameliorating, delaying the onset of, preventing, or ameliorating the disease, symptoms of the disease, a disease state secondary to the disease, or susceptibility to the disease.

[0203] "Effective amount" refers to the amount of active compound / drug required to provide a therapeutic effect to the subject being treated.As those skilled in the art will recognize, effective doses vary depending on the type of disease being treated, the route of administration, the use of excipients, and the possibility of co-use with other therapeutic treatments.The therapeutically effective amount of a combination for treating a tumor disease is, for example, the amount that causes a reduction in tumor size, a reduction in the number of tumor foci, or a slowdown in tumor growth, compared to untreated animals.

[0204] As disclosed herein, a number of numerical ranges are provided. Unless otherwise clearly indicated by the context, it is understood that each intermediate value between the upper and lower limit of the range, to the tenth of the unit of the lower limit, is also specifically disclosed. Each smaller range between any specified or intermediate value in a stated range and any other specified or intermediate value in the stated range is included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range that includes, excludes, or includes both the upper and lower limits in such smaller ranges is also included in the present invention, subject to any specifically excluded endpoints in the stated range. When a stated range includes one or both of the upper and lower limits, the range that excludes either or both of those included upper and lower limits is also included in the present invention.

[0205] The term "about" generally refers to plus or minus 10% of the indicated number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1" may mean 0.9 to 1.1. Other meanings of "about" may be apparent from the context, such as rounding, thus, for example, "about 1" may mean 0.5 to 1.4. EXAMPLES

[0206] The following examples are useful for a further understanding of the present invention, but are not intended to limit the scope of the invention in any way.

[0207] Example 1. Preparation of 30 km PEG-Lys(Mal)-3(Val-Cit-PAB-DEA-SN38) Preparation of Fmoc-Val-Cit-PAB-PNP (compound 5, Figure 1) Fmoc-Val-OSu(2): Fmoc-Val-OH (20.3 g, 60.0 mmol) and N-hydroxysuccinimide (NHS, 9.0 g, 78.0 mmol) were dissolved in a mixture of CHCl (120 mL) and THF (40 mL). Alone, EDCI (13.8 g, 72.0 mmol) was dissolved in CHCl (200 mL) and cooled to 0-5 °C. The solution of Fmoc-Val-OH and NHS was then added to the EDCI solution. The reaction was allowed to warm to room temperature and stirred at room temperature until the reaction was complete. The reaction mixture was then concentrated under reduced pressure and coevaporated twice with THF (100 mL). The concentrated residue was dissolved in THF (800 mL) and filtered to remove EDU. The filtrate was concentrated under reduced pressure and reslurried with n-heptane (800 mL) at 5-10 °C for 12 h. The solid was filtered, washed, and dried in vacuum to give Fmoc-Val-OSu(2) (23.8 g, 91%) as a white powder. 24 H 24 N2O6Na [M+Na] + The calculated HRMS (ESI) value was 459.1532 and the found value was 459.1523.

[0208] Fmoc-Val-Cit (3): Fmoc-Val-OSu (2) (9.8 g, 22.5 mmol) was dissolved in DME (150 mL) at room temperature. Sodium bicarbonate (2.1 g, 24.7 mmol) was dissolved in water (150 mL) at room temperature, followed by the addition of L-citrulline (4.3 g, 24.7 mmol) to obtain a homogeneous and clear solution. The prepared L-citrulline solution was then added to the Fmoc-Val-OSu solution, followed by the addition of THF (75 mL). The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was acidified with 15% citric acid (200 mL) and then concentrated in vacuum. The mixture was resuspended in water (500 mL) and the resulting mixture was stirred for 2 hours, followed by filtration and drying in vacuum. The dried solid was resuspended in methyl tert-butyl ether (500 mL) and stirred for 12 hours. The suspension was filtered and washed. The separated solid was dried in vacuum to give Fmoc-Val-Cit (3) (6.8 g, 61%) as a white powder. 26 H 33 N4O6[M+H] + The calculated HRMS (ESI) value was 497.2400 and the measured value was 497.2388.

[0209] Fmoc-Val-Cit-PAB-OH (4): EEDQ (4.95 g, 20.0 mmol) was added to a solution of compound 3 (4.96 g, 10.0 mmol) and 4-aminobenzyl alcohol (2.46 g, 20.0 mmol) in CHCl (350 mL) and MeOH (150 mL). The reaction mixture was stirred at room temperature for 24 h. An additional EEDQ (2.5 g, 10.0 mmol) was added and stirred for another 24 h. After the reaction was complete, the solvent was removed under reduced pressure and the resulting residue was reslurried in methyl tert-butyl ether (800 mL) for 12 h. The solid was filtered, washed, and dried under vacuum to give compound 4 (4.1 g, 69%) as a white powder. C 33 H 40 N5O6[M+H] + The calculated HRMS (ESI) value was 602.2979 and the found value was 602.2969.

[0210] Fmoc-Val-Cit-PAB-PNP (5): DIPEA (2.5 mL, 15.0 mmol) was added to a solution of compound 4 (5.2 g, 8.6 mmol) and bis(4-nitrophenyl)carbonate (4.9 g, 16.1 mmol) in DMF (52 mL) at room temperature. The reaction mixture was stirred at room temperature for 5 h. After the reaction was complete, the product was precipitated from the reaction mixture by adding anhydrous ethyl acetate (250 mL) and methyl tert-butyl ether (250 mL). The resulting slurry was stirred and cooled to 0 °C. After stirring at 0 °C for 30 min, the solid was isolated by filtration, followed by washing and drying in vacuum to give Fmoc-Val-Cit-PAB-PNP (5) (4.7 g, 72%) as a pale yellow powder. C 40 H 43 NO 10 [M+H] + The calculated HRMS (ESI) value was 767.3041 and the found value was 767.3045.

[0211] Preparation of Val-Cit-PAB-DEA-SN38 (Compound 10) Reaction Scheme A (Figure 2) Boc-DEA-SN38 (7): A solution of SN-38 (3.9 g, 10 mmol) in anhydrous THF (100 mL) is cooled to 0 °C under nitrogen atmosphere, then 4-nitrophenyl chloroformate (2.7 g, 13.4 mmol) and Et3N (7.0 mL, 50 mmol) are added. The mixture is stirred at 0 °C for 1.5 h. Boc-DEA (6) (9.4 g, 50 mmol) is added and the mixture is stirred for an additional 1 h. The reaction is allowed to warm slowly to room temperature. After the reaction is complete, the reaction mixture is concentrated and the crude is purified by column chromatography to give compound 7.

[0212] DEA-SN38(8): A solution of compound 7 (0.99 g, 1.63 mmol) in CH2Cl2 (10 mL) is cooled to 0 °C, then TFA (3 mL) is added. The mixture is stirred at 0 °C for 1 h, followed by the addition of CH2Cl2 (10 mL). The diluted mixture is concentrated to give crude 8.

[0213] Fmoc-Val-Cit-PAB-DEA-SN38 (9): Compound 8 (1.4 g, 2.8 mmol) and Fmoc-Val-Cit-PAB-PNP (5) (2.8 g, 3.6 mmol) are dissolved in DMF (20 mL). HOBt (0.75 g, 5.6 mmol) and pyridine (1.7 mL) are then added and the reaction mixture is stirred at room temperature for 24 h. After the reaction is complete, the reaction mixture is cooled to 0 °C and methyl tert-butyl ether (180 mL) is added. The resulting slurry is stirred for 3-5 h and filtered. The separated solid is washed and dried in vacuum. The crude is purified by column purification to give compound 9.

[0214] Val-Cit-PAB-DEA-SN38(10): Compound 9 (2.5 g, 2.2 mmol) is resuspended in anhydrous DMF (40 mL) and the resulting suspension is stirred at room temperature until a homogeneous suspension is formed. Diethylamine (10 mL) is then added and the reaction mixture is stirred at room temperature for 3 h. After the reaction is complete, methyl tert-butyl ether (100 mL) and ethyl acetate (50 mL) are then added over 60 min. The resulting mixture is stirred at 0° C. for 4 h. The solid is filtered and dried in vacuum to give compound 10.

[0215] Reaction Scheme B (Figure 3) Boc-DEA-SN38 (7): A solution of SN-38 (11.8 g, 30 mmol) and DIPEA (18.3 mL, 105 mmol) in anhydrous CHCl (500 mL) was cooled to 0° C. under nitrogen atmosphere, and then 4-nitrophenyl chloroformate (19.3 g, 960 mmol) was added. The mixture was stirred at room temperature for 16 h. After the reaction was complete, the solvent was removed under reduced pressure, and the resulting residue was reslurried in methyl tert-butyl ether (600 mL) for 1 h. The solid was filtered, washed, and dried in vacuum to give compound PNP-SN38 (16 g, 96%) as a pale yellow powder. MS (ESI) m / z [M+H] + 558.29

[0216] Compound 6 (5.31 g, 27.0 mmol) and PNP-SN38 (5.02 g, 9.0 mmol) were dissolved in DMF (50 mL). HOBt (2.43 g, 18.0 mmol) and pyridine (4.51 mL) were then added and the reaction mixture was stirred at room temperature for 24 h until the reaction was complete. The reaction mixture was cooled to 0° C. and added to methyl tert-butyl ether (90 mL). The resulting slurry was stirred for 3-5 h, filtered, washed, and dried in vacuum. The crude was purified by column purification to give compound 7 (4.5 g, 82%) as a pale yellow powder. MS (ESI) m / z [M+H] + 607.30, [M+Na] + 629.30

[0217] DEA-SN38(8): A solution of compound 7 (3.03 g, 5.0 mmol) in CH2Cl2 (20 mL) was stirred at room temperature and TFA (4 mL) was added dropwise. The mixture was stirred at room temperature for 0.5 h and the solvent was removed in vacuo. The residue was treated with methyl tert-butyl ether (60 mL) and the resulting slurry was stirred for 1 h, filtered, washed and dried to give pure compound 8 (2.42 g, 96%) as a pale yellow solid. MS (ESI) m / z [M+Na] + 529.25

[0218] Fmoc-Val-Cit-PAB-DEA-SN38 (9): Compound 8 (1.82 g, 3.6 mmol) and Fmoc-Val-Cit-PAB-PNP (5) (2.3 g, 3.0 mmol) were dissolved in DMF (20 mL). HOBt (0.81 g, 6.0 mmol) and pyridine (1.9 mL) were then added, and the reaction mixture was stirred at room temperature for 24 h. After the reaction was complete, the reaction mixture was cooled to 0° C., and methyl tert-butyl ether (150 mL) was added. The resulting slurry was stirred for 1 h and filtered. The separated solid was washed and dried in vacuum. The crude was purified by column purification to give compound 9 (2.9 g, 86%) as a white solid. MS (ESI) m / z [M+H] + 1134.57, [M+Na] +1156.47

[0219] Val-Cit-PAB-DEA-SN38 (10): Compound 9 (2.5 g, 2.2 mmol) was resuspended in anhydrous DMF (40 mL) and the resulting suspension was stirred at room temperature until a homogeneous suspension was formed. Diethylamine (10 mL) was then added and the reaction mixture was stirred at room temperature for 1 h. After the reaction was complete, methyl tert-butyl ether (100 mL) and ethyl acetate (50 mL) were then added over 60 min. The resulting mixture was stirred at 0° C. for 4 h. The solid was filtered and dried in vacuum to give compound 10 (2.0 g, 97%) as a pale yellow powder. MS (ESI) m / z [M+H] + 912.53, [M+Na] + 934.43

[0220] Branched intermediate NH 2 -PEG 6 Preparation of -3 (Val-Cit-PAB-DEA-SN38) (Compound 16, Figure 4) Compound 12: Reaction Scheme A: In 2.0 mL of freshly opened DMSO, compound 11 (1.21 g, 10.0 mmol) is cooled to 15 °C under N2. 0.2 mL of 5.0 M NaOH is injected with stirring, followed by dropwise injection of tert-butyl acrylate (5.0 mL, 34 mmol) (Note: a solvent mixture of 5-10% water in DMSO works best for this reaction). The reaction mixture is allowed to warm to room temperature and stirred for 24 h. At this point, excess reagents and solvents are removed in vacuo at room temperature and the residue is purified by column chromatography to give compound 12.

[0221] Reaction Scheme B: Compound 11 (2.43 g, 20.0 mmol) dissolved in 6.0 mL of freshly opened DMSO was cooled to 15° C. under argon. 0.6 mL of 5.0 M NaOH was injected with stirring, followed by dropwise injection of tert-butyl acrylate (8.72 g, 68 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 24 hours. At this point, excess reagents and solvents were removed in vacuo at room temperature and the residue was purified by column chromatography to give compound 12 (4.2 g, 43%) as a colorless oil. MS (ESI) m / z [M+H] + 506.35, [M+Na] + 528.40

[0222] Compound 13: To a stirred solution of FmocNH-PEG6-COOH (1.15 g, 2.0 mmol) in a mixture of anhydrous CHCl (20 mL) and DMF (20 mL) was added compound 12 (1.5 g, 2.2 mmol), EDCI (575 mg, 3.0 mmol), and HOBt (80 mg, 0.6 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was observed by HPLC. After the reaction was complete, the mixture was concentrated in vacuo. The crude reaction mixture was purified by silica gel chromatography to give product 13.

[0223] Compound 14: Compound 13 (1.25 g, 1.0 mmol) was dissolved in CH2Cl2 (10 mL) followed by the addition of TFA (3.0 mL). The mixture was stirred at room temperature for 3 h. The solvent was removed in vacuo as much as possible at <35 °C. The residue was purified by silica gel chromatography to give product 14.

[0224] Compound 15: To a stirred solution of compound 14 (865 mg, 0.8 mmol) in a mixture of anhydrous CHCl (10 mL) and DMF (10 mL) was added compound 10 (2.4 g, 2.64 mmol), EDCI (863 mg, 4.5 mmol), and HOBt (108 mg, 0.8 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was observed by HPLC. After the reaction was complete, the mixture was concentrated in vacuo. The crude reaction mixture was purified by silica gel chromatography to give product 15.

[0225] Compound 16: Diethylamine (2.0 ml) was added to a solution of compound 15 (0.41 g, 0.11 mmol) in DMF (5 ml) and the reaction was allowed to proceed at room temperature for 2 h. After the reaction, the reaction mixture was concentrated in vacuo and the residue was purified by preparative HPLC using a Welch Ultimate XB-C18 column to give product 16.

[0226] Preparation of 30 kmPEG-Lys(Mal)-3(Val-Cit-PAB-DEA-SN38) (Compound 22, Figure 5) Compound 18: H-Lys(boc)-OH (369 mg, 1.5 mmol) was added in anhydrous DMF (100 mL), followed by DIPEA (0.83 mL, 5.0 mmol), compound 30kmPEG-NHS (17) (15 g, 0.5 mmol), and anhydrous CHCl (150 mL). The mixture was stirred overnight at room temperature under argon. Insoluble material was filtered off. The solvent was removed and the residue was recrystallized from CHCl / methyl tert-butyl ether (45 mL / 300 mL). The separated solid was recrystallized again from MeCN / 2-propanol (30 mL / 450 mL). The product was dried in vacuum at 40° C. for 4 h to give product 18 (13.6 g, 91%) as a white powder.

[0227] Compound 19: Compound 18 (5.7 g, 0.19 mmol) was dissolved in anhydrous CHCl (57 mL) followed by the addition of TFA (29.5 mL). The mixture was stirred at room temperature for 1 h. The solvent was removed in vacuum as much as possible at <35° C. The residue was recrystallized twice from CHCl / methyl tert-butyl ether (14.5 mL / 115 mL). The isolated product was dried in vacuum at 40° C. to give product 19 (4.7 g, 84%) as a white powder.

[0228] Compound 21: To a stirred solution of compound 19 (5.5 g, 0.18 mmol) in anhydrous CHCl (55 mL) was added DIPEA (473 mg, 3.6 mmol) at 0 °C, followed by 5-maleimidovaleric acid-NHS (20) (138 mg, 0.47 mmol). The mixture was stirred at 0 °C for 1.5 h and then slowly warmed from 0 °C to room temperature. The reaction mixture was stirred overnight under argon atmosphere. The solvent was removed and the residue was recrystallized from CHCl / methyl tert-butyl ether (13.8 mL / 110 mL). The separated solid was recrystallized again from MeCN / 2-propanol (11 mL / 165 mL). The residue was dried in vacuum to give product 21 (5.0 g, 90%) as a white powder.

[0229] Compound 22: To a stirred solution of compound 21 (6.0 g, 0.2 mmol) in anhydrous CHCl (60 mL) was added compound 16 (2.1 g, 0.6 mmol), EDCI (230 mg, 1.2 mmol), and HOBt (243 mg, 1.8 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was observed by HPLC. The solvent was removed and the residue was recrystallized from CHCl / methyl tert-butyl ether (18 mL / 120 mL). The separated solid was recrystallized again from MeCN / 2-propanol (12 mL / 180 mL). The product was dried in vacuum at 40° C. for 4 h to give product 22.

[0230] Example 2. Preparation of 30 kmPEG-Lys(Mal)-6(Val-Cit-PAB-DEA-SN38) (Compound 28, FIG. 6) Compound 24: To a solution of compound FmocNH-PEG6-COOH (1.15 g, 2.0 mmol) in anhydrous CHCl (10 mL) was added di-tert-butyl 3,3'-azanediyldipropanoate (23) (0.64 mL, 2.2 mmol), EDCI (0.58 g, 3.0 mmol), and HOBt (54 mg, 0.4 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was observed by TLC. After the reaction was complete, the mixture was extracted with CHCl (30 mL x 2). The organic layer was washed with brine (20 mL), dried over NaSO, and concentrated in vacuo. The crude reaction mixture was purified by silica gel chromatography to give product 24.

[0231] Compound 25: Compound 24 (0.39 g, 0.47 mmol) was dissolved in CH2Cl2 (4.0 mL) followed by the addition of TFA (2.0 mL). The mixture was stirred at room temperature for 3 h. The solvent was removed in vacuo as much as possible at <35 °C. The residue was purified by silica gel chromatography to give product 25.

[0232] Compound 26: To a stirred solution of compound 25 (1.08 g, 1.5 mmol) in a mixture of anhydrous CHCl (20 mL) and DMF (20 mL) was added compound 16 (11.7 g, 3.3 mmol), EDCI (767 mg, 4.0 mmol), and HOBt (108 mg, 0.8 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was observed by HPLC. After the reaction was complete, the mixture was concentrated in vacuo. The crude reaction mixture was purified by silica gel chromatography to give product 26.

[0233] Compound 27: Diethylamine (5.0 ml) was added to a solution of compound 26 (2.4 g, 0.31 mmol) in DMF (20 ml) and the reaction mixture was allowed to proceed at room temperature for 2 h. After the reaction, the mixture was concentrated in vacuo and the residue was purified by preparative HPLC using a Welch Ultimate XB-C18 column to give product 27.

[0234] Compound 28: To a stirred solution of compound 21 (6.0 g, 0.2 mmol) in anhydrous CHCl (60 mL) was added compound 27 (4.5 g, 0.6 mmol), EDCI (230 mg, 1.2 mmol), and HOBt (243 mg, 1.8 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was observed by HPLC. The solvent was removed and the residue was recrystallized from CHCl / methyl tert-butyl ether (18 mL / 120 mL). The separated solid was recrystallized again from MeCN / 2-propanol (12 mL / 180 mL). The product was dried in vacuum at 40° C. for 4 h to give product 28.

[0235] Example 3. Preparation of 20 kmPEG-Glu(Mal)-4(Val-Cit-PAB-DEA-Duo-DM) Preparation of Val-Cit-PAB-DEA-Duo-DM (Compound 33, Figure 7) BocDEA-Duo-DM (30): A solution of duocarmycin DM (29) (4.6 g, 10 mmol) in anhydrous THF (100 mL) was cooled to 0 °C under nitrogen atmosphere, then 4-nitrophenyl chloroformate (2.7 g, 13.4 mmol) and Et3N (7.0 mL, 50 mmol) were added. The mixture was stirred at 0 °C for 1.5 h. Compound Boc-DEA (6) (9.4 g, 50 mmol) was added and the mixture was stirred for an additional 1 h. The reaction was allowed to warm slowly to room temperature and then concentrated. The crude reaction mixture was purified by silica gel chromatography to give product 30.

[0236] DEA-Duo-DM (31): A solution of compound 30 (1.1 g, 1.62 mmol) in CH2Cl2 (10 mL) was cooled to 0 °C, then TFA (3 mL) was added. The mixture was stirred at 0 °C for 1 h and diluted with CH2Cl2 (10 mL). The diluted solution was concentrated in vacuo to give crude 31.

[0237] Fmoc-Val-Cit-PAB-DEA-Duo-DM (32): Compound 31 (1.6 g, 2.8 mmol) and Fmoc-Val-Cit-PAB-PNP (5) (2.8 g, 3.6 mmol) were dissolved in DMF (20 mL). Then HOBt (0.75 g, 5.6 mmol) and pyridine (1.7 mL) were added. The reaction mixture was stirred at room temperature for 24 h. After the reaction was complete, the reaction mixture was cooled to 0 °C. Methyl tert-butyl ether (180 mL) was added. The resulting slurry was stirred for 3-5 h and filtered. The solid was washed and dried in vacuum. The crude was purified by silica gel chromatography to give product 32.

[0238] Val-Cit-PAB-DEA-Duo-DM (33): Compound 32 (2.0 g, 1.7 mmol) was resuspended in anhydrous DMF (40 mL) and the resulting suspension was stirred at room temperature until a homogeneous suspension was formed. Diethylamine (10 mL) was then added and the reaction mixture was stirred at room temperature for 3 h. After the reaction was complete, methyl tert-butyl ether (100 mL) and ethyl acetate (50 mL) were then added over 60 min. The resulting mixture was stirred at 0° C. for 4 h. The solid was filtered and dried in vacuum to give compound 33.

[0239] Branched intermediate NH 2 Preparation of -PEG6-4(Val-Cit-PAB-DEA-Duo-DM) (Compound 40) Reaction Scheme A (Figure 8) Compound 35: To a solution of compound 34 (0.68 g, 2.0 mmol) in anhydrous CHCl (10 mL) is added di-tert-butyl 3,3'-azanediyldipropanoate (23) (0.64 mL, 2.2 mmol), EDCI (0.58 g, 3.0 mmol), and HOBt (54 mg, 0.4 mmol) at room temperature under argon. The mixture is stirred at room temperature until complete conversion is observed by TLC. After the reaction is complete, the mixture is extracted with CHCl (30 mL x 2) and the combined organic layers are washed with brine (20 mL), dried over NaSO, and concentrated in vacuo. The crude is purified by silica gel chromatography to give product 35.

[0240] Compound 36: Compound 35 (0.5 g, 0.84 mmol) is dissolved in CH2Cl2 (6.0 mL) followed by the addition of TFA (3.0 mL). The mixture is stirred at room temperature for 3 h. The solvent is removed in vacuo as much as possible at <35 °C. The residue is purified by silica gel chromatography to give product 36.

[0241] Compound 37: To a solution of compound 36 (964 mg, 2.0 mmol) in a mixture of anhydrous CHCl (8 mL) and DMF (8 mL), add Val-Cit-PAB-DEA-Duo-DM (33) (4.3 g, 4.4 mmol), EDCI (575 mg, 3.0 mmol), and HOBt (135 mg, 1.0 mmol) at room temperature under argon. The mixture is stirred at room temperature until complete conversion is observed by HPLC. After the reaction is complete, the mixture is concentrated in vacuo. The crude reaction mixture is purified by silica gel chromatography to give product 37.

[0242] Compound 38: Diethylamine (2.0 ml) is added to a solution of compound 37 (0.5 g) in DMF (5 ml) and the reaction mixture is allowed to proceed at room temperature for 2 hours. After the reaction, the reaction mixture is concentrated in vacuo and the residue is purified by preparative HPLC using a Welch Ultimate XB-C18 column to give product 38.

[0243] Compound 39: To a stirred solution of compound 25 (546 mg, 0.76 mmol) in a mixture of anhydrous CHCl (20 mL) and DMF (20 mL) at room temperature under argon, compound 38 (3.7 g, 1.7 mmol), EDCI (430 mg, 2.3 mmol), and HOBt (40 mg, 0.3 mmol) are added. The mixture is stirred at room temperature until complete conversion is observed by HPLC. After the reaction is complete, the mixture is concentrated in vacuo. The crude reaction mixture is purified by silica gel chromatography to give product 39.

[0244] Compound 40: Diethylamine (2.0 ml) is added to a solution of compound 39 (0.51 g, 0.1 mmol) in DMF (5 ml) and the reaction mixture is allowed to proceed at room temperature for 2 hours. After the reaction, the reaction mixture is concentrated in vacuo and the residue is purified by preparative HPLC using a Welch Ultimate XB-C18 column to give product 40.

[0245] Reaction Scheme B (Figure 9) Compound 35: To a solution of compound 34 (0.62 g, 2.0 mmol) in anhydrous CHCl (15 mL) was added di-tert-butyl 3,3′-azanediyldipropanoate (23) (0.62 mL, 2.2 mmol), EDCI (0.58 g, 3.0 mmol), and HOBt (54 mg, 0.4 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was observed by TLC. After the reaction was complete, the mixture was extracted with CHCl (30 mL x 2) and the combined organic layers were washed with brine (20 mL), dried over NaSO, and concentrated in vacuo. The crude was purified by silica gel chromatography to give product 35 (1.1 g, 96%) as a colorless oil. C 32 H 43 N2O7[M+H] + The calculated HRMS (ESI) value was 567.3070 and the found value was 567.3062.

[0246] Compound 36: Compound 35 (0.5 g, 0.84 mmol) was dissolved in formic acid (3.0 mL). The mixture was stirred at room temperature for 16 h. The solvent was removed in vacuo as much as possible at <35° C. The residue was purified by silica gel chromatography to give the product 36 (1.5 g, 94%) as a colorless oil. 24 H 27 N2O7[M+H] + The calculated HRMS (ESI) value was 455.1818 and the found value was 455.1824.

[0247] Compound 37: To a solution of compound 36 (964 mg, 2.0 mmol) in a mixture of anhydrous CHCl (8 mL) and DMF (8 mL) was added Val-Cit-PAB-DEA-Duo-DM (33) (4.3 g, 4.4 mmol), EDCI (575 mg, 3.0 mmol), and HOBt (135 mg, 1.0 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. After the reaction was complete, the mixture was concentrated in vacuo. The crude was purified by silica gel chromatography to give product 37.

[0248] Compound 38: Diethylamine (2.0 mL) was added to a solution of compound 37 (0.5 g) in DMF (5.0 mL) and the reaction was allowed to proceed at room temperature for 2 hours. After the reaction, the reaction mixture was concentrated in vacuo and the residue was purified by preparative HPLC using a Welch Ultimate XB-C18 column to give product 38.

[0249] Compound 39: To a stirred solution of compound 25 (546 mg, 0.76 mmol) in anhydrous CHCl (20 mL) and DMF (20 mL) was added compound 38 (3.7 g, 1.7 mmol), EDCI (430 mg, 2.3 mmol), and HOBt (40 mg, 0.3 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. After the reaction was complete, the mixture was concentrated in vacuo. The crude was purified by silica gel chromatography to give product 39.

[0250] Compound 40: Diethylamine (2.0 ml) was added to a solution of compound 39 (0.51 g, 0.1 mmol) in DMF (5 mL) and the reaction was allowed to proceed at room temperature for 2 h. After the reaction, the reaction mixture was concentrated in vacuo and the residue was purified by preparative HPLC using a Welch Ultimate XB-C18 column to give product 40.

[0251] Preparation of 20kmPEG-Glu(Mal)-4(Val-Cit-PAB-DEA-Duo-DM) (Compound 46, Figure 10) Compound 42: H-Glu(OtBu)-OH (305 mg, 1.5 mmol) was added in anhydrous DMF (67 mL), followed by DIPEA (0.83 mL, 5.0 mmol), compound 20kmPEG-NHS (41) (10 g, 0.5 mmol), and anhydrous CHCl (100 mL). The mixture was stirred overnight at room temperature under argon. The solvent was removed and the residue was recrystallized from CHCl / methyl tert-butyl ether (30 mL / 200 mL). The separated solid was recrystallized again from MeCN / 2-propanol (20 mL / 300 mL). The product was dried in vacuum at 40 °C for 4 h to give product 42 as a white powder.

[0252] Compound 44: To a stirred solution of compound 42 (2.0 g, 0.1 mmol) in anhydrous CHCl (20 mL) was added compound 43 (66 mg, 0.3 mmol), EDCI (115 mg, 0.6 mmol), and HOBt (122 mg, 0.9 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. The solvent was removed and the residue was recrystallized from CHCl / methyl tert-butyl ether (5 mL / 40 mL). The separated solid was recrystallized again from MeCN / 2-propanol (4 mL / 60 mL). The product was dried in vacuum at 40° C. for 4 h to give product 44 as a white powder.

[0253] Compound 45: Compound 44 (5.8 g, 0.29 mmol) was dissolved in anhydrous CH2Cl2 (58 mL). TFA (29 mL) was added. The mixture was stirred at room temperature for 1 h. The solvent was removed in vacuum as much as possible at <35 °C. The residue was recrystallized twice from CH2Cl2 / methyl tert-butyl ether (14.5 mL / 115 mL). The isolated product was dried in vacuum at 40 °C to give product 45 as a white powder.

[0254] Compound 46: To a stirred solution of compound 45 (6.0 g, 0.3 mmol) in anhydrous CHCl (60 mL) was added compound 40 (4.4 g, 0.9 mmol), EDCI (345 mg, 1.8 mmol), and HOBt (365 mg, 2.7 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. The solvent was removed and the residue was recrystallized from CHCl / methyl tert-butyl ether (18 mL / 120 mL). The separated solid was recrystallized again from MeCN / 2-propanol (12 mL / 180 mL). The product was dried under vacuum at 40 °C to give compound 46.

[0255] Example 4. Preparation of Mal-20kPEG-3 (Val-Cit-PAB-DEA-Duo-DM) Branched intermediate NH 2 -PEG 6 Preparation of -3 (Val-Cit-PAB-DEA-Duo-DM) (Compound 48, FIG. 11) Compound 47: To a stirred solution of compound 14 (865 mg, 0.8 mmol) in a mixture of anhydrous CHCl (10 mL) and DMF (10 mL) was added compound 33 (2.6 g, 2.64 mmol), EDCI (863 mg, 4.5 mmol), and HOBt (108 mg, 0.8 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. After the reaction was complete, the mixture was concentrated in vacuo. The crude reaction mixture was purified by silica gel chromatography to give product 47.

[0256] Compound 48: Diethylamine (2.0 ml) was added to a solution of compound 47 (0.48 g, 0.12 mmol) in DMF (5.0 mL) and the reaction was allowed to proceed at room temperature for 2 h. After the reaction, the reaction mixture was concentrated in vacuo and the residue was purified by preparative HPLC using a Welch Ultimate XB-C18 column to give product 48.

[0257] Preparation of Mal-20kPEG-3(Val-Cit-PAB-DEA-Duo-DM) (Compound 51, FIG. 12) Compound 50: Amine-PEG20k-CO2H (49) (1.0 g, 0.05 mmol) was dissolved in anhydrous CHCl2 (10 mL) at 0 °C. DIPEA (83 μL, 0.5 mmol) was added, followed by compound 20 (46 mg, 0.15 mmol). The mixture was stirred at 0 °C for 1.5 h. After the reaction, the solution was slowly warmed from 0 °C to room temperature, followed by stirring overnight under argon atmosphere. The solvent was removed and the residue was recrystallized from CHCl2 / methyl tert-butyl ether (2.5 mL / 20 mL). The separated solid was recrystallized again from MeCN / 2-propanol (2 mL / 30 mL). The residue was dried in vacuum to give product 50.

[0258] Compound 51: To a stirred solution of compound 50 (6.0 g, 0.2 mmol) in anhydrous CHCl (60 mL) was added compound 48 (2.1 g, 0.6 mmol), EDCI (230 mg, 1.2 mmol), and HOBt (243 mg, 1.8 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. The solvent was removed and the residue was recrystallized from CHCl / methyl tert-butyl ether (18 mL / 120 mL). The separated solid was recrystallized again from MeCN / 2-propanol (12 mL / 180 mL). The product was dried in vacuum at 40° C. for 4 h to give product 51.

[0259] Example 5. Preparation of SCAPDL1xSCACD47 (Compound 52) Anti-PDL1 and anti-CD47 bispecific single chain antibodies (SCAs) (SCAPDL1xSCACD47) were prepared in mammalian cells (e.g., CHO using EasySelect™) or yeast (e.g., Pichia yeast expression kit with pPICZ vector) by recombinant DNA technology. To facilitate subsequent conjugation, a site-specific conjugation functional group thiol was inserted into the linker between the two PDL1 and CD47 SCAs by recombinant DNA technology. The DNA sequence of SCAPDL1xSCACD47 corresponding to the following amino acid sequence (SEQ ID NO:1) was synthesized, cloned into an expression vector, and introduced into host cells. After cell expression, the culture supernatant of host cells expressing SCAPDL1xSCACD47 was collected by centrifugation and loaded onto a Ni column (2.6 cmx13 cm) (Cat#AA207311, BestChrome, Shanghai, China) pre-equilibrated with 50 mM sodium phosphate, 100 mM NaCl, pH 7.0. The protein was eluted with a buffer of 50 mM sodium phosphate, 250 mM imidazole, 100 mM NaCl, pH 7.0, and aliquoted into 15 mL tubes. The captured protein was further purified using a CaptoL column (Cat#17-5478-02, GE Healthcare, NJ) (1.6 cmx8 cm) pre-equilibrated with 50 mM sodium phosphate and 100 mM NaCl (pH 7.0). The protein was eluted with 75 mM acetic acid (pH 3.0) to give the isolated product 52.

[0260] Amino acid sequence of SCAPDL1xSCACD47 (SEQ ID NO:1): DIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKGGSGGSGGGSGG SGGQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSGCGG SSGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGGSGGGGSGG GGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTHHHHHH

[0261] Example 6. Preparation of 30 kmPEG(SCAPDL1xSCACD47)-3(Val-Cit-PAB-DEA-SN38) (Compound 53, FIG. 13) Adjust the solution of protein 52 to pH 6.8 with a pH 4.12 stock solution of 500 mM sodium phosphate, followed by reduction with 3.5 mM TCEP-HCl for 30 min at room temperature. Adjust the reduced protein to 5 mg / mL. Perform PEGylation of SCAPDL1xSCACD47 with 5–10 equivalents of compound 22 [30kmPEG-Lys(Mal)-3(Val-Cit-PAB-DEA-SN38)] at room temperature, pH 6.8 for 3 h. Quench the reaction with 10 mM L-cystine for 10 min at room temperature. Purify the final product 53 [30kmPEG(SCAPDL1xSCACD47)-3(Val-Cit-PAB-DEA-SN38)] in 20 mM phosphate buffer at pH 6.5 using a cation exchange chromatography column (CM Fast Flow). Confirm target compound 53 with SEC-HPLC and cell-based activity assays.

[0262] Example 7. Preparation of 30 kmPEG-(SCAPDL1xSCACD47)-6(Val-Cit-PAB-DEA-SN38) (Compound 54, FIG. 14) Compound 54 is prepared by conjugating compound 28 [30 kmPEG-Lys(Mal)-6(Val-Cit-PAB-DEA-SN38)] to protein 52 using a procedure similar to the preparation of compound 53.

[0263] Example 8. Preparation of 20 kmPEG-(SCAPDL1xSCACD47)-4(Val-Cit-PAB-DEA-Duo-DM) (Compound 55, FIG. 15) Compound 55 is prepared by conjugating compound 46 [20kmPEG-Glu(Mal)-4(Val-Cit-PAB-DEA-Duo-DM)] to protein 52 using a procedure similar to the preparation of compound 53.

[0264] Example 9. Preparation of SCAPDL1xSCACD47-20kPEG-3(Val-Cit-PAB-DEA-Duo-DM) (Compound 56, FIG. 16) Compound 56 is prepared by conjugating compound 51 [Mal-20kPEG-3(Val-Cit-PAB-DEA-Duo-DM)] to protein 52 using a procedure similar to the preparation of compound 53.

[0265] Example 10. Branched intermediate NH 2 Preparation of -2 (Val-Cit-PAB-DEA-SN38) (Compound 58, FIG. 17) Compound 57: To a stirred solution of compound 36 (0.75 g, 1.55 mmol) in anhydrous DMF (30 mL) was added Val-Cit-PAB-DEA-SN38(10) (3.1 g, 3.42 mmol), EDCI (0.89 g, 4.66 mmol), and HOBt (0.21 g, 1.55 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was observed by TLC. After the reaction was complete, the mixture was concentrated in vacuo. The reaction mixture was cooled to 0° C. and added to methyl tert-butyl ether (90 mL). The resulting slurry was stirred for 1 h, filtered, washed, and dried in vacuum. The crude was purified by silica gel chromatography to give compound 57 (2.4 g, 68%) as a white solid. MS (ESI) m / z [M+2H] 2+ 1135.82

[0266] Compound 58: Diethylamine (4.0 mL) was added to a solution of compound 57 (1.2 g) in DMF (12 mL), and the reaction was then allowed to proceed at room temperature for 40 min. The reaction mixture was concentrated in vacuo, and the resulting residue was slurried in methyl tert-butyl ether (90 mL) for 2 h. The solid was filtered, washed, and dried in vacuo to give compound 58 (1.0 g, 93%) as a pale yellow powder. MS (ESI) m / z [M+2H] 2+ 1024.67, [M+2Na] 2+ 1046.63

[0267] Example 11. Branched intermediate N 3 -PEG 6 Preparation of -3 (Val-Cit-PAB-DEA-SN38) (Compound 62, FIG. 18) Compound 60: To a solution of compound 12 (1.5 g, 2.9 mmol) in anhydrous CHCl (20 mL) was added compound N-PEG-COH (59) (1.0 g, 2.64 mmol), EDCI (0.76 g, 4.0 mmol), and HOBt (110 mg, 0.8 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by TLC. After the reaction was complete, the mixture was extracted with CHCl (30 mL x 2), and the organic layer was washed with brine (20 mL), dried over NaSO, filtered, and concentrated in vacuo. The crude reaction mixture was purified by silica gel column to give product 60 (2.0 g, 88%) as a colorless oil. C 32 H 43 N2O7[M+H] + HRMS (ESI) calculated value was 567.3070, found value was 567.3062. MS (ESI) m / z [M+H] + 867.55, [M+Na] + 889.40

[0268] Compound 61: Compound 60 (2.0 g, 3.1 mmol) was dissolved in formic acid (30 mL) and the mixture was stirred at room temperature for 16 h. The solvent was removed in vacuo as much as possible at <35° C. The residue was purified on a silica gel column to give the product 61 (1.5 g, 98%) as a colorless oil. MS (ESI) m / z [M+H] + 699.24, [M+Na]+ 721.34

[0269] Compound 62: To a stirred solution of compound 61 (0.6 g, 0.86 mmol) in anhydrous DMF (25 mL) was added Val-Cit-PAB-DEA-SN38(10) (2.6 g, 2.8 mmol), EDCI (0.74 g, 3.86 mmol), and HOBt (0.12 g, 0.86 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. After the reaction was complete, the mixture was concentrated in vacuo. The reaction mixture was cooled to 0° C. and added to methyl tert-butyl ether (120 mL). The resulting slurry was stirred for 1 h, filtered, washed, and dried in vacuum. The crude was purified on a silica gel column to give compound 62 (1.7 g, 60%) as a white solid. MS (ESI) m / z [M+3H] 3+ 1127.53, [M+3Na] 3+ 1149.58

[0270] Example 12. Branched compound 65 [N 3 -PEG 6 -2(Val-Cit-PAB-DEA-SN38)] and 66[N 3 -PEG 6 Preparation of -4 (Val-Cit-PAB-DEA-SN38)] (Figure 19) Compound 63: To a solution of compound 59 (0.76 g, 2.0 mmol) in anhydrous CHCl (10 mL) was added di-tert-butyl 3,3'-azanediyldipropanoate (23) (0.64 mL, 2.2 mmol), EDCI (0.58 g, 3.0 mmol), and HOBt (54 mg, 0.4 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by TLC. After the reaction was complete, the mixture was extracted with CHCl (30 mL x 2), and the organic layer was washed with brine (20 mL), dried over NaSO, filtered, and concentrated in vacuo. The crude reaction mixture was purified by silica gel column to give product 63 (1.2 g, 99%) as a colorless oil. C 29 H 55 N4O 11 [M+H] + The calculated HRMS (ESI) value was 635.3867 and the found value was 635.3860.

[0271] Compound 64: Compound 63 (0.87 g, 1.37 mmol) was dissolved in CH2Cl2 (10 mL) followed by the addition of TFA (4.0 mL). The mixture was stirred at room temperature for 2 h. The solvent was removed in vacuo as much as possible at <35 °C. The residue was purified on a silica gel column to give the product 64 (0.62 g, 86%) as a colorless oil. 21 H 39 N4O 11 [M+H] + The calculated HRMS (ESI) value was 523.2615 and the found value was 523.2607.

[0272] Compound 65: To a stirred solution of compound 64 (0.6 g, 1.15 mmol) in anhydrous DMF (20 mL) was added Val-Cit-PAB-DEA-SN38(10) (2.3 g, 2.5 mmol), EDCI (0.66 g, 3.5 mmol), and HOBt (90 mg, 0.7 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. After the reaction was complete, the mixture was concentrated in vacuo. The reaction mixture was cooled to 0° C. and added to methyl tert-butyl ether (100 mL). The resulting slurry was stirred for 1 h, filtered, washed, and dried in vacuum. The crude was purified on a silica gel column to give compound 65 (1.2 g, 45%) as a white solid. MS (ESI) m / z [M+2H] 2+ 1155.92, [M+2Na] 2+ 1177.87

[0273] Compound 66: To a stirred solution of compound 64 (43 mg, 0.08 mmol) in a mixture of anhydrous CHCl (2 mL) and DMF (2 mL) was added compound 58 (0.42 g, 0.21 mmol), EDCI (47 mg, 0.25 mmol), and HOBt (5.4 mg, 0.04 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. After the reaction was complete, the mixture was concentrated in vacuo. The crude reaction mixture was purified by preparative HPLC using a Welch Ultimate XB-C18 column (eluent: A=0.1% TFA in water, B=MeCN) to give product 66 (230 mg, 61%) as a pale yellow solid. MS (ESI) m / z [M+3H] 3+ 1528.62, [M+4H] 4+ 1146.87

[0274] Example 13. 30 km PEG-Lys(PEG 2 Preparation of -Mal)-DBCO (Compound 68, FIG. 20) Compound 67: To a stirred solution of compound 19 (5.5 g, 0.18 mmol) in anhydrous CHCl (55 mL) was added DIPEA (473 mg, 3.6 mmol) at 0 °C, followed by NHS-PEG2-Mal (0.2 g, 0.47 mmol). The mixture was stirred at 0 °C for 1.5 h, then the solution was slowly warmed from 0 °C to room temperature, followed by stirring under argon atmosphere overnight. The solvent was removed and the residue was recrystallized from CHCl / methyl tert-butyl ether (13.8 mL / 110 mL). The separated solid was recrystallized again from MeCN / 2-propanol (11 mL / 165 mL). The residue was dried in vacuum to give product 67 (5.0 g, 90%) as a white powder.

[0275] Compound 68: To a stirred solution of compound 67 (3.0 g, 0.1 mmol) in anhydrous CHCl (30 mL) was added DBCO-NH (83 mg, 0.3 mmol), EDCI (115 mg, 0.6 mmol), and HOBt (122 mg, 0.9 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. The solvent was removed and the residue was recrystallized from CHCl / methyl tert-butyl ether (5 mL / 40 mL). The separated solid was recrystallized again from MeCN / 2-propanol (4 mL / 60 mL). The product was dried in vacuum at 40 °C for 4 h to give product 68 (2.7 g, 89%) as a white powder.

[0276] Example 14. 30 km PEG-Lys(PEG 2 Preparation of Val-Cit-PAB-DEA-SN38 (Compound 69, FIG. 21) Compound 69: To a stirred solution of compound 67 (0.9 g, 0.03 mmol) in a mixture of DMF / CHCl (5 mL / 5 mL) was added compound 58 (0.16 g, 0.08 mmol), EDCI (35 mg, 0.18 mmol), and HOBt (37 mg, 0.27 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. The solvent was removed and the residue was recrystallized from CHCl / methyl tert-butyl ether (2.5 mL / 20 mL). The separated solid was recrystallized again from MeCN / 2-propanol (2 mL / 30 mL). The product was dried in vacuum at 40 °C for 4 h to give product 69 (0.7 g, 80%) as a white powder. MS (MALDI-TOF) m / z 32065.3 Da

[0277] Example 15. 30 km PEG-Lys(PEG 2 Preparation of Val-Cit-PAB-DEA-SN38 (Compound 70, FIG. 22) Compound 70: To a stirred solution of compound 68 (1.5 g, 0.05 mmol) in anhydrous CHCl (10 mL) and MeOH (10 mL) was added compound 62 (0.5 g, 0.15 mmol). The mixture was stirred at room temperature overnight until complete conversion was confirmed by HPLC. The solvent was removed and the residue was recrystallized twice from MeCN / 2-propanol (3 mL / 45 mL). The residue was dried in vacuum to give product 70 (1.1 g, 73%) as a white powder.

[0278] Example 16. 30 km PEG-Lys(PEG 2 Preparation of Val-Cit-PAB-DEA-SN38 (Compound 71, FIG. 23) Compound 71: To a stirred solution of compound 68 (1.2 g, 0.04 mmol) in anhydrous CHCl (8 mL) and MeOH (8 mL) was added compound 66 (0.37 g, 0.08 mmol). The mixture was stirred at room temperature overnight until complete conversion was confirmed by HPLC. The solvent was removed and the residue was recrystallized twice from MeCN / 2-propanol (3 mL / 45 mL). The residue was dried in vacuum to give product 71 (0.91 g, 73%) as a white powder.

[0279] Example 17. 20 km PEG-Lys(PEG 2 Preparation of Val-Cit-PAB-DEA-SN38 (Compound 73, FIG. 24) Compound 73: To a stirred solution of compound 72 (2.0 g, 0.1 mmol) (see procedure for preparation of compound 68 for preparation of compound 72) in anhydrous CHCl (20 mL) was added compound 65 at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. The solvent was removed and the residue was recrystallized twice from MeCN / 2-propanol (4 mL / 60 mL). The product was dried in vacuum at 40° C. for 4 h to give product 73 (1.6 g, 82%) as a white powder.

[0280] Example 18. Mal-PEG 2 Preparation of -20kPEG-2(Val-Cit-PAB-DEA-SN38) (Compound 76, FIG. 25) Compound 74: To a stirred solution of amine-PEG20k-CO2H (49) (1.0 g, 0.05 mmol) in anhydrous CHCl2 (10 mL) was added DIPEA (83 μL, 0.5 mmol) at 0 °C, followed by NHS-PEG2-Mal (64 mg, 0.15 mmol). The mixture was stirred at 0 °C for 1.5 h, then the solution was slowly warmed from 0 °C to room temperature, followed by stirring under argon atmosphere overnight. The solvent was removed and the residue was recrystallized from CHCl2 / methyl tert-butyl ether (2.5 mL / 20 mL). The separated solid was recrystallized again from MeCN / 2-propanol (2 mL / 30 mL). The residue was dried in vacuum to give product 74 (0.92 g, 92%) as a white powder.

[0281] Compound 75: To a stirred solution of compound 74 (0.9 g, 0.045 mmol) in anhydrous CHCl (9 mL) was added DBCO-NH (37 mg, 0.14 mmol), EDCI (52 mg, 0.27 mmol), and HOBt (55 mg, 0.41 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. The solvent was removed and the residue was recrystallized from CHCl / methyl tert-butyl ether (2.5 mL / 20 mL). The separated solid was recrystallized again from MeCN / 2-propanol (2 mL / 30 mL). The product was dried in vacuum at 40° C. for 4 h to give product 75 (0.77 g, 86%) as a white powder.

[0282] Compound 76: To a stirred solution of compound 75 (0.7 g, 0.035 mmol) in anhydrous CHCl (8 mL) and MeOH (8 mL) was added compound 65 (0.17 g, 0.08 mmol). The mixture was stirred at room temperature overnight until complete conversion was observed by HPLC. The solvent was removed and the residue was recrystallized twice from MeCN / 2-propanol (2 mL / 30 mL). The residue was dried in vacuum to give product 76 (0.57 g, 81%) as a white powder.

[0283] Example 19. Preparation of Val-Cit-PAB-DEA-Dxd (Compound 81, FIG. 26) Compound 77: tert-Butyl 2-hydroxyacetate (2.0 g, 15.0 mmol) was added to a mixture of bis(4-nitrophenyl)carbonate (4.6 g, 15.0 mmol) and triethylamine (5.2 mL, 37.5 mmol) in 75 mL DMF at 0°C. After the mixture was stirred at room temperature for 2 h, compound 6 (1.7 g, 9.0 mmol) was added to the solution. The mixture was stirred at room temperature for another 2 h. The product was extracted with CHCl (100 mL x 3), and the organic layer was washed with water, dried over NaSO and concentrated. The crude was purified by column chromatography to give compound 77 (2.4 g, 76%) as a colorless oil. MS (ESI) m / z [M+Na] + 369.25

[0284] Compound 78: To a solution of compound 77 (2.2 g, 6.3 mmol) in anhydrous CH2Cl2 (20 mL) was added TFA (4.5 mL). The mixture was stirred at room temperature for 2 h. The solvent was removed in vacuo as much as possible at <35 °C. The residue was recrystallized from hexane (45 mL). The isolated product was dried in vacuo to give product 78 (1.6 g, 89%) as a white solid. MS (ESI) m / z [M+H] + 191.30

[0285] Compound 79: Compound 78 (3.3 g, 11.6 mmol) and compound 5 (3.6 g, 4.6 mmol) were dissolved in DMF (50 mL). HOBt (1.2 g, 9.2 mmol) and pyridine (2.5 mL) were then added and the reaction mixture was stirred at room temperature for 4 h until the reaction was complete. The reaction mixture was cooled to 0° C. and added to methyl tert-butyl ether (80 mL). The resulting slurry was stirred for 1 h, filtered, washed and dried in vacuum. The crude was purified by column purification to give compound 79 (2.9 g, 85%) as a pale yellow powder. MS (ESI) m / z [M+Na] + 840.43

[0286] Compound 80: To a stirred solution of exatecan mesylate (0.92 g, 1.7 mmol) and triethylamine (0.5 mL, 3.5 mmol) in anhydrous DMF (30 mL) was added compound 79 (1.4 g, 1.7 mmol) and HATU (0.83 g, 2.2 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. The mixture was concentrated in vacuo and the residue was purified on a silica gel column to give compound 80 (1.0 g, 78%) as a white solid. MS (ESI) m / z [M+H] + 1135.53, [M+Na] + 1157.43

[0287] Compound 81: Diethylamine (6.0 mL) was added to a solution of compound 80 (2.5 g) in DMF (30 mL), and the reaction was then allowed to proceed at room temperature for 50 min. The reaction mixture was concentrated in vacuo, and the resulting residue was slurried in methyl tert-butyl ether (90 mL) for 2 h. The solid was filtered, washed, and dried in vacuo. The crude was purified on a silica gel column to give compound 81 (1.6 g, 73%) as a white solid. MS (ESI) m / z [M+H] + 1013.63, [M+Na] + 1035.58

[0288] Example 20. 20 km PEG-Lys(PEG 2 Preparation of -Mal)-2(Val-Cit-PAB-DEA-Dxd) (Compound 83, FIG. 27) Compound 82: To a stirred solution of compound 64 (80 mg, 0.15 mmol) in CHCl / DMF (5 mL / 5 mL) was added Val-Cit-PAB-DEA-Dxd (81) (334 mg, 0.33 mmol), EDCI (92 mg, 0.48 mmol), and HOBt (20 mg, 0.15 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was confirmed by HPLC. After the reaction was complete, the mixture was concentrated in vacuo. The residue was recrystallized from CHCl / methyl tert-butyl ether to give compound 82 (360 mg, 93%) as a white solid. MS (ESI) m / z [M+2H] 2+ 1257.83, [M+2Na]2+ 1279.88

[0289] Compound 83: To a stirred solution of compound 68 (1.8 g, 0.06 mmol) in anhydrous CHCl (8 mL) and MeOH (8 mL) was added compound 82 (350 mg, 0.14 mmol). The mixture was stirred at room temperature overnight until complete conversion was confirmed by HPLC. The solvent was removed and the residue was recrystallized twice from MeOH / 2-propanol (5 mL / 40 mL). The residue was dried in vacuum to give product 83 (1.5 g, 81%) as a white powder.

[0290] Example 21. Preparation of SCAHer2(1)xSCAHer2(2) (Compound 84) Compound 84: Compound 84 was prepared using a procedure similar to the preparation of compound 52. The amino acid sequence of SCAHer2(1)xSCAHer2(2) (SEQ ID NO:2) is as follows: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTGGSGGSGGSGGSGGEVQ LVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGCGSGGGSGGSG GSGGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGG SGGSGGSGGSGGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTHHHHHH

[0291] Example 22. Preparation of SCAc-Met(1)xSCAc-Met(2) (Compound 85) Compound 85: Compound 85 was prepared using a procedure similar to the preparation of compound 52. The amino acid sequence of SCAc-Met(1)xSCAc-Met(2) (SEQ ID NO:6) is as follows: DIMMSQSPSSLTVSVGEKVTVSCKSSQSLLYTSSQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTITSVKADDLAVYYCQQYYAYPWTFGGGTKLEIKGGSGGSGGSGGSGGQV QLQQSGPELVRPGASVKMSCRASGYTFTSYWLHWVKQRPGQGLEWIGMIDPSNSDTRFNPNFKDKATLNVDRSSNTAYMLLSSLTSADSAVYYCATYGSYVSPLDYWGQGTSVTVSSGCGSGGSGGSGGS GGQVQLVESGPGLVKPSQTLSLTCAVSGGSITTNYYYWSWIRQSPGKGLEWMGVIAYDGSTDYSPSLKSRTSISRDTSKNQFSLQLSSVTPEDTAVYYCARDVRVIATGWATANALDAWGQGTLVTVSSG GSGGSGGSGGSGGQSVLTQPPSVSGSPGKTVTISCAGTSSDVGYGNYVSWYQQLPGTAPKLLIFAVSYRASGIPDRFSGSKSGNTAFLTISGLQSEDEADYYCASYRSSNNAAVFGGGTHLTVLHHHHHH

[0292] Example 23. Preparation of 30 kmPEG(SCAHer2(1)xSCAHer2(2))-2(Val-Cit-PAB-DEA-SN38) (Compound 86, FIG. 28) Protein SCAHer2(1)xSCAHer2(2)(84) (20 mg) was treated with reducing agent 2 mM TCEP in PBS buffer (pH=7.4) for 30 min at room temperature, and then the pH was adjusted with a stock solution of 500 mM sodium phosphate buffer at pH=4.12. Before PEGylation, the treated protein was concentrated to 5 mg / mL. PEGylation of SCAHer2(1)xSCAHer2(2) was carried out at room temperature for 3 h with 2-3 equivalents of compound 69. The reaction was quenched with 10 mM L-cystine for 10 min at room temperature. The final product compound 86 was purified using hydroxyapatite HA (TOSOH) in 20 mM sodium phosphate buffer at pH 6.8. Target compound 86 was confirmed by SEC-HPLC and cell-based activity assays.

[0293] Example 24. Preparation of 30 kmPEG(SCAPDL1xSCACD47)-2(Val-Cit-PAB-DEA-SN38) (Compound 87, FIG. 29) Compound 87 was prepared by conjugating compound 69 [30 kmPEG-Lys(PEG2-Mal)-2(Val-Cit-PAB-DEA-SN38)] with protein 52 using a procedure similar to the preparation of compound 86.

[0294] Example 25. Preparation of 30 kmPEG(SCAHer2(1)xSCAHer2(2))-3(Val-Cit-PAB-DEA-SN38) (Compound 88, FIG. 30) Compound 88 was prepared by conjugating compound 70 [30kmPEG-Lys(PEG2-Mal)-3(Val-Cit-PAB-DEA-SN38)] with protein 84 using a procedure similar to the preparation of compound 86.

[0295] Example 26. Preparation of 30 kmPEG(SCAPDL1xSCACD47)-3(Val-Cit-PAB-DEA-SN38) (Compound 89, FIG. 31) Compound 89 was prepared by conjugating compound 70 [30kmPEG-Lys(PEG2-Mal)-3(Val-Cit-PAB-DEA-SN38)] with protein 52 using a procedure similar to the preparation of compound 86.

[0296] Example 27. Preparation of 30 kmPEG(SCAPDL1xSCACD47)-4(Val-Cit-PAB-DEA-SN38) (Compound 90, FIG. 32) Compound 90 was produced by conjugating compound 71 [30 kmPEG-Lys(PEG2-Mal)-4(Val-Cit-PAB-DEA-SN38)] with protein 52 using a procedure similar to the preparation of compound 86.

[0297] Example 28. Preparation of 20 kmPEG(SCAPDL1xSCACD47)-2(Val-Cit-PAB-DEA-SN38) (Compound 91, FIG. 33) Compound 91 was prepared by conjugating compound 73 [20kmPEG-Lys(PEG2-Mal)-2(Val-Cit-PAB-DEA-SN38)] with protein 52 using a procedure similar to the preparation of compound 86.

[0298] Example 29. Preparation of SCAPDL1xSCACD47-20kPEG-2(Val-Cit-PAB-DEA-SN38) (Compound 92, FIG. 34) Compound 92 was produced by conjugating compound 76 [(PEG2-Mal)-20kPEG-2(Val-Cit-PAB-DEA-SN38)] with protein 52 using a procedure similar to the preparation of compound 86.

[0299] Example 30. Preparation of 30kmPEG(SCAPDL1xSCACD47)-2(Val-Cit-PAB-DEA-Dxd) (Compound 93, FIG. 35) Compound 93 was produced by conjugating compound 83 [30 kmPEG-Lys(PEG2-Mal)-2(Val-Cit-PAB-DEA-Dxd)] with protein 52 using a procedure similar to the preparation of compound 86.

[0300] Example 31. Preparation of 30 kmPEG(SCAc-Met(1)xSCAc-Met(1))-2(Val-Cit-PAB-DEA-SN38) (Compound 94, FIG. 36) Compound 94 was prepared by conjugating compound 69 [30 kmPEG-Lys(PEG2-Mal)-2(Val-Cit-PAB-DEA-SN38)] with protein 85 using a procedure similar to the preparation of compound 86.

[0301] Example 32. In vitro cytotoxicity of Compound 86 and Compound 88 against tumor cell lines (Figure 37) To verify the cytotoxic activity, the HER2 expression positive tumor cell line BxPC-3 was selected for in vitro viability analysis. The cells were cultured at 3 × 10 5Cells / well were seeded in 96-well plates and treated with Compound 86 and Compound 88 at the indicated doses. Cell viability was measured with Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. Cytotoxicity % was calculated according to the following formula: % cytotoxicity = (1-OD sample / OD control) x 100%. Data was analyzed with GraphPad Prism software and presented as % proliferation inhibition relative to untreated control. Results are shown in Figure 37.

[0302] Example 33. In vitro cytotoxicity of compounds 87, 89, 90, 91, and 92 against tumor cell lines (Figure 38) To verify cytotoxic activity, a series of CD47 / PD-L1-expressing tumor cell lines, BxPC-3, NCIH661, NCIH520, and HS746T, were selected for in vitro viability analysis. Cells were cultured at 3 × 10 5 Cells / well were seeded in 96-well plates and treated with compounds 87, 89, 90, 91, and 92 at the indicated doses. Cell viability was measured with Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. Cytotoxicity % was calculated according to the following formula: % cytotoxicity = (1-OD sample / OD control) x 100%. Data were analyzed with GraphPad Prism software and presented as % proliferation inhibition relative to untreated control. Results are shown in Figure 38.

[0303] Example 34. In vitro cytotoxicity of compound 93 against tumor cell lines (Figure 39) To verify cytotoxic activity, a series of CD47 / PD-L1-expressing tumor cell lines, BxPC-3, NCIH661, HS746T, U87.MG, T74D, and Calu6, were selected for in vitro viability analysis. Cells were cultured at 3 × 10 5Cells / well were seeded in 96-well plates and treated with Compound 93 at the indicated doses. Cell viability was measured with Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. Cytotoxicity % was calculated according to the following formula: % cytotoxicity = (1-OD sample / OD control) x 100%. Data were analyzed with GraphPad Prism software and presented as % proliferation inhibition relative to untreated control. Results are shown in Figure 39.

[0304] Example 35. In vitro cytotoxicity of compound 94 against tumor cell lines (Figure 40) To verify the cytotoxic activity, the c-MET expression positive tumor cell line BxPC-3 was selected for in vitro viability analysis. Cells were cultured at 3 × 10 5 Cells / well were seeded in 96-well plates and treated with Compound 94 at the indicated doses. Cell viability was measured with Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. Cytotoxicity % was calculated according to the following formula: % cytotoxicity = (1-OD sample / OD control) x 100%. Data were analyzed with GraphPad Prism software and presented as % proliferation inhibition relative to untreated control. Results are shown in Figure 40.

Claims

1. Compound of formula (Ib): 【Chemistry 1】 [In the formula, P is a non-immunogenic polymer; M is H or C １－５０ an end-capping group selected from alkyl and aryl, where one or more carbons of said alkyl may be optionally replaced with a heteroatom; y is an integer selected from 1 to 10; A is an antibody or antigen-binding fragment thereof; T is a multifunctional small molecule linker moiety; L １ and L ２ are each independently a hetero- or homobifunctional linker; a and b are each an integer selected from 0 to 10; B is a branched linker, where each branch has an amino acid sequence or carbohydrate moiety or disulfide bond attached to one or more self-immolating spacers, where cleavage of said amino acid sequence or carbohydrate moiety or disulfide bond by an enzyme triggers a self-immolative mechanism to release a hydroxyl-containing drug D, or each branch has a scissile bond, where cleavage of said scissile bond releases the hydroxyl-containing drug D; each D is independently a cytotoxic hydroxyl-containing small molecule or peptide, wherein the hydroxyl group of said D is attached to B; and n is an integer selected from 1 to 25.

2. T is a trifunctional linker derived from a molecule having three functional groups independently selected from hydroxyl, amino, hydrazinyl, azide, alkene, alkyne, carboxyl (aldehyde, ketone, ester, carboxylic acid, anhydride, acid halide), thiol, disulfide, nitrile, epoxide, imine, nitro, and halide, where T and (L １ ) ａ and T and (L ２ ) ｂ The compound of claim 1 , wherein the bonds to are the same or different.

3. 3. The compound of claim 2, wherein T is 1,3-diamino-2-propanol, triethanolamine, lysine, aspartic acid, glutamic acid, serine, or tyrosine.

4. (L １ ) ａ 4. The compound according to claim 1, wherein one of the functional groups at the linker terminus of is capable of site-specific conjugation with A and is selected from the group consisting of thiol, maleimide, 2-pyridyldithio variants, aromatic or vinyl sulfone, acrylate, bromo or iodoacetamide, azide, alkyne, dibenzocyclooctyl (DBCO), carbonyl, 2-amino-benzaldehyde or 2-amino-acetophenone group, hydrazide, oxime, potassium acyltrifluoroborate, O-carbamoylhydroxylamine, transcyclooctene, tetrazine, triarylphosphine, boronic acid, and iodine.

5. The compound according to any one of claims 1 to 4, wherein the antibody is a monospecific or multispecific full-length antibody, a monospecific or multispecific single-chain antibody, a monospecific or multispecific nanobody (single domain antibody), or a monospecific or multispecific antigen-binding domain thereof.

6. The compound according to any one of claims 1 to 5, wherein the antibody is a monospecific single chain antibody.

7. 7. The compound of claim 6, wherein the monospecific single chain antibody binds to a tumor associated antigen (TAA), such as Her2, cMet, PDL1, or CD47.

8. The compound of claim 7 , wherein the monospecific single chain antibody has two binding domains that bind to Her2.

9. The compound according to claim 8 , wherein the monospecific single chain antibody has the amino acid sequence shown in SEQ ID NO:

3.

10. The compound according to any one of claims 1 to 5, wherein the antibody is a bispecific antibody, such as a bispecific single chain antibody.

11. The compound of claim 10, wherein the two binding domains of the bispecific antibody bind to the same tumor-associated antigen (TAA), to two different TAAs, or to a TAA and an antigen expressed on T cells or NK cells (e.g. a component of the T cell receptor).

12. 12. The compound of claim 11 , wherein the antibody is an anti-PDL1 x anti-CD47 single chain bispecific antibody.

13. The compound of claim 12 , wherein the antibody has the amino acid sequence set forth in SEQ ID NO:

1.

14. 12. The compound of claim 11 , wherein the antibody is an anti-HER2(1) x anti-HER2(2) single chain bispecific antibody.

15. The compound of claim 14 , wherein the antibody has the amino acid sequence set forth in SEQ ID NO:

2.

16. 12. The compound of claim 11 , wherein the antibody is an anti-cMet(1) x anti-cMet(2) single chain bispecific antibody.

17. The compound of claim 16 , wherein the antibody has the amino acid sequence set forth in SEQ ID NO:

6.

18. The two binding domains of the monospecific single chain antibody are linked via a peptide linker, wherein the linker is １ ) ａ 10. The compound of any one of claims 6 to 9, comprising a cysteine ​​or unnatural amino acid residue for site-specific conjugation to.

19. The two binding domains of the bispecific single chain antibody are linked via a peptide linker, wherein the linker is １ ) ａ 18. The compound of any one of claims 10 to 17, comprising a cysteine ​​or unnatural amino acid residue for site-specific conjugation to.

20. The unnatural amino acid residue may be a genetically encoded alkene lysine, such as N6-(hex-5-enoyl)-L-lysine, 2-amino-8-oxononanoic acid, m- or p-acetylphenylalanine, an amino acid containing a β-diketone side chain, such as 2-amino-3-(4-(3-oxobutanoyl)phenyl)propanoic acid, (S)-2-amino-6-(((1R,2R)-2-azidocyclopentyloxy)carbonylamino)hexanoic acid, azidohomoalanine, the pyrrolidine analog N6-((prop-2-yn-1-yloxy)carbonyl)-L-lysine, (S)-2-amino-6-pent-4-ynamidohexanoic acid, (S)-2-amino-6-((prop-2-ynyl) 20. The compound of claim 18 or 19, wherein the compound is selected from the group consisting of Nε-acryloyl-l-lysine, Nε-5-norbornen-2-yloxycarbonyl-l-lysine, N-ε-(cyclooct-2-yn-1-yloxy)carbonyl)-L-lysine, N-ε-(2-(cyclooct-2-yn-1-yloxy)ethyl)carbonyl-L-lysine, and genetically encoded tetrazine amino acids such as 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine.

21. 21. The compound of any one of claims 1 to 20, wherein the hydroxyl-containing drug D is selected from a DNA crosslinking agent, a microtubule inhibitor, a DNA alkylating agent, a topoisomerase inhibitor, a protein degrading drug, a STING agonist, or a combination thereof.

22. The hydroxyl-containing drug D is selected from the group consisting of vinca alkaloids, laulimalide, colchicine, tubulysin, cryptophycin, hemiasterin, cemadotin, rhizoxin, discodermolide, taccalonolide A or B or AF or AJ, taccalonolide AI-epoxide, CA-4, epothilone A and B, taxanes, paclitaxel, docetaxel, epothilone, iSGD-1882, centanamycin, PNU-159682, uncialamycin, uncialamycin, indolinobenzodiazepine dimer, β-amanitin, amatoxin, thailanstatin, calicheamicin, anthracycline, daunomycin, larotaxel, tesetaxel, ortataxel, CC-1065, Dxd, SN38, topotecan, CPT-11, camptothecin, rubitecan, bryostatin, kallistatin, biceresin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin ( sarcodictyin), spongistatin, estramustine, prednimustine, chlorozotocin, ranimustine, calicheamicin, dynemicin, esperamicin, neocarzinostatin chromophore, aclacinomycin, azithromycin, bleomycin, caminomycin, carzinophilin, chromomycin, daunorubicin, detorubicin, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, myco Phenolic acid, nogalamycin, peplomycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, fludarabine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine (cytosine arabinoside, ara-C), gemcitabine, capecitabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calsterone, epithiostanol, trilostane,22. The compound of claim 21, selected from elliptinium acetate, maytansinoids, ansamitocins, mitoxantrone, mopidamol, pentostatin, pirarubicin, etoposide, podophyllotoxin, rhizoxin, tenuazonic acid, T-2 mycotoxin, verracurin, roridin A, anguidine, vindesine, mannomustine, mitobronitol, mitolactol, vinblastine, mitoxantrone, vincristine, vinorelbine, teniposide, xeloda, raloxifene, 4-hydroxytamoxifen, estradiol, trioxyphene, keoxifene, LY117018, onapristone, bicalutamide, leuprolide, goserelin, or a pharma- ceutically acceptable salt, acid, or derivative thereof, or a combination thereof.

23. 22. The compound of claim 21, wherein D is selected from duocarmycin, Dxd, SN38, topotecan, CPT-11, camptothecin, rubitecan, or derivatives thereof, or combinations thereof.

24. The compound according to any one of claims 1 to 23, wherein the non-immunogenic polymer is polyethylene glycol (PEG).

25. 25. The compound of claim 24, wherein the PEG is a linear PEG or a branched PEG.

26. 26. The compound of claim 24 or 25, wherein at least one end of the PEG is capped with methyl or a low molecular weight alkyl.

27. The compound according to any one of claims 24 to 26, wherein the total molecular weight of the PEG is from 3,000 to 100,000.

28. 28. The compound of any one of claims 24 to 27, wherein the PEG is attached to the tri- or tetra-functional or other cyclic or acyclic multifunctional moiety T (e.g., lysine) via a permanent or cleavable bond.

29. Compound of formula (Ic): 【Chemistry 2】 [In the formula, P is a linear PEG; A is an antibody or antigen-binding fragment thereof; L １ and L ２ are each independently a bifunctional linker; a and b are each an integer selected from 0 to 10; B is a branched linker, where each branch has an amino acid sequence or carbohydrate moiety or disulfide bond attached to one or more self-immolating spacers, where cleavage of said amino acid sequence or carbohydrate moiety or disulfide bond by an enzyme triggers a self-immolative mechanism to release the hydroxyl-containing drug D, or each branch has a scissile bond, where cleavage of said scissile bond releases the hydroxyl-containing drug D or a derivative thereof; each D is independently a cytotoxic hydroxyl-containing small molecule or peptide, wherein the hydroxyl group of said D is attached to B; and n is an integer selected from 1 to 25.

30. (L １ ) ａ 30. The compound of claim 29, wherein one of the functional groups at the linker terminus of is capable of site-specific conjugation with A and is selected from the group consisting of thiol, maleimide, 2-pyridyldithio variants, aromatic or vinyl sulfone, acrylate, bromo or iodoacetamide, azide, alkyne, dibenzocyclooctyl (DBCO), carbonyl, 2-amino-benzaldehyde or 2-amino-acetophenone group, hydrazide, oxime, potassium acyltrifluoroborate, O-carbamoylhydroxylamine, transcyclooctene, tetrazine, triarylphosphine, boronic acid, and iodine.

31. 31. The compound of claim 29 or 30, wherein the antibody is a monospecific or multispecific full-length antibody, a monospecific or multispecific single-chain antibody, a monospecific or multispecific nanobody (single domain antibody), or a monospecific or multispecific antigen-binding domain thereof.

32. 32. The compound of claim 31 , wherein the antibody is a monospecific single chain antibody, optionally wherein the monospecific single chain antibody binds to a tumor associated antigen (TAA), such as Her2, cMet, PDL1, or CD47.

33. 33. The compound of claim 32, wherein the monospecific single chain antibody has two binding domains that bind to Her2.

34. The compound of claim 33, wherein the monospecific single chain antibody has the amino acid sequence shown in SEQ ID NO:

3.

35. 32. The compound of claim 31 , wherein the antibody is a bispecific antibody, such as a bispecific single chain antibody.

36. The compound of claim 35, wherein the two binding domains of the bispecific antibody bind to the same tumor-associated antigen (TAA), to two different TAAs, or to a TAA and an antigen expressed on T cells or NK cells (e.g. a component of the T cell receptor).

37. 37. The compound of claim 36, wherein the antibody is an anti-PDL1x anti-CD47 single chain bispecific antibody.

38. The compound of claim 37, wherein the antibody has the amino acid sequence set forth in SEQ ID NO:

1.

39. 37. The compound of claim 36, wherein the antibody is an anti-HER2(1) x anti-HER2(2) single chain bispecific antibody.

40. The compound of claim 39 , wherein the antibody has the amino acid sequence set forth in SEQ ID NO:

2.

41. 37. The compound of claim 36, wherein the antibody is an anti-cMet(1) x anti-cMet(2) single chain bispecific antibody.

42. The compound of claim 41 , wherein the antibody has the amino acid sequence set forth in SEQ ID NO:

6.

43. The two binding domains of the monospecific single chain antibody are linked via a peptide linker, wherein the linker is １ ) ａ 35. The compound of any one of claims 33 to 34, comprising a cysteine ​​or unnatural amino acid residue for site-specific conjugation to.

44. The two binding domains of the bispecific single chain antibody are linked via a peptide linker, wherein the linker is １ ) ａ 43. The compound of any one of claims 37 to 42, comprising a cysteine ​​or unnatural amino acid residue for site-specific conjugation to.

45. The unnatural amino acid residue may be a genetically encoded alkene lysine, such as N6-(hex-5-enoyl)-L-lysine, 2-amino-8-oxononanoic acid, m- or p-acetylphenylalanine, an amino acid containing a β-diketone side chain, such as 2-amino-3-(4-(3-oxobutanoyl)phenyl)propanoic acid, (S)-2-amino-6-(((1R,2R)-2-azidocyclopentyloxy)carbonylamino)hexanoic acid, azidohomoalanine, the pyrrolidine analog N6-((prop-2-yn-1-yloxy)carbonyl)-L-lysine, (S)-2-amino-6-pent-4-ynamidohexanoic acid, (S)-2-amino-6-((prop-2-ynyl) 45. The compound of claim 43 or 44, wherein the compound is selected from the group consisting of Nε-acryloyl-l-lysine, Nε-5-norbornen-2-yloxycarbonyl-l-lysine, N-ε-(cyclooct-2-yn-1-yloxy)carbonyl)-L-lysine, N-ε-(2-(cyclooct-2-yn-1-yloxy)ethyl)carbonyl-L-lysine, and genetically encoded tetrazine amino acids such as 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine.

46. 46. ​​The compound of any one of claims 33 to 45, wherein the hydroxyl-containing drug D is selected from a DNA crosslinking agent, a microtubule inhibitor, a DNA alkylating agent, a topoisomerase inhibitor, a protein degrading drug, a STING agonist, or a combination thereof.

47. D is a vinca alkaloid, laulimalide, colchicine, tubulysin, cryptophycin, hemiasterin, cemadotin, rhizoxin, discodermolide, taccalonolide A or B or AF or AJ, taccalonolide AI-epoxide, CA-4, epothilone A and B, taxane, paclitaxel, docetaxel, epothilone, iSGD-1882, centanamycin, PNU-159682, uncialamycin ), indolinobenzodiazepine dimer, β-amanitin, amatoxin, thailanstatin, calicheamicin, anthracycline, daunomycin, larotaxel, tesetaxel, ortataxel, CC-1065, Dxd, SN38, topotecan, CPT-11, camptothecin, rubitecan, bryostatin, kallistatin, biceresin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spon Distatin, estramustine, prednimustine, chlorozotocin, ranimustine, calicheamicin, dynemicin, esperamicin, neocarzinostatin chromophore, aclacinomycin, azithromycin, bleomycin, caminomycin, carzinophilin, chromomycin, daunorubicin, detorubicin, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mycophenolic acid, nogalamycin, peplomycin cin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, fludarabine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine (cytosine arabinoside, ara-C), gemcitabine, capecitabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calsterone, epithiostanol, trilostane, elliptinium acetate,47. The compound of claim 46, selected from maytansinoids, ansamitocins, mitoxantrone, mopidamol, pentostatin, pirarubicin, etoposide, podophyllotoxin, rhizoxin, tenuazonic acid, T-2 mycotoxin, verracurin A, roridin A, anguidine, vindesine, mannomustine, mitobronitol, mitolactol, vinblastine, mitoxantrone, vincristine, vinorelbine, teniposide, xeloda, raloxifene, 4-hydroxytamoxifen, estradiol, trioxyphene, keoxifene, LY117018, onapristone, bicalutamide, leuprolide, goserelin, or a pharma- ceutically acceptable salt, acid, or derivative thereof, or a combination thereof.

48. 47. The compound of claim 46, wherein D is selected from duocarmycin, Dxd, SN38, topotecan, CPT-11, camptothecin, rubitecan, or derivatives thereof, or combinations thereof.

49. The compound according to any one of claims 33 to 48, wherein the total molecular weight of the PEG is from 3,000 to 100,000 Daltons.

50. L １ and L ２ However, each independently, -(CH ２ ) ａ XY(CH ２ ) ｂ -、 -X(CH ２ ) ａ O(CH ２ CH ２ O) ｃ (CH ２ ) ｂ Y-、 - (CH ２ ) ａ Heterocyclyl, -(CH ２ ) ａ X-、 -X(CH ２ ) ａ Y-、 -W １ -(CH ２ ) ａ C(O)NR １ (CH ２ ) ｂ O(CH ２ CH ２ O) ｃ (CH ２ ) ｄ C(O)-、 -C(O)(CH ２ ) ａ O (CH ２ CH ２ O) ｂ (CH ２ ) ｃ W ２ C(O)(CH ２ ) ｄ N.R. １ - and -W ３ -(CH ２ ) ａ C(O)NR １ (CH ２ ) ｂ O(CH ２ CH ２ O) ｃ (CH ２ ) ｄ W ２ C(O)(CH ２ ) ｅ C(O)- is selected from the group consisting of wherein a, b, c, d, and e are each independently an integer selected from 0 to 25; X and Y are each independently C(═O), NR ２ ,S,O,N3,CR ３ R ４、 R is selected from a DBCO-based moiety, or nothing; １ , R ２ , R ３ , and R ４ are each independently hydrogen, C １－１０ Alkyl, or (CH ２ ) １－１０ C(=O); W １ and / or W ３ is derived from the maleimide-based moiety, and W ２ represents a triazolyl- or tetrazolyl-containing group; and the heterocyclyl group is selected from a maleimide-derived moiety or a tetrazolyl- or triazolyl-based moiety.

51. (L １ ) ａ and (L ２ ) ｂ However, each independently, 【Chemistry 3】 is selected from 50. The compound of any one of claims 1 to 49, wherein i, m, and n are each independently an integer selected from 0 to 20.

52. 52. The compound of any one of claims 1 to 51, wherein the branched linker B comprises an extended spacer (optional), a trigger unit, one or more self-immolating spacers, or any combination thereof, wherein the trigger unit is an amino acid sequence cleavable by an enzyme (e.g., cathepsin B, plasmin, matrix metalloproteinase (MMP), β-glucuronidase, β-galactosidase, etc.) or a β-glucoronide or β-galactoside trigger moiety, a pH-sensitive linker capable of releasing the hydroxyl-containing drug D or its derivative under acidic pH conditions, or a disulfide bond linker capable of triggering the release of the hydroxyl-containing drug D or its derivative by glutathione, a thioredoxin family member (WCGH / PCK), or a thioreductase.

53. The branched linker B is 【Chemistry 4】 [In the formula, a, b, c, d, e, and f are each independently an integer selected from 1 to 25; (A) ｎ is a trigger unit of an amino acid sequence such as Val-Cit, Val-Ala, Val-Lys, Phe-Lys, Phe-Cit, Phe-Arg, Phe-Ala, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, D-Phe-Phe-Lys, Phe-Phe-Lys, Gly-Phe-Lys, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, or Ala-Leu-Ala-Leu; PAB is paraaminobenzyl alcohol; EDA is -NR １ (CH ２ ) ｍ N.R. ２ -, where m is 2 or 3, and R １ and R ２ are each independently H, a low molecular weight alkyl, or -(CH ２ CH ２ O) ｌ -CH ３ where l is an integer selected from 1 to 10; Each of Ex is as follows: -NR １ (CH ２ ) ｘ O(CH ２ CH ２ O) ｙ (CH ２ ) ｚ C(O)-、 -C(O)(CH ２ ) ｘ NR １ -、 -NR １ (CH ２ ) ｘ O(CH ２ CH ２ O) ｙ (CH ２ ) ｚ NR ２ -、 -NR １ (CH) ２ ) ｘ NR ２ -、 -NR １ (CH ２ ) ｘ O(CH ２ CH ２ O) ｙ (CH ２ ) ｚ O-、 -O(CH ２ ) ｘ NR １ -、 -C(O)(CH ２ ) ｘ O-、 -O(CH ２ ) ｘ O(CH ２ CH ２ O) ｙ (CH ２ ) ｚ C(O)-、 -C(O)(CH ２ ) ｘ O(CH ２ CH ２ O) ｙ (CH ２ ) ｚ C(O)-、 -C(O)(CH ２ ) ｘ C(O)-、 Or nothing (wherein x, y, and z are each independently an integer selected from 0 to 25; R １ and R ２ are each independently hydrogen or C １－１０ represents an alkyl group) is an extended spacer comprising a linker chain independently selected from 53. The compound of claim 52, selected from:

54. The branched linker B is 【Chemistry 5】 The compound according to any one of claims 1 to 51, selected from:

55. below: 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 [wherein Ab is a bispecific antibody or antigen-binding fragment thereof targeting PDL1 / CD47 or HER2(1) / HER2(2) or cMet(1) / cMet(2); 【Chemistry 9】 is] or a pharma- ceutically acceptable salt thereof.

56. 56. The compound of claim 55, wherein the antibody has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:

6.

57. the below described: 【Chemistry 10】 [wherein Ab is a bispecific antibody or antigen-binding fragment thereof targeting PDL1 / CD47 or HER2(1) / HER2(2) or cMet(1) / cMet(2); 【Chemistry 11】 is] or a pharma- ceutically acceptable salt thereof.

58. 50. The compound of claim 49, wherein the antibody has an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:

6.

59. Steps below: a) preparing a non-immunogenic modified (e.g., PEGylated) hydroxyl-containing drug conjugate having a free functional group for site-specific conjugation; b) site-specifically conjugating said non-immunogenic modified (e.g., PEGylated) hydroxyl-containing drug conjugate to an antibody to provide a compound of formula (Ib) or (Ic).

59. A method for preparing a compound according to any one of claims 1 to 58, comprising:

60. 60. A pharmaceutical formulation comprising an effective amount of a compound according to any one of claims 1 to 58 and a pharma- ceutically acceptable salt, carrier, or excipient.

61. 60. The compound of any one of claims 1 to 58 for use in the treatment of a cancer selected from the group consisting of non-Hodgkin's lymphoma, B-cell acute and chronic lymphocytic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemia, T-cell lymphoma and leukemia, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, renal cancer, bladder cancer, gastric cancer, colon cancer, colorectal cancer, salivary gland cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, head and neck cancer, and endometrial cancer.

62. 60. The compound of any one of claims 1 to 58 for use in combination with an effective amount of another anti-cancer agent or an immunosuppressant in the treatment of a cancer selected from the group consisting of non-Hodgkin's lymphoma, B-cell acute and chronic lymphocytic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemia, T-cell lymphoma and leukemia, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, renal cancer, bladder cancer, gastric cancer, colon cancer, colorectal cancer, salivary gland cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, and endometrial cancer.

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