PEG-based anti-CD47 / anti-PD-L1 bispecific antibody-drug conjugate

Abstract

The antibody-drug conjugates (ADCs), particularly PEG-based bispecific antibody-drug conjugates (P-BsADCs), contain antigen-binding domains that bind to CD47 and PD-L1. Methods for preparing P-BsADCs, compositions containing P-BsADCs, and their use in the treatment of diseases are described.

Description

[Technical Field]

[0001] This international patent application claims the benefit of International Patent Application No. PCT / CN2022 / 108570, filed July 28, 2022, the entire contents of which are incorporated herein by reference for all purposes.

[0002] FIELD OF THE INVENTION The present invention relates to polyethylene glycol (PEG)-based antibody-drug conjugates (ADCs), particularly PEG-based bispecific antibody-drug conjugates (P-BsADCs) that target two different receptors on tumor cells. In particular, the present invention relates to long-acting PEGylated single-chain bispecific antibody-drug conjugates that target CD47 and PD-L1. [Background technology]

[0003] Traditional small molecule cytotoxic drugs for killing rapidly dividing cells have been widely used in cancer treatment for decades, but the nonspecific action of such drugs can also attack proliferating healthy cells, causing chemotherapy-related toxicity and side effects (Baah, S. et al. Molecules, 2021, 26). Monoclonal antibodies can more accurately distinguish tumors from healthy tissue, but are not as potent as small molecule cytotoxic drugs (Shefet-Carasso, L. et al. Drug Resist Update, 2015, 18, 36-46). Antibody-drug conjugates (ADCs) have been developed to take advantage of both potent cytotoxic drugs and the tumor antigen recognition capabilities of antibodies (Khongorzul, P. et al. Molecular Cancer Research, 2020, 18, 3-19). Currently, 12 ADCs have been approved by the FDA in the United States, and over 100 ADC candidates are currently undergoing clinical trials (Coats, S. et al. Clin. Cancer Res., 2019, 25, 5441-5448). Unfortunately, all approved ADCs exhibit severe side effects, and the doses used to achieve clinical efficacy are often very close to the maximum tolerated dose (MTD), 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). Furthermore, the toxicity profile and dose-limiting toxicity of ADCs are usually associated with the cytotoxic head group (Fu, Z. et al. Signal Transduction and Targeted Therapy, 2022, 7, 93).

[0004] There is also heritable toxicity directly related to the design and structure of ADCs. For example, ADC toxicity can result from extracellular / extratumoral target binding to Fc receptors (FcγR) or lectin receptors (e.g., mannose receptor) on normal cells (Donaghy, H. et al. MAbs, 2016, 8, 659-671). While the Fc on antibodies typically does not cause such toxicity, ADCs can kill FcγR- or mannose-expressing cells by releasing their cytotoxic payload intracellularly (Gorovits, B. et al. Cancer Immunol Immunother, 2013, 62, 217-223). Another Fc-dependent toxicity is due to ADC aggregates, which can activate Fcγ receptors on immune cells, be internalized via FcγR, and ultimately kill such target-negative cells (Aoyama, M. et al. Pharmaceutical Research, 2022, 39, 89-103). It is clear that there are multiple mechanisms for Fc-dependent ADC toxicity.

[0005] To be effective, ADCs require efficient internalization and transport to lysosomes. The efflux of internalized conventional ADCs before reaching the lysosomes offsets their internalization efficiency, partially accounting for the proximity of the MTD to the clinical dose. To overcome this issue, biparatopic ADCs have been developed to significantly increase internalization efficiency, reduce efflux, and enhance tumor suppression (DaSilva, JO et al. Clinical Cancer Research, 2022, 26, 1408-1419; DaSilva, JO et al. Molecular Cancer Therapeutics, 20, 1966-1976; Gauzy-Lazo, L. et al. SLAS Discov., 2020, 25, 843-868). Furthermore, bispecific ADCs have been developed to enhance tumor selectivity, which can manipulate multiple mechanisms of action to synergistically improve efficacy (Kast, F. et al. Nature Communications, 2021, 12, 3790; Maruani, A. Drug Discov Today Technol, 2018, 30, 55-61).

[0006] Antibody drugs, including ADCs, face several barriers that affect their intratumoral distribution. The primary mode of antibody transport within tumors is diffusion-based, which is influenced by antibody size, binding affinity, tumor microenvironment, angiogenesis, and target antigen availability (Xenaki, KT et al. Front Immunol, 2017, 8, 1287). Due to their large size, antibodies or ADCs with molecular weights of approximately 150 kd have difficulty extravasating and penetrating deep into tumor tissue. Smaller antibody fragments have been shown to significantly increase tumor biodistribution (Li, Z. et al. MAbs, 2016, 8, 113-119). Binding site barriers (BSBs) are also an obstacle to antibody penetration into tumors (Miao, L. et al. ACS Nano, 2016, 10, 9243-9258). Because high affinity between antibodies and cellular targets is the primary cause of binding site barriers, a strategy based on temporary competitive inhibition of antibody-antigen binding has shown promising results in improving the efficacy of the ADC T-DM1 in solid tumors (Bordeau, BM et al. Cancer Res, 2021, 81, 4145-4154). A study co-administering an unconjugated competing antibody with the ADC found that the effect of the binding site barrier was reduced, resulting in more uniform distribution of the ADC (Evans, R. et al. Sci Rep., 2022, 12, 7677).

[0007] In recent years, antibody therapy using anti-PD-1 or anti-PD-L1 has achieved significant clinical and commercial success. Under normal circumstances, the PD-L1 / PD-1 signaling pathway is one of the immunosuppressive mechanisms to prevent autoimmunity. Unfortunately, tumor cells have exploited this pathway to evade immune surveillance. Therefore, blocking this signaling pathway with anti-PD-1 or anti-PD-L1 antibodies and restoring immunity may be useful in cancer treatment (Han, Y. et al. Am J Cancer Res., 2020, 10, 727-742). The PD-L1 (but not PD-1) antigen is expressed on tumor cells and therefore potentially can be targeted by ADCs. PD-L1 has been reported to be highly expressed in almost all types of hematological cancers and solid tumors. For example, PD-L1 has been reported to be expressed in up to 100% of melanoma tumor samples, up to 95% of NSCLC tumors, up to 54% of RCC tumors, up to 89% of ovarian cancers, and up to 93% of multiple myeloma (Patel, SP et al. Mol Cancer Ther., 2015, 14, 847-856; Gandini, S., et al. Critical reviews in oncology / hematology, 2016, 100, 88-98). On the other hand, PD-L1 is also expressed on normal cells and tissues, such as T cells, B cells, and antigen-presenting cells, as well as in several non-lymphoid tissues. It has also been detected in cardiac endothelium, placenta, and pancreatic islets (Qin, W. et al. Front Immunol., 2019, 10, 2298). Therefore, developing conventional ADCs targeting PD-L1 may present challenges.

[0008] Furthermore, anti-PD-L1 monotherapy can restore latent antitumor immunity and result in clinical responses of 43% in melanoma and approximately 20% in advanced NSCLC2 (Mahoney, KM et al. Clin Ther., 2015, 37, 764-782; Valecha, GK et al. Expert review of anticancer therapy, 2017, 17, 47-59; Malhotra, J. et al. Translational lung cancer research, 2017, 6, 196-211; Qiao, M. et al. Clinical lung cancer, 2017, 06.005; Emens, LA et al. European journal of cancer, 2017, 81, 116-129). Even when tumor samples are PD-L1 positive, some patients do not respond to anti-PD-L1 agents (Qiao, M. et al. Clinical lung cancer, 2017, 06.005; Emens, LA et al. European journal of cancer, 2017, 81, 116-129; Wang, Q. & Wu, X. International immunopharmacology, 46, 210-219). Some cancer patients who initially respond to anti-PD-L1 therapy subsequently develop resistance, potentially leading to disease progression after the initial response (Pathak, R. et al. Cancers (Basel), 2020, 12). The resistance mechanism has been reported to be related to additional immunosuppressive signaling or neoantigen mutations (Lei, Q. et al. Front Cell Dev Biol, 2020, 8, 672).

[0009] The CD47 / SIRPα signaling pathway is another immune checkpoint that has attracted researchers' attention in recent years. CD47 is a component of the innate immune checkpoint on tumor cells, acting as a "do not eat me" signal by interacting with the receptor signal-regulatory protein α (SIRPα) on professional phagocytes (e.g., macrophages and neutrophils). The CD47 antigen, like PD-L1, is overexpressed on tumor cells in almost all cancer types (Willingham, SB, et al., 2012. Proc Natl Acad Sci U S A. 109(17), p6662-7; Chao, MP, et al., 2012, Current opinion in immunology, 24(2), p225-232). Overexpression of CD47 is associated with poor prognosis or recurrence in clinical settings (Chan, KS, et al., 2009, Proc Natl Acad Sci USA, 106(33), p14016-21; Yuan, J., et al., 2019, Oncol Lett, 18(3), p3249-3255; Majeti, R., et al., 2009, Cell, 138(2), p286-99). Although CD47 is widely expressed at low levels on many normal cells, it is expressed at high levels on certain types of normal cells, such as T cells, NK cells, erythrocytes, and platelets (Strizova, Z., et al., 2020, Scientific reports, 10(1), p13936-13936; Olsson, M., et al., 2005, Blood, 105(9), p3577-82). This high expression level of CD47 poses a significant challenge for the development of antibody agents that block CD47 / SIRPα.

[0010] The present invention provides novel PEG-based single-chain bispecific antibody drug conjugates to address the above-mentioned problems. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention provides PEG-based bispecific antibody drug conjugates prepared by site-specific conjugation of a PEGylated drug conjugate to a bispecific antibody fragment or single-chain bispecific antibody engineered with site(s) for site-specific conjugation. [Means for solving the problem]

[0012] In one embodiment, the present invention provides a conjugate of formula I: [ka] [In the formula, P may be a non-immunogenic polymer; M is a proton or C 1~50 an end-capping group selected from alkyl and aryl, wherein one or more carbons of said alkyl may be 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, 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 each may independently be a bifunctional linker; Each of a and b may be an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; B may be a branched linker, each branch of which may have an amino acid sequence or an enzymatically cleavable trigger moiety, a pH-sensitive linker capable of releasing drug D or a derivative thereof under acidic pH conditions, or a disulfide bond linker capable of being enzymatically cleaved to release drug D; A may be any form of bispecific antibody or antigen-binding protein, including a single-chain bispecific antibody, a bispecific nanobody or other bispecific antigen-binding fragment thereof, that targets CD47 and PD-L1; D may be any cytotoxic small molecule or peptide or derivative thereof, and may be released from B either through enzymatic hydrolysis and / or autolysis mechanisms, or pH-induced hydrolysis, or any combination thereof; each 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.

[0013] Another aspect of the present invention is a conjugate of formula II: [ka] wherein each variable is as defined for Formula I. to provide.

[0014] In some embodiments, each branch of B comprises an extended spacer (optional), a trigger moiety, such as an amino acid sequence or a disulfide moiety, or a carbohydrate moiety, such as a β-glucoronide or β-galactoside, and is linked to the drug D via one or more autolytic spacers that are cleavable by, for example, cathepsin B, plasmin, matrix metalloproteinases (MMPs), glutathione, thioredoxin family members (WCGH / PCK), or thioreductase (Arunachalam, B. et. al. Proc. Natl. Acad. Sci. USA, 2000, 97, 745-750). Examples of autolytic spacers include the following: [ka] In particular, the following compounds are included: 1 , R 2 , R3 , R 4 is H or C 1~10 In such embodiments, D can be any small molecule or peptide or derivative thereof containing an active O or N or S functional group.

[0015] Other examples of one or two self-melting spacers include: [ka] where 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, and X=O, S, or N. In such embodiments, D can be any small molecule or peptide or derivative thereof containing an active OH functionality linked to an autolytic spacer.

[0016] In some embodiments, each branch of B may be a pH-sensitive linker that can release drug D or a derivative thereof under acidic pH conditions at the tumor site and / or within tumor cells. Examples of acid-sensitive linkers include, but are not limited to, the following formats: -CR 1 =N-NR 1 -, -CR 1 =NO-, -CR 1 =N-NR 2 -CO-, -N=N-CO-, -OCOO-, -NR 1 -COO-.

[0017] In some embodiments, each branch of B may be a disulfide bond linker that can be enzymatically cleaved to release the drug D or a derivative thereof at the tumor site and / or within the tumor cells.

[0018] In some embodiments, A is a single chain anti-CD47 / anti-PD-L1 bispecific antibody that binds to CD47 and PD-L1 expressed on cancer cells.

[0019] In some embodiments, D is monomethyl auristatin E (MMAE), an antimitotic drug or a derivative thereof, or SN38, a potent topoisomerase I inhibitor, or a derivative thereof, or a combination thereof.

[0020] In a further embodiment, D is MMAE, linked to an autolytic spacer, such as 4-aminobenzyl alcohol, via a trigger moiety, such as carbonate (PABC) and valine-citrulline.

[0021] In any of the above aspects and embodiments, the non-immunogenic polymer can be selected from the group consisting of polyethylene glycol (PEG), dextran, carbohydrate polymers, polyalkylene oxides, polyvinyl alcohol, hydroxypropyl methacrylamide (HPMA), and copolymers thereof. Preferably, the non-immunogenic polymer is PEG, such as branched or linear PEG. The total molecular weight of the PEG can be in the range of 3,000 to 100,000 daltons, e.g., 5,000 to 80,000, 10,000 to 60,000, and 20,000 to 40,000 daltons. The PEG can be linked to the multifunctional moiety via a permanent or cleavable bond.

[0022] (L 1 ) aThe functional group for site-specific conjugation that forms a bond between the amide and Protein A can be selected from the group consisting of thiol, maleimide, methylsulfonylpyrimidine, methylsulfonylbenzothiazole, vinylpyridine, ethyl P-ethynyl-N-(p-tolyl)phosphonamidate, 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, trans-cyclooctene, tetrazine, triarylphosphine, boronic acid, alkyne, iodine, and the like.

[0023] In some embodiments, (L 1 ) a One of the bonds may include a bond formed from an azide and an alkyne, or a methylsulfonylpyrimidine and a thiol, or a maleimide and a thiol. In some embodiments, the alkyne may be dibenzocyclooctyl (DBCO).

[0024] In some embodiments, T can be lysine, P can be PEG, y can be 1, and the alkyne can be dibenzocyclooctyl (DBCO).

[0025] In some embodiments, A may be derived from an azide-tagged bispecific antibody targeting CD47 and PD-L1, such as a single chain bispecific antibody, bispecific nanobody or other bispecific antigen-binding fragment thereof, or a combination thereof, where the azide is 1 ) a In other embodiments, Protein A may be derived from a thiol-tagged bispecific antibody targeting CD47 and PD-L1, such as a single-chain bispecific antibody, a bispecific nanobody or other bispecific antigen-binding fragment thereof, or a combination thereof, where the thiol is at the position of each (L 1) a The compound may be conjugated to maleimide or methylsulfonylpyrimidine in the following formula:

[0026] The above-described bispecific antibody drug conjugates can be produced according to a method comprising: (i) preparing a non-immunogenic polymer drug conjugate having a terminal functional group capable of site-specifically conjugating to a bispecific antibody or a modified form thereof; and (ii) site-specifically conjugating the non-immunogenic polymer drug conjugate to the bispecific antibody or a modified form thereof to form a compound of Formula I or II. In some examples, the bispecific antibody can be modified with a small molecule linker prior to the conjugation step.

[0027] The present invention also provides a pharmaceutical formulation comprising the above-described bispecific antibody drug conjugate, for example a PEGylated bispecific single chain antibody drug conjugate, and a pharmaceutically acceptable carrier.

[0028] 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 bispecific antibody drug conjugate, such as a PEGylated bispecific single chain antibody drug conjugate.

[0029] The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will become apparent from the description and from the claims.

[0030] A preferred embodiment of the present invention is as follows.

[0031] 1. Compounds of formula (I): [ka] [In the formula, P is a non-immunogenic polymer; M is a proton or C 1~50an end-capping group selected from alkyl and aryl, wherein one or more carbons of said alkyl may be replaced with a heteroatom; y is an integer selected from 1 to 10; A is a bispecific antibody or antigen-binding fragment thereof that targets two different antigens selected from a tumor-specific antigen (TSA) and a tumor-associated antigen (TAA); T is a trifunctional small molecule linker moiety; L 1 and L 2 each is independently a hetero- or homobifunctional linker; each of a and b is an integer selected from 0 to 10; B is a branched linker, each branch having an amino acid sequence or a carbohydrate moiety linked to an autolytic spacer, wherein cleavage of the amino acid sequence or carbohydrate moiety by an enzyme triggers an autolysis mechanism to release D, or each branch having a disulfide bond, wherein cleavage of the disulfide bond releases D or a derivative thereof, or each branch having a scissile bond, wherein cleavage of the scissile bond by a specific cleavage mechanism releases D; each of D is independently a cytotoxic small molecule or peptide; and and n is an integer selected from 1 to 25.

[0032] 2.L 1is capable of site-specific conjugation with A and is selected from the group consisting of thiol, maleimide, methylsulfonylpyrimidine, methylsulfonylbenzothiazole, vinylpyridine, ethyl P-ethynyl-N-(p-tolyl)phosphonamidate, 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, trans-cyclooctene, tetrazine, triarylphosphine, boronic acid, and iodine.

[0033] 3. The compound of any of embodiments 1-2, wherein the bispecific antibody is a single-chain bispecific antibody, a bispecific nanobody, or a bispecific antigen-binding domain thereof.

[0034] 4. The compound of embodiment 3, wherein the bispecific antibody comprises an antigen-binding domain that binds CD47 comprising a light chain variable region (VL) and a heavy chain variable region (VH), and an antigen-binding domain that binds PD-L1 comprising a VL and a VH.

[0035] 5. The compound of any one of embodiments 1-4, wherein the bispecific antibody is a single chain anti-CD47 / anti-PD-L1 bispecific antibody.

[0036] 6. The VL of the antigen-binding domain that binds to CD47 comprises the CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 2 to 4, respectively, and the VH of the antigen-binding domain that binds to CD47 comprises the CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 5 to 7, respectively; and The compound of embodiment 4 or 5, wherein the VL of the antigen-binding domain that binds to PD-L1 comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 8 to 10, respectively, and the VH of the antigen-binding domain that binds to PD-L1 comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 11 to 13, respectively.

[0037] 7. The VL of the antigen-binding domain that binds to CD47 comprises the amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15, and the VH of the antigen-binding domain that binds to CD47 comprises the amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16; and The compound of embodiment 6, wherein the VL of the antigen-binding domain that binds to PD-L1 comprises the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18, and the VH of the antigen-binding domain that binds to PD-L1 comprises the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17.

[0038] 8. The compound of any of embodiments 4-7, wherein the bispecific antibody has an amino acid sequence as set forth in SEQ ID NO: 1 or 14, or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 or 14.

[0039] 9. The antigen-binding domain that binds to CD47 and the antigen-binding domain that binds to PD-L1 are linked via a peptide linker or other chemical linker, and the linker is 1 9. The compound of any of embodiments 4-8, comprising a cysteine, azide, or unnatural amino acid residue for site-specific conjugation of a bispecific antibody to

[0040] 10. The compound of any one of embodiments 1-9, wherein D is selected from any DNA cross-linking agent, microtubule inhibitor, DNA alkylating agent, topoisomerase inhibitor, or combinations thereof.

[0041] 11. The compound of embodiment 10, wherein D is selected from MMAE, MMAF, SN38, DM1, DM4, a calicheamicin, a pyrrolobenzodiazepine, a duocarmycin, or a derivative thereof, or a combination thereof.

[0042] 12. The compound of any one of embodiments 1-11, wherein the non-immunogenic polymer is polyethylene glycol (PEG).

[0043] 13. The compound of embodiment 12, wherein PEG is linear PEG or branched PEG.

[0044] 14. The compound of embodiment 12 or 13, wherein at least one end of the polyethylene glycol is capped with methyl or a low molecular weight alkyl.

[0045] 15. The compound according to any one of embodiments 12 to 14, wherein the total molecular weight of PEG is 3,000 to 100,000.

[0046] 16. The compound according to any one of embodiments 12-15, wherein PEG is linked to a cyclic or acyclic trifunctional moiety T (e.g., lysine) via a permanent or cleavable bond.

[0047] 17. Compound of formula (II) [ka] [In the formula, P is a linear PEG; A is a bispecific antibody or antigen-binding fragment thereof that targets two different tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs); L 1 and L 2each is independently a bifunctional linker; each of a and b is an integer selected from 0 to 10; B is a branched linker, each branch having an amino acid sequence or a carbohydrate moiety linked to an autolytic spacer, wherein cleavage of the amino acid sequence or carbohydrate moiety by an enzyme triggers an autolysis mechanism to release D, or each branch having a disulfide bond, wherein cleavage of the disulfide bond releases D or a derivative thereof, or each branch having a scissile bond, wherein cleavage of the scissile bond by a specific cleavage mechanism releases D; each D is independently a cytotoxic small molecule or peptide; and n is an integer selected from 1 to 25.

[0048] 18.L 1 18. The compound of embodiment 17, wherein the functional group at the linker end of A is capable of site-specific conjugation with A and is selected from the group consisting of thiol, maleimide, methylsulfonylpyrimidine, methylsulfonylbenzothiazole, vinylpyridine, ethyl P-ethynyl-N-(p-tolyl)phosphonamidate, 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, trans-cyclooctene, tetrazine, triarylphosphine, boronic acid, and iodine.

[0049] 19. The compound of embodiment 17 or 18, wherein the bispecific antibody is a single-chain bispecific antibody, a bispecific nanobody, or a bispecific antigen-binding domain thereof.

[0050] 20. The compound of embodiment 19, wherein the bispecific antibody comprises an antigen-binding domain that binds to CD47 comprising a VL and a VH, and an antigen-binding domain that binds to PD-L1 comprising a VL and a VH.

[0051] 21. The compound of any one of embodiments 17-20, wherein the bispecific antibody is a single chain anti-CD47 / anti-PD-L1 bispecific antibody.

[0052] 22. The VL of the antigen-binding domain that binds to CD47 comprises CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 2 to 4, respectively, and the VH of the antigen-binding domain that binds to CD47 comprises CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 5 to 7, respectively; and The compound of embodiment 20 or 21, wherein the VL of the antigen-binding domain that binds to PD-L1 comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 8 to 10, respectively, and the VH of the antigen-binding domain that binds to PD-L1 comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 11 to 13, respectively.

[0053] 23. The VL of the antigen-binding domain that binds to CD47 comprises the amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15, and the VH of the antigen-binding domain that binds to CD47 comprises the amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16; and The compound of embodiment 22, wherein the VL of the antigen-binding domain that binds to PD-L1 comprises the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18, and the VH of the antigen-binding domain that binds to PD-L1 comprises the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17.

[0054] 24. The compound of any of embodiments 20-23, wherein the bispecific antibody has an amino acid sequence as set forth in SEQ ID NO: 1 or 14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 or 14.

[0055] 25. The antigen-binding domain that binds to CD47 and the antigen-binding domain that binds to PD-L1 are linked via a peptide linker or other chemical linker, and the linker is 1 25. The compound of any of embodiments 20-24, comprising a cysteine, azide or unnatural amino acid residue for site-specific conjugation of a bispecific antibody to

[0056] 26. The compound of any one of embodiments 17-25, wherein D is selected from any DNA crosslinking agent, microtubule inhibitor, DNA alkylating agent, topoisomerase inhibitor, or combinations thereof.

[0057] 27. The compound of embodiment 26, wherein D is selected from MMAE, MMAF, Dxd, SN38, DM1, DM4, calicheamicin, pyrrolobenzodiazepine, duocarmycin, or derivatives thereof, or combinations thereof.

[0058] 28. The compound according to any one of embodiments 17 to 27, wherein the total molecular weight of PEG is 3,000 to 100,000.

[0059] 29. The compound according to embodiment 28, wherein PEG is linked to the cyclic or acyclic tri- or polyfunctional moiety T (e.g. lysine) via a permanent or cleavable bond.

[0060] 30. The following formula: [ka] [ka] wherein BsAb is a bispecific antibody targeting PD-L1 and CD47, or an antigen-binding fragment thereof; or a pharmaceutically acceptable salt thereof.

[0061] 31. An antigen-binding domain that binds to CD47, wherein the bispecific antibody comprises a VL comprising CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 2 to 4, respectively, and a VH comprising CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 5 to 7, respectively; and 31. The compound of embodiment 30, comprising an antigen-binding domain that binds to PD-L1, wherein the VL comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 8-10, respectively, and a VH comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 11-13, respectively.

[0062] 32. The VL of the antigen-binding domain that binds to CD47 comprises the amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15, and the VH of the antigen-binding domain that binds to CD47 comprises the amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16; and The compound of embodiment 31, wherein the VL of the antigen-binding domain that binds to PD-L1 comprises the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18, and the VH of the antigen-binding domain that binds to PD-L1 comprises the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17.

[0063] 33. The compound of any of embodiments 30-32, wherein the bispecific antibody has an amino acid sequence as set forth in SEQ ID NO: 1 or 14, or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 or 14.

[0064] 34. A method for preparing a compound according to any one of embodiments 1 to 33, comprising: a) preparing a non-immunogenic modified (e.g., PEGylated) drug conjugate having a free functional group for site-specific conjugation; b) site-specifically conjugating a non-immunogenically modified (e.g., PEGylated) drug conjugate to the bispecific antibody to obtain a compound of formula (I) or (II).

[0065] 35. A pharmaceutical formulation comprising an effective amount of a compound according to any one of embodiments 1-33, and a pharmaceutically acceptable salt, carrier, or excipient.

[0066] 36. The compound of any one of embodiments 1-33 for use in treating 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, kidney cancer, bladder cancer, stomach cancer, colon cancer, colorectal cancer, salivary gland cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, head and neck cancer, and endometrial cancer.

[0067] 37. The compound of any one of embodiments 1-33 for use in combination with an effective amount of another anticancer agent or 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, kidney cancer, bladder cancer, stomach cancer, colon cancer, colorectal cancer, salivary gland cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, head and neck cancer, and endometrial cancer. [Brief explanation of the drawings]

[0068] [Figure 1] 1 shows a schematic reaction scheme for preparing compound 7 described in Example 1. [Figure 2] 1 shows a schematic reaction scheme for preparing compound 13 described in Example 1. [Figure 3] Schematic diagram of the reaction scheme for preparing compound 18 described in Example 1. [Figure 4] Schematic diagram of the reaction scheme for preparing compound 22 described in Example 1. [Figure 4a] Schematic diagram of the reaction scheme for preparing compound 22a described in Example 1. [Figure 5]Schematic diagram of the reaction scheme for preparing compound 27 described in Example 1. [Figure 6] Schematic reaction scheme for preparing compounds 32 and 32a described in Example 1a. [Figure 7] Schematic reaction scheme for preparing compound 35 described in Example 1b. [Figure 7a] Schematic reaction scheme for preparing compound 35a described in Example 1c. [Figure 7b] Schematic reaction scheme for preparing compound 35b described in Example 1d. [Figure 7c] Schematic reaction scheme for preparing compound 35c described in Example 1e. [Figure 8] Schematic reaction scheme for preparing compound 39 described in Example 1f. [Figure 9] Schematic diagram of the reaction scheme for preparing compound 41 described in Example 1g. [Figure 10] 1 is a schematic diagram of a reaction scheme for preparing JY207 described in Example 3. [Figure 10a] 1 shows a schematic reaction scheme for preparing JY207A described in Example 3a. [Figure 10b] 1 shows a schematic reaction scheme for preparing JY207A1 described in Example 3b. [Figure 10c] 1 shows a schematic reaction scheme for preparing JY207B described in Example 3c. [Figure 10d] 1 is a schematic diagram of a reaction scheme for preparing JY207W described in Example 4. [Figure 11] Cell line selectivity of JY207 as described in Example 5. [Figure 12-1] JY207 induces cytotoxicity against tumor cell lines expressing both CD47 and PD-L1, as described in Example 6. [Figure 12-2] JY207 induces cytotoxicity against tumor cell lines expressing both CD47 and PD-L1, as described in Example 6. [Figure 12a]JY207A did not exhibit cytotoxicity against CD47+ / PD-L1-MKN45 cells and CD47- / PD-L1+NCIN87 cells described in Example 6a, but selectively induced strong cytotoxicity against tumor cell lines expressing both CD47 and PD-L1. [Figure 12b] JY207A1 did not exhibit cytotoxicity against CD47+ / PD-L1-MKN45 cells and CD47- / PD-L1+NCIN87 cells described in Example 6a, but selectively induced strong cytotoxicity against tumor cell lines expressing both CD47 and PD-L1. [Figure 12c] JY207B did not exhibit cytotoxicity against CD47+ / PD-L1-MKN45 cells and CD47- / PD-L1+NCIN87 cells described in Example 6a, but selectively induced strong cytotoxicity against tumor cell lines expressing both CD47 and PD-L1. [Figure 13] Comparison of the internalization efficiency of JY207 and DS8201a as described in Example 7. [Figure 14] Selective binding of tumor cell lines by JY207 as described in Example 8. [Figure 15] In vivo efficacy of JY207 in pancreatic cancer xenograft tumor models as described in Example 9. [Figure 16] In vivo efficacy of JY207 in NCI-H1975 xenograft tumor model as described in Example 10. DETAILED DESCRIPTION OF THE INVENTION

[0069] In the present invention, a single-chain anti-PD-L1 / anti-CD47 bispecific antibody is used as an antibody module to construct a PEG-based ADC to target cancer cells that co-express two antigens (PD-L1 and CD47).

[0070] For normal cells (e.g., red blood cells) that express only CD47, conventional anti-CD47 / anti-PD-L1 bispecific ADCs could theoretically still recognize and bind to CD47 on red blood cells. However, because the anti-CD47 / anti-PD-L1 bispecific ADCs of the present invention are PEGylated single-chain bispecific ADCs, their affinity for CD47 is significantly reduced due to the structure of the single-chain antibody and steric hindrance caused by PEGylation (internal data). Therefore, PEG-based single-chain bispecific ADCs are safe for such normal cells. In fact, it is extremely rare for normal cells to express both PD-L1 and CD47, and co-expression of these two antigens is most often found in cancer cells. Therefore, the mechanism of action and structural characteristics of the PEGylated single-chain anti-CD47 / anti-PD-L1 bispecific ADCs dictate that they will only kill cancer cells and spare normal cells.

[0071] Both CD47 and PD-L1 are overexpressed in many types of cancer. Although there is little data available on the coexpression of these two proteins in the same tumor tissue, it is expected that the proportion of tumors coexpressing both proteins in clinical settings is not low. In a study of 430 clinical samples from non-small cell lung cancer (NSCLC), 96 cases (22.3%) expressed PD-L1 and 296 cases (68.8%) expressed CD47, of which 80 cases (18.6%) showed PD-L1 / CD47 coexpression. The PD-L1 / CD47 coexpression rate in the LUSC cancer subtype was 23.7%, significantly higher than that in the LUAD cancer subtype (14.6%) (p=0.018) (Yang, Z. et al. Thorac Cancer, 2021, 12, 1743-1751). In a study of 148 patients with pulmonary sarcomatoid carcinoma, 54 (36.5%) were PD-L1 positive and 78 (52.7%) were CD47 positive, with 36 (24.3%) of these showing co-expression of PD-L1 and CD47 (Yang, Z., et al. J Cancer Res Clin Oncol, 2019, 145 3055-3065). This co-expression rate in nearly all cancer types indicates that PEGylated single-chain anti-CD47 / anti-PD-L1 bispecific ADCs may benefit many patients with many different cancer types.

[0072] One of the significant drawbacks of conventional antibody-based drugs (including conventional ADCs) is their poor tumor penetration due to their large molecular size (150 kDa). As a result, antibodies may only exert limited and suboptimal effects within solid tumors. Some researchers have attempted to screen low-molecular-weight anti-PD-L1 peptides for cancer immunotherapy (Liu, H. et al. Journal for ImmunoTherapy of Cancer, 2019, 7, 270). However, such compounds generally have very short half-lives. In the present invention, the molecular weight (MW) of the PEGylated single-chain bispecific ADC is approximately 80–85 kD, which is much smaller than conventional antibody-based ADCs but is still large enough to maintain a good half-life of up to one week. Therefore, the present invention will have the advantage of improved solid tumor penetration.

[0073] Furthermore, the mechanism of action of PEGylated single-chain anti-PD-L1 / anti-CD47 bispecific ADCs does not depend on the inhibition of PD-L1 / PD-1 or CD47 / SIRPα signaling; instead, they use only PD-L1 and CD47 as tumor-specific antigens to induce specific binding and release the payload to exert cytotoxicity against cancer cells. Therefore, PEG-based single-chain anti-PD-L1 / anti-CD47 bispecific ADCs may be used to address the drug resistance issue associated with anti-PD-L1 and anti-CD47 drugs.

[0074] In summary, the present invention provides novel PEGylated single-chain bispecific antibody-drug conjugates that not only lack toxicity to megakaryocytes and other normal cells because they do not contain the Fc component that often causes Fc-associated toxicity in conventional full-length ADCs, but also have increased selectivity for tumor cells, improving the anti-tumor efficacy of the conjugates. Thus, the present invention expands upon current ADC technology and improves current cancer treatments.

[0075] I. Conjugates In one embodiment of the present invention, compounds of formula I and II: [ka] The present invention provides a compound of the formula:

[0076] In the conjugate of Formula I or Formula II, P can be a non-immunogenic polymer such as PEG; M is a proton or C 1~50 an end-capping group selected from alkyl and aryl, wherein one or more carbons of said alkyl may be 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 each may independently be a bifunctional linker; Each of a and b may 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 be an amino acid sequence or an enzymatically cleavable trigger moiety, a pH-sensitive linker capable of releasing drug D or a derivative thereof under acidic pH conditions, or a disulfide bond linker capable of releasing drug D upon enzymatic cleavage; A may be any bispecific antibody, including a single chain bispecific antibody, a bispecific nanobody or other bispecific antigen-binding fragment targeting CD47 and PD-L1, or a combination thereof; D may be any cytotoxic small molecule or peptide or derivative thereof, and may be released from B via either enzymatic hydrolysis and / or autolysis mechanisms, or pH-induced hydrolysis, or any combination thereof; each 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.

[0077] In some embodiments, each branch of B is linked to the drug D via one or more autolytic spacers that include an extended spacer (optional), a trigger moiety, such as an amino acid sequence or a disulfide moiety, or a carbohydrate moiety, such as a β-glucoronide or β-galactoside, and are 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), etc. Examples of autolytic spacers include the following: [ka] where R 1 , R 2 , R 3 , R 4 is H or C 1~10 It may also be alkyl. In such embodiments, D may be any small molecule or peptide or derivative thereof containing an active O or N or S functional group.

[0078] Other examples of one or two self-melting spacers include: [ka] where 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, and X=O, S, or N. In such embodiments, D can be any small molecule or peptide or derivative thereof containing a free -OH functional group.

[0079] In some embodiments, each branch of B may be a pH-sensitive linker that can release drug D or a derivative thereof under acidic pH conditions at the tumor site and / or within tumor cells. Examples of acid-sensitive linkers include, but are not limited to, the following formats: -CR 1 =N-NR 1 -, -CR 1 =NO-, -CR 1 =N-NR 2 -CO-, -N=N-CO-, -OCOO-, -NR 1 -COO-.

[0080] In some embodiments, each branch of B may be a disulfide bond linker that can be enzymatically cleaved to release the drug D or a derivative thereof at the tumor site and / or within the tumor cells.

[0081] In some embodiments, A is a single chain anti-CD47 / anti-PD-L1 bispecific antibody that binds to CD47 and PD-L1 expressed on cancer cells.

[0082] In some embodiments, A is a single chain bispecific antibody (SCAPD-L1'SCACD47) capable of binding to two different antigens, such as PD-L1 and CD47.

[0083] In some embodiments, the six complementarity determining regions (CDRs) that target CD47 may be as follows: RSSQSIVYSNGN (LCDR1, SEQ ID NO: 2), KVSNRFS (LCDR2, SEQ ID NO: 3), FQGSHVPYT (LCDR3, SEQ ID NO: 4), NYNMH (HCDR1, SEQ ID NO: 5), TIYPGNDDTSYNQKFK (HCDR2, SEQ ID NO: 6), and GGYRAMDY (HCDR3, SEQ ID NO: 7).

[0084] In some embodiments, the six complementarity determining regions (CDRs) that target PD-L1 may be as follows: GFTFSDSWIH (LCDR1, SEQ ID NO: 8), AWISPYGGSTYYADSVKG (LCDR2, SEQ ID NO: 9), RHWPGGFDY (LCDR3, SEQ ID NO: 10), RASQDVSTAVA (HCDR1, SEQ ID NO: 11), SASFLYS (HCDR2, SEQ ID NO: 12), and QQYLYHPAT (HCDR3, SEQ ID NO: 13).

[0085] In some embodiments, the amino acid sequence of a VL that targets CD47 may be as follows: DIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIK (SEQ ID NO: 15)

[0086] In some embodiments, the amino acid sequence of a VH that targets CD47 may be as follows: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 16)

[0087] In some embodiments, the amino acid sequence of a VH that targets PD-L1 may be as follows: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK (SEQ ID NO: 17)

[0088] In some embodiments, the amino acid sequence of a VL that targets PD-L1 may be as follows: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT (SEQ ID NO: 18)

[0089] In some embodiments, the amino acid sequence of SCAPD-L1 / SCACD47 may be as follows: DIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKGGSGGSGGSGGS GGQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSGCGGS SGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT (SEQ ID NO: 1)

[0090] In some embodiments, the amino acid sequence of SCAPD-L1 / SCACD47 may be as follows: DIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKGGSGGSGGSGSG GQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSGCGGSSGG SDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTHHHHHH (SEQ ID NO: 14)

[0091] In some embodiments, D can be released at the tumor site or within tumor cells either by enzymatic and / or autolytic mechanisms, or by pH-induced hydrolysis.

[0092] In some embodiments, agent D can be selected from any DNA cross-linking agent, microtubule inhibitor, DNA alkylating agent, topoisomerase inhibitor, or combinations thereof.

[0093] In some embodiments, agent D can be selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoids (DM1, DM4), SN38, Dxd, calicheamicin, pyrrolobenzodiazepines, sibiromycin, tomaymycin, duocarmycin, or any cytotoxic compound or analogue / derivative thereof, or a combination thereof.

[0094] In some embodiments, D is monomethyl auristatin E (MMAE), an antimitotic drug, or a derivative thereof.

[0095] In a further embodiment, D is linked to an autolytic spacer such as 4-aminobenzyl alcohol via a carbonate group (PABC) and a trigger moiety, eg, valine-citrulline, to form Val-Cit-PABC-D.

[0096] In one aspect of the invention, methods are provided for preparing PEGylated drug conjugates that can be site-specifically conjugated to bispecific antibody fragments or single-chain bispecific antibodies. In another aspect of the invention, methods are provided for preparing PEGylated single-chain BsADCs.

[0097] To synthesize a PEGylated single-chain BsADC targeting CD47 and PD-L1, the coding sequence of the single-chain bispecific antibody or a vector carrying the coding sequence can be synthesized and introduced into, for example, a CHO expression system. The protein can be expressed and purified as previously described (WO2018075308).

[0098] To synthesize PEGylated drug conjugates with side chains bearing site-specific conjugation functional groups, the terminal functional groups of PEG, such as hydroxyl or carboxyl groups, can be activated and conjugated with trifunctional small molecule moieties such as Boc- or Fmoc-protected lysine to form terminally branched heterobifunctional PEGs. The newly formed carboxyl groups can be coupled with a branched spacer to form PEG-Lys(Boc)-B. After coupling, the protecting groups can be removed, and the unprotected PEGylated branched linker can be coupled with a small molecule linker bearing a site-specific conjugation functional group, such as maleimide, 2-methylsulfonylpyrimidine, or DBCO, to form PEG-Lys(Mal)-B, PEG-Lys(pyrimidine)-B, or PEG-Lys(DBCO)-B. PEGylated drug conjugates such as PEG-lys(Mal)-B-Val-Cit-PABC-MMAE can be prepared by the coupling reaction of PEG-Lys(Mal)-B with Val-Cit-PABC-MMAE. The final step of the synthesis is the site-specific conjugation of the PEGylated drug conjugate to a thiol- or azide-tagged single-chain bispecific antibody.

[0099] Alternatively, Val-Cit-PABC-MMAE can be reacted with B to form B(Val-Cit-PABC-MMAE)n, which can then be conjugated with PEG-Lys(Mal)-COOH to give the PEGylated drug conjugate PEG-lys(Mal)-B(Val-Cit-PABC-MMAE)n, where n is an integer from 1 to 20.

[0100] II. Polyethylene glycol (PEG) moiety In one embodiment of the present invention, the linear PEG has the formula: [ka] may be.

[0101] where n may optionally be an integer from 60 to about 2300, preferably to provide a polymer with a total molecular weight of 3000 to 100,000 daltons or greater. M may be H, methyl, or other low molecular weight alkyl. Non-limiting examples of M include H, methyl, ethyl, isopropyl, propyl, butyl, or F(CH)CH. F and F may independently be terminal functional groups, such as hydroxyl, carboxyl, thiol, halide, amino, etc., which may be functionalized, activated, and / or conjugated to a small molecule spacer or linker. q and m may be any integer from 0 to 10.

[0102] In another embodiment of the invention, the method can also be practiced using alternative branched PEGs, which have the following formula: [ka] may be.

[0103] In this formula, PEG is polyethylene glycol. m may optionally be an integer from 2 to 10 to provide a branched PEG, preferably with a total molecular weight of 3,000 to 100,000 daltons or more. M may be methyl or other low molecular weight alkyl cap. L may be a functional linking moiety to which two or more PEGs are attached. Non-limiting examples of such linking moieties include: any amino acid, such as glycine, alanine, or lysine, or 1,3-diamino-2-propanol, triethanolamine, or any 5- or 6-membered aromatic or aliphatic ring with more than two functional groups attached. S is an optional non-cleavable spacer. F may be a terminal functional group such as a hydroxyl group, a carboxyl group, a thiol group, or an amino group. i is 0 or 1. When i is 0, the formula is: [ka] wherein each variable PEG, m, M, or L has the same definition as above.

[0104] The methods of the present invention can also be practiced with alternative polymeric materials, 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 exemplary and is not intended to limit the types of non-antigenic polymers suitable for use herein.

[0105] III. Trifunctional Linker T T is P, (L 1 ) a , and (L 2 ) b T represents a multifunctional linker, such as a trifunctional linker, which 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, carboxyl, thiol, and halide. The functional groups in the trifunctional linker can be the same or different. In some embodiments, one or two of the functional groups can be protected to achieve selective conjugation with other reaction partners. Various protecting groups are known in the art, for example, those shown in Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York). Functional groups can be converted to other groups before or after the reaction of T with another reaction partner. For example, a hydroxyl group can be converted to a mesylate or tosylate group. A halide can be replaced with an azide group. The acid functional group of T can be converted to an alkyne functional group by coupling with an amino group bearing a terminal alkyne.

[0106] In exemplary embodiments, T is derived from lysine, aspartic acid, glutamic acid, 1,3-diamino-2-propanol, or triethanolamine. One or more of the functional groups on these molecules may be protected for selective reaction. In some embodiments, T is derived from Boc-protected lysine.

[0107] IV. Bifunctional Linker L 1 and L 2 Both linkers L 1 and L 2 are independently the following: -(CH2) a XY(CH2) b -, -X(CH2) a O(CH2CH2O) c (CH2) b Y-, -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-, -W3-(CH2) a C(O)NR1(CH2) b O(CH2CH2O) c (CH2) d W2C(O)(CH2) e X-, -C≡C-, -CR1=CR2-, and a linker strand optionally selected from 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, and 25; each of X and Y is independently selected from C(=O), NR, S, O, CR, R, or null; R, R, R, and R are independently hydrogen, C alkyl, or (CH) 1~10 C(=O); W1 and / or W3 are derived from a maleimide-based moiety, a methylsulfonylpyrimidine-based moiety, a methylsulfonylbenzothiazole-based moiety, a 4-vinylpyridine-based moiety, or an ethyl P-ethynyl-N-phenylphosphonamidate-based moiety; W2 represents a triazolyl- or tetrazolyl-containing group; and the heterocyclyl group is selected from a maleimide-derived moiety or a tetrazolyl-based moiety, or a triazolyl-based moiety. Non-limiting examples of maleimide-based moieties include N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxybenzoate (EHCO), γ-maleimidobutyric ... Examples of suitable linker heterocyclyl groups include methyloxysuccinimide 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 pyrimidine, 4-vinylpyridine, tetrazolyl, or triazolyl.

[0108] In some exemplary embodiments, (L 1 ) a and (L 2 ) b is the following: [ka] where n and m are integers and are independently selected from 0 to 20.

[0109] In other exemplary embodiments, each linker unit may 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).

[0110] In some non-limiting exemplary embodiments, each linker unit can be derived from a sulfone-based moiety selected from vinyl sulfone reagents, mono sulfone reagents, and bis sulfone reagents. [ka]

[0111] In other non-limiting exemplary embodiments, each linker unit comprises the following moiety: [ka] It can be derived from.

[0112] V. Branched Linker B The branched linker B can comprise a branching unit, an extension spacer (optional), a trigger unit, one or more autolytic spacers, or any combination thereof.

[0113] In some embodiments, the branching unit is: [ka] and

[0114] In other embodiments, the branching unit is: [ka] and

[0115] In some embodiments, the extension spacer in each branch is: -X(CH2) a O(CH2CH2O) b (CH2) c Y-, -X(CH2) a X and Y are independently selected from NR 1 , N.R. 2 , C(O), O, or null, where R 1 and R 2 are independently hydrogen or C 1~10 represents an alkyl group.

[0116] In some embodiments, a branching unit (e.g., having two branches) can be combined with two or more branching units (e.g., having two branches), with or without an extended spacer, to form a branching unit with four branches.

[0117] In other embodiments, the trigger unit comprises any amino acid sequence, any carbohydrate moiety, or a disulfide bond, a pH-causing bond, or any cleavable bond that can be enzymatically or chemically cleaved.

[0118] In some embodiments, the autolytic spacer is: [ka] and where 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 may be NH or O or S.

[0119] In some embodiments, two autolytic spacers can be linked together, e.g. [ka] is.

[0120] In some embodiments, the branched linker B is: [ka] [ka] [ka] You can choose from where: each of a, b, c, d, e, and f is 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, where each A is an independent amino acid, and n is any integer from 1 to 25; PABC is 4-aminobenzyl alcohol carbonate; EDAC is null or -CONR 1 CH2CH2NR 2 -or- CONR 1 CH2CH2CH2NR 2 - may be where R 1 and R 2 are independently hydrogen, C1~10 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 null; 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.

[0121] In some other embodiments, the amino acid sequence trigger unit may 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.

[0122] In preferred embodiments, the amino acid sequence may be Val-Cit, Phe-Lys, Gly-Gly-Phe-Gly or Val-Lys.

[0123] In some exemplary embodiments, the branched linker B is: [ka] [ka] You can choose from: Here, n is an integer selected from 1 to 10.

[0124] VI. Linking group The different moieties of the conjugates of the present 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 the PEG moiety (P) can be activated and then coupled to lysine (T) to provide a desired point of attachment between P and T in Formula I or II. T and L 1 Between T and L 2 Between or L 2 The linking group between B and B is a linker L 2 The reaction of the amino group of α-Lysine with the carboxyl group of lysine (T), or L 1 Reaction of the carboxyl group of with the amino group of T, or L 2It may also be an amide resulting from the reaction of a carboxyl group of B with an amino group of B. Depending on the desired properties of the conjugate, the linker L adjacent to the antibody portion (A) may be 1 A suitable linking group can be incorporated between any two amino acids, or between an amino acid and a PABC.

[0125] In some embodiments, the linking group between different moieties of the conjugate may result from the coupling of a pair of functional groups that have a unique chemical affinity or selectivity for one another. These types of couplings or ring formations allow for site-specific conjugation for the introduction of protein or antibody moieties. Non-limiting examples of these functional groups that lead to site-specific conjugation include thiol, maleimide, 2'-pyridyldithio variants, methylsulfonylpyrimidine, methylsulfonylbenzothiazole, vinylpyridine, ethyl P-ethynyl-N-(p-tolyl)phosphonamidate, aromatic or vinyl sulfone, bissulfone, acrylate, bromo or iodoacetamide, azide, alkyne, dibenzocyclooctyl (DBCO), carbonyl, 2-amino-benzaldehyde or 2-amino-acetophenone groups, hydrazide, oxime, potassium acyltrifluoroborate, O-carbamoylhydroxylamine, trans-cyclooctene, tetrazine, and triarylphosphine, boronic acid, alkyne, iodine.

[0126] VII. Cytotoxic compounds In some embodiments, D includes maytansinoids (U.S. Pat. No. 5,208,020; U.S. Pat. No. 5,416,064; EP 0 425 235), auristatin derivatives such as monomethyl auristatin E (MMAE) and F (MMAF) (U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; U.S. Pat. No. 7,498,298), dolastatins, calicheamicins, pyrrolobenzodiazepines or derivatives thereof (U.S. Pat. No. 5,712,374; U.S. Pat. No. 5,714,586; U.S. Pat. No. 5,739,116; U.S. Pat. No. 5,767,285; U.S. Pat. No. 5,770,701; U.S. Pat. No. 5,770,710; U.S. Pat. No. 5,773,001; U.S. Pat. No. 5,877,296; Hinman, LM et al. Cancer Res., 1993, 53, 3336-3342; Lode, HN et al. Cancer Res., 1998, 58, 2925-2928), anthracyclines such as daunomycin or doxorubicin (Kratz, F. et al. Curr. Med. Chem., 2006, 13, 477-523; Jeffrey, SC et al. Bioorg. Med. Chem. Lett., 2006, 16, 358-362; Torgov, MY et al. Bioconjug. Chem., 2005, 16, 717-721; Nagy, A. et al. Proc. Natl. Acad. Sci. USA, 2000, 97, 829-834; Dubowchik, GM et al. Bioorg. Med. Chem. Lett., 2002, 12, 1529-1532; King, HD et al. J. Med. Chem., 2002, 45, 4336-4343; U.S. Pat. No. 6,630,579), methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel, trichothecenes, and CC-1065.

[0127] In other embodiments, D may be an enzymatically active toxin or fragment thereof, such as, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, aleurites fordii protein, dianthin protein, phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, and enomycin.

[0128] In yet some other embodiments, D may be a radioactive atom. A variety of radioisotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 When radioactive conjugates are used for detection, radioactive atoms for scintigraphic studies, such as Tc 99 Or I 123 , or spin labels for magnetic resonance imaging (MRI), e.g., again, I 123 , I 131 , In 111 , F 19 , C 13 , N 15 , O 17 , gadolinium, manganese or iron.

[0129] In some further embodiments, D is selected from alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturdopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaolamide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, caselesin, and bizelesin). cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictine; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as enediyne antibiotics, such as calicheamicin (Nicolaou, KC et al. Agnew Chem. Intl. Ed., 1994, 33 183-186), dynemicin, esperamicin, and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin), cyanomol doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, tetanol Stractone; antiadrenal drugs, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilon; oligomycin C; PNU-159682; valrubicin; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine;Maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenameth; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®) and docetaxel (TAXOTERE®); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; GDC-0068; mitomycin C; mitoxantrone; tubulysin (IM-2, tubulysin B); vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor rubitecan (9-nitrocamptothecin or RFS-2000); difluoromethylornithine; retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included within this definition are antihormonal agents that act to modulate or inhibit hormone action on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and toremifene; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0130] VIII. Bispecific antibodies In some embodiments, a BsADC comprises a bispecific single-chain antibody, and the two binding domains of the bispecific single-chain antibody are linked via a peptide linker or other chemical linker. In some embodiments, the linker comprises cysteine, azide, DBCO, or a non-natural amino acid residue that can be used for site-specific conjugation of the antibody to a non-immunogenic polymer drug conjugate, such as a PEGylated drug conjugate. In other embodiments, one or both of the two binding domains of the bispecific single-chain antibody comprises cysteine or a non-natural amino acid residue that can be used for site-specific conjugation of the antibody to a non-immunogenic polymer drug conjugate, such as a PEGylated drug conjugate.

[0131] In preferred embodiments, the bispecific antibody may be a single-chain bispecific antibody, a bispecific nanobody or other bispecific antigen-binding fragment targeting CD47 and PD-L1, or a combination thereof.

[0132] IX.Synthesis Once the desired size and number of branches of the PEG have been selected, the terminal functional groups of the PEG, such as hydroxyl groups, carboxyl groups, etc., can be converted to terminally branched heterobifunctional groups using any process recognized by those skilled in the art (WO 2018075308). Broadly speaking, terminally branched heterobifunctional PEGs can be prepared by activating the terminal hydroxyl or carboxyl groups of PEG bearing N-hydroxysuccinimide with reagents such as di(N-succinimidyl)carbonate (DSC) or triphosgene in the case of terminal hydroxyl groups, or with coupling reagents such as N,N-diisopropylcarbodiimide (DIPC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the case of terminal carboxyl groups, in the presence of a base such as 4-dimethylaminopyridine (DMAP) or pyridine to form the activated PEG.

[0133] The activated PEG can then be reacted with a trifunctional 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 bearing a free carboxyl group and a Boc-protected amino group, PEG-Lys(Boc)-COOH. Those skilled in the art will appreciate that other terminal functional groups on PEG, such as halide, amino, or thiol groups, as well as other trifunctional small molecules containing any combination of three functional groups from the list, such as -NH, -NHNH, -COOH, -OH, -C(O)X (X = halide), -N=C=O, -SH, anhydride, halide, maleimide, C=C, C≡C, or protected versions thereof, can alternatively be used for the same purpose, if desired.

[0134] The terminally branched heterobifunctional PEG-Lys(Boc)-COOH can then be used to form branched moieties, e.g. [ka] to give the PEGylated drug conjugate PEG-Lys(Mal)-(Val-Cit-PABC-MMAE). 2または4 can be formed.

[0135] The bispecific antibody SCACD47 / SCAPD-L1 can be prepared by genetic engineering of an expression system. DNA encoding the bispecific single-chain fragments of SCACD47 and SCAPD-L1 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.

[0136] To prepare PEGylated single-chain BsADCs, PEGylated drug conjugates bearing maleimide or DBCO functional groups were site-specifically reacted with free thiol or azide functional groups on the genetically engineered or derivatized bispecific antibody SCACD47 / SCAPD-L1 to form PEG-Lys(SCACD47 / SCAPD-L1)-(Val-Cit-PABC-MMAE). 2または4can be formed.

[0137] In addition to the thiol / maleimide or DBCO / azide site-specific conjugation group pairs exemplified herein, those skilled in the art will recognize that other known site-specific conjugation group pairs, such as thiol / methylsulfonylpyrimidine pair, thiol / methylsulfonylbenzothiazole pair, thiol / ethyl p-ethynyl-N-(p-tolyl)phosphonamidate pair, thiol / vinylpyridine pair, trans-cyclooctene / tetrazine pair, carbonyl / hydrazide, carbonyl / oxime, Suzuki-Miyaura cross-coupling reagent pair, Sonogashira cross-coupling reagent pair, Staudinger ligation reagent pair, Knoevangel-intra-Michael addition reagent pair, etc., can be similarly designed and, if desired, used as alternatives for the same purpose. 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.

[0138] X. Composition The present invention also provides compositions, e.g., pharmaceutical compositions, comprising compounds of the invention, e.g., formulated with a pharmaceutically acceptable carrier. For example, a pharmaceutical composition of the invention may comprise a compound that binds to two different receptors, CD47 and PD-L1 (e.g., a bispecific antibody-drug conjugate).

[0139] Therapeutic formulations of the present invention can be prepared in the form of lyophilized formulations or aqueous solutions by mixing the bispecific molecule-drug conjugates having the desired purity with any physiologically acceptable carrier, excipient, or stabilizer. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 amino acid residues) 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, Pluronic, or PEG.

[0140] The formulation may contain more than one active compound, preferably compounds with complementary activities that do not adversely affect each other, as needed for the particular indication being treated. For example, the formulation may further comprise another antibody or bispecific antibody, a cytotoxic agent, a chemotherapeutic agent, or an ADC. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0141] The active ingredient may be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, prepared by coacervation techniques or interfacial polymerization, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0142] The pharmaceutical compositions of the present invention can be administered in combination therapy, i.e., in combination with other drugs. Examples of therapeutic agents that can be used in combination therapy are described in more detail below.

[0143] The preparations used for in vivo administration must be sterile. This can be easily achieved by filtration through sterile filtration membranes. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the above-listed ingredients, as needed, and then sterilizing microfiltration. Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients listed above. For sterile powders to be prepared into sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which obtains a powder of the active ingredient and any additional desired ingredients from the solution that has previously been sterile-filtered.

[0144] XI. Dose The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular method of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the composition that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99%, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the active ingredient combined with a pharmaceutically acceptable carrier.

[0145] The dosage regimen is adjusted to obtain the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the exigencies of the therapeutic situation. Formulating parenteral compositions in dosage unit form is particularly advantageous in terms of ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a physically discrete unit suitable for unitary administration to the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are determined by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technology of formulating such active compounds for the treatment of individual sensitivities.

[0146] Dosages for administration of the bispecific molecule-drug conjugates of the invention range from about 0.0001 to 100 mg / kg, more usually 0.01 to 50 mg / kg, of host body weight. For example, dosages may be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, or 10 mg / kg body weight, or within the range of 1 to 10 mg / kg. Exemplary treatment regimens include daily, twice-weekly, weekly, biweekly, 3-weekly, 4-weekly, monthly, 3-monthly, or 3-6-monthly administration. Preferred dosing regimens for the bispecific drug conjugates of the present invention include 1 mg / kg body weight or 3 mg / kg body weight by intravenous administration, wherein the bispecific drug conjugates are administered using one of the following dosing schedules: (i) six doses every four weeks, then every three months; (ii) every three weeks; (iii) one dose of 3 mg / kg body weight, then 1 mg / kg body weight every three weeks.

[0147] Alternatively, the bispecific drug conjugate may be administered as a sustained-release formulation, in which case administration frequency may be reduced. The dosage and administration frequency depend on the half-life of the bispecific drug conjugate in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and administration frequency may vary depending on whether the treatment is preventive or therapeutic. For preventive applications, relatively low dosages are administered at relatively long intervals over a long period of time. Some patients may continue treatment for the rest of their lives. For therapeutic applications, relatively high dosages may be required at relatively short intervals until the progression of the disease is reduced or stopped, and preferably until the patient shows partial or complete improvement in the symptoms of the disease. Thereafter, the patient can be administered a preventive regimen.

[0148] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, health, and medical history of the patient being treated, and similar factors well known in the medical field.

[0149] A "therapeutically effective dose" of a bispecific molecule of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of disability or disabling illness due to the disease. For example, in the case of tumor treatment, a "therapeutically effective dose" preferably inhibits cell proliferation or tumor growth or metastasis by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, compared to untreated subjects. The ability of an agent or compound to inhibit tumor growth can be assessed in an animal model system predictive of efficacy against human tumors. Alternatively, this property of a composition can be assessed by examining the compound's inhibitory ability, e.g., in vitro, using assays known to those of skill in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size, metastasis, or otherwise ameliorate a subject's symptoms. One of ordinary skill in the art would be able to determine such amounts based on factors such as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0150] XII. Administration The compositions of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Preferred administration routes for the antibody-drug conjugates of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal, or other parenteral routes, for example, by injection or infusion. The term "parenteral administration" as used herein refers to modes of administration other than intestinal and topical administration, typically by injection, and includes, but is 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 bispecific molecule-drug conjugates of the present invention can be administered parenterally, for example, via topical, epidermal, or mucosal routes of administration, such as nasal, oral, vaginal, rectal, sublingual, or topical administration.

[0151] Active compound can be prepared with carrier that can protect compound from rapid release, for example, controlled release formulation, and includes implant, transdermal patch and microencapsulated delivery system.Biodegradable, biocompatible polymer can be used, for example, ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, polylactic acid, etc.Many of the methods for preparing such formulations are patented or generally known to those skilled in the art.For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0152] Therapeutic compositions can be administered using medical devices known in the art. For example, the therapeutic compositions of the present invention can be administered using needleless hypodermic injection devices such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556. Examples of well-known implants and modules useful in the present invention include those described in U.S. Patent Nos. 4,487,603, 4,486,194, 4,447,233, 4,447,224, 4,439,196, and 4,475,196. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0153] XIII. Treatment method In one aspect, the present invention relates to the in vivo treatment of a subject using the above-described bispecific molecule drug conjugate to inhibit the growth and / or metastasis of cancerous tumors. In one embodiment, the present 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 bispecific molecule drug conjugate.

[0154] Non-limiting examples of preferred cancers for treatment include chronic or acute leukemias, such as acute myeloid leukemia, chronic myelogenous 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 adenocarcinoma), colon cancer, and lung cancer (e.g., non-small cell lung carcinoma). Additionally, the present invention includes refractory or recurrent malignancies whose growth may be inhibited using the antibodies of the present invention. Other examples of cancers that may be treated with 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, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, 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, ureteral cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureteral cancer, renal pelvis carcinoma, tumors of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of the foregoing cancers.

[0155] As used herein, the term "subject" 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 an enhanced immune response. The method is particularly suitable for treating human patients with disorders that can be treated by enhancing the immune response.

[0156] The above-mentioned treatment can also be combined with standard cancer treatment. For example, it can be effectively combined with chemotherapy regimen. In such cases, it may be possible to reduce the dose of chemotherapy agent administered (Mokyr, M. et al. Cancer Res., 1998, 58, 5301-5304).

[0157] Other antibodies that can be used to activate host immune responsiveness can be used in or together with the bispecific molecule-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 can be used in combination with the bispecific molecule-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 (U.S. Pat. No. 5,811,097), 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 (U.S. Pat. No. 8,008,449) and PD-L1 (U.S. Pat. No. 7,943,743; U.S. Pat. No. 8,168,179) may also enhance the level of T cell activation. In another example, the bispecific molecule drug conjugates of the invention can be used in combination with anti-tumor antibodies such as RITUXAN (rituximab), HERCEPTIN (trastuzumab), BEXXAR (tositumomab), ZEVALIN (ibritumomab), CAMPATH (alemtuzumab), LYMPHOCIDE (epurtuzumab), AVASTIN (bevacizumab), and TARCEVA (erlotinib).

[0158] Definition of Terms As used herein, the term "alkyl" refers to a hydrocarbon chain, typically ranging from 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 usually 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. As used herein, "alkyl" includes cycloalkyl when three or more carbon atoms are referenced. Unless otherwise specified, alkyl may be substituted or unsubstituted.

[0159] As used herein, the term "functional group" refers to a group that may be used, under conventional conditions of organic synthesis, to form a covalent bond between the entity to which it is attached and another entity, usually bearing an additional functional group. A "bifunctional linker" refers to a linker that has two functional groups that form two bonds with other moieties of the conjugate.

[0160] The term "derivative" as used herein refers to a chemically modified compound having additional structural moieties for the purpose of introducing new functional groups or adjusting the properties of the original compound.

[0161] The term "protecting group" as used herein refers to a moiety that prevents or blocks the reaction of a specific chemically reactive functional group within a molecule under specific 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 Edition, Thieme Chemistry, 2003, and the references cited therein.

[0162] As used herein, the term "PEG" refers to polyethylene glycol. PEG as used in the present invention is typically -(CH2CH2O) n The PEG may have a variety of molecular weights, structures, or geometries. The PEG group may contain 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 alkyl and -Oaryl.

[0163] The term "linker" as used herein refers to an atom or collection of atoms used to connect interconnecting moieties, such as an antibody and a polymer moiety. Linkers can be cleavable or non-cleavable. The preparation of various linkers for conjugates is described in literature, including, for example, 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 incorporate groups or moieties that are cleavable under specific biological or chemical conditions. Examples include enzyme-cleavable disulfide linkers, 1,4- or 1,6-benzyl elimination, trimethyl lock systems, bicine-based self-cleavable systems, acid-labile silyl ether linkers, and other photolabile linkers.

[0164] As used herein, the term "linking group" or "linking group" refers to a functional group or moiety that connects different parts of a compound or conjugate. 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.

[0165] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to describe a sequence of amino acid residues in a polymer. A peptide, polypeptide, or protein 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 chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0166] A "recombinant" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein produced by recombinant DNA technology; i.e., produced from a cell transformed with an exogenous 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 reference to, for example, a cell, 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 the alteration of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell that has undergone such modification. Fusion proteins containing one or more of the aforementioned sequences and heterologous sequences are included within the scope of the present invention. A heterologous polypeptide, nucleic acid, or gene is derived from a foreign species or, if derived from the same species, is substantially modified from its native form. In a naturally occurring protein or nucleic acid, two fusion domains or sequences are heterologous to each other if they are not adjacent to each other.

[0167] An "isolated" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide / protein can constitute at least 10% of the dry weight of a purified preparation (i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%). Purity can be measured by any appropriate standard method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The isolated polypeptides / proteins described herein can be purified from natural sources, produced by recombinant DNA technology, or produced by chemical methods.

[0168] "Antigen" refers to a substance that elicits an immune response or binds to the products of that response. The term "epitope" refers to the region of an antigen to which an antibody or T cell binds.

[0169] The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragments or single chains thereof. Whole antibodies are glycoproteins comprising 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 C H 1. C H 2, and C H Each light chain is composed of three domains: the light chain variable region (V L ) and the light chain constant region (C L ), and the light chain constant region is composed of one domain: V H and V L The region can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V Lis composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs and FRs of the heavy chain variable region are HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, HFR4. The CDRs and FRs of the light chain variable region are LFR1, LCDR1, 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 of the classical complement system (C1q).

[0170] As used herein, an "antibody fragment" may comprise a portion of an intact antibody, generally comprising the antigen-binding and / or variable regions of the intact antibody. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0171] As used herein, the term "antigen-binding fragment or portion" of an antibody (or simply "antibody fragment or portion") 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 also be performed 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) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with part of the hinge region; (iv) a V H Domain and C H (v) a single-arm V fragment of an antibody; L(vi) dAbs consisting of a VH domain; (vii) isolated complementarity-determining regions (CDRs); and (viii) nanobodies, which are heavy chain variable regions comprising a single variable domain and two constant domains. Furthermore, the V of an Fv fragment L and V H The two domains are encoded by separate genes, but can be joined by a synthetic linker using recombinant methods, resulting in V L and V H These regions can be produced as a single protein chain (known as a single-chain Fv (scFv)) in which they pair to form a monovalent molecule; see, e.g., Bird et al. Science 1988, 242, 423-426; and Huston et al. Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment or portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0172] As used herein, the term "Fc fragment" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain.

[0173] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically 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 nature of the antibody as obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein (Kohler, G. et al. Nature, 1975, 256, 495-497), incorporated herein by reference, or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567), incorporated herein by reference. Monoclonal antibodies can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 1991, 352, 624-628 and Marks et al., J Mol Biol, 1991, 222, 581-597, each of which is incorporated herein by reference.

[0174] The term "monoclonal antibodies" as used herein specifically includes "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies 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 U.S. Pat. No. 4,816,567; Morrison et al., Proc Natl Acad Sci USA, 1984, 81, 6851-6855; Neuberger et al., Nature, 312, 1984, 604-608; Takeda et al., Nature, 1985, 314, 452-454; International Patent Application No. PCT / GB85 / 00392, each of which is incorporated herein by reference).

[0175] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing 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 comprise residues that are not found in the recipient or donor antibody. These modifications are made to further improve antibody performance. Generally, 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. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For details, see Jones et al., Nature, 1986, 321, 522-525; Riechmann et al., Nature, 1988, 332, 323-329; Presta, Curt Op Struct Biol, 1992, 2, 593-596; and U.S. Patent No. 5,225,539, each of which is incorporated herein by reference.

[0176] "Human antibody" refers to any antibody with fully human sequences, such as those obtained from human hybridomas, human phage display libraries, or transgenic mice expressing human antibody sequences.

[0177] The term "pharmaceutical composition" refers to a combination of an active agent with a carrier, inert or active, making the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0178] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, absorption delaying agents, and the like. A "pharmaceutically acceptable carrier" does not cause undesired physiological effects after or upon administration to a subject. A carrier in a pharmaceutical composition must also be "acceptable" in the sense of being compatible with and capable of stabilizing the active ingredient. One or more solubilizing agents may be utilized as pharmaceutical carriers for delivery of active agents. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a usable composition in dosage form. Other examples of carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as the necessary pharmaceutical agents for their use, are described in Remington's Pharmaceutical Sciences. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, intraspinal, or epidermal administration (e.g., by injection or infusion). Therapeutic compounds may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects (see, e.g., Berge, SM, et al. J. Pharm. Sci. 1997, 66, 1-19).

[0179] As used herein, "treating" or "treatment" refers to the administration of a compound or agent to a subject having or at risk of developing a disorder, with the intent to cure, alleviate, relieve, ameliorate, delay the onset of, prevent, or ameliorate the disorder, symptoms of the disorder, pathology resulting from the disorder, or predisposition to the disorder.

[0180] "Effective amount" refers to the amount of active compound / drug required to produce a therapeutic effect in the treated subject.As will be recognized by those skilled in the art, the effective amount varies depending on the type of condition to be treated, the route of administration, the use of excipients, and the possibility of use in combination with other therapeutic treatments.The therapeutically effective amount of a combination for treating a tumor condition is, for example, an amount that causes a decrease in tumor size, a decrease in the number of tumor foci, or a delay in tumor growth, compared with untreated animals.

[0181] As disclosed herein, several ranges of values are provided. Each intermediate value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, is also understood to be specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any stated value or intermediate value in a stated range and any other stated or intermediate value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded, and each range in which either limit, neither limit, or both limits are included in the smaller range is also encompassed within the invention, unless the limit included in the stated range is explicitly excluded. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0182] The term "about" generally refers to ±10% of the indicated numerical value. For example, "about 10%" may refer to 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; for example, "about 1" may also mean 0.5 to 1.4. [Example]

[0183] The following examples are presented to provide a further understanding of the present invention and are not intended to limit the scope of the invention in any way. [Example 1]

[0184] Preparation of intermediates Preparation of Val-Cit-PABC-MMAE (Compound 7, Figure 1): Fmoc-Val-OSu(2): Fmoc-Val-OH (20.3 g, 60.0 mmol) and N-hydroxysuccinimide (9.0 g, 78.0 mmol) were dissolved in a mixture of CHCl (120 mL) and THF (40 mL). EDCI (13.8 g, 72.0 mmol) was separately dissolved in CHCl (200 mL) and the solution was cooled to 0-5 °C. The Fmoc-Val-OH / NHS solution was then added to the EDCI solution, and the reaction mixture was warmed to room temperature. The reaction mixture was stirred at room temperature until the reaction was complete. The reaction mixture was then concentrated under reduced pressure and azeotroped 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 under vacuum to give Fmoc-Val-OSu(2) (23.8 g, 91%) as a white powder. 24 H 24 N2O6Na[M+Na] + Calculated value: 459.1532, measured value: 459.1523.

[0185] Fmoc-Val-Cit(3):Fmoc-Val-OSu (9.8 g, 22.5 mmol) was dissolved in DME (150 mL) at room temperature. Separately, sodium bicarbonate (2.1 g, 24.7 mmol) was dissolved in water (150 mL) at room temperature, and then L-citrulline (4.3 g, 24.7 mmol) was added to obtain a homogeneous, clear solution. The prepared L-citrulline solution was then added to the Fmoc-Val-OSu solution, followed by THF (75 mL). The reaction mixture was stirred at room temperature for 16 hours until the reaction was complete. The reaction mixture was acidified with 15% citric acid (200 mL) and then concentrated in vacuo. The mixture was suspended in water (500 mL), and the resulting mixture was stirred for 2 hours, filtered, and dried in vacuo. The solid was resuspended in methyl tert-butyl ether (500 mL) and stirred for 12 h, then filtered, washed, and dried under vacuum to give Fmoc-Val-Cit (3) (6.8 g, 61%) as a white powder. HRMS (ESI) calculated for C26H33N4O6 [M+H]+: 497.2400, found: 497.2388.

[0186] Fmoc-Val-Cit-PABOH(4): 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) was added EEDQ (4.95 g, 20.0 mmol), and the reaction mixture was stirred at room temperature for 24 h. Subsequently, 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. HRMS (ESI) analysis revealed a C 33 H 40 N5O6[M+H] + Calculated value: 602.2979, measured value 602.2969.

[0187] Fmoc-Val-Cit-PABC-PNP(5):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, DIPEA (2.5 mL, 15.0 mmol) was added. The reaction mixture was stirred at room temperature for 5 hours until 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, then cooled to 0°C and stirred for 30 minutes. The solid was isolated by filtration, washed, and dried in vacuo to give Fmoc-Val-Cit-PABC-PNP (5) (4.7 g, 72%) as a pale yellow powder. HRMS (ESI) showed C 40 H 43 NO 10 [M+H] + Calculated value: 767.3041, measured value 767.3045.

[0188] Fmoc-Val-Cit-PABC-MMAE(6): Compound MMAE (2.0 g, 1.8 mmol) and Fmoc-Val-Cit-PABC-PNP (5) (2.8 g, 3.6 mmol) were dissolved in DMF (20 mL). After adding HOBt (0.75 g, 5.6 mmol) and pyridine (1.7 mL), the reaction mixture was stirred at room temperature for 24 hours until the reaction was complete. The reaction mixture was cooled to 0 °C, and methyl tert-butyl ether (180 mL) was added. The resulting slurry was stirred for 3-5 hours, filtered, washed, and dried under vacuum. The crude product was purified by column chromatography to give Fmoc-Val-Cit-PABC-MMAE (6) (3.0 g, 80%) as a yellow powder. HRMS (ESI) analysis revealed a C 73 H 105 N 10 O 14 [M+H] + Calculated value: 1345.7812, measured value 1345.7820.

[0189] Val-Cit-PABC-MMAE(7):Compound 6 (3.0 g, 2.2 mmol) was suspended in anhydrous DMF (40 mL), and the resulting suspension was stirred at room temperature until a uniform suspension was formed. Diethylamine (10 mL) was then added, and the reaction mixture was stirred at room temperature for 3 hours until the reaction was complete. Methyl tert-butyl ether (100 mL) and ethyl acetate (50 mL) were then added over 60 minutes. The resulting mixture was stirred at 0°C for 4 hours. The solid was filtered and dried under vacuum to give Val-Cit-PABC-MMAE (7) (2.3 g, 92%) as a pale yellow powder. HRMS (ESI) showed C 58 H 95 N 10 O 12 [M+H] + The calculated value was 1123.7131 and the measured value was 1123.7142.

[0190] Preparation of branched linker intermediate compound 13 (Figure 2) Compound 10: To a solution of compound 8 (0.62 g, 2.0 mmol) in anhydrous CHCl (15 mL) was added di-tert-butyl 3,3′-azanediyldipropanoate (9) (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 × 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 chromatography to give compound 10 (1.1 g, 96%) as a colorless oil. HRMS (ESI) showed C 32 H 43 N2O7[M+H] + Calculated value: 567.3070, measured value: 567.3062.

[0191] Compound 11:Compound 10 (5.2 g, 9.2 mmol) was dissolved in CHCl (100 mL) and then TFA (25 mL) was added. The mixture was stirred at room temperature for 3 hours. The solvent was removed in vacuo as much as possible below 35°C. The residue was purified by silica gel chromatography to give compound 11 (3.4 g, 83%) as a colorless oil. HRMS (ESI) showed C 24 H 27 N2O7[M+H] + Calculated value: 455.1818, measured value: 455.1824.

[0192] Compound 12: To a stirred solution of compound 11 (41 mg, 0.091 mmol) in anhydrous CHCl (2 mL) and DMF (2 mL) at room temperature under argon was added Val-Cit-PABC-MMAE (7) (224 mg, 0.2 mmol), EDCI (52 mg, 0.27 mmol), and HOBt (5 mg, 0.04 mmol). The mixture was stirred at room temperature until complete conversion was observed by TLC. After completion of the reaction, 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 compound 12 (74 mg, 31%) as a pale yellow solid. HRMS (ESI) showed C 140 H 212 N 22 O 29 [M+2H] 2+ Calculated value: 1333.2912, measured value: 1333.2907.

[0193] Compound 13: Diethylamine (0.6 mL) was added to a solution of compound 12 (73 mg) in DMF (3 mL), and the reaction was allowed to proceed at room temperature for 4 hours. The reaction mixture was concentrated in vacuo, and the residue was purified by preparative HPLC using a Welch Ultimate XB-C18 column (eluent: A = 0.1% TFA in water, B = MeCN) to give compound 13 (71 mg, 99%) as a pale yellow solid. HRMS (ESI) showed C 125 H 202 N 22 O 27 [M+2H] 2+Calculated value: 1222.2572, measured value: 1222.2560.

[0194] Preparation of branched linker intermediate compound 18 (Figure 3) Compound 15: To a solution of compound 14 (0.68 g, 2.0 mmol) in anhydrous CHCl (10 mL) was added di-tert-butyl 3,3′-azanediyldipropanoate (9) (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 × 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 chromatography to give compound 15 (1.2 g, 99%) as a colorless oil. HRMS (ESI) showed C 34 H 47 N2O7[M+H] + Calculated value: 595.3383, measured value: 595.3380.

[0195] Compound 16: Compound 15 (0.5 g, 0.84 mmol) was dissolved in CHCl (6.0 mL), and then TFA (3.0 mL) was added. The mixture was stirred at room temperature for 3 hours. The solvent was removed as much as possible under vacuum below 35°C. The residue was purified by silica gel chromatography to give compound 16 (0.34 g, 85%) as a colorless oil. HRMS (ESI) showed C 26 H 31 N2O7[M+H] + Calculated value: 483.2131, measured value: 483.2127.

[0196] Compound 17:To a solution of compound 16 (185 mg, 0.383 mmol) in a mixture of anhydrous CHCl (8 mL) and DMF (8 mL) was added Val-Cit-PABC-MMAE (7) (947 mg, 0.843 mmol), EDCI (238 mg, 1.23 mmol), and HOBt (26 mg, 0.19 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 preparative HPLC using a Welch Ultimate XB-C18 column (eluent: A = 0.1% TFA in water, B = MeCN) to give compound 17 (0.56 g, 54%) as a pale yellow solid. HRMS (ESI) analysis revealed a C 142 H 216 N 22 O 29 [M+H] + Calculated value: 2694.6137, measured value: 2694.6146.

[0197] Compound 18: Diethylamine (2.0 mL) was added to a solution of compound 17 (0.62 g) in DMF (5 mL), and the reaction mixture was allowed to proceed at room temperature for 2 hours. The reaction mixture was concentrated in vacuo, and the residue was purified by preparative HPLC using a Welch Ultimate XB-C18 column (eluent: A = 0.1% TFA in water, B = MeCN) to give compound 18 (0.51 g, 89%) as a pale yellow solid. HRMS (ESI) showed C 127 H 205 N 22 O 27 [M+H] + Calculated value: 2471.5378, Measured value: 2471.5369; C 127 H 206 N 22 O 27 [M+2H] 2+ Calculated value: 1236.2728, measured value: 1236.2744.

[0198] Preparation of branched linker intermediate compound 22 (Figure 4) Compound 20:To a solution of compound 19 (0.76 g, 2.0 mmol) in anhydrous CHCl (10 mL) was added di-tert-butyl 3,3′-azanediyldipropanoate (9) (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 × 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 chromatography to give compound 20 (1.2 g, 99%) as a colorless oil. HRMS (ESI) showed C 29 H 55 N4O 11 [M+H] + Calculated value: 635.3867, measured value: 635.3860.

[0199] Compound 21: Compound 20 (0.3 g, 0.47 mmol) was dissolved in CHCl (4.0 mL), and then TFA (2.0 mL) was added. The mixture was stirred at room temperature for 3 hours. The solvent was removed as much as possible under vacuum below 35°C. The residue was purified by silica gel chromatography to give compound 21 (0.34 g, 85%) as a colorless oil. HRMS (ESI) showed C 21 H 39 N4O 11 [M+H] + Calculated value: 523.2615, measured value: 523.2607.

[0200] Compound 22:To a stirred solution of compound 21 (39 mg, 0.076 mmol) in a mixture of anhydrous CHCl (2 mL) and DMF (2 mL) at room temperature under argon was added compound 18 (0.41 g, 0.17 mmol), EDCI (43 mg, 0.23 mmol), and HOBt (4.0 mg, 0.03 mmol). 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 preparative HPLC using a Welch Ultimate XB-C18 column (eluent: A = 0.1% TFA in water, B = MeCN) to give compound 22 (81 mg, 20%) as a pale yellow solid. HRMS (ESI) showed C 275 H 445 N 48 O 63 [M+3H] 3+ Calculated value: 1810.1053, measured value: 1810.1061; C 275 H 446 N 48 O 63 [M+4H] 4+ Calculated value: 1357.8310, measured value 1357.8346.

[0201] Preparation of branched linker intermediate compound 22a (Figure 4a) Compound 22a: To a stirred solution of compound 21 (0.6 g, 1.15 mmol) in anhydrous DMF (20 mL) at room temperature under argon, Val-Cit-PAB-MMAE (7) (2.8 g, 2.5 mmol), EDCI (0.66 g, 3.5 mmol), and HOBt (90 mg, 0.7 mmol) were added. 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 cooled to 0 °C, and then methyl tert-butyl ether (100 mL) was slowly added. The resulting slurry was stirred for 1 h, filtered, washed, and dried in vacuo. The crude product was purified on a silica gel column to give compound 22a (2.4 g, 78%) as a white solid. MS (ESI) m / z [M+2H] 2+ 1366.94, [M+2Na] 2+ 1389.02.

[0202] Preparation of branched linker intermediate compound 27 (Figure 5) Compound 24: To a solution of compound 21 (0.57 g, 1.1 mmol) in anhydrous CHCl (10 mL) was added compound 23 (0.51 g, 2.4 mmol), EDCI (0.67 g, 3.5 mmol), HOBt (74 mg, 0.55 mmol), and DIPEA (0.78 mL, 4.4 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 × 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 chromatography to give compound 24 (0.7 g, 79%) as a colorless oil. HRMS (ESI) showed C 39 H 73 NO 13 [M+H] + Calculated value: 833.5236, measured value: 833.5231.

[0203] Compound 25: Compound 24 (0.52 g, 0.62 mmol) was dissolved in CHCl (5.0 mL), and then TFA (2.0 mL) was added. The mixture was stirred at room temperature for 3 hours. The solvent was removed as much as possible under vacuum below 35°C. The residue was purified by silica gel chromatography to give compound 25 (0.42 g, 93%) as a colorless oil. HRMS (ESI) showed C 31 H 57 NO 13 [M+H] + Calculated value: 721.3984, measured value: 721.3997.

[0204] Compound 26:To a solution of compound 25 (77 mg, 0.11 mmol) in DMF (5 mL) at room temperature under argon, compound 18 (0.58 g, 0.24 mmol), EDCI (82 mg, 0.43 mmol), and HOBt (14 mg, 0.11 mmol) were added. 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 preparative HPLC using a Welch Ultimate XB-C18 column (eluent: A = 0.1% TFA in water, B = MeCN) to give compound 26 (0.23 g, 38%) as a pale yellow solid. HRMS (ESI) showed C 285 H 463 N 50 O 65 [M+3H] 3+ Calculated value: 1876.4854, measured value: 1876.4851; C 285 H 464 N 50 O 65 [M+4H] 4+ Calculated value: 1407.6160, measured value 1407.6158.

[0205] Compound 27: Lindlar's catalyst (130 mg, 5 wt%) was added to a stirred solution of azide 26 (180 mg, 0.03 mmol) in methanol (10 mL). The reaction flask was evacuated and flushed with hydrogen gas. The resulting mixture was stirred under a hydrogen atmosphere (balloon) at room temperature for 5 h. After the reaction was complete, the catalyst was filtered through a pad of Celite, the filter cake was washed with methanol (10 mL), and the filtrate was concentrated under reduced pressure. 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 compound 27 (130 mg, 74%) as a pale yellow solid. HRMS (ESI) showed C 285 H 465 N 48 O 65 [M+3H] 3+ Calculated value: 1867.8219, measured value: 1867.8217; C 285 H 466 N 48 O 65 [M+4H]4+ Calculated value: 1401.1184, measured value: 1401.1181. Example 1a

[0206] 30kmPEG-Lys(Mal)-(MMAE) 2 Preparation of compounds 32 and 32a (Figure 6) Compound 29: H-Lys(boc)-OH (369 mg, 1.5 mmol) was added to anhydrous DMF (100 mL), followed by DIPEA (0.83 mL, 5.0 mmol), compound 28 (15 g, 0.5 mmol), and anhydrous CHCl (150 mL). The mixture was stirred overnight at room temperature under argon. Insoluble material was removed by filtration. The solvent was removed, and the residue was recrystallized from CHCl / methyl tert-butyl ether (45 mL / 300 mL). The isolated solid was recrystallized again from MeCN / 2-propanol (30 mL / 450 mL). The product was dried under vacuum at 40 °C for 4 hours to give compound 29 (13.6 g, 91%) as a white powder.

[0207] Compound 30: To a solution of compound 29 (5.7 g, 0.19 mmol) in anhydrous CHCl (57 mL) was added TFA (29.5 mL). The mixture was stirred at room temperature for 1 hour. The solvent was removed as much as possible under vacuum below 35°C. The residue was recrystallized twice from CHCl / methyl tert-butyl ether (14.5 mL / 115 mL). The isolated product was dried under vacuum at 40°C to give compound 30 (4.7 g, 84%) as a white powder.

[0208] Compound 31:To a stirred solution of compound 30 (5.5 g, 0.18 mmol) in anhydrous CHCl (55 mL) at 0 °C, DIPEA (473 mg, 3.6 mmol) was added, followed by NHS-PEG-Mal (0.2 g, 0.47 mmol). The mixture was stirred at 0 °C for 1.5 h, after which the solution was slowly warmed from 0 °C to room temperature and then stirred overnight under an argon atmosphere. The solvent was removed, and the residue was recrystallized from CHCl / methyl tert-butyl ether (13.8 mL / 110 mL). The isolated solid was recrystallized again from MeCN / 2-propanol (11 mL / 165 mL). The residue was dried under vacuum to give compound 31 (5.0 g, 90%) as a white powder.

[0209] Compound 32: To a stirred solution of compound 31 (0.76 g, 0.025 mmol) in a mixture of DMF / CHCl (5 mL / 5 mL) was added compound 13 (0.12 g, 0.05 mmol), DCC (31 mg, 0.15 mmol), and DMAP (28 mg, 0.23 mmol) at room temperature under argon. The mixture was stirred at room temperature until complete conversion was observed by HPLC. After completion of the reaction, the mixture was concentrated in vacuo. The crude reaction mixture was purified by preparative HPLC using a Phenomenex Jupiter C column (eluent: A = 0.1% TFA in water, B = MeCN) to give compound 32 (0.36 g, 47%) as a white solid.

[0210] Compound 32a: Compound 32a was prepared similarly to compound 32, using compound 18 instead of compound 13. Example 1b

[0211] 20k mPEG-Lys(Mal)-(MMAE) 4 Preparation of (Compound 35, Figure 7) Compound 34:To a stirred solution of compound 33 (2.0 g, 0.1 mmol) (see the procedure for preparing compound 31 for the synthesis of compound 33) in anhydrous CHCl (20 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 observed by HPLC. The solvent was removed, and the residue was recrystallized from CHCl / methyl tert-butyl ether (5 mL / 40 mL). The isolated solid was recrystallized again from MeCN / 2-propanol (4 mL / 60 mL). The product was dried in vacuo at 40 °C for 4 h to give compound 34 (1.9 g, 89%) as a white powder.

[0212] Compound 35: Compound 34 (147 mg, 0.007 mmol) was dissolved in anhydrous MeOH (3 mL), followed by the addition of compound 22 (40 mg, 0.007 mmol). The mixture was stirred at room temperature for 24 hours. The mixture was concentrated in vacuo, and the crude reaction mixture was purified by preparative HPLC using a Phenomenex Jupiter® C18 column (eluent: A = 0.1% TFA in water, B = MeCN) to give compound 35 (41 mg, 22%) as a white solid. Example 1c

[0213] 30kmPEG-Lys(Mal)DBCO-(MMAE) 2 Preparation of (compound 35a, Figure 7a) Compound 34a was prepared in the same manner as compound 34, except that 30 km PEG was used instead of 20 km PEG.

[0214] Compound 35a: To a stirred solution of compound 34a (12.0 g, 0.4 mmol) in anhydrous CHCl (60 mL) and MeOH (60 mL) was added compound 22a (1.31 g, 0.48 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 (30 mL / 450 mL). The residue was dried in vacuo to give compound 35a (11.2 g, 94%) as a white powder. [Example 1d]

[0215] 2arm-40kmPEG-Lys(Mal)DBCO-(MMAE) 2 Preparation of (compound 35b, Figure 7b) Compound 29a: To a stirred solution of 2arm-40kmPEG-CO2H (4.0 g, 0.1 mmol) in anhydrous CHCl2 (40 mL) was added compound H-Lys(boc)-OtBu·HCl (102 mg, 0.3 mmol), followed by DIPEA (0.1 mL, 5.0 mmol), EDCI (115 mg, 0.6 mmol), and HOBt (122 mg, 0.9 mmol) at room temperature. The mixture was stirred under argon at room temperature for 16 h. The solvent was removed, and the residue was recrystallized from CHCl2 / methyl tert-butyl ether (12 mL / 90 mL). The isolated solid was recrystallized again from MeCN / 2-propanol (9 mL / 140 mL). The product was dried under vacuum at 40 °C for 4 h to give compound 29a (3.8 g, 96%) as a white powder.

[0216] Compound 30a: Compound 29a (1.5 g, 0.19 mmol) was dissolved in anhydrous CHCl (15 mL), followed by the addition of TFA (10 mL). The mixture was stirred at room temperature for 4 h. The solvent was removed under vacuum below 35 °C as much as possible, and the residue was recrystallized twice from CHCl / methyl tert-butyl ether (6.0 mL / 45 mL). The isolated product was dried under vacuum at 35 °C to give compound 30a (1.4 g, 95%) as a white powder.

[0217] Compound 33a:To a stirred solution of compound 30a (1.9 g, 0.05 mmol) in anhydrous CHCl (20 mL) at 0 °C, DIPEA (0.16 mL, 0.9 mmol) was added, followed by 5-maleimidovaleric acid-NHS (62 mg, 0.14 mmol). The mixture was stirred at 0 °C for 1.5 h and then slowly warmed from 0 °C to room temperature. The reaction was stirred overnight under argon. The solvent was removed, and the residue was recrystallized from CHCl / methyl tert-butyl ether (6.0 mL / 45 mL). The isolated solid was recrystallized again from MeCN / 2-propanol (4.5 mL / 70 mL). The residue was dried under vacuum to give compound 33a (1.8 g, 93%) as a white powder.

[0218] Compound 34a: To a stirred solution of compound 33a (1.8 g, 0.045 mmol) in anhydrous CHCl (25 mL) at room temperature under argon was added DBCO-NH (38 mg, 0.14 mmol), EDCI (53 mg, 0.28 mmol), and HOBt (56 mg, 0.4 mmol). 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 (6.0 mL / 45 mL). The isolated solid was recrystallized again from MeCN / 2-propanol (4.5 mL / 70 mL). The product was dried in vacuo at 40 °C for 4 h to give compound 34a (1.6 g, 91%) as a white powder.

[0219] Compound 35b: To a stirred solution of compound 34a (1.4 g, 0.035 mmol) in anhydrous CHCl (10 mL) and MeOH (10 mL) was added compound 22a (124 mg, 0.045 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 (6.0 mL / 45 mL). The residue was dried in vacuo to give compound 35b (1.2 g, 88%) as a white powder. [Example 1e]

[0220] 2arm-40kmPEG-Lys(pyrimidine)DBCO-(MMAE) 2 Preparation of (compound 35c, Figure 7c) Compound 33b: A solution of compound 30a (1.6 g, 0.04 mmol) in 16 mL of anhydrous CHCl was treated with DIPEA (0.13 mL, 0.8 mmol) at room temperature. Then, compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (32 mg, 0.12 mmol), EDCI (46 mg, 0.24 mmol), and HOBt (48 mg, 0.36 mmol) were added to the mixture. The resulting 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 (6.0 mL / 45 mL). The isolated solid was recrystallized again from MeCN / 2-propanol (4.5 mL / 70 mL). The product was dried under vacuum at 40 °C for 4 hours to give compound 33b (1.5 g, 91%) as a white powder.

[0221] Compound 34b: To a stirred solution of compound 33b (1.3 g, 0.033 mmol) in anhydrous CHCl (13 mL) at room temperature under argon was added DBCO-NH (27 mg, 0.1 mmol), EDCI (37 mg, 0.2 mmol), and HOBt (40 mg, 0.3 mmol). 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 (6.0 mL / 45 mL). The isolated solid was recrystallized again from MeCN / 2-propanol (4.5 mL / 70 mL). The product was dried in vacuo at 40 °C for 4 h to give compound 34b (1.2 g, 93%) as a white powder.

[0222] Compound 35c: To a stirred solution of compound 34b (1.1 g, 0.028 mmol) in anhydrous CHCl (7 mL) and MeOH (7 mL) was added compound 22a (99 mg, 0.036 mmol). The mixture was stirred overnight at room temperature until complete conversion was observed by HPLC. The solvent was removed, and the residue was recrystallized twice from MeCN / 2-propanol (6.0 mL / 45 mL). The isolated wet solid was dried under vacuum to give compound 35c (1.0 g, 91%) as a white powder. [Example 1f]

[0223] Mal-PEG(20K)-(MMAE) 4 Preparation of (Compound 39, Figure 8) Compound 37: To a stirred solution of amine-PEG20k-CO2H (36) (1.0 g, 0.05 mmol) in anhydrous CHCl2 (10 mL) was added DIPEA (83 μL, 0.5 mmol) followed by 6-maleimidohexanoic acid-NHS (46 mg, 0.15 mmol) at 0 °C. The mixture was stirred at 0 °C for 1.5 h, after which the solution was slowly warmed from 0 °C to room temperature and then stirred overnight under argon. The solvent was removed, and the residue was recrystallized from CHCl2 / methyl tert-butyl ether (2.5 mL / 20 mL). The isolated solid was recrystallized again from MeCN / 2-propanol (2 mL / 30 mL). The residue was dried under vacuum to give compound 37 (0.95 g, 95%) as a white powder.

[0224] Compound 38: To a stirred solution of compound 37 (0.9 g, 0.045 mmol) in anhydrous CHCl (9 mL) at room temperature under argon was added DBCO-NH (37 mg, 0.14 mmol), EDCI (52 mg, 0.27 mmol), and HOBt (55 mg, 0.41 mmol). 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 (2.5 mL / 20 mL). The isolated solid was recrystallized again from MeCN / 2-propanol (2 mL / 30 mL). The product was dried in vacuo at 40 °C for 4 h to give compound 38 (0.86 g, 89%) as a white powder.

[0225] Compound 39:Compound 38 (166 mg, 0.008 mmol) was dissolved in anhydrous MeOH (3 mL), followed by the addition of compound 22 (30 mg, 0.006 mmol). The mixture was stirred at room temperature for 24 hours. The mixture was concentrated in vacuo, and the crude reaction mixture was purified by preparative HPLC using a Phenomenex Jupiter® C18 column (eluent: A = 0.1% TFA in water, B = MeCN) to give compound 39 (37 mg, 27%) as a white solid. [Example 1g]

[0226] DBCO-PEG(20K)-(MMAE) 4 Preparation of (Compound 41, Figure 9) Compound 40: To a stirred solution of amine-PEG20k-CO2H (36) (1.0 g, 0.05 mmol) in anhydrous CHCl2 (10 mL) at 0 °C, DIPEA (83 μL, 0.5 mmol) was added, followed by DBCO-NHS (60 mg, 0.15 mmol). The mixture was stirred at 0 °C for 1.5 h, after which the solution was slowly warmed from 0 °C to room temperature and stirred overnight under an argon atmosphere. The solvent was removed, and the residue was recrystallized from CHCl2 / methyl tert-butyl ether (2.5 mL / 20 mL). The isolated solid was recrystallized again from MeCN / 2-propanol (2 mL / 30 mL). The residue was dried under vacuum to give compound 40 (0.91 g, 91%) as a white powder.

[0227] Compound 41: Under an argon atmosphere, compound 40 (120 mg, 0.006 mmol) was dissolved in a mixture of CHCl / DMF (2 mL / 2 mL). Compound 27 (50 mg, 0.009 mmol), EDCI (6.9 mg, 0.036 mmol), and HOBt (7.3 mg, 0.054 mmol) were then added sequentially to the solution. The resulting reaction mixture was stirred at room temperature for 24 hours. The mixture was concentrated in vacuo, and the crude reaction mixture was purified by preparative HPLC using a Phenomenex Jupiter® C18 column (eluent: A = 0.1% TFA in water, B = MeCN) to give compound 41 (53 mg, 36%) as a white solid. [Example 2]

[0228] Preparation of bispecific antibody SCACD47 / SCAPD-L1: Bispecific single-chain antibody (SCA) fragments of SCACD47 and SCAPD-L1 can be prepared by recombinant DNA technology in mammalian cells (e.g., CHO cells using EasySelect™) or yeast (e.g., a Pichia pastori expression kit containing the pPICZ vector). The DNA sequence of SCACD47 / SCAPD-L1 corresponding to the following amino acid sequence (SEQ ID NO: 14) was synthesized, cloned into an expression vector, and transformed into host cells. The expressed protein was purified using Ni-chelate resin or protein L resin. To facilitate subsequent conjugation, a site-specific conjugation functional group, thiol, was inserted into the linker between the two SCAs by recombinant DNA technology.

[0229] Amino acid sequence of SCACD47 / SCAPD-L1: DIVMTQSPLSLPVTPGEPASISC RSSQSIVYSNGN TYLGWYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC FQGSHVPYT FGQGTKLEIKGGSGGSGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTFT NYNMH WVRQAPGQRLEWMG TIYPGNDDTSYNQKFK DRVTITADTSASTAYMELSSLRSEDTAVYYCAR GGYRAMDY WGQGTLVTVSSGCGGSSGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLY HPATFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTHHHHHH (SEQ ID NO: 14); Here, the CDR sequences targeting CD47 are shown in bold and underlined, and the CDR sequences targeting PD-L1 are shown in bold and italic. [Example 3]

[0230] Preparation of JY207 (Figure 10) Compound JY207: The protein SCACD47 / SCAPD-L1 was treated with the reducing agent TCEP-HCl in PBS buffer (pH = 7.4) for 30 min at room temperature, then adjusted to pH 6.8 with a 500 mM sodium phosphate stock solution (pH = 4.12) or 2 M Tris-HCl stock solution. TCEP-HCl was removed by ultrafiltration, and the treated protein was concentrated to 5 mg / mL before PEGylation. PEGylation of SCACD47 / SCAPD-L1 was carried out at room temperature for 3 h using 1.5–5 molar equivalents of compound 32a [30kJ mPEG-Lys(Mal)-(Val-Cit-PABC-MMAE)]. The reaction was quenched with 10 mM L-cysteine for 10 min at room temperature. The final product, 30 kmPEG-Lys(SCACD47 / SCAPD-L1)-(Val-Cit-PABC-MMAE)2 (JY207), was first purified by chromatography using Ca++ Pure-HA™ (Part No. 45039, TOSOH) and then by cation exchange chromatography (CM Fast Flow). The target compound was confirmed by SEC-HPLC and cell-based activity assays. Example 3a

[0231] Preparation of JY207A (Figure 10a) Compound JY207A:The protein SCAPDL1 / SCACD47 (20 mg) was treated with 2 mM TCEP in PBS buffer (pH 7.4) at room temperature for 30 minutes, after which the pH was adjusted with a 500 mM sodium phosphate buffer pH 4.12 stock solution. The treated protein was concentrated to 5 mg / mL and then PEGylated. PEGylation of SCAPDL1 / SCACD47 was carried out using 2-3 equivalents of compound 35a at room temperature for 3 hours. The reaction was quenched with 10 mM L-cysteine at room temperature for 10 minutes. The final product compound was purified using hydroxyapatite HA (TOSOH) in 20 mM sodium phosphate buffer (pH 6.8). The target compound JY207A was confirmed by SEC-HPLC and cell-based activity assays. Example 3b

[0232] Preparation of JY207A1 (Figure 10b) Compound JY207A1: Compound JY207A1 was prepared in the same manner as compound JY207A, except that compound 35b was used instead of compound 35a. Example 3c

[0233] Preparation of JY207B (Figure 10c) Compound JY207B: Compound JY207B was prepared in the same manner as compound JY207A, except that compound 35c was used instead of compound 35a. [Example 4]

[0234] Preparation of SCACD47 / SCAPD-L1-20kPEG-4MMAE(JY207W) (Figure 10d) Compound Mal-PEG 11 -N 3 : 15 mg (1.0 equivalent) of N-succinimidyl-4-maleimidobutyrate was dissolved in azido-dPEG 11 The resulting compound, azide-PEG, was reacted with 38 mg (1.2 equivalents) of α-amine in 200 μL of DMSO at room temperature for 30 minutes. 11 -Maleimide (Mal-PEG) 11 -N3) was used immediately in the next step without further purification.

[0235] Compound JY207W: The protein SCACD47 / SCAPD-L1 was treated with the reducing agent TCEP-HCl in PBS buffer (pH = 7.4) at room temperature for 30 minutes, after which the pH was adjusted to 6.8 with a stock solution of 500 mM sodium phosphate, pH = 4.12, or a stock solution of 2 M Tris-HCl. The TCEP-HCl was removed by ultrafiltration, and the treated protein was concentrated to 5 mg / mL. 50 mol of aliquots of Mal-PEG were added. 11 -N3 was added to the reduced protein with stirring at room temperature. The crude protein-linker product was passed through a chromatography column using Ca++Pure-HA™ (Part No. 45039, TOSOH) and eluted with a linear gradient (CV, 0-50% Buffer B) from Buffer A (5 mM phosphate, pH 6.8) to Buffer B (200 mM phosphate, 200 mM arginine, pH 6.8). Protein-linker conjugation was carried out at room temperature for 3 hours using 1.5-5 molar equivalents of compound 41 (DBCO-20kPEG-4MMAE). The final product, 20 kmPEG-Lys(SCACD47 / SCAPD-L1)-(Val-Cit-PABC-MMAE)4 (JY207W), was first purified by chromatography using Ca++Pure-HA™ (Part No. 45039, TOSOH) and then by cation exchange chromatography (CM Fast Flow). The target compound was confirmed by SEC-HPLC and cell-based activity assays. [Example 5]

[0236] JY207 is not cytotoxic to cell lines expressing only CD47 or PD-L1 JY207 is a PEGylated single-chain bispecific ADC targeting CD47 and PD-L1. We hypothesize that JY207 is selective for cell lines expressing both PD-L1 and CD47. To prove this hypothesis, we performed a PD-L1 + / CD47 - and PD-L1 - / CD47 + The cytotoxicity of JY207 against cell lines with different PD-L1 and CD47 expression patterns, including leukocytes, was measured.

[0237] To evaluate the cytotoxicity of JY207, Raji cells (Type Culture Collection of Chinese Academy of Science, Shanghai, China), MKN45 cells, and NCIN87 cells (Procell, Wuhan, China) under standard maintenance conditions were cultured at 3 × 10 6 Cells / well were seeded into 96-well plates and treated with the indicated doses of JY207. Cell viability (OD490) was measured 3 days after treatment using an MTS (Promega Corporation, Catalog No. G3581) cell viability assay according to the manufacturer's instructions. Data were analyzed using GraphPad Prism software and expressed as percent growth inhibition relative to the untreated control using the following formula: 1 − (OD control − ODJY207) / OD control) × 100%.

[0238] Among the three single antigen-positive cell lines, Raji showed the highest PD-L1 activity. - and CD47 + These are leukemia cell lines. As can be seen in Figure 11, JY207 showed no cytotoxicity even at a high concentration of 10 μg / ml. In the MKN45 cell line, which has very low PD-L1 expression but is CD47 positive, JY207 did not induce cytotoxicity at almost any concentration tested, except at the highest concentration of 10 μg / ml, which induced 10% cytotoxicity. In the NCIN87 cell line, which expresses only "physiological" levels of CD47 (similar to the CD47 levels of normal cells) and is considered CD47 negative and PD-L1 positive, JY207 again did not induce cytotoxicity, except at the highest concentration of 10 μg / ml, which induced 10% cytotoxicity. The results shown in Figure 11 demonstrate that JY207 does not induce cytotoxicity against tumor cells that express only CD47 or PD-L1. [Example 6]

[0239] JY207 induces cytotoxicity against tumor cell lines expressing both CD47 and PD-L1 The cytotoxic activity of JY207 was further evaluated using the same method as in Example 5 using a panel of human cancer cell lines expressing both CD47 and PD-L1: NCIH1975, NCIH661, U87, Calu6, NCIH520, SKBR-3, BxPC-3 (Procell, Wuhan, China), HCC827 (GuangZhou Jennio Biotech Co., Ltd., China), and JIMT-1 (Shanghai Model Organisms Center, Inc.).

[0240] The results are shown in Figure 12 as mean and SD (n = 3), demonstrating that JY207 can induce drug-specific cytotoxicity against different CD47 / PD-L1 double-positive tumor cells. In general, the EC 50 was found to be at the μg / mL level. Example 6a

[0241] In vitro cytotoxicity of compounds JY207A, JY207A1, and JY207B against tumor cell lines To verify cytotoxic activity, CD47 + / PD-L1 + Tumor cell lines BXPC-3, SKBR-3, NCIH661, and NCIH520, and CD47 + / PD-L1 - MKN45 and CD47 - / PD-L1 + NCIN87 was selected for in vitro viability analysis. Cells were grown at 3 × 10 cells per well in a 96-well plate. 5 Cells were seeded at 1000 cells / well and treated with compounds JY207A, JY207A1, or JY207B at the indicated doses. Cell viability was measured using Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. Data were analyzed using GraphPad Prism software and expressed as the percentage of growth inhibition relative to the untreated control using the following formula: % Cytotoxicity = (1-OD sample / OD control) x 100%.

[0242] The results for JY207A, JY207A1, and JY207B are shown in Figures 12a, 12b, and 12c, respectively. Figures 12a to 12c demonstrate that JY207A, JY207A1, and JY207B induced drug-specific cytotoxicity against different CD47 / PD-L1 double-positive tumor cells, but did not induce cytotoxicity against tumor cells expressing only CD47 or PD-L1. [Example 7]

[0243] JY207-induced internalization The efficacy of ADCs depends on the efficiency of antigen-targeted internalization to deliver the payload into tumor cells. To examine the internalization rate of JY207, BxPC-3 cells were treated with 3 μg / mL Flour647-labeled DS8201a (Daiichi Sankyo, product code: WJA0005) or JY207 overnight at 4°C, after which unbound antibody was removed by washing. Aliquots of cells were stored at 4°C, and the remaining cells were incubated at 37°C for the indicated times to allow for internalization. Cells were analyzed using flow cytometry and FlowJo software. Internalization of the antigen-antibody complex was calculated as the mean percentage decrease in fluorescence intensity (MFI) at 37°C relative to the value at 4°C, after subtracting the background MFI obtained from the untreated control.

[0244] Surprisingly, as shown in Figure 13, JY207 induced a much faster and higher level of receptor internalization than the approved ADC, DS8201a. [Example 8]

[0245] JY207 in vitro selective binding assay For a CD47-associated antibody drug to be successful, it must minimize or eliminate toxicity to its target cells, red blood cells (RBCs). This experiment evaluates whether JY207 binds to RBCs.

[0246] Fluorescence-activated cell sorting (FACS) was used to detect the binding of JY207 to target cells. Briefly, freshly prepared red blood cells (from healthy donors) and BxPC-3 tumor cells (Procell, Wuhan, China) were used as target cells. Cells were incubated with the indicated doses of Flour647-labeled JY207 or FITC-labeled Hu5F9-G4 (anti-CD47 antibody, expressed and prepared in-house) for 30 minutes at 4°C in the dark. Cells were washed at least twice with PBS and analyzed by flow cytometry after incubation.

[0247] The results shown in Figure 14 demonstrated that both Hu5F9-G4 and JY207 could specifically bind to BxPC-3. For red blood cells, only Hu5F9-G4 bound with high affinity, and JY207 binding to RBCs from the same donor was undetectable. These data suggest that JY207 specifically binds to CD47 + and PD-L1 + It can selectively bind to tumor cells, but not to CD47 + and PD-L1 - It was confirmed that the antibody did not bind to RBCs. [Example 9]

[0248] In vivo efficacy of JY207 in a pancreatic cancer xenograft tumor model The in vivo antitumor activity of JY207 was evaluated in a CD47- and PD-L1-positive BxPC-3 xenograft model in NOD / SCID mice. BxPC-3 cells (2 × 10 6 ) was injected subcutaneously into the backs of mice. The average tumor volume was approximately 100 mm 3 At the time of tumor growth, mice were randomly assigned to one of four groups and administered vehicle (control) or JY207 (grouping and administration schedules are shown in Table 1; Q2D × 10 means 10 administrations every other day). Tumor volume and mouse weight were measured twice weekly or at the indicated time points.

[0249] [Table 1]

[0250] The results shown in Figure 15 indicate that JY207, even at a low dose of 0.1 mg / kg, can significantly inhibit the growth of BxPC-3 xenograft tumors. No significant difference in body weight was observed between the experimental and control groups, suggesting that the experimental dose of JY207 did not cause toxic effects in the animals. [Example 10]

[0251] In vivo efficacy of JY207 in NCI-H1975 xenograft tumor model The in vivo antitumor activity of JY207 was also evaluated in another CD47- and PD-L1-positive NCI-H1975 xenograft model in NOD / SCID mice. NCI-H1975 cells (2 × 10 6 ) was injected subcutaneously into the backs of mice. The average tumor volume was approximately 100 mm 3 At the time of tumor growth, mice were randomly assigned to one of two groups (n = 6 per group) and administered vehicle (control) or 3 mg / kg JY207 on a Q2D x 10 schedule (Q2D x 10 refers to 10 doses administered every other day). Tumor volume and mouse weight were measured periodically.

[0252] The results shown in Figure 16 indicate that JY207 at 3 mg / kg can effectively inhibit the growth of NCI-H1975 xenograft tumors. No significant difference in body weight was observed between the experimental and control groups, suggesting that the experimental dose of JY207 did not cause toxic effects in the animals.

[0253] The foregoing examples and description of the preferred embodiment should be construed as illustrative, rather than limiting, of the invention as defined in the claims. As will be readily appreciated, numerous variations and combinations of the features described above can be utilized without departing from the invention as set forth in the claims. Such variations are not considered a departure from the scope of the invention, and all such variations are intended to be encompassed within the scope of the following claims.

Claims

1. Compounds of formula (I) 【Chemical 1】 [In the formula, P is a non-immunogenic polymer; M is a proton or C １～５０ an end-capping group selected from alkyl and aryl, wherein one or more carbons of said alkyl may be replaced with a heteroatom; y is an integer selected from 1 to 10; A is a bispecific antibody or antigen-binding fragment thereof that targets two different antigens selected from a tumor-specific antigen (TSA) and a tumor-associated antigen (TAA); T is a trifunctional small molecule linker moiety; L １ and L ２ each is independently a hetero- or homobifunctional linker; each of a and b is an integer selected from 0 to 10; B is a branched linker, each branch having an amino acid sequence or carbohydrate moiety linked to an optional extension spacer, an autolytic spacer, wherein cleavage of said amino acid sequence or carbohydrate moiety by an enzyme triggers an autolysis mechanism to release D, or each branch having a disulfide bond, wherein cleavage of said disulfide bond releases D or a derivative thereof, or each branch having a scissile bond, wherein cleavage of said scissile bond by a specific cleavage mechanism releases D; each D is independently a cytotoxic small molecule or peptide; and n is an integer selected from 1 to 25.

2. L １ 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, trans-cyclooctene, tetrazine, triarylphosphine, boronic acid, and iodine.

3. L １ the functional group at the end of the linker of is capable of site-specific conjugation with A and is selected from the group consisting of thiol, maleimide, methylsulfonylpyrimidine, methylsulfonylbenzothiazole, vinylpyridine, ethyl P-ethynyl-N-(p-tolyl)phosphonamidate, 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, trans-cyclooctene, tetrazine, triarylphosphine, boronic acid, and iodine; where both linkers L １ and L ２ are independently the following: -(CH ２ ) ａ XY(CH ２ ) ｂ -、 -X(CH ２ ) ａ O(CH ２ CH ２ O) ｃ (CH ２ ) ｂ Y-、 -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 X-、 -X(CH ２ ) ａ O(CH ２ CH ２ O) ｂ (CH ２ ) ｃ W ２ C(O)(CH ２ ) ｄ Y-、 -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 X-、 -C≡C-, -CR １ =CR ２ -、 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; and each of X and Y is independently selected from C(═O), NR ２ ,S.O.,CR. ３ R ４ , or null; R １ , R ２ , R ３ , and R ４ are independently hydrogen, C1-10 alkyl, or (CH ２ ) １～１０ C(=O); W １ and / or W ３ is derived from a maleimide-based moiety, a methylsulfonylpyrimidine-based moiety, a methylsulfonylbenzothiazole-based moiety, a 4-vinylpyridine-based moiety, an ethyl P-ethynyl-N-phenylphosphonamidate-based moiety; W ２ represents a triazolyl- or tetrazolyl-containing group; said heterocyclyl group is selected from a maleimide-derived moiety or a tetrazolyl- or triazolyl-based moiety. and 【Chemistry 2】 wherein n and m are integers and are independently selected from 0 to 20. The compound of claim 1 , comprising a linker chain that can be selected from:

4. The compound of any one of claims 1 to 3, wherein the bispecific antibody is a single-chain bispecific antibody, a bispecific nanobody, or a bispecific antigen-binding domain thereof.

5. The compound of claim 4, wherein the bispecific antibody comprises an antigen-binding domain that binds to CD47, the antigen-binding domain comprising a light chain variable region (VL) and a heavy chain variable region (VH), and an antigen-binding domain that binds to PD-L1, the antigen-binding domain comprising a VL and a VH.

6. The compound of any one of claims 1 to 5, wherein the bispecific antibody is a single chain anti-CD47 / anti-PD-L1 bispecific antibody.

7. the VL of the antigen-binding domain that binds to CD47 comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 2 to 4, respectively, and the VH of the antigen-binding domain that binds to CD47 comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 5 to 7, respectively; The compound according to claim 5 or 6, wherein the VL of the antigen-binding domain that binds to PD-L1 comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 8 to 10, respectively, and the VH of the antigen-binding domain that binds to PD-L1 comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 11 to 13, respectively.

8. the VL of the antigen-binding domain that binds to CD47 comprises the amino acid sequence shown in SEQ ID NO: 15, and the VH of the antigen-binding domain that binds to CD47 comprises the amino acid sequence shown in SEQ ID NO: 16; The compound of claim 7, wherein the VL of the antigen-binding domain that binds to PD-L1 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the VH of the antigen-binding domain that binds to PD-L1 comprises the amino acid sequence set forth in SEQ ID NO:

17.

9. The compound of any one of claims 5 to 8, wherein the bispecific antibody has the amino acid sequence shown in SEQ ID NO: 1 or 14.

10. The antigen-binding domain that binds to CD47 and the antigen-binding domain that binds to PD-L1 are linked via a peptide linker or other chemical linker, and the linker is a linker that binds to the L of the bispecific antibody. １ 10. The compound of any one of claims 5 to 9, comprising a cysteine, an azide, or an unnatural amino acid residue for site-specific conjugation to

11. 11. The compound of any one of claims 1 to 10, wherein D is selected from any DNA crosslinking agent, microtubule inhibitor, DNA alkylating agent, topoisomerase inhibitor, or combinations thereof.

12. D is selected from MMAE, MMAF, SN38, DM1, DM4, calicheamicin, pyrrolobenzodiazepines, duocarmycin, or derivatives thereof, or combinations thereof; or 12. The compound of claim 11, wherein D is Dxd.

13. The compound of any one of claims 1 to 12, wherein the non-immunogenic polymer is polyethylene glycol (PEG).

14. The compound of claim 13 , wherein the PEG is a linear PEG or a branched PEG.

15. 15. The compound of claim 12 or 14, wherein at least one end of the polyethylene glycol is capped with methyl or a low molecular weight alkyl.

16. The compound according to any one of claims 13 to 15, wherein the total molecular weight of the PEG is 3,000 to 100,000.

17. 17. The compound of any one of claims 13 to 16, wherein the PEG is linked to a cyclic or acyclic trifunctional moiety T (e.g., lysine) via a permanent or cleavable bond.

18. Compound of formula (II) 【Chemistry 3】 [In the formula, P is a linear PEG; A is a bispecific antibody or antigen-binding fragment thereof that targets two different tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs); L １ and L ２ each is independently a bifunctional linker; each of a and b is an integer selected from 0 to 10; B is a branched linker, each branch having an amino acid sequence or carbohydrate moiety linked to an optional extension spacer, an autolytic spacer, wherein cleavage of said amino acid sequence or carbohydrate moiety by an enzyme triggers an autolysis mechanism to release D, or each branch having a disulfide bond, wherein cleavage of said disulfide bond releases D or a derivative thereof, or each branch having a scissile bond, wherein cleavage of said scissile bond by a specific cleavage mechanism releases D; each D is independently a cytotoxic small molecule or peptide; and n is an integer selected from 1 to 25.

19. L １ 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, trans-cyclooctene, tetrazine, triarylphosphine, boronic acid, and iodine.

20. L １ the functional group at the end of the linker of is capable of site-specific conjugation with A and is selected from the group consisting of thiol, maleimide, methylsulfonylpyrimidine, methylsulfonylbenzothiazole, vinylpyridine, ethyl P-ethynyl-N-(p-tolyl)phosphonamidate, 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, trans-cyclooctene, tetrazine, triarylphosphine, boronic acid, and iodine; where both linkers L １ and L ２ are independently the following: -(CH ２ ) ａ XY(CH ２ ) ｂ -、 -X(CH ２ ) ａ O(CH ２ CH ２ O) ｃ (CH ２ ) ｂ Y-、 -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 X-、 -X(CH ２ ) ａ O(CH ２ CH ２ O) ｂ (CH ２ ) ｃ W ２ C(O)(CH ２ ) ｄ Y-、 -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 X-、 -C≡C-, -CR １ =CR ２ -、 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; and each of X and Y is independently selected from C(═O), NR ２ ,S.O.,CR. ３ R ４ , or null; R １ , R ２ , R ３ , and R ４ are independently hydrogen, C1-10 alkyl, or (CH ２ ) １～１０ C(=O); W １ and / or W ３ is derived from a maleimide-based moiety, a methylsulfonylpyrimidine-based moiety, a methylsulfonylbenzothiazole-based moiety, a 4-vinylpyridine-based moiety, an ethyl P-ethynyl-N-phenylphosphonamidate-based moiety; W ２ represents a triazolyl- or tetrazolyl-containing group; said heterocyclyl group is selected from a maleimide-derived moiety or a tetrazolyl- or triazolyl-based moiety. and 【Chemistry 4】 wherein n and m are integers and are independently selected from 0 to 20.

20. The compound of claim 18, comprising a linker chain that may be selected from:

21. The compound of any one of claims 18 to 20, wherein the bispecific antibody is a single-chain bispecific antibody, a bispecific nanobody, or a bispecific antigen-binding domain thereof.

22. 22. The compound of claim 21, wherein the bispecific antibody comprises an antigen-binding domain that binds to CD47 comprising a VL and a VH, and an antigen-binding domain that binds to PD-L1 comprising a VL and a VH.

23. The compound of any one of claims 18 to 22, wherein the bispecific antibody is a single chain anti-CD47 / anti-PD-L1 bispecific antibody.

24. the VL of the antigen-binding domain that binds to CD47 comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 2 to 4, respectively, and the VH of the antigen-binding domain that binds to CD47 comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 5 to 7, respectively; The compound of claim 22 or 23, wherein the VL of the antigen-binding domain that binds to PD-L1 comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 8 to 10, respectively, and the VH of the antigen-binding domain that binds to PD-L1 comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 11 to 13, respectively.

25. the VL of the antigen-binding domain that binds to CD47 comprises the amino acid sequence shown in SEQ ID NO: 15, and the VH of the antigen-binding domain that binds to CD47 comprises the amino acid sequence shown in SEQ ID NO: 16; The compound of claim 24, wherein the VL of the antigen-binding domain that binds to PD-L1 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the VH of the antigen-binding domain that binds to PD-L1 comprises the amino acid sequence set forth in SEQ ID NO:

17.

26. The compound of any one of claims 22 to 25, wherein the bispecific antibody has the amino acid sequence shown in SEQ ID NO: 1 or 14.

27. The antigen-binding domain that binds to CD47 and the antigen-binding domain that binds to PD-L1 are linked via a peptide linker or other chemical linker, and the linker is a linker that binds to the L of the bispecific antibody. １ 27. The compound of any one of claims 22 to 26, comprising a cysteine, an azide, or an unnatural amino acid residue for site-specific conjugation to

28. 28. The compound of any one of claims 18 to 27, wherein D is selected from any DNA crosslinking agent, microtubule inhibitor, DNA alkylating agent, topoisomerase inhibitor, or combinations thereof.

29. D is selected from MMAE, MMAF, SN38, DM1, DM4, calicheamicin, pyrrolobenzodiazepines, duocarmycin, or derivatives thereof, or combinations thereof; or 29. The compound of claim 28, wherein D is Dxd.

30. The compound according to any one of claims 18 to 29, wherein the total molecular weight of the PEG is 3,000 to 100,000.

31. 31. The compound of claim 30, wherein the PEG is linked to any cyclic or acyclic trifunctional or polyfunctional moiety T (e.g., lysine) via a permanent or cleavable bond.

32. The following formula: 【Chemistry 5】 【Chemistry 6】 [wherein BsAb is a bispecific antibody or antigen-binding fragment thereof that targets PD-L1 and CD47] or a pharmaceutically acceptable salt thereof.

33. the bispecific antibody an antigen-binding domain that binds to CD47, comprising a VL comprising CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:2-4, respectively, and a VH comprising CDR1, CDR2, and CDR3 set forth in SEQ ID NOs:5-7, respectively; and An antigen-binding domain that binds to PD-L1, comprising a VL comprising CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 8 to 10, respectively, and a VH comprising CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 11 to 13, respectively.

33. The compound of claim 32, comprising:

34. the VL of the antigen-binding domain that binds to CD47 comprises the amino acid sequence shown in SEQ ID NO: 15, and the VH of the antigen-binding domain that binds to CD47 comprises the amino acid sequence shown in SEQ ID NO: 16; The compound of claim 33, wherein the VL of the antigen-binding domain that binds to PD-L1 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the VH of the antigen-binding domain that binds to PD-L1 comprises the amino acid sequence set forth in SEQ ID NO:

17.

35. The compound of any one of claims 32 to 34, wherein the bispecific antibody has the amino acid sequence shown in SEQ ID NO: 1 or 14.

36. A method for preparing a compound according to any one of claims 1 to 35, comprising: a) preparing a non-immunogenic modified (e.g., PEGylated) drug conjugate having a free functional group for site-specific conjugation; b) site-specifically conjugating the non-immunogenically modified (e.g., PEGylated) drug conjugate to the bispecific antibody to obtain a compound of formula (I) or (II). A method comprising:

37. 36. A pharmaceutical formulation comprising an effective amount of a compound of any one of claims 1 to 35 and a pharmaceutically acceptable salt, carrier, or excipient.

38. 36. The compound of any one of claims 1 to 35 for use in treating 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, kidney cancer, bladder cancer, stomach cancer, colon cancer, colorectal cancer, salivary gland cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, head and neck cancer, and endometrial cancer.

39. 36. The compound of any one of claims 1 to 35 for use in combination with an effective amount of another anti-cancer agent or 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, kidney cancer, bladder cancer, stomach cancer, colon cancer, colorectal cancer, salivary gland cancer, thyroid cancer, skin cancer, bone cancer, brain cancer, head and neck cancer, and endometrial cancer.

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