Tumor microenvironment-activated drug-conjugate and uses relating thereto

Abstract

Disclosed are extracellularly activated binder-drug conjugates, wherein both the binder and the free drug moiety have pharmacological activity.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 680,300, filed June 4, 2018. Background Technology

[0003] It is known that the PD-1-PD-L1 interaction drives T cell dysfunction, which can be blocked by anti-PD-1 / PD-L1 antibodies. However, studies have also shown that the function of the PD-1-PD-L1 axis is influenced by a complex immune regulatory network. In most advanced cancers, except for Hodgkin lymphoma (which has high PD-L1 / L2 expression) and melanoma (which has a high tumor mutational burden), the objective response rate of anti-PD-1 / PD-L1 monotherapy is only about 20%, and immune-related toxicity and excessive progression can occur in a small subset of patients during PD-1 / PD-L1 blockade therapy. The lack of efficacy in up to 80% of patients is not necessarily associated with negative PD-1 and PD-L1 expression, suggesting that the role of PD-1 / PD-L1 in immunosuppression and the mechanism of antibody action still need to be better defined. Similarly, similar limitations have been observed under CTLA-4 and other checkpoint pathways. Therefore, it is necessary to target important synergistic immunomodulatory mechanisms within or outside the PD-1 / PD-L1, CTLA-4, and other checkpoint networks to increase the response rate of immune checkpoint blockade therapy and reduce its toxicity in some conditions.

[0004] In this regard, agents believed to induce innate immune responses, such as STING, RIG-I, and TLR agonists, have the potential to enhance the efficacy of immune tumor checkpoint inhibitors. However, these types of agents are often too toxic for systemic use because dose-limiting toxicity is a product of innate immune activation throughout the body, and the maximum tolerated dose is not reached in many patients.

[0005] This invention is based on a novel system, particularly for the co-delivery of two classes of therapeutic agents: an innate immune inducer that triggers a local inflammatory event in the tumor to elicit an effective immune response, and one or more checkpoint inhibitors or co-stimulatory agonists that promote or maintain an adaptive immune response. This novel system addresses the systemic toxicity of either component, particularly the innate immune inducer, while maintaining its pharmacologically inactive form until release via proteases in the tumor microenvironment. Simply put, one agent induces an anti-tumor immune response, while the other ensures its effectiveness upon reaching the tumor. The checkpoint inhibitor or co-stimulatory agonist, along with TME enzyme release, helps the drug localize within the tumor and improves the therapeutic index relative to the individual component drugs. Summary of the Invention

[0006] One aspect of the present invention relates to a binder-drug conjugate comprising:

[0007] (i) A cell-binding portion that binds to a cell surface feature on a target cell in a diseased tissue, the cell surface feature being slowly internalized upon binding by the binder-drug conjugate;

[0008] (ii) A drug fraction having pharmacological effect on bystander cells adjacent to the target cells, wherein the EC50 of the drug fraction, when it is part of the binder-drug conjugate, is at least twice as weak as that of the free drug fraction released from the binder-drug conjugate; and

[0009] (iii) Covalently linking a polypeptide binding portion to a linker portion of the drug portion, the linker portion including a substrate recognition sequence cleavable by an enzyme present extracellularly in diseased tissue, wherein the linker portion is cleavable in the presence of the enzyme and releases the free drug portion.

[0010] In some embodiments, the EC50 of the drug portion, when it is part of a binder-drug conjugate, for achieving the pharmacological effect is reduced by at least 5 times relative to the free drug portion released from the binder-drug conjugate, and more preferably, by at least 10 times, 20 times, 30 times, 40 times, 50 times, 75 times, 100 times, 250 times, 500 times, or even 1000 times.

[0011] In some embodiments, the diseased tissue is a tumor. In some embodiments, the target cells are tumor cells. In some embodiments, the target cells are macrophages, monocyte-derived suppressor cells (MDSCs), dendritic cells, fibroblasts, T cells, NK cells, mast cells, granulocytes, eosinophils, and B cells.

[0012] In some embodiments, the binder-drug conjugate has an internalization half-life of at least 6 hours, more preferably at least 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 75 hours or even 100 hours when it binds to the surface features on the target cells.

[0013] In some embodiments, the cell surface features are proteins selectively expressed or upregulated by target cells in diseased tissue relative to normal cells from healthy tissue. For example, the proteins are detectable on the surface of target cells at levels 2 times higher than those from normal cells from the tissue, or even more preferably at levels at least 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, or even 1000 times higher than those from normal cells from the tissue.

[0014] In some embodiments, the cell surface features are proteins that are selectively expressed or upregulated by target cells in diseased tissues relative to cells from other tissues, particularly cells from critical organs. For example, the proteins are detectable on the surface of target cells at levels that are 2 times higher than those from cells from other tissues, or even more preferably at levels that are at least 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, or even 1000 times higher than those from cells from other tissues.

[0015] In some embodiments, the cell surface feature is a checkpoint protein and the binding portion is a checkpoint antagonist. Examples of checkpoint proteins include those selected from the group consisting of: CTLA-4, PD-1, LAG-3, BTLA, KIR, TIM-3, PD-L1, PD-L2, B7-H3, B7-H4, HVEM, GAL9, CD160, VISTA, BTNL2, TIGIT, PVR, BTN1A1, BTN2A2, BTN3A2, and CSF-1R, more preferably CTLA-4, PD-1, LAG-3, TIM-3, BTLA, VISTA, HVEM, TIGIT, PVR, PD-L1, and CD160.

[0016] In some embodiments, the cell surface is characterized as a co-stimulatory receptor and the binding portion is a co-stimulatory agonist of the receptor. Examples include a surface characteristic of a co-stimulatory receptor or ligand selected from the group consisting of: 4-1BB, 4-1BB-L, OX40, OX40-L, GITR, CD28, CD40, CD40-L, ICOS, ICOS-L, LIGHT, and CD27, more preferably 4-1BB, OX40, GITR, CD40, and ICOS.

[0017] In some implementations, the cell-binding portion is an antibody, such as a humanized antibody, a human antibody, or a chimeric antibody, or includes an antigen-binding portion that binds to cell surface features, such as Fab, F(ab)2, F(ab'), F(ab')2, F(ab')3, Fd, Fv, disulfide-linked Fv, dAb or sdAb (or nanobodies), CDR, scFv, (scFv)2, di-scFv, bi-scFv, tascFv (tandem scFv), AVIBODY (e.g., bifunctional, trifunctional, tetrafunctional antibodies), T-cell conjugating molecules (BiTE), scFv-Fc, Fcab, mAb2, small modular immunopharmaceutical (SMIP), Genmab / monoantibody or bispecific antibody (duobody), V-NAR domain, IgNAR, microantibody, IgGACH2, DVD-Ig, antibody precursor, intracellular antibody, or multispecific antibody.

[0018] In other embodiments, the binding agent portion is a non-antibody scaffold selected from the group consisting of: affinity antibodies, affinity molecules, affilin, anticarrier proteins, atrimer, high-affinity multimers, DARPin, FN3 scaffolds (e.g., adnectin and centyrin), fenomer, Kunitz domains, nanofitin, pronectin, OBodies, trifunctional antibodies, high-affinity multimers, bicyclic peptides, and Cys knots.

[0019] In some embodiments, the linker portion includes two, three, or even four substrate recognition sequences that are cleavable by the same or different enzymes (at least one of which is present extracellularly) present in the diseased tissue, wherein the linker portion can be completely cleaved to release the free drug moiety in the simultaneous or sequential presence of multiple enzymes. For example, a linker having two different substrate recognition sequences can be constructed, requiring cleavage by both MMP and FAPα. In a preferred embodiment, cleavage by one of these two enzymes requires cleavage by the other enzyme first—that is, by constructing a linker that is a poor substrate for FAPα when intact and is modified into a substrate cleaved by FAPα after MMP cleavage, MMP cleavage may be required before FAPα cleavage.

[0020] To further illustrate, a binder-drug conjugate can be represented by one of the following formulas:

[0021]

[0022] in

[0023] CBM indicates that the cell binding portion may be the same or different each time it appears;

[0024] L 1 Indicates a spacer base or a straight bond;

[0025] SRS stands for Substrate Recognition Sequence;

[0026] L 2 Indicates a trigger-activated self-degrading connector or a straight key;

[0027] DM indicates the drug component;

[0028] m represents an integer from 1 to 6; and

[0029] n represents an integer from 1 to 500, more preferably from 1 to 100, 1 to 10, or 1 to 5.

[0030] In some implementations, L 1 It is a hydrocarbon (straight-chain or cyclic), such as 6-maleimide hexanoyl, maleimide propionyl, and maleimide methylcyclohexane-1-carboxylic acid ester, or L 1 It is N-succinimide-4-(2-pyridylthio)valerate, N-succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate, and N-succinimide-(4-iodo-acetyl)aminobenzoate.

[0031] In some implementations, L 1 It is a polyether, such as poly(ethylene glycol) or other hydrophilic connector. For example, in the case where the CBM includes thiols (such as cysteine ​​residues), L 1 It can be a poly(ethylene glycol) coupled to a thiol group via a maleimide moiety, such as shown in the following formula:

[0032]

[0033] Where p represents an integer from 1 to 100, preferably from 6 to 50, and more preferably from 6 to 12.

[0034] In other embodiments, CBM includes thiols and L 1 In the case of a hydrocarbon moiety coupled via a maleimide moiety and a thiol group, L 1 It can be shown in the following formula:

[0035]

[0036] Where p represents an integer from 1 to 20, preferably from 1 to 4.

[0037] In some embodiments of the binder-drug conjugate of the present invention, the substrate recognition sequence is cleaved by an extracellular protease with protease activity, preferably a serine protease, metalloproteinase or cysteine ​​protease, located in the extracellular domain of the target tissue, i.e., an extracellular protease with protease activity that is a cell surface protease or a secreted / released protease.

[0038] In some implementations, the level of the protease present in the extracellular space of diseased tissues in patients is at least 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, or even 1,000 times higher than the level of the protease present in healthy tissues in patients.

[0039] In some implementations, the protease is present in the extracellular space at levels at least 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, or even 1,000 times higher in diseased tissues of the patient than in other tissues of the patient.

[0040] In some embodiments, the protease is a matrix metalloproteinase. The matrix metalloproteinase may be a membrane-bound matrix metalloproteinase (such as MMP14-17 and MMP24-25) or a secreted matrix metalloproteinase (such as MMP1-13, MMP18-23, and MMP26-28). In some embodiments, the metalloproteinase is MMP1, MMP2, MMP3, MMP4, MMP9, MMP11, MMP13, MMP14, MMP17, or MMP19, and more preferably MMP2, MMP9, or MMP14.

[0041] In some embodiments, the protease is an A deintegrin and metalloproteinase (ADAM), or an A deintegrin or metalloproteinase with a platelet-reactive protein motif (ADAMTS).

[0042] In some embodiments, the protease is pod protein, protein lyase (MT-SP1), neutrophil elastase, TMPRSS, thrombin, u-type plasminogen activator (uPA, also known as urokinase), PSMA, or CD10 (CALLA).

[0043] In some implementations, the protease is a proline-cleaving protease, such as fibroblast activation protein α (FAPα).

[0044] In some embodiments of the subject binder-drug conjugate, the substrate recognition sequence is cleaved via fibroblast activation protein α (FAPα) and represented by the following formula:

[0045]

[0046] in

[0047] R 2 It represents H or (C1-C6) alkyl, and preferably H;

[0048] R 3 The symbol represents H or (C1-C6) alkyl, preferably methyl, ethyl, propyl or isopropyl, and more preferably methyl;

[0049] R 4 It does not exist or represents (C1-C6) alkyl, -OH, -NH2 or halogen;

[0050] X represents O or S; and

[0051] If L 2 For a triggered self-degrading connector, -NH- indicates that it is an L 2 Partial amines or if L 2 If it is a straight bond, then -NH- represents an amine that is part of DM.

[0052] In some implementations, R 2 For H, R 3 For methyl, R 4 It does not exist and X is 0.

[0053] In some implementations, L 2 The triggered self-degrading linker is selected from the group consisting of: -NH-(CH2)4-C(=O)-, -NH-(CH2)3-C(=O)-, p-aminobenzyloxycarbonyl (PABC), and 2,4-bis(hydroxymethyl)aniline. In some embodiments, L 2 For p-aminobenzyloxycarbonyl (PABC), especially when the subject recognition sequence is cleaved by FAPα, because p-aminobenzyloxycarbonyl (PABC) satisfies the special requirement of P'1 for FAPα.

[0054] In some embodiments, the free drug fraction interacts with an intracellular target, and the pharmacological effect of the drug fraction depends on the free drug fraction being cell-permeable, i.e., capable of interacting with its intracellular target, whereas when it is a portion of a binder-drug conjugate, the drug fraction is substantially impermeable to cells. For example, the rate of accumulation of the binder-drug conjugate in cells is less than 50% of the rate of the free drug fraction, more preferably less than 25%, 10%, 5%, 1%, or even less than 0.1% of the rate of the free drug fraction. For example, the EC50 of the pharmacological effect of the free drug fraction is at least 2 times smaller (more effective) than that of the binder-drug conjugate, more preferably at least 5, 10, 20, 30, 40, 50, 100, 250, 500, or even 1000 times smaller (more effective) than that of the binder-drug conjugate.

[0055] In some implementations, the free drug moiety interacts with the extracellular target and when covalently linked to L 1 The pharmacological effect of the drug moiety is substantially reduced. For example, the EC50 of the pharmacological effect of the free drug moiety is at least 2 times smaller (more effective) than that of the binder-drug conjugate, preferably at least 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 250 times, 500 times or even 1000 times smaller.

[0056] In some embodiments, when delivered systemically, the therapeutic index of the binder-drug conjugate is at least 2 times greater than that of the free drug fraction, and even more preferably at least 5, 10, 20, 30, 40, 50, 100, 250, 500, or even 1000 times greater than that of the free drug fraction.

[0057] In some embodiments, the free drug portion is an immunomodulator, comprising a drug portion that acts as an immune activator and / or an inducer of innate immune pathway responses. In some embodiments, the free drug portion induces the production of IFN-α. In some embodiments, the free drug portion induces the production of pro-inflammatory cytokines. In some embodiments, the free drug portion induces the production of IL-1β. In some embodiments, the free drug portion induces the production of IL-18.

[0058] In some implementations, the free drug portion promotes the expansion and survival of effector cells, including NK, γδT, and CD8+T cells.

[0059] In some embodiments, the free drug portion is an immune-DASH inhibitor that inhibits the enzymatic activity of DPP8 and DPP9 and induces pyroptosis of macrophages in vitro and / or in vivo.

[0060] In some embodiments, the free drug portion is a damage-associated molecular pattern molecule. In some embodiments, the free drug portion is a pathogen-associated molecular pattern molecule.

[0061] In some implementations, the free drug portion is a STING agonist.

[0062] In some implementations, the free drug portion is a RIG-1 agonist.

[0063] In some embodiments, the free drug portion is a Toll-like receptor (TLR) agonist, such as selected from the group consisting of TLR1 / 2 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 / 2 agonists, TLR7 agonists, TLR7 / 8 agonists, TLR7 / 9 agonists, TLR8 agonists, TLR9 agonists, and TLR11 agonists, preferably selected from the group consisting of TLR3 agonists, TLR7 agonists, TLR7 / 8 agonists, and TLR9 agonists.

[0064] In some implementations, the free drug portion is a cyclic dinucleotide.

[0065] In some implementations, the free drug portion is ADU-S100.

[0066] In some embodiments, the free drug portion is a RIG-I agonist, wherein the RIG-I agonist is KIN700, KIN1148, KIN600, KIN500, KIN100, KIN101, KIN400, KIN2000, or SB-9200.

[0067] In some implementations, the free drug portion is selected from the group consisting of: S-27609, CL307, UC-IV150, imiquimod, gardiquimod, resiquimod, motolimod, VTS-1463GS-9620, GSK2245035, TMX-101, TMX-201, TMX-202, isatoribine, AZD8848, MEDI9197, 3M-051, 3M-852, 3M-052, 3M-854A, S-34240, KU34B, or CL663.

[0068] In some implementations, the free drug fraction is cytotoxic to cancer-associated fibroblasts (CAFs).

[0069] In some implementations, the free drug portion biases the tumor-associated macrophage population toward M1 macrophages and / or inhibits the immunosuppressive activity of M2 macrophages.

[0070] In some implementations, the free drug portion accelerates T cell initiation and / or dendritic cell transport.

[0071] In some implementations, the free drug portion inhibits or depletes Treg cells, for example, by blocking immunosuppressive function or migrating to lymph nodes and / or the tumor microenvironment.

[0072] In some embodiments, the therapeutic index (TI) of the binder-drug conjugate is at least 5 times greater than that of the free drug fraction when administered systemically, more preferably at least 10, 20, 30, 40, 50, 75, or even 100 times greater.

[0073] In some embodiments, the free drug portion is a low molecular weight inhibitor, i.e., a molecular weight of less than 5000 amu, preferably less than 2500 amu, and even more preferably less than 1500 amu.

[0074] Another aspect of the present invention provides a binder-drug conjugate comprising a polypeptide sequence (such as an antibody fragment or a non-antibody scaffold) including one or more small domains binding to a cell surface protein on tumor cells, preferably one or more affinity sequences and linked to one or more drug-conjugate moieties, said drug-conjugate moieties being as shown in the following formula:

[0075]

[0076] in

[0077] L 1 Indicates a spacer base or a straight bond;

[0078] SRS represents the substrate recognition sequence of extracellular proteases expressed in the extracellular space of tumor cells;

[0079] L 2 Indicates a trigger-activated self-degrading connector or a straight key;

[0080] DM indicates the drug component;

[0081] m represents an integer from 1 to 6, preferably 1, 2, or 3; and

[0082] n represents an integer from 1 to 500, more preferably from 1 to 100, 1 to 10, or 1 to 5.

[0083] The binder-drug conjugate has an internalization half-life of at least 6 hours, more preferably at least 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 75 hours or even 100 hours when it binds to the surface features on the target cells.

[0084] In some embodiments, at least one small domain binding polypeptide sequence is a PD-L1 binding moiety.

[0085] In some embodiments, the peptide of the binder-drug conjugate is in the form of 1 × 10 -6 M or lower Kd (and more preferably 1×10 -7 M, 1×10-8 M, 1×10 -9 M, 1×10 -10 M or even 1×10 -11 M or lower Kd, especially in embodiments where the peptide is divalent or higher-order polyvalent for PD-L1 binding, binds to PD-L1 and inhibits the interaction between the bound PD-L1 and PD-1.

[0086] Another aspect of the present invention provides a multispecific binder-drug conjugate comprising

[0087] (i) A polypeptide comprising a polypeptide sequence having two or more distinct binding domains that selectively bind to two different cell surface proteins on two different cell types in a tumor, and

[0088] (ii) One or more drug-conjugate moieties linked to the polypeptide, the drug-conjugate moieties being as shown in the following formula:

[0089]

[0090] in

[0091] L 1 Indicates a spacer base or a straight bond;

[0092] SRS represents the substrate recognition sequence of extracellular proteases expressed in the extracellular space of tumor cells;

[0093] L 2 Indicates a trigger-activated self-degrading connector or a straight key;

[0094] DM indicates the drug component;

[0095] m represents an integer from 1 to 6, preferably 1, 2, or 3; and

[0096] n represents an integer from 1 to 500, more preferably from 1 to 100, 1 to 10, or 1 to 5.

[0097] The multispecific binder-drug conjugate has an internalization half-life of at least 6 hours, more preferably at least 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 75 hours or even 100 hours when bound to any of the surface proteins.

[0098] In certain embodiments of the multispecific binder-drug conjugate, the polypeptide includes a first binding domain polypeptide sequence that selectively binds to tumor cell antigens and a second binding domain polypeptide sequence that selectively binds to cells selected from the group consisting of macrophages, monocyte-derived suppressor cells (MDSCs), dendritic cells, fibroblasts, NK cells, mast cells, granulocytes, eosinophils, and B cells.

[0099] In certain embodiments of the multispecific binder-drug conjugate, the polypeptide comprises a first binding domain polypeptide sequence that acts as a checkpoint inhibitor or costimulatory agonist and binds to a checkpoint protein or costimulatory receptor protein expressed on tumor-infiltrating lymphocytes (such as LAG-3, TIM-3, TIGIT, PD-1, BTLA, or CTLA-4 in the case of checkpoints, and CD28, ICOS, OX40, GITR, CD137, or CD27 in the case of costimulatory proteins) and a second binding domain polypeptide sequence that acts as a checkpoint inhibitor and binds to checkpoints expressed on tumor cells (such as PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, CD155, HVEM, or galactohemagglutinin-9).

[0100] Another aspect of the invention relates to a combination PD-L1 inhibitor / innate immune stimulant comprising a PD-L1-binding polypeptide and a pharmaceutical portion conjugated thereto as a sterile inducer of an innate immune response (such as an immune-DASH inhibitor, a STING agonist, a TRL7 / 8 agonist, or a RIG-1 agonist), wherein, relative to other tissues of the patient, the PD-L1-binding polypeptide causes the PD-L1 inhibitor / innate stimulant to accumulate in the tumor, and wherein, relative to other tissues of the patient, the pharmaceutical portion is selectively released from the PD-L1-binding polypeptide in the tumor microenvironment.

[0101] In some embodiments of the drug-conjugate of the present invention, the molecule includes a PD-L1 binding moiety, which is 1 × 10 -6 M or lower Kd (and more preferably 1×10) -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 An affinity polypeptide sequence of PD-L1 (M or lower Kd) that binds to PD-L1 and inhibits its binding to PD-L1 and the interaction between PD-L1 and PD-1.

[0102] In some embodiments, the PD-L1-binding affinity peptide binds to human PD-L1 and blocks interaction with human PD-1. In some embodiments, the PD-L1-binding affinity peptide is expressed at a concentration of 1 × 10⁻⁶. -7M or lower Kd, 1×10 -8 M or lower Kd, 1×10 -9 M or lower Kd or even 1×10 -10 M or lower Kd binds to PD-L1. In some embodiments, the affinity peptide for PD-L1 binds at 10 -3 s -1 Or slower, 10 -4 s -1 Or slower or even 10 -5 s -1 Or even slower K off Binding to PD-L1. In some embodiments, the affinity peptide for PD-L1 binds at 10... 3 M -1 s -1 Or faster, 10 4 M -1 s -1 Or faster, 10 5 M - 1 s -1 Or faster or even 10 6 M -1 s -1 Or faster K on Binding to PD-L1. In some embodiments, in a competitive binding assay with human PD-1, the affinity peptide that binds to PD-L1 binds to PD-L1 at an IC50 of 1 μM or less, 100 nM or less, 40 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or even 0.1 nM or less.

[0103] In some embodiments, the affinity peptide that binds to PD-L1 has a Tm of 65°C or higher and 70°C or higher, 75°C or higher, 80°C or higher, or 85°C or higher. In some embodiments, the protein has a Tm of 65°C or higher and 70°C or higher, 75°C or higher, 80°C or higher, or 85°C or higher.

[0104] In some embodiments, the affinity polypeptide that binds to PD-L1 has the amino acid sequence represented in general formula (I):

[0105] FR1-(Xaa) n -FR2-(Xaa) m -FR3 (I)

[0106] in

[0107] FR1 is a polypeptide sequence represented by MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID No. 1) or a polypeptide sequence having at least 70% homology with it;

[0108] FR2 is a polypeptide sequence represented by GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID No. 2) or a polypeptide sequence having at least 70% homology with it;

[0109] FR3 is a polypeptide sequence represented by EDLVLTGYQV DKNKDDELTG F (SEQ ID No. 3) or a polypeptide sequence having at least 70% homology with it; and

[0110] Each time Xaa appears, it is an individual amino acid residue; and

[0111] n and m are each an independent integer from 3 to 20.

[0112] In some embodiments, FR1 may be a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% homology with SEQ ID No. 1. In some embodiments, FR2 may be a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% homology with SEQ ID No. 2. In some embodiments, FR3 may be a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% homology with SEQ ID No. 2.

[0113] For embodiments in which at least one drug-conjugate moiety is linked to the affinity sequence via a thiol side chain of a cysteine ​​residue introduced into the affinity sequence, the cysteine ​​will preferably be provided in a portion of the affinity sequence region corresponding to FR1, FR2, and / or FR3, and more preferably, will replace the side chain in the affinity sequence with a solvent-accessible amino acid residue that is independent of hydrogen bonding of other parts of the affinity sequence. Generally, the cysteine ​​will not introduce a ring (Xaa). n Or (Xaa) m middle.

[0114] In some embodiments, the affinity peptide that binds to PD-L1 has an amino acid sequence represented by the following general formula:

[0115] MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(Xaa) n -Xaa2-TNYYIKVRAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa)m -Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF(SEQID No.4)

[0116] in

[0117] Each time Xaa appears, it is an individual amino acid residue;

[0118] n and m are each independent integers from 3 to 20;

[0119] Xaa1 can be Gly, Ala, Val, Arg, Lys, Asp, or Glu;

[0120] Xaa2 is Gly, Ala, Val, Ser, or Thr;

[0121] Xaa3 can be Arg, Lys, Asn, Gln, Ser, or Thr;

[0122] Xaa4 is Gly, Ala, Val, Ser, or Thr;

[0123] Xaa5 can be Ala, Val, Ile, Leu, Gly, or Pro;

[0124] Xaa6 is Gly, Ala, Val, Asp, or Glu; and

[0125] Xaa7 can be Ala, Val, Ile, Leu, Arg, or Lys.

[0126] For some embodiments, Xaa1 is Gly, Ala, Arg, or Lys, more preferably Gly or Arg. For some embodiments, Xaa2 is Gly or Ser. For some embodiments, Xaa3 is Arg, Arg, Lys, Asn, or Gln, more preferably Lys or Asn. For some embodiments, Xaa4 is Gly or Ser. For some embodiments, Xaa5 is Ala, Val, Ile, Leu, Gly, or Pro, more preferably Ile, Leu, or Pro, and even more preferably Leu or Pro. For some embodiments, Xaa6 is Ala, Val, Asp, or Glu, even more preferably Ala or Glu. For some embodiments, Xaa7 is Ile, Leu, or Arg, more preferably Leu or Arg.

[0127] For those embodiments in which at least one drug-conjugate moiety is linked to the affinity sequence via a thiol side chain of a cysteine ​​residue introduced into the affinity sequence, the cysteine ​​residue will preferably be provided in the circumcyclic sequence (Xaa). n Or (Xaa) mThe sequence is part of the affinity sequence outside the compound. Therefore, SEQ ID No. 4 may include 1 to 5 cysteine ​​residues replacing amino acid residues at the changed positions in the sequence.

[0128] In some embodiments, the affinity peptide that binds to PD-L1 has an amino acid sequence represented by the following general formula:

[0129] MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(Xaa) n -STNYYIKVRAGDNKYMHLKVFNGP-(Xaa) m -ADRVLTGYQVDKNKDDELTGF(SEQ ID No.5)

[0130] Each occurrence of Xaa represents an individual amino acid residue; and n and m are each an independent integer from 3 to 20.

[0131] For those embodiments in which at least one drug-conjugate moiety is linked to the affinity sequence via a thiol side chain of a cysteine ​​residue introduced into the affinity sequence, the cysteine ​​residue will preferably be provided in the circumcyclic sequence (Xaa). n Or (Xaa) m The sequence is part of the affinity sequence outside the compound. Therefore, SEQ ID No. 5 may include 1 to 5 cysteine ​​residues replacing amino acid residues at the modified positions in the sequence.

[0132] In some implementations of the above sequence, (Xaa) n (“Ring 2”) is the amino acid sequence represented in general formula (II):

[0133] -aa1-aa2-aa3-Gly-Pro-aa4-aa5-Trp-aa6- (II)

[0134] in

[0135] aa1 represents an amino acid residue with a basic side chain;

[0136] aa2 represents an amino acid residue, preferably an amino acid residue having a neutral polar or nonpolar side chain or a charged (acidic or basic) side chain, more preferably an amino acid residue with a small aliphatic side chain, a neutral polar side chain or a basic or acidic side chain;

[0137] aa3 indicates an amino acid residue with an aromatic or basic side chain;

[0138] aa4 indicates an amino acid residue having a neutral polar or nonpolar side chain or a charged (acidic or basic) side chain, preferably a neutral polar side chain or a charged (acidic or basic) side chain;

[0139] aa5 indicates an amino acid residue having a neutral polar or charged (acidic or basic) or small aliphatic or aromatic side chain, preferably a neutral polar or charged side chain; and

[0140] aa6 represents an amino acid residue with an aromatic or acidic side chain.

[0141] For some embodiments, aa1 represents Lys, Arg, or His, more preferably Lys or Arg. For some embodiments, aa2 represents Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg, or His, more preferably Ala, Gln, Asp, or Glu. For some embodiments, aa3 represents Phe, Tyr, Trp, Lys, Arg, or His, preferably Phe, Tyr, or Trp, more preferably His or Tyr, Trp, or His. For some embodiments, aa4 represents Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg, or His, more preferably Gln, Lys, Arg, His, Asp, or Glu. For some embodiments, aa5 represents Ser, Thr, Asn, Gln, Asp, Glu, Arg, or His, more preferably Ser, Asn, Gln, Asp, Glu, or Arg. For some implementations, aa6 represents Phe, Tyr, Trp, Asp, or Glu; preferably Trp or Asp; more preferably Trp.

[0142] In some other implementations of the above sequence, (Xaa) n (“Ring 2”) is the amino acid sequence represented in general formula (III):

[0143] -aa1-aa2-aa3-Phe-Pro-aa4-aa5-Phe-Trp- (III)

[0144] in

[0145] aa1 represents an amino acid residue with a basic or aromatic side chain;

[0146] aa2 represents an amino acid residue, preferably an amino acid residue having a neutral polar or nonpolar side chain or a charged (acidic or basic) side chain, more preferably an amino acid residue with a small aliphatic side chain, a neutral polar side chain or a basic or acidic side chain;

[0147] aa3 represents an amino acid residue having an aromatic or basic side chain, preferably Phe, Tyr, Trp, Lys, Arg or His, more preferably Phe, Tyr, Trp or His, and even more preferably Tyr, Trp or His.

[0148] aa4 indicates an amino acid residue having a neutral polar or nonpolar side chain or a charged (acidic or basic) side chain, preferably a neutral polar side chain or a charged (acidic or basic) side chain; more preferably Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg, or His, and even more preferably Gln, Lys, Arg, His, Asp, or Glu; and

[0149] aa5 indicates an amino acid residue having a neutral polar or charged (acidic or basic) or small aliphatic or aromatic side chain, preferably a neutral polar or charged side chain; more preferably Ser, Thr, Asn, Gln, Asp, Glu, Arg or His, and even more preferably Ser, Asn, Gln, Asp, Glu or Arg.

[0150] For some embodiments, aa1 represents Lys, Arg, His, Ser, Thr, Asn, or Gln, more preferably Lys, Arg, His, Asn, or Gln, and even more preferably Lys or Asn. For some embodiments, aa2 represents Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg, or His, more preferably Ala, Gln, Asp, or Glu. For some embodiments, aa3 represents Phe, Tyr, Trp, Lys, Arg, or His, more preferably Phe, Tyr, Trp, or His, and even more preferably Tyr, Trp, or His. For some embodiments, aa4 represents Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg, or His, and even more preferably Gln, Lys, Arg, His, Asp, or Glu. For some implementations, aa5 represents Ser, Thr, Asn, Gln, Asp, Glu, Arg, or His, and even more preferably Ser, Asn, Gln, Asp, Glu, or Arg.

[0151] In some implementations of the above sequence, (Xaa) n (“Ring 2”) is an amino acid sequence selected from SEQ ID No. 6 to 40 or an amino acid sequence having at least 80% homology with it, and more preferably an amino acid sequence having at least 85%, 90%, 95% or even 98% homology with it.

[0152] In some implementations of the above sequence, (Xaa) n (“Ring 2”) is an amino acid sequence selected from SEQ ID No. 6 to 40 or an amino acid sequence having at least 80% identity with it, and more preferably an amino acid sequence having at least 85%, 90%, 95% or even 98% identity with it.

[0153] In some implementations of the above sequence, (Xaa) m (“Ring 4”) is the amino acid sequence represented in general formula (IV):

[0154] -aa7-aa8-aa9-aa10-aa11-aa12-aa13-aa14-aa15- (IV)

[0155] in

[0156] aa7 indicates an amino acid residue with a neutral polar or nonpolar side chain or an acidic side chain;

[0157] aa8 represents an amino acid residue, preferably an amino acid residue having a neutral polar or nonpolar side chain, a charged (acidic or basic) side chain, or an aromatic side chain, more preferably an amino acid residue with a charged (basic or acidic) side chain.

[0158] aa9 represents an amino acid residue, preferably an amino acid residue having a neutral polar or nonpolar side chain, a charged (acidic or basic) side chain, or an aromatic side chain, more preferably an amino acid residue with a neutral polar side chain or an acidic side chain.

[0159] aa10 represents an amino acid residue, preferably an amino acid residue having a neutral polar or nonpolar side chain, a charged (acidic or basic) side chain, or an aromatic side chain, more preferably an amino acid residue with a neutral polar side chain, a basic side chain, or an acidic side chain.

[0160] aa11 represents an amino acid residue, preferably an amino acid residue having a neutral polar side chain, a charged (acidic or basic) side chain, a nonpolar aliphatic side chain, or an aromatic side chain, more preferably an amino acid residue with a neutral polar side chain, a basic side chain, or an acidic side chain.

[0161] aa12 represents an amino acid residue, preferably an amino acid residue having a neutral polar side chain, a charged (acidic or basic) side chain, a nonpolar aliphatic side chain, or an aromatic side chain, and more preferably an acidic side chain.

[0162] aa13 represents an amino acid residue, preferably an amino acid residue having a neutral polar side chain, a charged (acidic or basic) side chain, a nonpolar aliphatic side chain, or an aromatic side chain, and more preferably an acidic side chain.

[0163] aa14 represents an amino acid residue, preferably an amino acid residue with a neutral polar side chain or a charged (acidic or basic) side chain; and

[0164] aa15 represents an amino acid residue, preferably an amino acid residue having a neutral polar or neutral nonpolar side chain or a charged (acidic or basic) side chain.

[0165] For some embodiments, aa7 represents Gly, Ala, Val, Pro, Trp, Gln, Ser, Asp, or Glu, and even more preferably Gly, Ala, Trp, Gln, Ser, Asp, or Glu. For some embodiments, aa8 represents Asp, Glu, Lys, Arg, His, Gln, Ser, Thr, Asn, Ala, Val, Pro, Gly, Tyr, or Phe, and even more preferably Asp, Glu, Lys, Arg, His, or Gln. For some embodiments, aa9 represents Gln, Ser, Thr, Asn, Asp, Glu, Arg, Lys, Gly, Leu, Pro, or Tyr, and even more preferably Gln, Thr, or Asp. For some embodiments, aa10 represents Asp, Glu, Arg, His, Lys, Ser, Gln, Asn, Ala, Leu, Tyr, Trp, Pro, or Gly, and even more preferably Asp, Glu, His, Gln, Asn, Leu, Trp, or Gly. For some embodiments, aa11 represents Asp, Glu, Ser, Thr, Gln, Arg, Lys, His, Val, Ile, Tyr, or Gly, and even more preferably Asp, Glu, Ser, Thr, Gln, Lys, or His. For some embodiments, aa12 represents Asp, Glu, Ser, Thr, Gln, Asn, Lys, Arg, Val, Leu, Ile, Trp, Tyr, Phe, or Gly, and even more preferably Asp, Glu, Ser, Tyr, Trp, Arg, or Lys. For some embodiments, aa13 represents Ser, Thr, Gln, Asn, Val, Ile, Leu, Gly, Pro, Asp, Glu, His, Arg, Trp, Tyr, or Phe, and even more preferably Ser, Thr, Gln, Asn, Val, Ile, Leu, Gly, Asp, or Glu. For some embodiments, aa14 represents Ala, Ile, Trp, Pro, Asp, Glu, Arg, Lys, His, Ser, Thr, Gln, or Asn, and even more preferably Ala, Pro, Asp, Glu, Arg, Lys, Ser, Gln, or Asn. For some embodiments, aa15 represents His, Arg, Lys, Asp, Ser, Thr, Gln, Asn, Ala, Val, Leu, Gly, or Phe, and even more preferably His, Arg, Lys, Asp, Ser, Thr, Gln, or Asn.

[0166] In some implementations of the above sequence, (Xaa) n(“Ring 4”) is an amino acid sequence selected from SEQ ID No. 41 to 75 or an amino acid sequence having at least 80% homology with it, and more preferably an amino acid sequence having at least 85%, 90%, 95% or even 98% homology with it.

[0167] In some implementations of the above sequence, (Xaa) n (“Ring 4”) is an amino acid sequence selected from SEQ ID No. 41 to 75 or an amino acid sequence having at least 80% identity with it, and more preferably an amino acid sequence having at least 85%, 90%, 95% or even 98% identity with it.

[0168] In some embodiments, the affinity polypeptide that binds to PD-L1 has an amino acid sequence selected from SEQ ID No. 76 to 84, or has at least 70% homology with it, and even more preferably has an amino acid sequence with at least 75%, 80%, 85%, 90%, 95% or even 98% homology with it.

[0169] In some embodiments, the affinity polypeptide that binds to PD-L1 has an amino acid sequence selected from SEQ ID No. 76 to 84, or has at least 70% identity with it, and even more preferably has an amino acid sequence with at least 75%, 80%, 85%, 90%, 95% or even 98% identity with it.

[0170] In some embodiments, the affinity polypeptide that binds to PD-L1 has an amino acid sequence that can be encoded by a nucleic acid having a coding sequence of nucleotides 1-336 corresponding to one of SEQ ID Nos. 85 to 92, or a coding sequence having at least 70% identity with it, and even more preferably a coding sequence having at least 75%, 80%, 85%, 90%, 95% or even 98% identity with it.

[0171] In some embodiments, the PD-L1-binding affinity peptide has an amino acid sequence encoded by a nucleic acid having a coding sequence that can hybridize with any one of SEQ ID No. 85 to 92 under stringent conditions of 6X sodium chloride / sodium citrate (SSC) at 45°C followed by washing in 0.2X SSC at 65°C.

[0172] In some embodiments, the PD-L1-binding affinity described herein binds to PD-L1 in a manner that competes with anti-PD-L1 antibodies atezolizumab, avelumab, and / or durvalumab for PD-L1 binding.

[0173] In some embodiments, the PD-L1-binding affinity polypeptide forms a crystal structure with PD-L1, the crystal structure comprising an interface involving at least 10 residues of PD-L1 selected from the following: Ile-54, Tyr-56, Glu-58, Glu-60, Asp-61, Lys-62, Asn-63, Gln 66, Val-68, Val-76, Val-111, Arg-113, Met-115, Ile-116, Ser-117, Gly-120, Ala-121, Asp-122, Tyr-123, and Arg-125.

[0174] In some embodiments, the binding of the PD-L1 affinity peptide to PD-L1 (a) increases T cell proliferation in a mixed lymphocyte response (MLR) assay; (b) increases interferon-γ production in an MLR assay; and / or (c) increases interleukin-2 (IL-2) secretion in an MLR assay.

[0175] In some embodiments, the binder-drug conjugate of the present invention is a fusion protein that, in addition to the affinity polypeptide that binds to PD-L1 or other target-binding moieties, may include, for example, one or more additional amino acid sequences selected from the group consisting of: secretion signaling sequences, peptide linker sequences, affinity tags, transmembrane domains, cell surface retention sequences, substrate recognition sequences for post-translational modification, polymeric domains that generate protein polymers aggregated via protein-protein interactions, polypeptide moieties that extend half-life, polypeptide sequences for altering antibody tissue localization and antigen binding sites, and one or more additional affinity polypeptide sequences that bind to other and different targets.

[0176] In some embodiments, the fusion protein includes a polypeptide moiety with an extended half-life, such as selected from the group consisting of: an Fc domain or a portion thereof, albumin or a portion thereof, a polypeptide moiety that binds to albumin, transferrin or a portion thereof, a polypeptide moiety that binds to transferrin, fibronectin or a portion thereof, or a polypeptide moiety that binds to fibronectin.

[0177] In cases where the fusion protein includes an Fc domain or a portion thereof, in some embodiments, it is an Fc domain that retains FcN ​​binding.

[0178] When the fusion protein includes an Fc domain or a portion thereof, in some embodiments, the Fc domain or a portion thereof is derived from IgA, IgD, IgE, IgG, and IgM or their subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0179] In some embodiments, the fusion protein has the amino acid sequence of SEQ ID No. 108 or SEQ ID No. 109, or a sequence having at least 70% homology with it, and even more preferably having at least 75%, 80%, 85%, 90%, 95% or even 98% identity with it.

[0180] In cases where the fusion protein includes an Fc domain or a portion thereof, in some embodiments, the Fc domain or a portion thereof retains effector functions selected from C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of B cell receptors, or combinations thereof.

[0181] In some embodiments, where the fusion protein includes a polypeptide portion with an extended half-life, the portion increases the serum half-life of the protein by at least 5 times, more preferably 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 500 times, or even 1000 times, relative to the absence of the protein.

[0182] In some embodiments, the fusion protein of the present invention is provided as a pharmaceutical formulation suitable for therapeutic use in human patients, said pharmaceutical formulation further comprising one or more pharmaceutically acceptable excipients, buffers, salts, etc.

[0183] Another aspect of the invention relates to pharmaceutical formulations suitable for therapeutic use in human patients, comprising (i) the binder-drug conjugates or combinations of PD-L1 inhibitors / innate immune stimulants described herein, and (ii) one or more pharmaceutically acceptable excipients, buffers, salts, etc.

[0184] In some embodiments of the drug-conjugate of the present invention, the free drug portion is an immuno-DASH inhibitor. In some embodiments, the immuno-DASH inhibitor exhibits an in vitro intracellular IC50 of less than 200 nM in human macrophages for inhibiting DPP8 and DPP9. In some embodiments, the in vitro cell-free IC50 for inhibiting DPP8 and / or DPP9 (and preferably both DPP8 and DPP9) is less than 100 nM, 10 nM, 1.0 nM, 0.1 nM, 0.01 nM, or even 0.001 nM. In some embodiments, the EnPlex IC50 for inhibiting DPP8 and / or DPP9 (and preferably both DPP8 and DPP9) is less than 100 nM, 10 nM, 1.0 nM, 0.1 nM, 0.01 nM, 0.001 nM (1 picomolar), or even 0.0001 nM (100 femtomolar). In some embodiments, the Ki for inhibiting DPP8 and / or DPP9 (and preferably both DPP8 and DPP9) is less than 100 nM, 10 nM, 1.0 nM, 0.1 nM, 0.01 nM, 0.001 nM (1 picomolar) or even 0.0001 nM (100 femtomolar).

[0185] In some implementations, the subject immuno-DASH inhibitor also inhibits fibroblast activation protein (FAP) within the concentration range of drugs that are effective antitumor agents. For example, the Ki of FAP inhibition by the immuno-DASH inhibitor can be less than 100 nM, 10 nM, 1.0 nM, 0.1 nM, 0.01 nM, 0.001 nM (1 picomol) or even 0.0001 nM (100 femtomoles).

[0186] In some embodiments, the subject immune-DASH inhibitor inhibits human fibroblast activation protein (FAP) with an IC50 at least 2 times higher than that of human macrophages, more preferably at least 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times or even at least 100 times higher, i.e. immune-DASH is a more effective pyroptosis inducer than FAP inhibition.

[0187] In some embodiments, the immune-DASH inhibitor exhibits slow binding inhibitory kinetics. In some embodiments, the koff rate of interaction between the immune-DASH inhibitor and DPP4 is less than 1 × 10⁻⁴ / sec, and preferably less than 5 × 10⁻⁵ / sec, 3 × 10⁻⁵ / sec, or even less than 1 × 10⁻⁵ / sec.

[0188] In some implementations, the immune-DASH inhibitor is administered to the patient as a binder-drug conjugate in an amount sufficient to reduce the number of tumor-associated macrophages.

[0189] In some implementations, the immune-DASH inhibitor is administered to the patient as a binder-drug conjugate sufficient to reduce the amount of mononuclear myeloid-derived suppressor cells in the tumor.

[0190] In some implementations, the immune-DASH inhibitor is administered to the patient as a binder-drug conjugate in an amount sufficient to reduce the T-cell suppressive activity of granulocytic myeloid-derived suppressor cells in the tumor.

[0191] In some implementations, the immune-DASH inhibitor is administered to the patient as a binder-drug conjugate at a therapeutically effective dose that causes complete tumor regression, and the therapeutically effective dose is less than the maximum tolerated dose of the binder-drug conjugate.

[0192] In some implementations, the immune-DASH inhibitor is administered to the patient as a binder-drug conjugate, either alone or in combination with a PGE2 inhibitor, such as a cPLA-2 inhibitor.

[0193] In some implementations, the immune-DASH inhibitor is administered to the patient as a conjugate drug, either alone or in combination with DPP4 inhibitors such as sitagliptin, vildagliptin, saxagliptin, linagliptin, and alogliptin. Attached Figure Description

[0194] Figure 1A , Figure 1B and Figure 1C Structure and characterization of the AVA04-182 Fc fusion protein.

[0195] Figure 2 The binding kinetics of AVA04-182 Fc to mouse PD-L1 as evaluated by Biacore.

[0196] Figure 3 : By ELISA, AVA04-182 Fc competes with mouse PD-L1 / mouse PD-1.

[0197] Figure 4 : Mouse mixed lymphocyte response by ELISA AVA04-182 Fc.

[0198] Figure 5A and Figure 5B Structure and characterization of the AVA04-251 Fc fusion protein.

[0199] Figure 6 The binding dynamics of AVA04-251 Fc to human PD-L1 as evaluated by Biacore.

[0200] Figure 7 The inhibition of PD-1 / PD-L1 interaction by AVA04-251 Fc was evaluated by NFAT gene reporter assay (Promega).

[0201] Figure 8A , Figure 8B and Figure 8C Structure and characterization of AVA04-251 BH cys inline fusion protein.

[0202] Figure 9 Chemical structure of compound 6323.

[0203] Figure 10 Synthesis scheme of compound 6323.

[0204] Figure 11 Chemical structure of compound 6325.

[0205] Figure 12 Synthesis scheme of compound 6325.

[0206] Figure 13 Synthetic scheme using maleimide chemistry AVA04-251 BH cys-6323.

[0207] Figure 14 The synthesis scheme of AVA04-183 Fc-6325 from NHS Chemicals was used.

[0208] Figure 15 Effects of combined treatment (AVA04-182Fc+VbP) on tumor growth in a genotype murine bladder cancer (MB49) model.

[0209] Figure 16 The effect of tumor growth after tumor stimulation in a genotype murine bladder cancer (MB49) model.

[0210] Figure 17 Effects of combined treatment (AVA04-251Fc+VbP) on tumor growth in a humanized homologous colorectal cancer model (MC38 HuPD-L1).

[0211] Figure 18 Effects of combined treatment (AVA04-251Fc+VbP) on tumor growth in a humanized homologous colorectal cancer model (MC38 HuPD-L1).

[0212] Figure 19 The effect of tumor stimulation on tumor growth in a humanized homologous colorectal cancer model (MC38 HuPD-L1).

[0213] Figure 20 Comparison of the binding of AVA04-251 BH cys to human PD-L1 before and after conjugation with maleimide chemistry and IR Dye 800CW.

[0214] Figure 21 Comparison of the binding of AVA04-251Fc to human PD-L1 before and after conjugation with NHS Chemistry and IR Dye 800CW.

[0215] Figure 22 Biodistribution of AVA04-251 Fc-800 in the A375 mouse xenograft model.

[0216] Figure 23 Tumor infiltration of AVA04-251 Fc-800 in an A375 mouse xenograft model.

[0217] Figure 24 In vitro rhFAPα cleavage of affinity-connector-VbP prodrug.

[0218] Figure 25 In vitro rhFAPα cleavage kinetics of affinity-connector-VbP prodrug.

[0219] Figure 26 Evaluation of the connector-VbP prodrug compared to VbP in an acute toxicity study of Sprague Dawley rats.

[0220] Figure 27 Pyroptosis induced by in vitro affinity-adaptor-VbP prodrug in the J774 mouse macrophage cell line.

[0221] Figure 28 G-CSF stimulation induced in vivo by Cys-modified linker-VbP prodrug in BALB / c mice.

[0222] Figure 29 Ipilimumab (a biosimilar) / AVA04-141, which is transiently expressed in Expi293 cells, has a purified yield of approximately 160 mg / L after protein A purification.

[0223] Figure 30 Bevacizumab (a biosimilar) / AVA04-251, transiently expressed in Expi293 cells, could be purified to a yield of over 97%, and Biacore demonstrated that the bispecific antibody-affinity fusion was able to bind to both targets regardless of whether the construct included a flexible linker [(G4S)3] or a rigid linker [A(EAAAK)3].

[0224] Figure 31 Illustrative examples of antiPD-L1 affinity formats that can be used to generate the antiPD-L1-drug conjugates of the present invention include Fc fusions (showing a bivalent PD-L1 binder format and a bispecific bivalent PD-L1 binder and target X binder format), various formats of inline antibody fusions, BiTE formats, and inline fusions of antiPD-L1 affinity and receptor trap domains. Each of these formats can be derivatized with one or more drug-conjugates.

[0225] Figure 32 The affinity can be formatted at various sites on the Fc and should therefore be translated into IgG-affinity fusions. Typical (unoptimized) expression yields are in the range of 400-800 mg / L. Purity was assessed using analytical SEC-HPLC.

[0226] Figure 33 This demonstrates the cleavage selectivity of the FAPα substrate recognition sequence, even among tightly packed enzymes such as FAPα and PREP. Only FAPa is able to cleave and release the free drug moiety.

[0227] Figure 34 This describes a FAPα cleavable linker designed to increase DAR while preserving enzyme release of each drug moiety. Using this linker design, DAR can exceed 25, 50, or even 100.

[0228] Figure 35A and Figure 35B This indicates that FAPα is selectively overexpressed in the tumor microenvironment of most solid tumors. For example, analysis using mRNA (...) Figure 35A ), histochemistry Figure 35A ) and detection of enzyme activity ( Figure 35B The results showed that FAPα was upregulated in malignant human epithelial tissues relative to normal epithelial tissues.

[0229] Figure 36 FAPα-activated linkers are selectively activated only by FAPα.

[0230] Figure 37A and Figure 37BThe free drug fraction of Val-boroPro induced pyroptosis in AML cell lines in vitro. Human PDX models demonstrated the efficacy of Val-boroPro itself in vivo against MV4-11 AML cells (a human acute monocytic leukemia model). One million MV4-11 cells were injected into the tail vein of 10-week-old female NOD-SCID Il2rg- / - mice. Val-boroPro was administered intraperitoneally at a dose of 20 mg / mouse once daily on a 5-day cycle with a 2-day interruption.

[0231] Figure 38 Derivation of the crystal-derived structure of ACA04-261, an anti-PD-L1 affinity that binds to human PD-L1. Figure 16 Provide a list of amino acid residues involved in the interface between the two proteins. Detailed Implementation

[0232] I. Overview

[0233] One aspect of the present invention relates to a binder-drug conjugate comprising:

[0234] (i) A cell-binding portion of a cell surface feature that binds to a target cell in a diseased tissue, the cell surface feature being slowly internalized when bound by a binder-drug conjugate;

[0235] (ii) A drug fraction that has pharmacological activity on bystander cells near target cells, wherein the EC50 of the drug fraction, when acting as a portion of a binder-drug conjugate, is at least twice as low as that of the free drug fraction released from the binder-drug conjugate; and

[0236] (iii) Covalently linking a polypeptide binding portion to a linker portion of a drug portion, the linker portion comprising a substrate recognition sequence that is cleavable by an enzyme present in the extracellular space of diseased tissue, wherein the linker portion is cleavable in the presence of the enzyme and releases the free drug portion.

[0237] II. definition

[0238] To facilitate understanding of the invention, several terms and phrases are defined below.

[0239] a. Affinity

[0240] The term "Stefin polypeptide" refers to a subgroup of proteins in the cystatin superfamily, which is a family of proteins containing multiple cystatin-like sequences.

[0241] The Stefin subgroup of the cystatin family consists of relatively small (approximately 100 amino acids) single-domain proteins. They do not undergo known post-translational modifications and are free of disulfide bonds, suggesting they will fold identically across a wide range of extracellular and intracellular environments. Stefin A itself is a monomeric, single-chain, single-domain protein of 98 amino acids. The structure of Stefin A has been resolved, facilitating the rationale for mutagenesis of Stefin A into an affinity scaffold. The only known biological activity of cystatin is inhibition of cathepsin activity, which allows us to thoroughly test the residual biological activity of engineered proteins.

[0242] The term "affinity" (or "affinity scaffold" or "affinity polypeptide") refers to a highly stable, small protein that is a recombinantly engineered variant of a Stefin polypeptide. The affinity protein exhibits two peptide loops and an N-terminal sequence, both of which can be randomized to bind to the desired target protein with high affinity and specificity in a manner similar to that of a monoclonal antibody. The stabilization of these two peptides by the Stefin protein scaffold restricts the possible conformations that the peptides can take, increasing binding affinity and specificity compared to a library of free peptides. These engineered non-antibody-binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in various applications. Other portions of the Stefin polypeptide sequence can be mutated, wherein said mutations improve the properties of these affinity agents, such as increasing stability, making them robust under a range of conditions such as temperature and pH. Preferably, the affinity comprises a sequence derived from Stefin A that shares substantial identity with the wild-type Stefin A sequence (such as human Stefin A). Those skilled in the art will appreciate that modifications can be made to the scaffold sequence without departing from the invention. In detail, the affinity scaffold may have an amino acid sequence that is at least 25%, 35%, 45%, 55%, or 60% identical to the corresponding sequence of human Stefin A, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 92%, preferably at least 94%, preferably at least 95% identical to it, for example, wherein sequence variations do not adversely affect the scaffold's ability to bind to a desired target (such as PD-L1), and, for example, do not restore or produce biological functions, such as those present in wild-type Stefin A but eliminated in the mutational changes described herein.

[0243] "Binding-drug conjugate" refers to a polypeptide that includes an affinity polypeptide sequence and has any other modifications (e.g., conjugation, post-translational modifications, etc.) to present a therapeutically active protein intended for delivery to a patient.

[0244] "Programmed cell death ligand 1," also known as "PD-L1," "differentiation cluster 274," "CD274," "B7 homolog 1," or "B7-H1," refers to the protein encoded by the CD274 gene in the human case. Human PD-L1 is a 40 kDa type 1 transmembrane protein that plays a major role in suppressing the immune system under various conditions. A representative human PD-L1 sequence is provided by UniProtKB major register number Q9NZQ7, and other human isoforms of the sequence will be included. PD-L1 binds to its receptor PD-1, found on activated T cells, B cells, and bone marrow cells, to regulate activation or suppression. PD-L1 also has a significant affinity for the co-stimulatory molecule CD80 (B7-1). The binding of PD-L1 to its receptor PD-1 ("programmed cell death protein 1" or "CD279") on T cells delivers a signal that inhibits TCR-mediated IL-2 production and T cell proliferation. In this regard, PD-L1 is considered a checkpoint, and its upregulation in tumors helps suppress T cell-mediated antitumor responses. Although PD-L1 will generally be used when referring to PD-L1 from various mammalian species, it should be understood that throughout this application, any reference to PD-L1 includes human PD-L1 and preferably refers to human PD-L1 itself.

[0245] "PD-L1 binder-drug conjugate" refers to a drug that has at least one component with a concentration of at least 10... -6 The dissociation constant (Kd) of M binds to PD-L1, especially the affinity peptide of human PD-L1, in a drug conjugate.

[0246] b. Polypeptide

[0247] The terms “polypeptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. These polymers may be linear or branched, may contain modified amino acids, and may be interspersed with non-amino acids. The term also covers amino acid polymers that have been modified naturally or through intervention; said intervention is, for example, the formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation with a labeled component. The definition also includes, for example, polypeptides containing one or more amino acids (including, for example, non-natural amino acids) and other modified polypeptides known in the art.

[0248] The terms “amino acid residue” and “amino acid” are used interchangeably and, in the case of polypeptides, refer to an amino acid involved in one or more peptide bonds of the polypeptide. Generally, the abbreviations used herein to designate amino acids are based on the recommendations of the IUPAC-IUB Committee on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). For example, Met, Ile, Leu, Ala, and Gly represent “residues” of methionine, isoleucine, leucine, alanine, and glycine, respectively. A residue signifies a group derived from the corresponding α-amino acid by eliminating the OH portion of the carboxyl group and the H portion of the α-amino group. The term “amino acid side chain” refers to the portion of an amino acid other than the -CH(NH2)COOH portion, as defined by KDKopple, “Peptides and Amino Acids”, WABenjamin Inc., New York and Amsterdam, 1966, pp. 2 and 33.

[0249] In most cases, the amino acids used in this application are those naturally occurring amino acids found in proteins, or naturally occurring synthetic or catabolitic products of said amino acids containing amino and carboxyl groups. Particularly suitable amino acid side chains include side chains selected from the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan, as well as those amino acids and amino acid analogs identified as components of peptidoglycan bacterial cell walls.

[0250] Amino acid residues with "basic side chains" include Arg, Lys, and His. Amino acid residues with "acidic side chains" include Glu and Asp. Amino acid residues with "neutral polar side chains" include Ser, Thr, Asn, Gln, Cys, and Tyr. Amino acid residues with "neutral nonpolar side chains" include Gly, Ala, Val, Ile, Leu, Met, Pro, Trp, and Phe. Amino acid residues with "nonpolar aliphatic side chains" include Gly, Ala, Val, Ile, and Leu. Amino acid residues with "hydrophobic side chains" include Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp. Amino acid residues with "small hydrophobic side chains" include Ala and Val. Amino acid residues with "aromatic side chains" include Tyr, Trp, and Phe.

[0251] The term "amino acid residue" also includes analogs, derivatives, and congeners of any particular amino acid mentioned herein. For example, the subject affinity (especially if produced by chemical synthesis) may include amino acid analogs such as cyanoalanine, canavanine, quinoline, leucine, 3-phosphoserine, homoserine, dihydroxyphenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid. Those skilled in the art will recognize other naturally occurring amino acid metabolites or precursors having side chains suitable for use herein and which are included within the scope of this invention.

[0252] When the structure of an amino acid allows for stereoisomerism, it also includes (D) and (L) stereoisomers of the amino acid. The configurations of amino acids and amino acid residues herein are designated by appropriate symbols (D), (L), or (DL), and when no configuration is specified, an amino acid or residue may have configurations (D), (L), or (DL). It should be noted that the structures of some compounds of this invention include asymmetric carbon atoms. Therefore, it should be understood that isomers resulting from such asymmetry are included within the scope of this invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by sterically controlled synthesis. For the purposes of this application, unless expressly stated to the contrary, the named amino acid should be considered to include (D) or (L) stereoisomers.

[0253] The term "identical" or "percentage of identity" in the context of two or more nucleic acids or peptides refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotide or amino acid residues when compared and aligned according to maximum correspondence (with gaps introduced as needed), regardless of any conserved amino acid substitutions as part of sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are well known in the art for obtaining alignments of amino acid or nucleotide sequences. These algorithms and software include (but are not limited to) BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, the two nucleic acids or peptides of the present invention are substantially identical, meaning that when compared and aligned according to maximum correspondence, as measured using sequence comparison algorithms or by visual inspection, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity. In some embodiments, identity exists within amino acid sequence regions of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or any integer value therebetween. In some embodiments, identity exists within regions longer than 60-80 residues (e.g., at least about 80-100 residues), and in some embodiments, the sequences are substantially identical across the entire length of the compared sequences (e.g., the coding region of a target protein or antibody). In some embodiments, identity exists within nucleotide sequence regions of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases, or any integer value therebetween. In some embodiments, identity exists within regions longer than 60-80 bases (e.g., at least about 80-1000 bases or more), and in some embodiments, the sequences are substantially identical across the entire length of the compared sequences (e.g., the nucleotide sequence encoding a target protein).

[0254] A “conservative amino acid substitution” is a substitution in which one amino acid residue is replaced by another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, replacing tyrosine with phenylalanine is a conserved substitution. Generally, conserved substitutions in the sequences of the peptides, soluble proteins, and / or antibodies of the present invention do not eliminate the binding of the peptide, soluble protein, or antibody containing the said amino acid sequence to the target binding site. Methods for identifying conserved amino acid substitutions that do not eliminate binding are well known in the art.

[0255] The "isolated" polypeptide, soluble protein, antibody, polynucleotide, carrier, cell, or composition is a polypeptide, soluble protein, antibody, polynucleotide, carrier, cell, or composition in a form not found in nature. The isolated polypeptide, soluble protein, antibody, polynucleotide, carrier, cell, or composition includes polypeptides, soluble proteins, antibodies, polynucleotides, carriers, cells, or compositions purified to the point that they no longer exist in their naturally occurring form. In some embodiments, the isolated polypeptide, soluble protein, antibody, polynucleotide, carrier, cell, or composition is substantially pure.

[0256] As used herein, the term “substantially pure” means a material that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0257] As used herein, the term "fusion protein" or "fusion polypeptide" refers to a hybrid protein expressed by a nucleic acid molecule containing nucleotide sequences of at least two genes.

[0258] As used herein, the term "linker" or "linker region" refers to a linker inserted between a first polypeptide (e.g., a copy of an affinity peptide) and a second polypeptide (e.g., another affinity peptide, an Fc domain, a ligand-binding domain, etc.). In some embodiments, the linker is a peptide linker. The linker should not adversely affect the expression, secretion, or biological activity of the polypeptide. Preferably, the linker is non-antigenic and does not elicit an immune response.

[0259] "Affinity-antibody fusion" refers to a fusion protein comprising an affinity polypeptide moiety and an antibody variable region. An affinity-antibody fusion includes a full-length antibody having, for example, one or more affinity polypeptide sequences linked to one or more of the VH and / or VL chains at a C-terminus or N-terminus; that is, at least one chain of the assembled antibody is a fusion protein having an affinity polypeptide. An affinity-antibody fusion also includes embodiments in which one or more affinity polypeptide sequences are provided as part of a fusion protein having an antigen-binding site or a variable region of an antibody fragment.

[0260] As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or any combination thereof) via at least one antigen-binding site, wherein the antigen-binding site is typically located within a variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies (provided these fragments are formatted to include an Fc or other FcγRIII binding domain), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigen-binding site (formatted to include an Fc or other FcγRIII binding domain), and any other modified immunoglobulin molecule containing an antigen-binding site, provided that the antibody exhibits the desired biological activity.

[0261] Furthermore, based on the identity of the antibody’s heavy chain constant domains, known as α, δ, ε, γ, and μ, antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0262] The term "variable region" of an antibody refers to the variable region of the antibody light chain, either alone or in combination, or the variable region of the antibody heavy chain. Typically, the variable regions of both the heavy and light chains consist of four frame regions (FRs) and three complementarity-determining regions (CDRs, also known as "hypervariable regions"). The CDRs in each chain are held together by the frame regions and, together with CDRs from other chains, facilitate the formation of the antigen-binding site of the antibody. At least two techniques exist for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., National Institutes of Health, Bethesda Md.); and (2) methods based on crystallization studies of antigen-antibody complexes (Al Lazikani et al. (1997 J. Mol. Biol. 273: 927-948)). Furthermore, combinations of these two methods are sometimes used in the art to determine CDRs.

[0263] As used herein, the term "humanized antibody" refers to a form of non-human (e.g., murine) antibody that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing a minimal non-human sequence. Typically, a humanized antibody is a human immunoglobulin in which residues of the CDR are replaced by residues of a CDR from a non-human species (e.g., mouse, rat, rabbit, or hamster) to achieve the desired specificity, affinity, and / or binding ability. In some cases, Fv frame region residues of the human immunoglobulin are replaced by corresponding residues from an antibody from a non-human species. Humanized antibodies can be further modified by substitution of additional residues within the Fv frame region and / or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and / or binding ability. Humanized antibodies may contain a variable domain containing all or substantially all of the CDR corresponding to the non-human immunoglobulin, and the frame region being the frame region of the human immunoglobulin sequence. In some embodiments, the variable domain contains the frame region of the human immunoglobulin sequence. In some implementations, the variable domain comprises a frame region of a common sequence of human immunoglobulins. Humanized antibodies may also comprise a constant region or Fc domain of an immunoglobulin, typically at least a portion of the constant region or Fc domain of a human immunoglobulin. Humanized antibodies are generally considered distinct from chimeric antibodies.

[0264] The terms “antigenic determinant” and “antigenic determinant cluster” are used interchangeably herein and refer to the portion of an antigen that can be recognized and specifically bound by a particular antibody, a particular affinity, or other specific binding domain. When the antigen is a polypeptide, the antigenic determinant can be formed by both adjacent and non-adjacent amino acids arranged side-by-side through the ternary folding of the protein. Antigenic determinants formed by adjacent amino acids (also known as linear antigenic determinants) are generally retained during protein denaturation, while antigenic determinants formed by ternary folding (also known as conformational antigenic determinants) are generally lost during protein denaturation. Antigenic determinants typically include at least three, and more commonly at least five, six, seven, or eight to ten amino acids in a distinctive spatial conformation.

[0265] As used herein, the terms “specifically bound to” or “specific to” refer to measurable and reproducible interactions, such as the binding between a target and an affinity, antibody, or other binding partner, which determine the presence of the target in the presence of a non-uniform population of molecules, including biomolecules. For example, an affinity that specifically binds to a target is an affinity that binds to the target with greater affinity, affinity (if polymorphic), ease of binding, and / or longer duration of binding compared to its binding to other targets.

[0266] c. Checkpoint inhibitors, co-stimulatory agonists, and chemotherapy agents

[0267] "Checkpoint molecules" are proteins expressed by tissues and / or immune cells that reduce the effectiveness of the immune response in a manner dependent on the expression level of the checkpoint molecule. When these proteins are blocked, "brakes" on the immune system are released, and, for example, T cells are able to kill cancer cells more effectively. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2, PD-L2, NKG2A, KIR, LAG-3, TIM-3, CD96, VISTA, and TIGIT.

[0268] "Checkpoint inhibitors" are pharmacological entities that reverse immunosuppressive signaling from checkpoint molecules.

[0269] "Co-stimulatory molecules" refer to molecules that specifically bind to co-stimulatory ligands, thereby mediating co-stimulation, such as (but not limited to) proliferating immune cells, such as T cell homologous binding partners. Co-stimulatory molecules are cell surface molecules that promote an effective immune response, other than antigen receptors or ligands. Co-stimulatory molecules include (but are not limited to) MHCI molecules, BTLA receptors and Toll ligands, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Examples of co-stimulatory molecules include (but are not limited to): CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18 , LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244,2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL 1. CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and CD83 ligands.

[0270] "Costimulatory agonists" refer to pharmaceutical entities that, like costimulatory ligands, activate (excite) costimulatory molecules and generate immunostimulatory signals or otherwise enhance the efficacy or effectiveness of immune responses.

[0271] "Chemotherapy agents" are compounds that can be used to treat cancer. Examples of chemotherapy agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; and ethyleneimine and methylmelamine, including altretamine and triethylate. Lemonelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylol melamine; polyacetyl (especially bullatacin and bullatacinone); delnabinol (marinol); β-lappaone; laparol; colchicine; betulinic acid; camptothecin (including the synthetic analogue topotecan) (hycamtin) CPT-11 (irinotecan (CAMPTOSAR)), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; pemetrexed; callystatin; CC-1065 (including synthetic analogs of adozelesin, carzelesin, and bizelesin); podophyllotoxin; and more. Methyl monoxide; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral α-4 integrin inhibitor; sarcodictyin; spongistatin;Nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, and uracil. Mustard); nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as ethynylene antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ΩI1 (see, for example, Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); dynemicin (including dynemicin A); esperamycin;And new carcinogen chromophores and related chromogens (enediyne antibiotic chromophores), aclacinomysin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin C, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin D, daunorubicin Rubicin, detorubicin, 6-diazo-5-oxo-L-leucine, doxorubicin (including ADRIAMYCIN, N-morpholino-doxorubicin, cyano-N-morpholino-doxorubicin, 2-pyrrolinyl-doxorubicin, doxorubicin HCl liposome injection (DOXIL), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid (acid), nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tuberculin, ubenimex, zinostatin, zorubicin cin); antimetabolites (such as methotrexate, gemcitabine (GEMZAR), tegafur (UFTORAL), capecitabine (XELODA), epothilone, and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimetrexate;Purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, deoxyfluorouridine, enocitabine, fluxuridine, and imatinib (a 2-phenylaminopyrimidine derivative) and other c-Kit inhibitors; antiadrenergic agents, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as folinic acid; aceglatone; and aldophosphamide. glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate); etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine-like substances such as maytansine and ansamitocins, mitoguazone, mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazine; procarbazine; PSK polysaccharide complex (JHS Natural) Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonicacid; triaziquone; 2,2',2″-trichlorotriethylamine;Trichothecene (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine (ELDISINE, FILDESIN); dacarbazine; mannomustine; mitobronitol; mitolactalol; pipebroman; gasitocin (g acytosine; arabinoside (Ara-C); thiotepa; taxoids, such as paclitaxel (TAXOL), albumin-engineered nanoparticle formulations of paclitaxel (ABRAXANE), and docetaxel (TAXOTERE); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate. xate); platinum analogs, such as cisplatin and carboplatin; vinblastine (VELBAN); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN); oxaliplatin; leucovovin; vinorelbine (NAVELBINE); novantrone; idatraxa (edatrexate); daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP (an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone) and FOLFOX (an abbreviation for a treatment regimen of oxaliplatin (ELOXATIN) combined with 5-FU and levofloxacin).

[0272] The definition also includes antihormonal agents used to regulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth, and these agents are typically used in the form of systemic or whole-body treatments. They may themselves be hormones. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen (including NOLVADEX), raloxifene (EVISTA), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); anti-progesterone; estrogen receptor down-regulators (ERDs); estrogen receptor antagonists, such as fulvestrant (FASLODEX); and agents used to suppress or shut down the ovaries, such as luteinizing hormone-releasing hormone (LHRH) agonists, such as leuprolide. Acetates (LUPRON and ELIGARD), goserelin acetate, buserelin acetate, and tripterelin; antiandrogens, such as fiutamide, nilutamide, and bicalutamide; and aromatase inhibitors, which inhibit aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate (MEGASE), exemestane (AROMASIN), formestanie, fadrozole, vorozole (RIVISOR), letrozole (FEMARA), and anastrozole (ARIMIDEX).Additionally, the definition of chemotherapeutic agents includes: bisphosphonates such as clodronate (e.g., BONEFOS or OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid / zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (AREDIA), tiludronate (SKELID), or risedronate (ACTONEL); and troxacitabine (a 1,3-dioxane cyclopentane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOPE vaccines and gene therapy vaccines, such as ALLOVECTIN vaccines. Vaccines, LEUVECTIN vaccines, and VAXID vaccines; topoisomerase 1 inhibitors (e.g., LURTOTECAN); anti-estrogens, such as fulvestrant; Kit inhibitors, such as imatinib or EXEL-0862 (tyrosine kinase inhibitors); EGFR inhibitors, such as erlotinib or cetuximab; anti-VEGF inhibitors, such as bevacizumab; irinotecan; rmRH (e.g., ABARELIX); lapatinib and lapatinib dimethylbenzenesulfonate (small molecule inhibitors of dual tyrosine kinases of ErbB-2 and EGFR, also known as GW572016); 17AAG (geldanamycin derivative, which is a heat shock protein (Hsp) 90 toxin); and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0273] As used herein, the term "cytokine" generally refers to a protein released by a population of cells that acts as an intercellular mediator to another cell or has an autocrine effect on the cell that produces the protein. Examples of such cytokines include lymphokines, monocyte hormones; interleukins (“ILs”), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, including PROLEUKIN rIL-2; tumor necrosis factors, such as TNF-α or TNF-β, TGF-β1-3; and other polypeptide factors, including leukemia suppressor factor (“LIF”), ciliary neurotrophic factor (“CNTF”), CNTF-like cytokines (“CLC”), cardiotrophins (“CT”), and kit ligands (“KL”).

[0274] As used herein, the term "chemokine" refers to a soluble factor (e.g., cytokine) that can selectively induce chemotaxis and activation of leukocytes. It also triggers processes such as angiogenesis, inflammation, wound healing, and tumorigenesis. Exemplary chemokines include IL-8, a human homologue of the mouse keratinocyte chemoattractant (KC).

[0275] d. Treatment

[0276] As used herein, the term “functional disorder” also includes impaired antigen recognition or failure to respond to antigen recognition, particularly a diminished ability to translate antigen recognition into downstream T cell effector functions such as proliferation, cytokine production (e.g., IL-2), and / or target cell killing.

[0277] The term "loss of capacity" refers to a state of unresponsiveness to antigenic stimulation caused by incomplete or insufficient signaling delivered via T cell receptors (e.g., intracellular Ca2+ in the absence of ras activation). +2 (Elevated). T cell dysfunction can also occur upon antigen stimulation in the absence of co-stimulation, causing cells to become subsequently difficult to activate by antigens even under co-stimulatory conditions. This unresponsive state can often be resolved by the presence of interleukin-2. Dysfunctional T cells do not undergo clonal expansion and / or acquire effector function.

[0278] The term "exhaustion" refers to T cell exhaustion, a state of T cell dysfunction caused by persistent TCR signaling that occurs during many chronic infections and cancers. It differs from impaired capacity in that it is not caused by incomplete or insufficient signaling, but by persistent signaling. It is defined by adverse effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors.

[0279] "Enhancing T cell function" means inducing, eliciting, or stimulating T cells to acquire sustained or expanded biological functions, or renewing or reactivating exhausted or inactive T cells. Examples of enhancing T cell function include: increased secretion of interferon-gamma from CD8+ T cells compared to pre-intervention levels, increased proliferation, and increased antigen responses (e.g., clearance of viruses, pathogens, or tumors). In one embodiment, the enhancement level is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. The methods for measuring this enhancement are known to those skilled in the art.

[0280] "T-cell dysfunction syndrome" is a condition or symptom characterized by a reduced response of T cells to antigen stimulation. In one particular embodiment, T-cell dysfunction syndrome is specifically associated with inappropriately elevated PD-1 levels. T-cell dysfunction syndrome may also be associated with inappropriately elevated PD-L1 levels in tumors, which cause suppression of one or more T-cell antitumor functions. In another embodiment, T-cell dysfunction syndrome is a condition in which T cells lack capacity or have reduced ability to secrete cytokines, proliferate, or perform cytolytic activities. In one particular aspect, the reduced response results in ineffective control of pathogens or tumors expressing immunogens. Examples of T-cell dysfunction syndromes characterized by T-cell dysfunction include acute infections of unknown origin, chronic infections, and tumor immunity.

[0281] "Tumor immunity" refers to the process by which tumors evade immune recognition and elimination. Therefore, as a therapeutic concept, tumor immunity is "cured" when this evasion is weakened and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor elimination.

[0282] "Sustained response" refers to the continued effect of reducing tumor growth after treatment has been discontinued. For example, the tumor size may remain the same or smaller compared to the size at the start of the application phase. In some embodiments, the duration of the sustained response is at least the same as the duration of treatment, being at least 1.5, 2.0, 2.5, or 3.0 times the duration of treatment.

[0283] As used herein, the terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals characterized by unregulated cell growth. Examples of cancer include (but are not limited to) carcinomas, blastomas, sarcomas, and blood cancers such as lymphomas and leukemias.

[0284] As used herein, the terms “tumor” and “necrotic tumor” refer to any mass of tissue resulting from excessive cell growth or proliferation, whether benign (non-cancerous) or malignant (cancerous), including precancerous lesions. Tumor growth is typically uncontrolled and progressive, neither inducing nor inhibiting the proliferation of normal cells. Tumors can affect a variety of cells, tissues, or organs, including (but not limited to) organs or tissues or corresponding cells selected from the bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestines, kidneys, liver, lungs, lymph nodes, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid gland, trachea, urethra, urethra, uterus, and vagina. Tumors include cancers such as sarcomas, carcinomas, plasmacytomas, or (malignant plasma cells). The tumors of this invention may include (but are not limited to) leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myeloid-monocytic leukemia, acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (e.g., Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia, heavy chain disease, and solid tumors such as sarcomatous cancers (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor). Tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, carcinoma, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver tumor, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma, esophageal cancer, gallbladder cancer, kidney cancer, multiple myeloma. Preferably, "tumor" includes (but is not limited to): pancreatic cancer, liver cancer, lung cancer, stomach cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer, and glioma.

[0285] As used in this article, the term "metastasis" refers to the spread or metastasis of cancer from its primary site to other areas of the body, where it creates cancer-like lesions in the new location. "Metastatic" or "metastatic" cells are those that have lost their adhesive contact with neighboring cells and migrate from the primary site of the disease via the bloodstream or lymphatic system to invade adjacent body structures.

[0286] The terms “cancer cell” and “tumor cell” refer to the total population of cells originating from cancer, tumors, or precancerous lesions, including both non-tumorigenic cells, which constitute the majority of the cancer cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term “cancer cell” or “tumor cell” will be modified by the term “non-tumorigenic” when referring only to those cells that lack the capacity for renewal and differentiation to distinguish tumor cells from cancer stem cells.

[0287] As used in this article, the term "effective amount" refers to the amount that provides therapeutic or preventative benefits.

[0288] As used in this article, “complete response” or “CR” means that all target lesions have disappeared; “partial response” or “PR” means that the sum of the longest diameters (SLD) of the target lesions has decreased by at least 30% with reference to the baseline SLD; and “stable disease” or “SD” means that the target lesions have neither shrunk sufficiently to be considered PR nor increased sufficiently to be considered PD with reference to the minimum SLD at the start of treatment.

[0289] As used in this article, “progression-free survival” (PFS) refers to the length of time during and after treatment during which the treated disease (e.g., cancer) does not worsen. PFS can include the amount of time a patient experiences a complete or partial response, as well as the amount of time a patient experiences stable disease.

[0290] As used in this article, “Total Reaction Rate” (ORR) refers to the sum of the complete reaction rate (CR) and the partial reaction rate (PR).

[0291] As used in this article, “overall survival” refers to the percentage of subjects in a group who are likely to survive after a certain duration.

[0292] As used herein, the term "treatment" refers to a process or individual attempt to alter a clinical disease caused by cellular intervention (which may be a preventative intervention process in clinicopathology). This includes (but is not limited to) treatments to prevent the onset or recurrence of disease, alleviate symptoms, reduce the direct or indirect pathological consequences of any disease, prevent cancer metastasis, slow the rate of disease progression, improve or alleviate disease, or improve prognosis.

[0293] The term "subject" refers to any animal (e.g., a mammal) that is a recipient of a particular treatment, including (but not limited to) humans, non-human primates, canines, felines, rodents, etc. Generally, the terms "subject" and "patient" are used interchangeably in this document when referring to human subjects.

[0294] As used herein, the terms “agonist” and “activator” refer to an agent that can directly or indirectly significantly induce, activate, promote, increase, or enhance the biological activity of a target or target pathway. The term “agonist” as used herein includes any agent that partially or completely induces, activates, promotes, increases, or enhances the activity of a protein or other target.

[0295] As used herein, the terms “antagonist” and “antagonist” refer to or describe an agent that can directly or indirectly partially or completely block, inhibit, reduce, or neutralize the biological activity of a target and / or pathway. The term “antagonist” as used herein includes any agent that partially or completely blocks, inhibits, reduces, or neutralizes the activity of a protein or other target.

[0296] As used herein, the terms “modulation” and “modulate” refer to alterations or changes in biological activity. Modulation includes (but is not limited to) stimulatory or inhibitory activity. Modulation can be an increase or decrease in activity, a change in binding characteristics, or any other change in biological, functional, or immune properties related to the activity of a protein, pathway, system, or other target biological organism.

[0297] As used herein, the term "immune response" includes responses from both the innate and acquired immune systems. It includes cell-mediated and / or humoral immune responses. It includes T-cell and B-cell responses, as well as responses from other cells of the immune system, such as natural killer (NK) cells, monocytes, macrophages, etc.

[0298] The term “pharmaceutically acceptable” means a substance that has been approved or is permitted by a federal or state regulatory agency or is listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, including humans.

[0299] The terms "pharmaceuticalally acceptable excipient, carrier, or adjuvant" or "acceptable drug carrier" refer to an excipient, carrier, or adjuvant that can be administered to a subject together with at least one agent of the present invention and that, when administered in a dose sufficient to deliver a therapeutic effect, does not impair the pharmacological activity of said agent and is non-toxic. Generally, those skilled in the art and the USFDA consider pharmaceutically acceptable excipients, carriers, or adjuvants to be inactive ingredients in any formulation.

[0300] The terms “effective amount” or “therapeutic effective amount” or “therapeutic effect” refer to the amount of the PD-L1 ...

[0301] The terms “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” mean both of the following: (1) a therapeutic measure of the cure, reduction, symptom relief, and / or cessation of progression of a diagnosed pathological symptom or condition; and (2) a preventive or therapeutic measure of preventing or slowing the development of a target pathological symptom or condition. Therefore, those who require treatment include those who already have the condition; those who are prone to the condition; and those who need to prevent the condition. In the case of cancer or tumor, the method according to the invention successfully “treats” a subject if the patient exhibits one or more of the following: increased immune response; increased anti-tumor response; increased cytolytic activity of immune cells; increased killing of tumor cells by immune cells; reduced number of cancer cells or their complete absence; reduced tumor size; inhibited or absent cancer cell infiltration into peripheral organs, including cancer cell spread to soft tissues and bones; inhibited or absent tumor or cancer cell metastasis; inhibited or absent cancer growth; alleviated one or more symptoms associated with a specific cancer; reduced morbidity and mortality; improved quality of life; reduced tumorigenicity; reduced number or frequency of cancer stem cells; or a combination of said effects.

[0302] e. Other (Miscellaneous)

[0303] The term "alkyl" refers to a group with a saturated aliphatic group, including straight-chain alkyl, branched alkyl, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl, and cycloalkyl-substituted alkyl. In some embodiments, the straight-chain or branched alkyl has 30 or fewer carbon atoms in its main chain (e.g., straight-chain C1-C1). 30 C3-C of the branch chain 30(e.g., 20 or fewer). Similarly, some cycloalkyl groups have 3-10 carbon atoms in their ring structure, such as 5, 6, or 7 carbon atoms in the ring structure. As used throughout this specification and the scope of the claims, "alkyl" (or "lower alkyl") is intended to include both "unsubstituted alkyl" and "substituted alkyl".

[0304] As used herein, the term "aralkyl" refers to an alkyl group that has been substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

[0305] The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups that are similar in length and possible substituents to the alkyl groups described above, but each contains at least one double or triple bond.

[0306] Unless otherwise specified, as used herein, "lower alkyl" means an alkyl group as defined above but having one to ten carbon atoms, for example one to four or one to six carbon atoms, in the main chain structure. Similarly, "lower alkenyl" and "lower alkynyl" have similar chain lengths. In some embodiments, the alkyl group is a lower alkyl group. In some embodiments, the substituents specified herein as alkyl groups are lower alkyl groups.

[0307] As used herein, the term "aryl" includes 5-, 6-, and 7-membered monocyclic aromatic groups that may include zero to four heteroatoms, such as benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. Those aryl groups having heteroatoms in their ring structure may also be referred to as "aryl heterocycles" or "heteroaromatic compounds." The aromatic ring may be substituted at one or more ring positions with substituents as described above, such as halogens, azides, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, thioalkyl, imino, amide, phosphonate, phosphonite, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclic group, aromatic or heteroaromatic moiety, -CF3, -CN, etc. The term "aryl" also includes polycyclic systems having two or more rings, wherein two or more carbons are shared by two adjacent rings (the rings are "fused rings"), wherein at least one ring is an aromatic ring, and other rings may be cycloalkyl, cycloalkenyl, cycloynyl, aryl and / or heterocyclic.

[0308] The term "heterocyclyl" or "heterocyclic group" refers to a 3- to 10-membered ring structure containing one to four heteroatoms, such as 3- to 7-membered rings. Heterocycles can also be polycyclic. Heterocyclic groups include, for example, thiophene, thiathrone, furan, pyran, isobenzofuran, chromene, xanthene, phenoxazine, pyrrole, imidazole, pyrazole, isothiazine, isoxazine, pyridine, pyrazine, pyrimidine, pyridazine, indoleazine, isoindole, indole, indazole, purine, quinazine, isoquinoline, quinoline, phthalazine, naphthidine, quinoxaline, quinazolin, cinnamoline, pteridine, carbazole, caroline, phenanthridine, acridine, pyrimidine, phenanthrene-rhein, phenazine, phenopyrazine, phenothiazine, furazine, phenothiazine, pyrrole, oxacyclopentane, thiocyclopentane, oxazole, piperidine, piperazine, morpholine, lactone, lactam (such as azacyclobutanone and pyrrolidone), sulfonamide, sulfonyl lactone, etc. The heterocycle may be substituted at one or more ring positions with substituents as described above, such as halogens, alkyl groups, aralkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, hydroxyl groups, amino groups, nitro groups, thioalkyl groups, imino groups, amide groups, phosphonates, phosphonites, carbonyl groups, carboxyl groups, silyl groups, ethers, alkylthio groups, sulfonyl groups, ketones, aldehydes, esters, heterocyclic groups, aromatic or heteroaromatic moieties, -CF3, -CN, etc.

[0309] The term "heteroaryl" refers to a monovalent aromatic monocyclic ring system in which at least one ring atom is a heteroatom independently selected from the group consisting of O, N, and S. The term "5-membered heteroaryl" refers to a heteroaryl group with 5 ring atoms. Examples of 5-membered heteroaryl groups include pyrrole, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, furazolyl, imidazolinyl, and triazolyl.

[0310] The term "heterocyclic alkyl" refers to a monocyclic or bicyclic monovalent saturated or non-aromatic unsaturated ring system in which one to four ring atoms are heteroatoms independently selected from the group consisting of O, N, and S. The term "3- to 10-membered heterocyclic alkyl" refers to a heterocyclic alkyl system with 3 to 10 ring atoms. Examples of 3- to 10-membered heterocyclic alkyl systems include 3- to 6-membered heterocyclic alkyl systems. Bicyclic systems include fused, bridged, and spirocyclic ring systems. More specific examples of heterocyclic alkyl systems include azirmonyl heptyl, azirmonyl butyl, azirmonyl propaneyl, imidazolyl, morpholinyl, oxazolyl, oxazolyl, piperazine, piperidinyl, pyrazolyl, pyrrolidinyl, quininecycloyl, and thiomorpholinyl.

[0311] The terms "polycyclic group" and "polycyclic compound" refer to two or more rings in which two or more carbon atoms are shared by two adjacent rings, such as two or more rings that are "fused rings" (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic groups). Rings joined by non-adjacent atoms are called "bridged" rings. Each ring of a polycyclic compound can be substituted with substituents as described above, such as halogens, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, thioalkyl, imino, amide, phosphonate, phosphonite, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic group, aromatic or heteroaromatic moiety, -CF3, -CN, etc.

[0312] As used in this article, the term "carbon ring" refers to an aromatic or non-aromatic ring in which every atom of the ring is carbon.

[0313] As used herein, the term "heteroatom" refers to an atom of any element other than carbon or hydrogen. Exemplary heteroatoms include nitrogen, oxygen, sulfur, and phosphorus.

[0314] As used herein, the term “nitro” means -NO2; the term “halogen” refers to -F, -Cl, -Br or -I; the term “sulfhydryl” means -SH; the term “hydroxyl” means -OH; and the term “sulfonyl” means -SO2-.

[0315] "Halogen" or "halogen group" itself or as another substituent refers to fluorine, chlorine, bromine and iodine, or fluorine group, chlorine group, bromine group and iodine group.

[0316] It should be understood that “substitution” or “substituted” includes implied limitations, namely that the substitution conforms to the permissible valences of the substituted atom and the substituent, and that the substitution produces a stable compound, such as a compound that does not spontaneously transform, for example, through rearrangement, cyclization, elimination, etc.

[0317] As used herein, the term "substituted" is intended to include all permissible substituents of an organic compound. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. Illustrative substituents include, for example, those described above. For a suitable organic compound, permissible substituents may be one or more and may be the same or different. Substituents may include, for example, halogens, hydroxyl groups, carbonyl groups (such as carboxyl, ester, formyl, or ketone), thiocarbonyl groups (such as thioesters, thioacetic acids, or thiocarbamates), alkoxy groups, phosphoryl groups, phosphonates, phosphonites, amino groups, amide groups, amidine groups, imine groups, cyano groups, nitro groups, azide groups, thiohydrothio groups, alkylthio groups, sulfate groups, sulfonates, aminosulfonyl groups, sulfonamide groups, sulfonyl groups, heterocyclic groups, aralkyl groups, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that the substituted portion of a hydrocarbon chain itself may be substituted where appropriate. For example, substituents of substituted alkyl groups may include substituted and unsubstituted forms of amino, azide, imino, amide, phosphoryl (including phosphonates and phosphonites), sulfonyl (including sulfates, sulfonamides, aminosulfonyls, and sulfonates), and silyl, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylic esters, and esters), -CF3, -CN, etc. Exemplary substituted alkyl groups are described below. Cycloalkyl groups may be further substituted with alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl groups, -CF3, -CN, etc. For the purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds that satisfy the heteroatom valences described herein. This invention is not intended to be limited in any way by permissible substituents of organic compounds.

[0318] The terms “amino acid residue” and “peptide residue” refer to an amino acid or peptide molecule that does not have a carboxyl group (-OH). Generally, the abbreviations used here to designate amino acids and protecting groups are based on the recommendations of the IUPAC-IUB Committee on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). For example, Met, Ile, Leu, Ala, and Gly represent “residues” of methionine, isoleucine, leucine, alanine, and glycine, respectively. A residue signifies a group derived from the corresponding α-amino acid by removing the OH portion of the carboxyl group and the H portion of the α-amino group. The term “amino acid side chain” refers to the portion of an amino acid other than the -CH(NH2)COOH portion, as defined by KDKopple, “Peptides and Amino Acids”, WABenjamin Inc., New York and Amsterdam, 1966, pp. 2 and 33.

[0319] In most cases, the amino acids used in this application are those naturally occurring amino acids found in proteins, or naturally occurring synthetic or catabolitic products of said amino acids containing amino and carboxyl groups. Particularly suitable amino acid side chains include side chains selected from the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan, as well as those amino acids and amino acid analogs identified as components of peptidoglycan bacterial cell walls.

[0320] The term "amino acid residue" also includes analogs, derivatives, and congeners of any particular amino acid mentioned herein. For example, the subject compound may include amino acid analogs such as cyanoalanine, canavanine, quinoline, leucine, 3-phosphoserine, homoserine, dihydroxyphenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid. Other naturally occurring amino acid metabolites or precursors having side chains suitable for use herein are recognized by those skilled in the art and are included within the scope of this invention.

[0321] When the structure of an amino acid allows for stereoisomerism, it also includes (D) and (L) stereoisomers of the amino acid. The configurations of amino acids and amino acid residues herein are designated by appropriate symbols (D), (L), or (DL), and when no configuration is specified, an amino acid or residue may have configurations (D), (L), or (DL). It should be noted that the structures of some compounds of this invention include asymmetric carbon atoms. Therefore, it should be understood that isomers resulting from said asymmetry are included within the scope of this invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by sterically controlled synthesis. For the purposes of this application, unless expressly stated to the contrary, the named amino acid should be considered to include (D) or (L) stereoisomers.

[0322] As noted above, some of the compounds of this invention may exist in specific geometric or stereoisomeric forms. This invention covers all such compounds within its scope, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are intended to be included in this invention.

[0323] For example, if a specific enantiomer of the compound of the present invention is required, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary agent, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, in the case where the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with a suitable optically active acid or base, and the resulting diastereomeric salt is then resolved by stepwise crystallization or chromatographic means well known in the art, and the pure enantiomer is subsequently recovered.

[0324] The term "IC" 50 "Ki value" refers to the concentration of the inhibitor that reduces the reaction (or binding) by half, and can be measured in whole-cell, animal, or cell-free (purified enzyme) systems in vitro. In some formal kinetic measurements, inhibition of cell-free enzymes can also be reported as a Ki value.

[0325] The term "ICIC" 50 "or "IIC" 50 "This is a measure of the inhibition of DPP8 and DPP9, which convert cell permeability into factors, in the whole-cell state (DPP8 and DPP9 are cell permeable and are used to measure IC50)." 50 (A purified enzyme that does not require cell permeability).

[0326] The term "DPP8" refers to dipeptidyl peptidase 8.

[0327] The term "DPP9" refers to dipeptidyl peptidase 9.

[0328] For the purposes of this invention, chemical elements are identified according to the periodic table, CAS edition, Handbook of Chemistry and Physics, 67th edition, 1986-87, enclosed. Also for the purposes of this invention, the term "hydrocarbon" is used to encompass all permissible compounds having at least one hydrogen atom and one carbon atom. In a broad aspect, permissible hydrocarbons include substituted or unsubstituted acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic compounds.

[0329] In the case of dipeptides (or dipeptide analogs), the terms "P1 position" and "P2 position" refer to the carboxyl-terminal residue and the amino-terminal residue, respectively. In the case of Subject I-DASH inhibitors, the P1 position is the amino acid (or amino acid analog) that replaces the carboxyl-terminal residue with boric acid.

[0330] It should be understood that when the embodiments are described herein with the language “comprising”, other similar embodiments described with the terms “consisting of” and / or “substantially consisting of” are also provided. It should also be understood that when the embodiments are described herein with the language “substantially consisting of”, other similar aspects described with the term “consisting of” are also provided.

[0331] As used herein, references to “about” or “approximately” values ​​or parameters include (and describe) embodiments associated with said value or parameter. For example, a description relating to “about X” includes a description of “X”.

[0332] As used in phrases such as “A and / or B”, the term “and / or” is intended herein to include A and B; A or B; A (alone); and B (alone). Similarly, as used in phrases such as “A, B and / or C”, the term “and / or” is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0333] III. Exemplary Implementation

[0334] One aspect of the present invention provides a binder-drug conjugate comprising (i) a cell-binding moiety of a cell surface feature, such as a protein, upregulated or otherwise selectively displayed on cells in a tumor, such as an antibody, antibody fragment, non-antibody scaffold, or other polypeptide entity; and (ii) one or more drug-conjugate moieties attached thereto, said drug-conjugate moieties being as shown in the following formula:

[0335]

[0336] in

[0337] L 1 Indicates a spacer base or a straight bond;

[0338] SRS represents the substrate recognition sequence of extracellular proteases expressed in the extracellular space of tumor cells;

[0339] L 2 Indicates a trigger-activated self-degrading connector or a straight key;

[0340] DM indicates the drug component;

[0341] m represents an integer from 1 to 6, preferably 1, 2, or 3; and

[0342] n represents an integer from 1 to 500, more preferably from 1 to 100, 1 to 10, or 1 to 5.

[0343] The binder-drug conjugate has an internalization half-life of at least 6 hours, more preferably at least 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 75 hours or even 100 hours when it binds to the surface features on the target cells.

[0344] a. Substrate recognition sequence

[0345] In some embodiments, the substrate recognition sequence is a portion (typically a peptide or peptide base) cleaved by an enzyme expressed in the target cells by binding in the tissue. "Selectively cleavable cleavage site near the target cells" means a site that can be cleaved only by an agent selectively present near the target cells, thereby reducing the release of free drug portions from the diseased tissue. Preferably, the enzyme cleaving the substrate recognition sequence is present near the target cells at a concentration at least five or ten times higher than that of enzymes outside the vicinity of the target cells, and more preferably at a concentration at least 100, 500, or 1000 times higher. Most preferably, the enzyme cleaving the substrate recognition sequence is found only near the target cells. For example, when the target cells are specific tumor cells (e.g., breast tumor cells), the substrate recognition sequence may be a substrate recognition sequence cleaved by an enzyme selectively present in the specific tumor (e.g., breast tumor) but not present outside the vicinity of the specific tumor (e.g., breast tumor).

[0346] "Near cell" means on the cell surface or in the interstitial fluid within the tissue or both, or in the environment that directly surrounds the cell, such as blood, lymph, and other bodily fluids.

[0347] The substrate recognition sequence is selectively cleaved near the target cells, thereby allowing the free drug moiety to be preferentially released from the self-conjugate near the target cells, so as to exert its pharmacological activity preferentially on cells / tissues adjacent to the target cells rather than on the desired (healthy) cells. Therefore, preferably, the substrate recognition sequence is selectively cleaved such that the drug moiety is released near the target cells at least five or ten times, and more preferably at least 100 or 500 or 1000 times more, than the release of the free drug moiety near healthy cells / tissues.

[0348] For a given target cell, a technician will be able to identify appropriate substrate recognition sequences that are selectively cleavable near the target cell using methods recognized in the art. For example, which proteases cleave which peptides can be assessed by consulting peptide libraries and studying MS analysis of the fragmentation profile after cleavage. Furthermore, published literature on protease cleavage motifs and peptide cleavage data can be further described below.

[0349] Generally, substrate recognition sequences are cleavage sites of proteases. Therefore, when the target cell is a tumor cell, the substrate recognition sequence can be selectively cleaved by proteases present near the tumor cell. In other words, the substrate recognition sequence can be a substrate recognition sequence that can be cleaved by tumor-associated proteases. It is well known that during tumor development, tumors abnormally express proteases that allow them to invade local tissues and eventually metastasize.

[0350] The protease may be a metalloproteinase (MMP1-28), including membrane-bound forms (MMP14-17 and MMP24-25) and secreted forms (MMP1-13, MMP18-23, and MMP26-28). The protease may belong to the A deintegrin and metalloproteinase (ADAM) families of proteases and the A deintegrin or metalloproteinase (ADAMTS) families containing platelet-reactive protein motifs. Other examples include CD10 (CALLA) and prostate-specific antigen (PSA). In some preferred embodiments, the protease is a fibroblast activation protein (FAPα). It should be understood that the protease may or may not be membrane-bound.

[0351] The protease cleavage site is well known in the scientific literature and can be readily used as the basis for a given substrate recognition sequence included in the drug-conjugate portion using recognized synthetic techniques known in the art.

[0352] For proteases whose extracellular concentration in target tissues is upregulated / increased due to changes in expression, cellular transport, or when intracellular enzymes become extracellular enzymes due to cell lysis caused by disease states, substrate recognition sequences designed to be selectively cleavable by one or a selected subgroup of human proteases can be utilized (MEROPS peptidase database number provided in parentheses; Rawlings ND, Morton FR, Kok, CY, Kong, J. and Barrett AJ (2008) MEROPS: the peptidase database. Nucleic Acids Res. 36 Database). Issue D320-325): Pepsin A (MER000885), pepsinin (MER000894), membrane aspartic protease-2 (MER005870), renin (MER000917), cathepsin D (MER000911), cathepsin E (MER000944), membrane aspartic protease-1 (MER005534), aspartic protease A (MER004981), Mername-AA034 peptidase (MER014038), pepsin A4 (MER037290), pepsin A5 (Homo sapiens) (MER037291), hCG1733572 (Homo sapiens) type putative peptidase (MER107386), aspartic protease B pseudogene (MER004982), CYMP gp(Homo sapiens) (MER002929), subfamily A1A unspecified peptidase (MER181559), mouse mammary tumor virus retroviral protease (MER048030), rabbit endogenous retroviral endopeptidase (MER043650), S71-associated human endogenous retroviral protease (MER001812), RTVL-H type putative peptidase (MER047117), RTVL-H type putative peptidase (MER047133), RTVL-H type putative peptidase (MER047160), RTVL-H type putative peptidase (MER047206), RTVL-H type putative peptidase (M ER047253), RTVL-H type putative peptidase (MER047260), RTVL-H type putative peptidase (MER047291), RTVL-H type putative peptidase (MER047418), RTVL-H type putative peptidase (MER047440), RTVL-H type putative peptidase (MER047479), RTVL-H type putative peptidase (MER047559), RTVL-H type putative peptidase (MER047583), RTVL-H type putative peptidase (MER015446), human endogenous retroviral retroviral protease homolog 1 (MER015479)Human endogenous retrovirus retrovirus protease homolog 2 (MER015481), endogenous retrovirus retrovirus protease pseudogene 1 (Homo sapiens chromosome 14) (MER029977), endogenous retrovirus retrovirus protease pseudogene 2 (Homo sapiens chromosome 8) (MER029665), endogenous retrovirus retrovirus protease pseudogene 3 (Homo sapiens chromosome 17) (MER002660), endogenous retrovirus retrovirus protease pseudogene 3 (Homo sapiens chromosome 17) (MER030286), endogenous retrovirus retrovirus protease pseudogene 3 (Homo sapiens chromosome 17) (MER047144), endogenous retrovirus... Endogenous retrovirus retrovirus protease pseudogene 5 (Homo sapiens chromosome 12) (MER029664), endogenous retrovirus retrovirus protease pseudogene 6 (Homo sapiens chromosome 7) (MER002094), endogenous retrovirus retrovirus protease pseudogene 7 (Homo sapiens chromosome 6) (MER029776), endogenous retrovirus retrovirus protease pseudogene 8 (Homo sapiens chromosome Y) (MER030291), endogenous retrovirus retrovirus protease pseudogene 9 (Homo sapiens chromosome 19) (MER029680), endogenous retrovirus retrovirus protease pseudogene 10 (Homo sapiens chromosome 12) (MER002848), endogenous retrovirus retrovirus protease pseudogene 10 (Homo sapiens chromosome 12) (MER002848), endogenous retrovirus retrovirus protease pseudogene 10 (Homo sapiens chromosome 12) (MER002848), endogenous retrovirus retrovirus protease pseudogene 10 (Homo sapiens chromosome 12), endogenous ... Endogenous retrovirus retrovirus protease pseudogene 11 (Homo sapiens chromosome 17) (MER004378), endogenous retrovirus retrovirus protease pseudogene 12 (Homo sapiens chromosome 11) (MER003344), endogenous retrovirus retrovirus protease pseudogene 13 (Homo sapiens chromosome 2 and similar) (MER029779), endogenous retrovirus retrovirus protease pseudogene 14 (Homo sapiens chromosome 2) (MER029778), endogenous retrovirus retrovirus protease pseudogene 15 (Homo sapiens chromosome 4) (MER047158), endogenous retrovirus retrovirus protease pseudogene 15 (Homo sapiens chromosome 4) (MER0473) 32) Endogenous retrovirus pseudogene 15 (Homo sapiens chromosome 4) (MER003182), Endogenous retrovirus pseudogene 16 (MER047165), Endogenous retrovirus pseudogene 16 (MER047178), Endogenous retrovirus pseudogene 16 (MER047200), Endogenous retrovirus pseudogene 16 (MER047315), Endogenous retrovirus pseudogene 16 (MER047405), Endogenous retrovirus pseudogene 16 (MER030292)Endogenous retrovirus retrovirus protease pseudogene 17 (Homo sapiens chromosome 8) (MER005305), endogenous retrovirus retrovirus protease pseudogene 18 (Homo sapiens chromosome 4) (MER030288), endogenous retrovirus retrovirus protease pseudogene 19 (Homo sapiens chromosome 16) (MER001740), endogenous retrovirus retrovirus protease pseudogene 21 (Homo sapiens) (MER047222), endogenous retrovirus retrovirus protease pseudogene 21 (Homo sapiens) (MER047454), endogenous retrovirus retrovirus protease pseudogene 21 (Homo sapiens) (MER047477), endogenous retrovirus retrovirus... Viral protease pseudogene 21 (Homo sapiens) (MER004403), endogenous retrovirus retrovirus protease pseudogene 22 (Homo sapiens chromosome X) (MER030287), subfamily A2A non-peptidase homolog (MER047046), subfamily A2A non-peptidase homolog (MER047052), subfamily A2A non-peptidase homolog (MER047076), subfamily A2A non-peptidase homolog (MER047080), subfamily A2A non-peptidase homolog (MER047088), subfamily A2A non-peptidase homolog (MER047089), subfamily A2A non-peptidase homolog (MER047091), subfamily A2A non-peptidase homolog Subfamily A2A non-peptidase homolog (MER047092), subfamily A2A non-peptidase homolog (MER047093), subfamily A2A non-peptidase homolog (MER047094), subfamily A2A non-peptidase homolog (MER047097), subfamily A2A non-peptidase homolog (MER047099), subfamily A2A non-peptidase homolog (MER047101), subfamily A2A non-peptidase homolog (MER047102), subfamily A2A non-peptidase homolog (MER047107), subfamily A2A non-peptidase homolog (MER047108), subfamily A2A non-peptidase homolog (MER047109), subfamily A2A non-peptidase homolog (MER047109). Subfamily A2A non-peptidase homologs (MER047110), (MER047111), (MER047114), (MER047118), (MER047121), (MER047122), (MER047126), (MER047129), (MER047130), (MER047134), and (MER047135) are all subfamily A2A non-peptidase homologs.Subfamily A2A non-peptidase homolog (MER047137), Subfamily A2A non-peptidase homolog (MER047140), Subfamily A2A non-peptidase homolog (MER047141), Subfamily A2A non-peptidase homolog (MER047142), Subfamily A2A non-peptidase homolog (MER047148), Subfamily A2A non-peptidase homolog (MER047149), Subfamily A2A non-peptidase homolog (MER047151), Subfamily A2A non-peptidase homolog (MER047154), Subfamily A2A non-peptidase homolog (MER047155), Subfamily A2A non-peptidase homolog (MER047156), Subfamily A2A Non-peptidase homolog (MER047157), subfamily A2A non-peptidase homolog (MER047159), subfamily A2A non-peptidase homolog (MER047161), subfamily A2A non-peptidase homolog (MER047163), subfamily A2A non-peptidase homolog (MER047166), subfamily A2A non-peptidase homolog (MER047171), subfamily A2A non-peptidase homolog (MER047173), subfamily A2A non-peptidase homolog (MER047174), subfamily A2A non-peptidase homolog (MER047179), subfamily A2A non-peptidase homolog (MER047183), subfamily A2A non-peptidase homolog (MER047186), Subfamily A2A non-peptidase homolog (MER047190), Subfamily A2A non-peptidase homolog (MER047191), Subfamily A2A non-peptidase homolog (MER047196), Subfamily A2A non-peptidase homolog (MER047198), Subfamily A2A non-peptidase homolog (MER047199), Subfamily A2A non-peptidase homolog (MER047201), Subfamily A2A non-peptidase homolog (MER047202), Subfamily A2A non-peptidase homolog (MER047203), Subfamily A2A non-peptidase homolog (MER047204), Subfamily A2A non-peptidase homolog (MER04) 7205), Subfamily A2A non-peptidase homolog (MER047207), Subfamily A2A non-peptidase homolog (MER047208), Subfamily A2A non-peptidase homolog (MER047210), Subfamily A2A non-peptidase homolog (MER047211), Subfamily A2A non-peptidase homolog (MER047212), Subfamily A2A non-peptidase homolog (MER047213), Subfamily A2A non-peptidase homolog (MER047215), Subfamily A2A non-peptidase homolog (MER047216), Subfamily A2A non-peptidase homolog (MER047218), Subfamily A2A non-peptidase homolog (MER047219)Subfamily A2A non-peptidase homolog (MER047221), Subfamily A2A non-peptidase homolog (MER047224), Subfamily A2A non-peptidase homolog (MER047225), Subfamily A2A non-peptidase homolog (MER047226), Subfamily A2A non-peptidase homolog (MER047227), Subfamily A2A non-peptidase homolog (MER047230), Subfamily A2A non-peptidase homolog (MER047232), Subfamily A2A non-peptidase homolog (MER047233), Subfamily A2A non-peptidase homolog (MER047234), Subfamily A2A non-peptidase homolog (MER047236), Subfamily A2A Non-peptidase homolog (MER047238), subfamily A2A non-peptidase homolog (MER047239), subfamily A2A non-peptidase homolog (MER047240), subfamily A2A non-peptidase homolog (MER047242), subfamily A2A non-peptidase homolog (MER047243), subfamily A2A non-peptidase homolog (MER047249), subfamily A2A non-peptidase homolog (MER047251), subfamily A2A non-peptidase homolog (MER047252), subfamily A2A non-peptidase homolog (MER047254), subfamily A2A non-peptidase homolog (MER047255), subfamily A2A non-peptidase homolog (MER047263), Subfamily A2A non-peptidase homolog (MER047265), Subfamily A2A non-peptidase homolog (MER047266), Subfamily A2A non-peptidase homolog (MER047267), Subfamily A2A non-peptidase homolog (MER047268), Subfamily A2A non-peptidase homolog (MER047269), Subfamily A2A non-peptidase homolog (MER047272), Subfamily A2A non-peptidase homolog (MER047273), Subfamily A2A non-peptidase homolog (MER047274), Subfamily A2A non-peptidase homolog (MER047275), Subfamily A2A non-peptidase homolog (MER04) 7276), Subfamily A2A non-peptidase homolog (MER047279), Subfamily A2A non-peptidase homolog (MER047280), Subfamily A2A non-peptidase homolog (MER047281), Subfamily A2A non-peptidase homolog (MER047282), Subfamily A2A non-peptidase homolog (MER047284), Subfamily A2A non-peptidase homolog (MER047285), Subfamily A2A non-peptidase homolog (MER047289), Subfamily A2A non-peptidase homolog (MER047290), Subfamily A2A non-peptidase homolog (MER047294), Subfamily A2A non-peptidase homolog (MER047295)Subfamily A2A non-peptidase homolog (MER047298), Subfamily A2A non-peptidase homolog (MER047300), Subfamily A2A non-peptidase homolog (MER047302), Subfamily A2A non-peptidase homolog (MER047304), Subfamily A2A non-peptidase homolog (MER047305), Subfamily A2A non-peptidase homolog (MER047306), Subfamily A2A non-peptidase homolog (MER047307), Subfamily A2A non-peptidase homolog (MER047310), Subfamily A2A non-peptidase homolog (MER047311), Subfamily A2A non-peptidase homolog (MER047314), Subfamily A2A Non-peptidase homolog (MER047318), subfamily A2A non-peptidase homolog (MER047320), subfamily A2A non-peptidase homolog (MER047321), subfamily A2A non-peptidase homolog (MER047322), subfamily A2A non-peptidase homolog (MER047326), subfamily A2A non-peptidase homolog (MER047327), subfamily A2A non-peptidase homolog (MER047330), subfamily A2A non-peptidase homolog (MER047333), subfamily A2A non-peptidase homolog (MER047362), subfamily A2A non-peptidase homolog (MER047366), subfamily A2A non-peptidase homolog (MER047369), Subfamily A2A non-peptidase homolog (MER047370), Subfamily A2A non-peptidase homolog (MER047371), Subfamily A2A non-peptidase homolog (MER047375), Subfamily A2A non-peptidase homolog (MER047376), Subfamily A2A non-peptidase homolog (MER047381), Subfamily A2A non-peptidase homolog (MER047383), Subfamily A2A non-peptidase homolog (MER047384), Subfamily A2A non-peptidase homolog (MER047385), Subfamily A2A non-peptidase homolog (MER047388), Subfamily A2A non-peptidase homolog (MER04) 7389), Subfamily A2A non-peptidase homolog (MER047391), Subfamily A2A non-peptidase homolog (MER047394), Subfamily A2A non-peptidase homolog (MER047396), Subfamily A2A non-peptidase homolog (MER047400), Subfamily A2A non-peptidase homolog (MER047401), Subfamily A2A non-peptidase homolog (MER047403), Subfamily A2A non-peptidase homolog (MER047406), Subfamily A2A non-peptidase homolog (MER047407), Subfamily A2A non-peptidase homolog (MER047410), Subfamily A2A non-peptidase homolog (MER047411)Subfamily A2A non-peptidase homolog (MER047413), Subfamily A2A non-peptidase homolog (MER047414), Subfamily A2A non-peptidase homolog (MER047416), Subfamily A2A non-peptidase homolog (MER047417), Subfamily A2A non-peptidase homolog (MER047420), Subfamily A2A non-peptidase homolog (MER047423), Subfamily A2A non-peptidase homolog (MER047424), Subfamily A2A non-peptidase homolog (MER047428), Subfamily A2A non-peptidase homolog (MER047429), Subfamily A2A non-peptidase homolog (MER047431), Subfamily A2A Non-peptidase homolog (MER047434), subfamily A2A non-peptidase homolog (MER047439), subfamily A2A non-peptidase homolog (MER047442), subfamily A2A non-peptidase homolog (MER047445), subfamily A2A non-peptidase homolog (MER047449), subfamily A2A non-peptidase homolog (MER047450), subfamily A2A non-peptidase homolog (MER047452), subfamily A2A non-peptidase homolog (MER047455), subfamily A2A non-peptidase homolog (MER047457), subfamily A2A non-peptidase homolog (MER047458), subfamily A2A non-peptidase homolog (MER047459), Subfamily A2A non-peptidase homolog (MER047463), Subfamily A2A non-peptidase homolog (MER047468), Subfamily A2A non-peptidase homolog (MER047469), Subfamily A2A non-peptidase homolog (MER047470), Subfamily A2A non-peptidase homolog (MER047476), Subfamily A2A non-peptidase homolog (MER047478), Subfamily A2A non-peptidase homolog (MER047483), Subfamily A2A non-peptidase homolog (MER047488), Subfamily A2A non-peptidase homolog (MER047489), Subfamily A2A non-peptidase homolog (MER04) 7490), Subfamily A2A non-peptidase homolog (MER047493), Subfamily A2A non-peptidase homolog (MER047494), Subfamily A2A non-peptidase homolog (MER047495), Subfamily A2A non-peptidase homolog (MER047496), Subfamily A2A non-peptidase homolog (MER047497), Subfamily A2A non-peptidase homolog (MER047499), Subfamily A2A non-peptidase homolog (MER047502), Subfamily A2A non-peptidase homolog (MER047504), Subfamily A2A non-peptidase homolog (MER047511), Subfamily A2A non-peptidase homolog (MER047513)Subfamily A2A nonpeptidase homolog (MER047514), Subfamily A2A nonpeptidase homolog (MER047515), Subfamily A2A nonpeptidase homolog (MER047516), Subfamily A2A nonpeptidase homolog (MER047520), Subfamily A2A nonpeptidase homolog (MER047533), Subfamily A2A nonpeptidase homolog (MER047537), Subfamily A2A nonpeptidase homolog (MER047569), ...5), Subfamily A2A nonpeptidase homolog (MER047516), Subfamily A2A nonpeptidase homolog (MER047520), Subfamily A2A nonpeptidase homolog (MER047533), Subfamily A2A nonpeptidase homolog (MER047537), Subfamily A2A nonpeptidase homolog (MER047569), Subfamily A2A nonpeptidase homolog (MER047515), Subfamily A2A nonpeptidase homolog (MER047515), Subfamily A2A nonpeptidase homolog (MER047516), Subfamily A2A nonpeptidase homolog (MER047515), Subfamily A2A nonp Family A2A non-peptidase homolog (MER047570), subfamily A2A non-peptidase homolog (MER047584), subfamily A2A non-peptidase homolog (MER047603), subfamily A2A non-peptidase homolog (MER047604), subfamily A2A non-peptidase homolog (MER047606), subfamily A2A non-peptidase homolog (MER047609), subfamily A2A non-peptidase homolog (MER047616), subfamily A2A non-peptidase homolog (MER047619), subfamily A2A non-peptidase homolog (MER047648), subfamily A2A non-peptidase homolog (MER047649), subfamily A2A non-peptidase homolog (MER047662), subfamily A2A non-peptidase homolog (MER048004), subfamily A2A non-peptidase homolog (MER048018), subfamily A2A non-peptidase homolog (MER048019), subfamily A2 A non-peptidase homolog (MER048023), subfamily A2A non-peptidase homolog (MER048037), subfamily A2A unspecified peptidase (MER047164), subfamily A2A unspecified peptidase (MER047231), subfamily A2A unspecified peptidase (MER047386), skin aspartic protease (MER057097), presenilin 1 (MER005221), presenilin 2 (MER005223), impas Impas 1-peptidase (MER019701), impas 1-peptidase (MER184722), impas 4-peptidase (MER019715), impas 2-peptidase (MER019708), impas 5-peptidase (MER019712), impas 3-peptidase (MER019711), possible family A22 pseudogene (Homo sapiens chromosome 18) (MER029974), possible family A22 pseudogene (Homo sapiens chromosome 11) (MER023159), cathepsin V (MER004437), cathepsin X (MER004508), cathepsin F (MER004980), cathepsin L (MER000622), cathepsin S (MER000633), cathepsin O (MER001690), cathepsin K (MER000644).Cathepsin W (MER003756), cathepsin H (MER000629), cathepsin B (MER000686), dipeptidyl peptidase I (MER001937), bleomycin hydrolase (animal) (MER002481), tubulointerstitial nephritis antigen (MER016137), tubulointerstitial nephritis antigen-associated protein (MER021799), cathepsin L-like pseudogene 1 (Homo sapiens) (MER002789), cathepsin B-like pseudogene (chromosome 4, Homo sapiens) (MER029469), cathepsin B-like pseudogene (chromosome 1, Homo sapiens) (MER029457), CTSLL2 gp(Homo sapiens) (MER005210), CTSLL3g.p.(Homo sapiens) (MER005209), Caloplasmin-1 (MER000770), Caloplasmin-2 (MER000964), Caloplasmin-3 (MER001446), Caloplasmin-9 (MER004042), Caloplasmin-8 (MER021474), Caloplasmin-15 (MER004745), Caloplasmin-5 (MER002939), Caloplasmin-11 (MER00584) 4) Caloplasmin-12 (MER029889), Caloplasmin-10 (MER013510), Caloplasmin-13 (MER020139), Caloplasmin-14 (MER029744), Mername-AA253 peptidase (MER005537), Calmodulin (MER000718), putative protein flj40251 (MER003201), ubiquitin-L1 hydrolase (MER000832), ubiquitin-L3 hydrolase (MER000836) Ubiquitin-based hydrolase-BAP1 (MER003989), Ubiquitin-based hydrolase-UCH37 (MER005539), Ubiquitin-specific peptidase 5 (MER002066), Ubiquitin-specific peptidase 6 (MER000863), Ubiquitin-specific peptidase 4 (MER001795), Ubiquitin-specific peptidase 8 (MER001884), Ubiquitin-specific peptidase 13 (MER002627), Ubiquitin-specific peptidase 2 (MER004834), Ubiquitin-specific peptidase 11 (MER002693) Ubiquitin-specific peptidase 14 (MER002667), ubiquitin-specific peptidase 7 (MER002896), ubiquitin-specific peptidase 9X (MER005877), ubiquitin-specific peptidase 10 (MER004439), ubiquitin-specific peptidase 1 (MER004978), ubiquitin-specific peptidase 12 (MER005454), ubiquitin-specific peptidase 16 (MER005493), ubiquitin-specific peptidase 15 (MER005427), ubiquitin-specific peptidase 17 (MER002900).Ubiquitin-specific peptidase 19 (MER005428), ubiquitin-specific peptidase 20 (MER005494), ubiquitin-specific peptidase 3 (MER005513), ubiquitin-specific peptidase 9Y (MER004314), ubiquitin-specific peptidase 18 (MER005641), ubiquitin-specific peptidase 21 (MER006258), ubiquitin-specific peptidase 22 (MER012130), ubiquitin-specific peptidase 33 (MER014335), ubiquitin-specific peptidase 29 (MER012093), ubiquitin-specific peptidase 25 (MER011115), ubiquitin-specific peptidase 36 (MER014033), ubiquitin-specific peptidase 32 (MER014290) Ubiquitin-specific peptidase 26 (Homo sapiens type) (MER014292), Ubiquitin-specific peptidase 24 (MER005706), Ubiquitin-specific peptidase 42 (MER011852), Ubiquitin-specific peptidase 46 (MER014629), Ubiquitin-specific peptidase 37 (MER014633), Ubiquitin-specific peptidase 28 (MER014634), Ubiquitin-specific peptidase 47 (MER014636), Ubiquitin-specific peptidase 38 (MER014637), Ubiquitin-specific peptidase 44 (MER014638), Ubiquitin-specific peptidase 50 (MER030315), Ubiquitin-specific peptidase 35 (MER014646), Ubiquitin-specific peptidase 3 ... R014649), Mername-AA091 peptidase (MER014743), ubiquitin-specific peptidase 45 (MER030314), ubiquitin-specific peptidase 51 (MER014769), ubiquitin-specific peptidase 34 (MER014780), ubiquitin-specific peptidase 48 (MER064620), ubiquitin-specific peptidase 40 (MER015483), ubiquitin-specific peptidase 41 (MER045268), ubiquitin-specific peptidase 31 (MER015493), Mername-AA129 peptidase (MER016485), ubiquitin-specific peptidase 49 (MER016486), Mername-AA187 peptidase (MER014649). 052579), USP17-like peptidase (MER030192), ubiquitin-specific peptidase 54 (MER028714), ubiquitin-specific peptidase 53 (MER027329), ubiquitin-specific endogenous peptidase 39 [misunderstood] (MER064621), Mername-AA090 non-peptidase homolog (MER014739), ubiquitin-specific peptidase [misunderstood] (MER030140), ubiquitin-specific peptidase 52 [misunderstood] (MER030317), NEK2 pseudogene (MER014736), C19 pseudogene (Homo sapiens: chromosome 5) (MER029972), Mername-AA088 peptidase (MER014750).Autophagy-associated protein-2 (MER013564), autophagy-associated protein-1 (MER013561), autophagy-associated protein-3 (MER014316), autophagy-associated protein-4 (MER064622), Cezanne deubiquitinating peptidase (MER029042), Cezanne-2 peptidase (MER029044), tumor necrosis factor α-inducible protein 3 (MER029050), trabid peptidase (MER029052), VCIP135 deubiquitinating peptidase (MER152304), otubai n-1 (MER029056), otubain-2 (MER029061), CyID protein (MER030104), UfSP1 peptidase (MER042724), UfSP2 peptidase (MER060306), DUBA deubiquitinase (MER086098), KIAA0459 (Homo sapiens)-like protein (MER122467), Otud1 protein (MER125457), 1 isoform of CRA_c (Homo sapiens)-like protein containing glycosyltransferase 28 domain (MER123606), hin1L gp (Homo sapiens) (MER139816), ataxia protein-3 (MER099998), ATXN3L putative peptidase (MER115261), Josephin domain-containing 1 (Homo sapiens) (MER125334), Josephin domain-containing 2 (Homo sapiens) (MER124068), YOD1 peptidase (MER116559), pod protein (plant α form) (MER044591), pod protein (MER001800), glycosylphosphatidylinositol: protein transamidase (MER002479), pod protein pseudogene (Homo sapiens) (MER029741), family C13 unspecified peptidase (MER175813), cysteine ​​protease-1 (MER000850), cysteine ​​protease-3 (MER000853). Cysteine ​​protease-7 (MER002705), cysteine ​​protease-6 (MER002708), cysteine ​​protease-2 (MER001644), cysteine ​​protease-4 (MER001938), cysteine ​​protease-5 (MER002240), cysteine ​​protease-8 (MER002849), cysteine ​​protease-9 (MER002707), cysteine ​​protease-10 (MER002579), cysteine ​​protease-14 (MER012083), paracysteine ​​protease (MER019325), Mername-AA143 peptidase (MER021304), Mername-AA186 peptidase (MER020516), and putative cysteine ​​protease (Homo sapiens) (MER021463).FLIP protein (MER003026), Mername-AA142 protein (MER021316), cysteine ​​protease-12 pseudogene (Homo sapiens) (MER019698), Mername-AA093 cysteine ​​protease pseudogene (MER014766), subfamily C14A non-peptidase homolog (MER185329), subfamily C14A non-peptidase homolog (MER179956), cleavage enzyme (Homo sapiens type) (MER011775), cleavage enzyme-like pseudogene (MER014797), SENP1 peptidase (MER011012), SENP3 peptidase (MER011019), SENP6 peptidase (MER0014797). R011109), SENP2 peptidase (MER012183), SENP5 peptidase (MER014032), SENP7 peptidase (MER014095), SENP8 peptidase (MER016161), SENP4 peptidase (MER005557), pyroglutamyl-peptidase I (vertebrates) (MER011032), Mername-AA073 peptidase (MER029978), Hedgehog protein (MER002539), Indian hedgehog protein (MER002538), Desert hedgehog protein (MER012170), dipeptidyl-peptidase III (MER004252), Mername-AA164 protein ( MER020410), LOC138971g.p. (Homo sapiens) (MER020074), Atp23 peptidase (MER060642), isoprenyl peptidase 1 (MER004246), aminopeptidase N (MER000997), aminopeptidase A (MER001012), leukotriene A4 hydrolase (MER001013), pyroglutamyl-peptidase II (MER012221), cytoplasmic alanyl aminopeptidase (MER002746), cysteyl aminopeptidase (MER002060), aminopeptidase B (MER001494), aminopeptidase PILS (MER005331), arginyl aminopeptidase-like 1 (M ER012271), Leukocyte-derived arginine aminopeptidase (MER002968), Aminopeptidase Q (MER052595), Aminopeptidase O (MER019730), Ta-binding protein-related factor (MER026493), Angiotensin-converting peptidase unit 1 (MER004967), Angiotensin-converting peptidase unit 2 (MER001019), Angiotensin-converting enzyme-2 (MER011061), Mername-AA153 protein (MER020514), Phenylephrine oligopeptidase (MER001737), Neurolysin (MER010991), Mitochondrial intermediate peptidase (MER003665)Mername-AA154 protein (MER021317), Leishmaniazin-2 (MER014492), Leishmaniazin-3 (MER180031), Matrix metallopeptidase-1 (MER001063), Matrix metallopeptidase-8 (MER001084), Matrix metallopeptidase-2 (MER001080), Matrix metallopeptidase-9 (MER001085), Matrix metallopeptidase-3 (MER001068), Matrix metallopeptidase-10 (Homo sapiens type) (MER001072), Matrix metallopeptidase-11 (MER001075), Matrix metallopeptidase-7 (MER001092), Matrix metallopeptidase-12 (MER001092), Matrix metallopeptidase-12 (MER001084), Matrix metallopeptidase-2 (MER001085), Matrix metallopeptidase-12 (MER001084 ... 001089), matrix metallopeptidase-13 (MER001411), membrane matrix metallopeptidase-1 (MER001077), membrane matrix metallopeptidase-2 (MER002383), membrane matrix metallopeptidase-3 (MER002384), membrane matrix metallopeptidase-4 (MER002595), matrix metallopeptidase-20 (MER003021), matrix metallopeptidase-19 (MER002076), matrix metallopeptidase-23B (MER004766), membrane matrix metallopeptidase-5 (MER005638), membrane matrix matrix metallopeptidase-6 (MER012071), matrix metallopeptidase-21 (MER006101), matrix Metallopeptidase-22 (MER014098), matrix metallopeptidase-26 (MER012072), matrix metallopeptidase-28 (MER013587), matrix metallopeptidase-23A (MER037217), macrophage elastase homolog (chromosome 8, Homo sapiens) (MER030035), Mername-AA156 protein (MER021309), matrix metallopeptidase-like 1 (MER045280), subfamily M10A non-peptidase homolog (MER175912), subfamily M10A non-peptidase homolog (MER187997), subfamily M10A non-peptidase homolog (MER187998), subfamily M10A non-peptidase Homologous protein (MER180000), meprin α subunit (MER001111), meprin β subunit (MER005213), procollagen C-peptidase (MER001113), mammalian tolloid-like 1 protein (MER005124), mammalian tolloid-like 2 protein (MER005866), ADAMTS9 peptidase (MER012092), ADAMTS14 peptidase (MER016700), ADAMTS15 peptidase (MER017029), ADAMTS16 peptidase (MER015689), ADAMTS17 peptidase (MER016302).ADAMTS18 peptidase (MER016090), ADAMTS19 peptidase (MER015663), ADAMTS peptidase (MER003902), ADAM9 peptidase (MER001140), ADAM10 peptidase (MER002382), ADAM12 peptidase (MER005107), ADAM19 peptidase (MER012241), ADAM15 peptidase (MER002386), ADAM17 peptidase (MER003094), ADAM20 peptidase (MER004725), ADAMTS1 peptidase (MER000743), ADAMTS3 peptidase (MER005100), ADAMTS4 peptidase (MER005101), ADAMTS1 peptidase (MER005546) ADAM28 peptidase (Human type) (MER005495), ADAMTS5 peptidase (MER005548), ADAMTS8 peptidase (MER005545), ADAMTS6 peptidase (MER005893), ADAMTS7 peptidase (MER005894), ADAM30 peptidase (MER006268), ADAM21 peptidase (Human type) (MER004726), ADAMTS10 peptidase (MER014331), ADAMTS12 peptidase (MER014337), ADAMTS13 peptidase (MER015450), ADAM33 peptidase (MER015143), astaxanthin (MER029996), ADAMTS20 peptidase (Human type) (MER026906), procollagen I N-peptidase (MER004985), ADAM2 protein (MER003090), ADAM6 protein (MER047044), ADAM7 protein (MER005109), ADAM18 protein (MER012230), ADAM32 protein (MER026938), non-peptidase homolog (Homo sapiens chromosome 4) (MER029973), family M12 non-peptidase homolog (Homo sapiens chromosome 16) (MER047654), family M12 non-peptidase homolog (Homo sapiens chromosome 15) (MER047250). ADAM3B protein (Homo sapiens) (MER005199), ADAM11 protein (MER001146), ADAM22 protein (MER005102), ADAM23 protein (MER005103), ADAM29 protein (MER006267), protein similar to ADAM21 peptidase progenitor (Homo sapiens) (MER026944), Mername-AA225 peptidase homolog (Homo sapiens) (MER047474), and hypothetical ADAM pseudogene (chromosome 4, Homo sapiens) (MER029975).ADAM3A gp (Homo sapiens) (MER005200), ADAM1 gp (Homo sapiens) (MER003912), subfamily M12B non-peptidase homolog (MER188210), subfamily M12B non-peptidase homolog (MER188211), subfamily M12B non-peptidase homolog (MER188212), subfamily M12B non-peptidase homolog (MER188220), enkephalin (MER001050), endothelin-converting enzyme 1 (MER001057), endothelin-converting enzyme 2 (MER004776), DINE peptidase (MER005197), enkephalin-2 (MER013406), Kerr blood group (Kell Blood-group protein (MER001054), PHEX peptidase (MER002062), i-AAA peptidase (MER001246), i-AAA peptidase (MER005755), paraplegia protein (MER004454), Afg3-like protein 2 (MER005496), Afg3-like protein 1A (MER014306), pregnancy-associated plasma protein-1 (MER002217), pregnancy-associated plasma protein-2 (MER014521) Farnesyl-converting protein 1 (MER002646), metalloproteinase-associated protein-1 (MER030873), aminopeptidase AMZ2 (MER011907), aminopeptidase AMZ1 (MER058242), carboxypeptidase A1 (MER001190), carboxypeptidase A2 (MER001608), carboxypeptidase B (MER001194), carboxypeptidase N (MER001198), carboxypeptidase E (MER001199), carboxypeptidase M (MER001199) 001205), Carboxypeptidase U (MER001193), Carboxypeptidase A3 (MER001187), Metallocarboxypeptidase D peptidase unit 1 (MER003781), Metallocarboxypeptidase Z (MER003428), Metallocarboxypeptidase D peptidase unit 2 (MER004963), Carboxypeptidase A4 (MER013421), Carboxypeptidase A6 (MER013456), Carboxypeptidase A5 (MER017121), Metallocarboxypeptidase O (MER016044), Cytoplasmic carboxyl group Peptidase-like protein 5 (MER033174), cytoplasmic carboxypeptidase 3 (MER033176), cytoplasmic carboxypeptidase 6 (MER033178), cytoplasmic carboxypeptidase 1 (MER033179), cytoplasmic carboxypeptidase 2 (MER037713), metallocarboxypeptidase D non-peptidase unit (MER004964), adipocyte enhancer-binding protein 1 (MER003889), carboxypeptidase-like protein X1 (MER013404), carboxypeptidase-like protein X2 (MER078764).Cytoplasmic carboxypeptidase (MER026952), family M14 non-peptidase homolog (MER199530), insulin lysozyme (MER001214), mitochondrial processing peptidase β-subunit (MER004497), phenylephrine lyase (MER003883), eupitrilysin (MER004877), mitochondrial processing peptidase non-peptidase α-subunit (MER001413), ubiquitin-cytochrome c reductase core protein I (MER003543), ubiquitin-cytochrome c reductase core protein II (MER003544), ubiquitin-cytochrome c reductase core protein domain 2 (MER043998), insulin Lysozyme Unit 2 (MER046821), Phenylhexyl lyase Unit 2 (MER046874), Insulin Lysozyme Unit 3 (MER078753), Mitochondrial Processing Peptidase Subunit α Unit 2 (MER124489), Phenylhexyl lyase Unit 3 (MER142856), LOC133083 g.p. (Homo sapiens) (MER021876), Subfamily M16B Non-peptidase Homolog (MER188757), Leucylaminopeptidase (Animal) (MER003100), Mername-AA040 Peptidase (MER003919), Leucylaminopeptidase-1 (Caenorhabditis type) (MER003919) 013416), Methionyl aminopeptidase 1 (MER001342), Methionyl aminopeptidase 2 (MER001728), Aminopeptidase P2 (MER004498), Xaa-Pro dipeptidase (eukaryote) (MER001248), Aminopeptidase P1 (MER004321), Mitochondrial intermediate cleavage peptidase 55kDa (MER013463), Mitochondrial methionyl aminopeptidase (MER014055), Mername-AA020 peptidase homolog (MER010972), Proliferation-associated protein 1 (MER005497), Chromosomal-specific transcription elongation factor 140kDa subunit (MER026495) The following proteins were detected: a proliferation-associated protein (Homo sapiens chromosome X) (MER029983), a Mername-AA226 peptidase homolog (Homo sapiens) (MER056262), a Mername-AA227 peptidase homolog (Homo sapiens) (MER047299), a subfamily M24A non-peptidase homolog (MER179893), an aspartic aminopeptidase (MER003373), a Gly-Xaa carboxypeptidase (MER033182), a carnosine dipeptidase II (MER014551), a carnosine dipeptidase I (MER015142), a Mername-AA161 protein (MER021873), and an aminoamidase (MER001271).Glutamate carboxypeptidase II (MER002104), NAALADASE L peptidase (MER005239), Glutamate carboxypeptidase III (MER005238), Plasma glutamate carboxypeptidase (MER005244), Mername-AA103 peptidase (MER015091), Fxna peptidase (MER029965), Transferrin receptor protein (MER002105), Transferrin receptor 2 protein (MER005152), Glutamine acyl cyclase (MER015095), Glutamate carboxypeptidase II (Homo sapiens) type non-peptidase homolog (MER026971), nicalin (MER044627), Membrane dipeptidase (MER001260), Membrane-bound dipeptidase -2 (MER013499), membrane-bound dipeptidase-3 (MER013496), dihydroorotase (MER005767), dihydropyrimidinease (MER033266), dihydropyrimidinease-associated protein-1 (MER030143), dihydropyrimidinease-associated protein-2 (MER030155), dihydropyrimidinease-associated protein-3 (MER030151), dihydropyrimidinease-associated protein-4 (MER030149), dihydropyrimidinease-associated protein-5 (MER030136), putative protein-like protein 5730457F11RIK (MER033184), 1300019j08rik protein (MER033186) ), guanine aminohydrolase (MER037714), Kae1 putative peptidase (MER001577), OSGEPL1-like protein (MER013498), S2P peptidase (MER004458), subfamily M23B non-peptidase homolog (MER199845), subfamily M23B non-peptidase homolog (MER199846), subfamily M23B non-peptidase homolog (MER199847), subfamily M23B non-peptidase homolog (MER137320), subfamily M23B non-peptidase homolog (MER201557), subfamily M23B non-peptidase homolog (MER199417), subfamily M23 M23B nonpeptidase homolog (MER199418), subfamily M23B nonpeptidase homolog (MER199419), subfamily M23B nonpeptidase homolog (MER199420), subfamily M23B nonpeptidase homolog (MER175932), subfamily M23B nonpeptidase homolog (MER199665), Poh1 peptidase (MER020382), Jab1 / MPN domain metalloenzyme (MER022057), Mername-AA165 peptidase (MER021865), Brcc36 isopeptidase (MER021890), histone H2A deubiquitinase MYSM1 (MER021887).AMSH deubiquitinated peptidase (MER030146), putative peptidase (Homo sapiens chromosome 2) (MER029970), Mername-AA168 protein (MER021886), COP9 signal transducer subunit 6 (MER030137), 26S proteasome non-ATPase regulatory subunit 7 (MER030134), eukaryotic translation initiation factor 3 subunit 5 (MER030133), 1FP38 peptidase homolog (MER030132), subfamily M67A non-peptidase homolog (MER191181), subfamily M67A unspecified peptidase (MER191144), granzyme B (Homo sapiens type) (MER000168), testosterone White (MER005212), trypsin β (MER000136), kallikrein-associated peptidase 5 (MER005544), corin (MER005881), kallikrein-associated peptidase 12 (MER006038), DESC1 peptidase (MER006298), trypsin γ1 (MER011036), kallikrein-associated peptidase 14 (MER011038), hyaluronic acid-binding peptidase (MER003612), transmembrane peptidase, serine 4 (MER011104), intestinal serine peptidase (rodents) (MER016130), adrenal secretory serine peptidase (MER003734), trypsin δ1 (Homo sapiens) (MER005948), protein lyase-3 (MER029902), marapsin (MER006119), trypsin-6 (MER006118), ovalbumin-1 domain 1 (MER099182), transmembrane peptidase, serine 3 (MER005926), kallikrein-associated peptidase 15 (MER000064), Mername-AA031 peptidase (MER014054), TMPRSS13 peptidase (MER014226), Mername-AA038 peptidase (MER062848), Mername-AA204 peptidase (MER029980) Cationic trypsin (Homo sapiens type) (MER000020), elastase-2 (MER000118), mannan-binding lectin-associated serine peptidase-3 (MER031968), cathepsin G (MER000082), myeloblast protease (MER000170), granzyme A (MER001379), granzyme M (MER001541), chymotrypsin (Homo sapiens type) (MER000123), trypsin α (MER000135), granzyme K (MER001936), granzyme H (MER000166), chymotrypsin B (MER000001), elastase-1 (MER003733).Pancreatic endogenous peptidase E (MER000149), pancreatic elastase II (MER000146), enterokinase (MER002068), chymotrypsin C (MER000761), prostaglandin (MER002460), kallikrein 1 (MER000093), kallikrein-related peptidase 2 (MER000094), kallikrein-related peptidase 3 (MER000115), mesotrypsin (MER000022), complement component C1r-like peptidase (MER016352), complement factor D (MER000130), complement component activated C1r (MER000238), complement component activated C1s (MER000... 239), complement component C2a (MER000231), complement factor B (MER000229), mannan-binding lectin-associated serine peptidase 1 (MER000244), complement factor I (MER000228), pancreatic endopeptidase E form B (MER000150), pancreatic elastase IIB (MER000147), coagulation factor XIIa (MER000187), plasma kallikrein (MER000203), coagulation factor Xia (MER000210), coagulation factor IXa (MER000216), coagulation factor Vila (MER000215), coagulation factor Xa (MER000212), coagulation factor C2a (MER000231), coagulation factor C2a (MER000212), coagulation factor C2a (MER000212), coagulation factor C2a (MER000212), coagulation factor C2a (MER000212), coagulation factor C2a (MER000231), complement factor C2a (MER000229), complement factor C2a (MER000244), complement factor I (MER000228), complement factor C2a (MER000212 ... Enzyme (MER000188), Protein C (Activation) (MER000222), Acrosomal enzyme (MER000078), Hepsin (MER000156), Hepatocyte growth factor activator (MER000186), Mannan-binding lectin-associated serine peptidase 2 (MER002758), Urokinase plasminogen activator (MER000195), Tissue plasminogen activator (MER000192), Plasmin (MER000175), Kallikrein-associated peptidase 6 (MER002580), Neurotrypsin (MER004171), Kallikrein-associated peptidase 8 (MER005400), Kallikrein Related peptidase 10 (MER003645), epitheliasin (MER003736), kallikrein-related peptidase 4 (MER005266), prosemin (MER004214), chymopasin (MER001503), kallikrein-related peptidase 11 (MER004861), kallikrein-related peptidase 11 (MER216142), trypsin type 2 A (MER000021), HtrA1 peptidase (Homo sapiens type) (MER002577), HtrA2 peptidase (MER208413), HtrA2 peptidase (MER004093)HtrA3 peptidase (MER014795), HtrA4 peptidase (MER016351), Tysnd1 peptidase (MER050461), TMPRSS12 peptidase (MER017085), HAT-like putative peptidase 2 (MER021884), trypsin C (MER021898), kallikrein-associated peptidase 7 (MER002001), protein lyase (MER003735), kallikrein-associated peptidase 13 (MER005269), kallikrein-associated peptidase 9 (MER005270), protein lyase-2 (MER005278), umbilical vein peptidase (MER005421), LCLP peptidase ( MER001900), spinal protein (MER014385), Malawi protein-2 (MER021929), complement factor D-like putative peptidase (MER056164), ovalbumin-2 (MER022410), HAT-like tetrapeptidase (MER044589), ovalbumin-1 domain 1 (MER022412), epidermal-specific SP-like putative peptidase (MER029900), testis serine peptidase 5 (MER029901), Mername-AA258 peptidase (MER000285), polyserase-IA unit 1 (MER030879), polyserase-IA unit 2 (MER0 ...001900), complement factor D-like putative peptidase (MER056164), ovalbumin-2 (MER022410), testis serine peptidase 5 (MER022410), testis serine peptidase 5 (MER022410), testis serine peptidase 5 (MER02 030880), testicular serine peptidase 2 (human type) (MER033187), presumed acrosomal enzyme-like peptidase (Homo sapiens) (MER033253), HAT-like pentapeptidase (MER028215), polyserine enzyme-3 unit 1 (MER061763), polyserine enzyme-3 unit 2 (MER061748), tryptophan / serine protease-like peptidase (MER056263), polyserine enzyme-2 unit 1 (MER061777), Mername-AA123 peptidase (MER021930), HAT-like dipeptidase (MER099184), hCG2041452-like protein (MER099172), h CG22067 (Homo sapiens) (MER099169), Brain rescue factor-1 (Homo sapiens) (MER098873), hCG2041108 (Homo sapiens) (MER099173), Polyserine enzyme-2 unit 2 (MER061760), Polyserine enzyme-2 unit 3 (MER065694), Mername-AA201 (peptidase homolog) MER099175, Secretory trypsin-like serine peptidase homolog (MER030000), Polyserine enzyme-1A unit 3 (MER029880), Azurocidin (MER000119), Globin-1 (MER000233)Globin-associated protein (MER000235), macrophage-stimulating protein (MER001546), hepatocyte growth factor (MER000185), protein Z (MER000227), TESP1 protein (MER047214), LOC136242 protein (MER016132), plasma kallikrein-like protein 4 (MER016346), PRSS35 protein (MER016350), DKFZp586H2123-like protein (MER066474), apolipoprotein (MER000183), psi-KLK1 pseudogene (Homo sapiens) (MER033287), trypsin pseudogene I (MER01) 5077), trypsin pseudogene II (MER015078), trypsin pseudogene III (MER015079), subfamily S1A unspecified peptidase (MER216982), subfamily S1A unspecified peptidase (MER216148), amide phosphoribosyltransferase precursor (MER003314), glutamine-fructose-6-phosphoaminotransferase 1 (MER003322), glutamine:fructose-6-phosphoamidase (MER012158), Mername-AA144 protein (MER021319), asparagine synthase (MER033254), family C44 non-peptidase homolog (MER159286). Family C44 unspecified peptidase (MER185625), Family C44 unspecified peptidase (MER185626), isolated protein 1 (MER045376), isolated protein 2 (MER064573), isolated protein 3 (MER064582), acidic ceramide precursor (MER100794), N-acylethanolamine amidase precursor (MER141667), proteasome catalytic subunit 1 (MER000556), proteasome catalytic subunit 2 (MER002625), proteasome catalytic subunit 3 (MER002149), proteasome catalytic subunit 1i (MER000552), proteasome catalytic subunit 2i (MER000552). 01515), proteasome catalytic subunit 3i (MER000555), proteasome catalytic subunit 5t (MER026203), protein serine kinase c17 (MER026497), proteasome subunit α6 (MER000557), proteasome subunit α2 (MER000550), proteasome subunit α4 (MER000554), proteasome subunit α7 (MER033250), proteasome subunit α5 (MER000558), proteasome subunit α1 (MER000549), proteasome subunit α3 (MER000553), proteasome subunit XAPC7 (MER004372).Proteasome subunit β3 (MER001710), proteasome subunit β2 (MER002676), proteasome subunit β1 (MER000551), proteasome subunit β4 (MER001711), Mername-AA230 peptidase homolog (Homo sapiens) (MER047329), Mername-AA231 pseudogene (Homo sapiens) (MER047172), Mername-AA232 pseudogene (Homo sapiens) (MER047316), glycosyl asparaginase precursor (MER003299), isoaspartic dipeptidase (threonine type) (MER031622), threonine aspartate enzyme-1 (MER001710). 16969), γ-glutamyltransferase 5 (mammalian type) (MER001977), γ-glutamyltransferase 1 (mammalian type) (MER001629), γ-glutamyltransferase 2 (Homo sapiens) (MER001976), γ-glutamyltransferase-like protein 4 (MER002721), γ-glutamyltransferase-like protein 3 (MER016970), γ-glutamyltransferase 1 precursor similar to Homo sapiens (MER026204), γ-glutamyltransferase 1 precursor similar to Homo sapiens (MER026205), Mername-AA211 putative peptidase (MER026207), γ-glutamyltransferase 6 (MER001977), γ-glutamyltransferase 1 (MER0016969), γ-glutamyltransferase 5 (mammalian type) (MER001977), γ-glutamyltransferase 1 (mammalian type) (MER001629), γ-glutamyltransferase 2 (Homo sapiens) (MER001976), γ-glutamyltransferase 6 (MER001977), γ-glutamyltransferase 1 (Mammalian type) (MER001629), γ-glutamyltransferase 2 (Homo sapiens) (MER001976), γ-glutamyltransferase 3 (MER0016970), γ-glutamyltransferase 1 precursor similar to Homo sapiens (MER0026205), γ-glutamyltransferase 1 (MER0016969), γ-glutamyltransferase 5 (mammalian type) (MER001977), γ-glutam 159283), γ-glutamyl transpeptidase homolog (chromosome 2, Homo sapiens) (MER037241), polycystic protein-1 (MER126824), KIAA1879 protein (MER159329), polycystic kidney disease 1-like 3 (MER172554), γ-glutamyl hydrolase (MER002963), guanine 5″-monophosphate synthase (MER043387), carbamoyl-phosphoryl synthase (Homo sapiens type) (MER078640), dihydroorotase (N-terminal unit) (Homo sapiens type) (MER060647), DJ-1 putative peptidase (MER003390), Mername-AA100 putative peptidase (MER014) 802), Mername-AA101 nonpeptidase homolog (MER014803), KIAA0361 protein (Homo sapiens type) (MER042827), F1134283 protein (Homo sapiens) (MER044553), nonpeptidase homolog chromosome 21 open reading frame 33 (Homo sapiens) (MER160094), family C56 nonpeptidase homolog (MER177016), family C56 nonpeptidase homolog (MER176613), family C56 nonpeptidase homolog (MER176918), mucin-like hormone receptor-like 2 containing EGF-like module (MER037230), CD97 antigen (human type) (MER037286).Cadmin-like hormone receptor-like 3 (MER037288) containing an EGF-like module, mucin-like hormone receptor-like 1 (MER037278) containing an EGF-like module, mucin-like hormone receptor-like 4 (MER037294) containing an EGF-like module, cadherin EGF LAG heptaton G receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus musculus) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), arachnid receptor 2 (MER122199), arachnid receptor-1 (MER126380), arachnid receptor 3 (MER124612), and procadherin Flamingo 2 (MER124239), ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), seven-transmembrane helical receptor (MER125448), Gpr114 protein (MER159320), GPR126 angiogenic G protein-coupled receptor (MER140015), GPR125 (Homo sapiens) protein (MER159279), GPR116 (Homo sapiens) G protein-coupled receptor (MER159280), GPR128 (Homo sapiens) G protein-coupled receptor (MER162015), GPR133 (Homo sapiens) protein (MER159334), GPR110 G protein-coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773), KPG_008 protein (MER161835), KPG_009 protein (MER159335), unspecified homolog (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD autoprocessing protein unit 1 (MER020001), PIDD autoprocessing protein unit 2 (MER063690), MUC1 self-cleaving mucin (MER074260), dystrophin (MER0547) 41) Proprotein convertase 9 (MER022416), site-1 peptidase (MER001948), furin (MER000375), proprotein convertase 1 (MER000376), proprotein convertase 2 (MER000377), proprotein convertase 4 (MER028255), PACE4 proprotein convertase (MER000383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl peptidase II (MER000355), subfamily S8A nonpeptidase homolog (MER201339), subfamily S8A nonpeptidase homolog (MER191613)Subfamily S8A unspecified peptidase (MER191611), Subfamily S8A unspecified peptidase (MER191612), Subfamily S8A unspecified peptidase (MER191614), Tripeptidyl-peptidase I (MER003575), Prolyl oligopeptidase (MER000393), Dipeptidyl-peptidase IV (eukaryotes) (MER000401), Acylaminoacyl-peptidase (MER000408), Fibroblast activating protein α subunit (MER000399), PREPL Protein A (MER004227), dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Mername-AA194 putative peptidase (MER017353), Mername-AA195 putative peptidase (MER017367), Mername-AA196 putative peptidase (MER017368), Mername-AA197 putative peptidase (MER017371), C14orf29 protein (MER004227). 033244), putative protein (MER033245), putative esterase / lipase / thioesterase (MER047309), protein bat5 (MER037840), putative protein flj40219 (MER033212), putative protein flj37464 (MER033240), putative protein flj33678 (MER033241), dipeptidyl peptidase homolog DPP6 (MER000403), dipeptidyl peptidase homolog DPP10 (MER005988), similar to mouse chromosome 20 open reading frame Protein 135 (MER037845), kynurenine formamidinase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198), carboxylesterase D1 (MER033213), hepatic carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile salt-dependent lipase (MER033227), and carboxylesterase-associated protein (MER033231). Neural connexin 3 (MER033232), X-linked neural connexin 4 (MER033235), Y-linked neural connexin 4 (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274), neural connexin 1 (MER033280), neural connexin 2 (MER033283), and family S9 nonpeptidase homolog (MER212939).Family S9 non-peptidase homolog (MER211490), subfamily S9C unspecified peptidase (MER192341), family S9 unspecified peptidase (MER209181), family S9 unspecified peptidase (MER200434), family S9 unspecified peptidase (MER209507), family S9 unspecified peptidase (MER209142), serine carboxypeptidase A (MER000430), egg yolk carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), family S15 unspecified peptidase (MER199442), family S15 unspecified peptidase (MER200437), family S15 unspecified peptidase (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl-peptidase I I (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), protein 4 containing an autohydrolase domain (MER031616), epoxide hydrolase (MER000432), mesodermal-specific transcript (MER199890), mesodermal-specific transcript (MER017123), cytoplasmic epoxide hydrolase (MER029997), cytoplasmic epoxide hydrolase (MER213866), similar putative protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome The following proteins are involved in the syndrome: epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), putative protein flj22408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), putative protein (MER033249), valacyclovir hydrolase (MER033259), Ccg1 interaction factor b (MER210738), glycosyl asparaginase precursor (MER003299), and isoaspartic diacetate. Peptidase (threonine type) (MER031622), threonine aspartate enzyme-1 (MER016969), gamma-glutamyl transferase 5 (mammalian type) (MER001977), gamma-glutamyl transferase 1 (mammalian type) (MER001629), gamma-glutamyl transferase 2 (Homo sapiens) (MER001976), gamma-glutamyl transferase-like protein 4 (MER002721), gamma-glutamyl transferase-like protein 3 (MER016970), and a precursor similar to gamma-glutamyl transferase 1 (Homo sapiens) (MER026204).Similar to γ-glutamyltransferase 1 precursor (Homo sapiens) (MER026205), Mername-AA211 putative peptidase (MER026207), γ-glutamyltransferase 6 (MER159283), γ-glutamyl transpeptidase homolog (chromosome 2, Homo sapiens) (MER037241), polycystic protein-1 (MER126824), KIAA1879 protein (MER159329), and polycystic kidney disease 1-like 3 (MER172554). γ-Glutamyl hydrolase (MER002963), Guanine 5″-monophosphate synthase (MER043387), Carbamoyl-phosphoryl synthase (Homo sapiens type) (MER078640), Dihydroorotase (N-terminal unit) (Homo sapiens type) (MER060647), DJ-1 putative peptidase (MER003390), Mername-AA100 putative peptidase (MER014802), Mername-AA101 non-peptidase homolog (MER00 14803), KIAA0361 protein (Homo sapiens type) (MER042827), FLJ34283 protein (Homo sapiens) (MER044553), non-peptidase homolog chromosome 21 open reading frame 33 (Homo sapiens) (MER160094), family C56 non-peptidase homolog (MER177016), family C56 non-peptidase homolog (MER176613), family C56 non-peptidase homolog (MER176918), mucin-like protein containing EGF-like modules Hormone receptor-like 2 (MER037230), CD97 antigen (human type) (MER037286), mucin-like hormone receptor-like 3 containing an EGF-like module (MER037288), mucin-like hormone receptor-like 1 containing an EGF-like module (MER037278), mucin-like hormone receptor-like 4 containing an EGF-like module (MER037294), cadherin EGFLAG heptacort G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus The following proteins were identified: GPR56 (Homo sapiens) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), arachnoid receptor 2 (MER122199), arachnoid receptor-1 (MER126380), arachnoid receptor 3 (MER124612), procadherin Flamingo 2 (MER124239), ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), seven-transmembrane helical receptor (MER125448), Gpr114 protein (MER159320), GPR126 angiogenic G protein-coupled receptor (MER140015), GPR125 (Homo sapiens) type protein (MER159279), and GPR116 (Homo sapiens) type G protein-coupled receptor (MER159280).GPR128 (Homo sapiens) type G protein-coupled receptor (MER162015), GPR133 (Homo sapiens) type protein (MER159334), GPR110 G protein-coupled receptor (MER159277), GPR97 protein (MER159322), KPG_006 protein (MER161773), KPG_008 protein (MER161835), KPG_009 protein (MER159335), unspecified homolog (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD autoprocessing protein unit 1 (MER020001), PIDD autoprocessing protein unit 2 (MER063690), MUC1 self-cleaving mucin (MER074260), dystrophin (MER054741), proprotein convertase 9 (MER022416), site-1 peptidase (MER001948), furin protease (MER000375), proprotein convertase 1 (MER000376), proprotein convertase 2 (MER0 00377), proprotein convertase 4 (MER028255), PACE4 proprotein convertase (MER000383), proprotein convertase 5 (MER002578), proprotein convertase 7 (MER002984), tripeptidyl-peptidase II (MER000355), subfamily S8A non-peptidase homolog (MER201339), subfamily S8A non-peptidase homolog (MER191613), subfamily S8A unspecified peptidase (ME R191611), Subfamily S8A Unspecified Peptidase (MER191612), Subfamily S8A Unspecified Peptidase (MER191614), Tripeptidyl-Peptidase I (MER003575), Prolyl Oligopeptidase (MER000393), Dipeptidyl-Peptidase IV (Eukaryotes) (MER000401), Acylaminoacyl-Peptidase (MER000408), Fibroblast Activator Protein α Subunit (MER000399), PREPL Protein A (MER004227), dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Mername-AA194 putative peptidase (MER017353), Mername-AA195 putative peptidase (MER017367), Mername-AA196 putative peptidase (MER017368), Mername-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), putative protein (MER033245), putative esterase / lipase / thioesterase (MER047309)Protein bat5 (MER037840), putative protein flj40219 (MER033212), putative protein flj37464 (MER033240), putative protein flj33678 (MER033241), dipeptidyl peptidase homolog DPP6 (MER000403), dipeptidyl peptidase homolog DPP10 (MER005988), protein resembling mouse chromosome 20 open reading frame 135 (MER037845), kynurenine formamidinase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198) Carboxylesterase D1 (MER033213), hepatic carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile salt-dependent lipase (MER033227), carboxylesterase-associated protein (MER033231), neural connexin 3 (MER033232), X-linked neural connexin 4 (MER033235), Y-linked neural connexin 4 (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER03). 3274), Neural Connectin 1 (MER033280), Neural Connectin 2 (MER033283), Family S9 Non-peptidase Homolog (MER212939), Family S9 Non-peptidase Homolog (MER211490), Subfamily S9C Unspecified Peptidase (MER192341), Family S9 Unspecified Peptidase (MER209181), Family S9 Unspecified Peptidase (MER200434), Family S9 Unspecified Peptidase (MER209507), Family S9 Unspecified Peptidase (MER209142), Serine Carboxypeptidase A (MER000430), Egg Yolk Carboxypeptidase-like Protein (MER005492), RISC Peptidase (MER0109) 60) Family S15 unspecified peptidase (MER199442), Family S15 unspecified peptidase (MER200437), Family S15 unspecified peptidase (MER212825), Lysosomal Pro-Xaa carboxypeptidase (MER000446), Dipeptidyl-peptidase II (MER004952), Thymus-specific serine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER031614), Loc328574-like protein (MER033246), Protein 4 containing an autohydrolase domain (MER031616), epoxide hydrolase (MER000432), Mesodermal-specific transcription protein (MER199890).Mesodermal-specific transcription protein (MER017123), cytoplasmic epoxide hydrolase (MER029997), cytoplasmic epoxide hydrolase (MER213866), similar putative protein FLJ22408 (MER031608), CGI-58 putative peptidase (MER030163), Williams-Boyren syndrome key region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), putative protein flj22408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), putative protein (MER033249), valacyclovir hydrolase (MER033259), Ccg1 interacting factor b (MER210738).

[0353] In some embodiments, the substrate recognition sequence is a peptide moiety of up to 15 amino acids in length. The substrate recognition sequence is cleaved by a protease. In some embodiments, the protease and the target of the cell-binding moiety coexist in the tissue, and the protease cleaves the substrate recognition sequence in the drug-conjugate moiety when the conjugate is exposed to the protease. In some embodiments, the protease is inactive, or has significantly reduced activity in tissues that do not significantly express cell surface features. In some embodiments, the protease is inactive, or has significantly reduced activity in healthy, e.g., disease-free tissues.

[0354] In some implementations, the substrate recognition sequence is cleaved by a protease selected from the following:

[0355] · ADAMS or ADAMTS, such as ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4 or ADAMTS5.

[0356] • Aspartic proteases, such as BACE or renin.

[0357] • Aspartate cathepsins (in cases where they are upregulated or released in the extracellular space via cell lysis), such as cathepsin D or cathepsin E.

[0358] • Cysteine ​​proteases (upregulated or released in the extracellular space via cell lysis), such as cysteine ​​protease 1, cysteine ​​protease 2, cysteine ​​protease 3, cysteine ​​protease 4, cysteine ​​protease 5, cysteine ​​protease 6, cysteine ​​protease 7, cysteine ​​protease 8, cysteine ​​protease 9, cysteine ​​protease 10, or cysteine ​​protease 14.

[0359] • Cysteine ​​cathepsins, such as cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, and cathepsin X / Z / P.

[0360] • Cysteine ​​proteases, such as Cruzipain, pod protein, or Otubain-2.

[0361] • KLK, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 or KLK14.

[0362] Metalloproteinases, such as transmembrane peptidase, enkephalinase, PSMA, or BMP-1,

[0363] ·MMPs such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27.

[0364] • Serine proteases, such as activated protein C, cathepsin A, cathepsin G, rennet, coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidinylbenzoic acidase, HtrA1, human neutrophil elastase, lactoferrin, Malagasycin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, trypsin-like proteases, or uPA.

[0365] Type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, hepatic serine enzymes, protein lyase-2, MT-SP1 / protein lyase, TMPRSS2, TMPRSS3, and TMPRSS4.

[0366] For example, a suitable substrate recognition sequence, or SRS, that may be included in a binder-drug conjugate is a peptide motif selected from the group consisting of: TGRGPSWV, SARGPSRW, TARGPSFK, LSGRSDNH, GGWHTGRN, HTGRSGAL, PLTGRSGG, AARGPAIH, RGPAFNPM, SSRGPAYL, RGPATPIM, RGPA, GGQPSGMWGW, FPRPLLGITGL, VHMPLGFLGP, SPLTGRSG, SAGFSLPA, LAPLGLQRR, SGGPLGVR, PLGL, GPRSFGL, and GPRSFG.

[0367] In some implementations, the substrate identification sequence is an MMP substrate, such as sequences selected from the group consisting of: ISSGLLSS, QNQALRMA, AQNLLGMV, STPPFGMF, PVGYTSSL, DWLYWPGI, MIAPVAYR, RPSPMWAY, WATPRPMR, FRLLDWQW, LKAAPRWA, GPSHLVLT, LPGGLSPW, MGLFSEAG, SPLPLRVP, RMHLRSLG, LAAPLGLL, AVGLLAPP, LLAPSHRA, PAGLWLDP, and ISSGLSS.

[0368] In some implementations, the substrate identification sequence is an MMP substrate, such as sequences selected from the group consisting of: ISSGLSS, QNQALRMA, AQNLLGMV, STPPFGMF, PVGYTSSL, DWLYWPGI, ISSGLLSS, LKAAPRWA, GPSHLVLT, LPGGLSPW, MGLFSEAG, SPLPLRVP, RMHLRSLG, LAAPLGLL, AVGLLAPP, LLAPSHRA, and PAGLWLDP.

[0369] In some implementations, the substrate recognition sequence is a substrate of thrombin, such as GPRSFGL or GPRSFG.

[0370] In some embodiments of the subject binder-drug conjugate, the substrate recognition sequence is cleaved via fibroblast activation protein α (FAPα) and represented by the following formula:

[0371]

[0372] in

[0373] R 2 It represents H or (C1-C6) alkyl, and preferably H;

[0374] R 3 The symbol represents H or (C1-C6) alkyl, preferably methyl, ethyl, propyl or isopropyl, and more preferably methyl;

[0375] R 4 It does not exist or represents (C1-C6) alkyl, -OH, -NH2 or halogen;

[0376] X represents O or S; and

[0377] If L 2 For a triggered self-degrading connector, -NH- indicates that it is an L 2 A portion of the amine, or if L2 If it is a straight bond, then -NH- represents an amine that is part of DM.

[0378] In some implementations, R 2 For H, R 3 For methyl, R 4 It does not exist and X is 0.

[0379] b. Triggered self-degradation

[0380] The binder-drug conjugate of the present invention may employ a heterocyclic triggered self-degrading moiety covalently linked to a drug moiety and a cleavable substrate recognition sequence moiety. A triggered self-degrading moiety can be defined as a bifunctional chemical group capable of covalently linking two spaced-apart chemical moieties together into a generally stable molecule, releasing one of the spaced-apart chemical moieties from the molecule by enzymatic cleavage, and subsequently, spontaneously cleaving from the remaining portion of the bifunctional chemical group to release the other of the spaced-apart chemical moieties. According to the invention, the triggered self-degrading moiety is covalently linked to a ligand via an amide bond at one of its ends, directly or indirectly via a spacer unit, and covalently linked at the other end to a chemical reaction site (functional group) of the drug. Derivatization of the drug moiety with the triggered self-degrading moiety can result in pharmacologically less active (e.g., less toxic) or no activity of the drug until the drug cleaves.

[0381] Binder-drug conjugates are generally stable in cycling, or at least should be so in the absence of enzymes that can cleave the amide bond between the substrate recognition sequence and the triggered self-degradation moiety. However, when a binder-drug conjugate is exposed to a suitable enzyme, the amide bond cleaves, initiating a spontaneous triggered self-degradation reaction that results in the cleavage of the covalently linked triggered self-degradation moiety to the drug, thereby affecting the release of the free drug moiety in its underivated or pharmacologically active form.

[0382] The conjugates of the present invention contain a triggered self-degrading portion and one or more heteroatoms, thereby providing improved solubility, improved cleavage rate, and reduced tendency for conjugate aggregation. These improvements of the heterocyclic triggered self-degrading linker constructs of the present invention over non-heterocyclic PAB-type linkers can produce unexpected and unintended biological properties, such as increased efficacy, reduced toxicity, and more desirable pharmacokinetics.

[0383] In some implementations, L 2 It is a benzyloxycarbonyl group.

[0384] In some implementations, L 2 for

[0385]

[0386] Where R1 Hydrogen, unsubstituted or substituted C 1-3 Alkyl or unsubstituted or substituted heterocyclic groups. In some embodiments, R 1 It is hydrogen. In some cases, R 1 It is a methyl group.

[0387] In some implementations, L 2 Selected from

[0388] and and

[0389] In some implementations, the triggered self-degradation portion L2 is selected from...

[0390]

[0391] in

[0392] U is O, S, or NR 6 ;

[0393] Q is CR 4 Or N;

[0394] V 1 V 2 and V 3 Independent for CR 4 Or N, provided that for equations II and III, Q and V 1 and V 2 At least one of them is N;

[0395] T represents the NH and NR components of the drug component. 6 , O or S;

[0396] R 1 R 2 R 3 and R 4 Independently selected from H, F, Cl, Br, I, OH, -N(R) 5 )2、-N(R 5 )3 + C1-C8 alkyl halides, carboxylic acid esters, sulfate esters, aminosulfonates, sulfonates, -SO2R 5 -S(=O)R 5 -SR 5 -SO2N(R) 5 )2、-C(=O)R 5 -CO2R 5 -C(=O)N(R) 5)2, -CN, -N3, -NO2, C1-C8 alkoxy, C1-C8 halosubstituted alkyl, polyoxyethylene, phosphonate, phosphate, C1-C8 alkyl, C1-C8 substituted alkyl, C2-C8 alkenyl, C2-C8 substituted alkenyl, C2-C8 alkynyl, C2-C8 substituted alkynyl, C6-C 20 Aryl, C6-C 20 Substituted aryl, C1-C 20 Heterocyclic rings and C1-C 20 Substituted heterocyclic rings; or when used together, R 2 and R 3 Forms a carbonyl group (=O) or a spirocarbocyclic ring with 3 to 7 carbon atoms; and

[0397] R 5 and R 6 Independently selected from H, C1-C8 alkyl, C1-C8 substituted alkyl, C2-C8 alkenyl, C2-C8 substituted alkenyl, C2-C8 ynyl, C2-C8 substituted ynyl, C6-C 20 Aryl, C6-C 20 Substituted aryl, C1-C 20 Heterocyclic rings and C1-C 20 Substituted heterocycles;

[0398] Among them, C1-C8 substituted alkyl, C2-C8 substituted alkenyl, C2-C8 substituted alkynyl, C6-C 20 Substituted aryl and C2-C 20 The substituted heterocycle is independently substituted by one or more substituents selected from the following: F, Cl, Br, I, OH, -N(R) 5 )2、-N(R 5 )3 + C1-C8 alkyl halides, carboxylic acid esters, sulfate esters, aminosulfonates, sulfonates, C1-C8 alkylsulfonates, C1-C8 alkylamino, 4-dialkylaminopyridine, C1-C8 alkylhydroxy, C1-C8 alkylthiols, -SO2R 5 -S(=O)R 5 -SR 5 -SO2N(R) 5 )2、-C(=O)R 5 -CO2R 5 -C(=O)N(R) 5 )2, -CN, -N3, -NO2, C1-C8 alkoxy, C1-C8 trifluoroalkyl, C1-C8 alkyl, C3-C 12 Carbon rings, C6-C 20 Aryl, C2-C 20 Heterocyclic compounds, polyoxyethylene compounds, phosphonates, and phosphate esters.

[0399] It should be understood that when T is NH, it is derived from the primary amine (-NH2) of the drug-side portion (before coupling to the triggered self-degradation portion), and when T is N, it is derived from the secondary amine (-NH-) of the drug-side portion (before coupling to the triggered self-degradation portion). Similarly, when T is O or S, it is derived from the hydroxyl (-OH) or thiosulfate (-SH) groups of the drug-side portion, respectively, before coupling to the triggered self-degradation portion.

[0400] In some implementations, the trigger-activated self-degrading connector L 2 It is -NH-(CH2)4-C(=O)- or -NH-(CH2)3-C(=O)-.

[0401] In some implementations, the trigger-activated self-degrading connector L 2 It is p-aminobenzyloxycarbonyl (PABC).

[0402] In some implementations, the trigger-activated self-degrading connector L 2 It is 2,4-bis(hydroxymethyl)aniline.

[0403] Other exemplary trigger-based self-degrading linker teachings readily applicable to the present invention are found in, for example, U.S. Patents US7754681, WO2012074693A1, US9089614, EP1732607A2, and WO2015038426A1 (all incorporated herein by reference); Walther et al., “Prodrugs in medicinal chemistry and enzyme prodrugtherapies”, Adv. Drug Deliv Rev., September 1, 2017; 118:65-77; and Tranoy-Opalinski et al., “Design of self-immolative linkers for tumor-activated prodrug therapy”, Anticancer Agents Med Chem., August 2008; 8(6):618-37; the teachings of which are incorporated herein by reference.

[0404] c. Pharmaceutical Section

[0405] Various drug entities can be used as the drug component of a drug conjugate, which is a subject binder.

[0406] In some embodiments, the free drug portion is an immunomodulator, comprising a drug portion that acts as an immune activator and / or an inducer of innate immune pathway responses. In some embodiments, the free drug portion induces the production of IFN-α. In some embodiments, the free drug portion induces the production of pro-inflammatory cytokines. In some embodiments, the free drug portion induces the production of IL-1β. In some embodiments, the free drug portion induces the production of IL-18.

[0407] In some implementations, the free drug portion promotes the expansion and survival of NK, γδT, and CD8+ T cells.

[0408] In some implementations, the free drug partially induces macrophage pyroptosis.

[0409] (i) Exemplary immune-DASH inhibitors

[0410] In some embodiments, the immune-DASH inhibitor used in the method of the present invention is represented by the following general formula;

[0411]

[0412] in

[0413] A indicates a 4-8 membered heterocycle including N and Cα carbons;

[0414] Z represents C or N;

[0415] W indicates -CN, -CH = NR5.

[0416]

[0417] R'1 represents a C-terminal amino acid residue or amino acid analog or a C-terminal peptide or peptide analog, whose amine terminus forms a covalent bond with L1 or, if L1 is a straight bond, forms a covalent bond with the substrate recognition sequence.

[0418] R'2 may be absent or may represent one or more substitutions of ring A, each of which may independently be halogen, lower alkyl, lower alkenyl, lower alkynyl, carbonyl (such as carboxyl, ester, formate, or ketone), thiocarbonyl (such as thioester, thioacetate, or thiocarbamate), amino, acylamino, amide, cyano, nitro, azide, sulfate, sulfonate, sulfonamide, -(CH2). m -R7、-(CH2) m -OH, -(CH2) m -O-lower alkyl group, -(CH2) m -O-lower alkenyl group, -(CH2) n -O-(CH2) m -R7、-(CH2)m -SH, -(CH2) m -S-lower alkyl group, -(CH2) m -S-lower alkenyl groups, -(CH2) n -S-(CH2) m -R7;

[0419] If X is N, then R'3 represents hydrogen; if X is C, then R'3 represents hydrogen or halogen, lower alkyl, lower alkenyl, lower alkynyl, carbonyl (such as carboxyl, ester, formate, or ketone), thiocarbonyl (such as thioester, thioacetate, or thiocarbamate), amino, acylamino, amide, cyano, nitro, azide, sulfate, sulfonate, sulfonamide, -(CH2) m -R7、-(CH2) m -OH, -(CH2) m -O-lower alkyl group, -(CH2) m -O-lower alkenyl group, -(CH2) n -O-(CH2) m -R7、-(CH2) m -SH, -(CH2) m -S-lower alkyl group, -(CH2) m -S-lower alkenyl groups, -(CH2) n -S-(CH2) m -R7;

[0420] R4 represents hydrogen, lower alkyl, lower alkenyl, lower alkynyl, or -(CH2). m -R3、-(CH2) n -OH, -(CH2) n -O-lower alkyl group, -(CH2) n -O-alkenyl, -(CH2) n -O-acetylinyl group, -(CH2) n -O-(CH2) m -R7、-(CH2) n -SH, -(CH2) n -S-lower alkyl group, -(CH2) n -S-lower alkenyl groups, -(CH2) n -S-lower alkyne group, -(CH2) n -S-(CH2) m -R3, -C(O)C(O)NH2 or -C(O)C(O)OR8;

[0421] R5 represents H, alkyl, alkenyl, alkynyl, -C(X1)(X2)X3, or -(CH2). m -R7、-(CH2)n -OH, -(CH2) n -O-alkyl, -(CH2) n -O-alkenyl, -(CH2) n -O-acetylinyl group, -(CH2) n -O-(CH2) m -R7、-(CH2) n -SH, -(CH2) n -S-alkyl, -(CH2) n -S-alkenyl, -(CH2) n -S-alkynyl group, -(CH2) n -S-(CH2) m -R7, -C(O)C(O)NH2 or -C(O)C(O)OR'7;

[0422] R6 represents hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, or -(CH2). m -R7、-(CH2) m -OH, -(CH2) m -O-lower alkyl group, -(CH2) m -O-lower alkenyl group, -(CH2) n -O-(CH2) m -R7、-(CH2) m -SH, -(CH2) m -S-lower alkyl group, -(CH2) m -S-lower alkenyl groups, -(CH2) n -S-(CH2) m -R7,

[0423] R7 indicates substituted or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocyclic compounds each time it appears;

[0424] R'7, each time it appears, indicates hydrogen or a substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocyclic group; and

[0425] Y1 and Y2 can be OH or groups capable of hydrolysis into hydroxyl groups, either independently or together, including cyclic derivatives (such as pinacol) in which Y1 and Y2 are linked via a ring structure having 5 to 8 atoms.

[0426] R50 indicates O or S;

[0427] R51 represents N3, SH2, NH2, NO2, or O-R'7;

[0428] R52 represents hydrogen, a lower alkyl group, an amine, OR'7, or a pharmaceutically acceptable salt, or R51 and R52 together with the phosphorus atom they are attached to form a heterocycle having 5 to 8 atoms in the ring structure.

[0429] X1 represents halogen;

[0430] X2 and X3 each represent hydrogen or halogen.

[0431] m is an integer in the range of 0 or 1 to 8; and

[0432] n is an integer in the range of 1 to 8.

[0433] In a preferred embodiment, ring A is a 5-, 6-, or 7-membered ring, for example, represented by the following formula:

[0434]

[0435] More preferably, it is a 5- or 6-membered ring (i.e., n is 1 or 2, but n can also be 3 or 4). The ring can be chosen to be further replaced.

[0436] In the preferred embodiment, W represents

[0437] In the preferred embodiment, R'1 is

[0438]

[0439] Where R36 is a small hydrophobic group, such as a lower alkyl or halogen, and R38 is hydrogen, or R36 and R37 together form a 4-7 membered heterocycle including N and Cα carbons, as defined above for A.

[0440] In a preferred embodiment, R'2 is absent or represents a small hydrophobic group, such as a lower alkyl group or a halogen.

[0441] In a preferred embodiment, R'3 is hydrogen or a small hydrophobic group, such as a lower alkyl group or a halogen.

[0442] In a preferred embodiment, R'5 is hydrogen or a halogenated lower alkyl group.

[0443] In a preferred embodiment, X1 is fluorine and X2 and X3 are fluorine if they are halogens.

[0444] Any compound that can be hydrolyzed into any of the aforementioned compounds, including borate esters and halides, as well as carbonyl equivalents including acetals, hemiacetals, ketals and hemiketals, and cyclic dipeptide analogs, are also considered equivalents.

[0445] In some preferred embodiments, the subject method utilizes boronic acid analogs of amino acids as immuno-DASH inhibitors. For example, the present invention covers the use of boron-prolyl derivatives in the subject method. Exemplary boronic acid-derived inhibitors of the present invention are represented by the following general formula:

[0446]

[0447] in

[0448] R'1 represents a C-terminal linked amino acid residue or amino acid analog, or a C-terminal linked peptide or peptide analog, whose amine terminus forms a covalent bond with L1, or if L1 is a straight bond, it forms a covalent bond with the substrate recognition sequence; and

[0449] R11 and R12 each independently represent hydrogen, alkyl, or pharmaceutically acceptable salts, or R11 and R12 together with their attached OBO atoms form a heterocycle having 5 to 8 atoms in a ring structure.

[0450] In some embodiments, the immuno-DASH inhibitor is a peptide or peptide analogue thereof comprising a prolyl group or an analogue thereof at a P1-specific position and a nonpolar (and preferably hydrophobic) amino acid, such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, or methionine, at a P2-specific position. In other embodiments, the P2 position is an amino acid having a charged side chain, such as arginine, lysine, aspartic acid, or glutamic acid. For example, the immuno-DASH inhibitor may comprise an Ala-Pro or Val-Pro dipeptide sequence or equivalents thereof, and is represented as follows:

[0451]

[0452] In a preferred embodiment, ring A is a 5-, 6-, or 7-membered ring, for example, represented by the following formula:

[0453]

[0454] In some preferred embodiments, R32 is a small hydrophobic group, such as a lower alkyl group or a halogen.

[0455] In some preferred embodiments, R32 is a lower alkyl-guanidine, a lower alkyl-amine, a lower alkyl-C(O)OH, such as (CH2). m -NH-C(=N)(NH2), -(CH2) m -NH2 or -(CH2) m -COOH, where m is 1-6 and preferably 1-3.

[0456] In a preferred embodiment, R'2 is absent or represents a small hydrophobic group, such as a lower alkyl group or a halogen.

[0457] In a preferred embodiment, R'3 is hydrogen or a small hydrophobic group, such as a lower alkyl group or a halogen.

[0458] Another aspect of the present invention relates to an immune-DASH inhibitor represented by Formula III or a pharmaceutical salt thereof:

[0459]

[0460] in

[0461] The Z-ring represents a 4-10 membered heterocycle including N and Cα carbons;

[0462] W represents a functional group such as -CN, -CH=NR4, or that reacts with the active site residues of the target.

[0463]

[0464] 7X is either O or S;

[0465] X2 is H, halogen, or a lower alkyl group;

[0466] Y1 and Y2 are OH independently, or together with the boron atom attached to them, they represent groups that can be hydrolyzed to boric acid, or together with the boron atom attached to them, they form a 5-8 membered ring that can be hydrolyzed to boric acid.

[0467] R1, each time it appears, independently represents a halogen, lower alkyl, lower alkenyl, lower alkynyl, carbonyl, thiocarbonyl, amino, acylamino, amide, cyano, nitro, azide, sulfate, sulfonate, sulfonamide, -CF3, or -(CH2). m -R3、-(CH2) m OH, -(CH2) m -O-lower alkyl group, -(CH2) m -O-lower alkenyl group, -(CH2) n -O-(CH2) m -R3、-(CH2) m -SH, -(CH2) m -S-lower alkyl group, -(CH2) m -S-lower alkenyl groups or -(CH2) n -S-(CH2) m -R3;

[0468] R2 indicates hydrogen, lower alkyl, lower alkynyl, or -(CH2) each time it appears. m-R3, -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-ynyl or -C(=O)-(CH2) m -R3;

[0469] R3 indicates hydrogen or a substituted or unsubstituted lower alkyl, lower alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocyclic ring each time it appears;

[0470] R4 represents hydrogen, lower alkyl, lower alkenyl, lower alkynyl, or -(CH2). m -R3、-(CH2) n -OH, -(CH2) n -O-lower alkyl group, -(CH2) n -O-alkenyl, -(CH2) n -O-acetylinyl group, -(CH2) n -O-(CH2) m -R7、-(CH2) n -SH, -(CH2) n -S-lower alkyl group, -(CH2) n -S-lower alkenyl groups, -(CH2) n -S-lower alkyne group, -(CH2) n -S-(CH2) m -R3, -C(O)C(O)NH2 or -C(O)C(O)OR8;

[0471] R5 represents O or S;

[0472] R6 represents N3, SH, NH2, NO2, or OR8;

[0473] R7 represents hydrogen, a lower alkyl group, an amine, OR8 or a pharmaceutically acceptable salt, or R5 and R6 together with the phosphorus atom to which they are attached to form a heterocycle having 5 to 8 atoms in the ring structure.

[0474] R8 represents hydrogen, substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl or heterocyclic groups;

[0475] R10 either does not exist or represents one to three substitutions of the ring Z it is linked to, each of which can independently be a halogen, lower alkyl, lower alkenyl, lower alkynyl, carbonyl (such as carboxyl, ester, formate, or ketone), thiocarbonyl (such as thioester, thioacetate, or thiocarbamate), amino, acylamino, amide, cyano, isocyano, cyanothio, isocyanothio, cyanoxy, nitro, azide, sulfate, sulfonate, sulfonylamino, lower alkyl -C(O)OH, -O-lower alkyl -C(O)OH, -guanidinyl, -(CH2). m -R7、-(CH2) m-OH, -(CH2) m -O-lower alkyl group, -(CH2) m -O-lower alkenyl group, -(CH2) n -O-(CH2) m -R3、-(CH2) m -SH, -(CH2) m -S-lower alkyl group, -(CH2) m -S-lower alkenyl groups, -(CH2) n -S-(CH2) m -R3;

[0476] n is 0, 1, 2, or 3; and

[0477] m can be 0, 1, 2, or 3.

[0478] Another aspect of the present invention relates to an immune-DASH inhibitor represented by formula IV or a pharmaceutical salt thereof:

[0479]

[0480] in

[0481] Ring A represents a 3-10 element ring structure including N;

[0482] The Z-ring represents a 4-10 membered heterocycle including N and Cα carbons;

[0483] W represents a functional group such as -CN, -CH=NR4, or that reacts with the active site residues of the target.

[0484]

[0485] X is either O or S;

[0486] X1 represents halogen;

[0487] Y1 and Y2 are OH independently, or together with the boron atom attached to them, they represent groups that can be hydrolyzed to boric acid, or together with the boron atom attached to them, they form a 5-8 membered ring that can be hydrolyzed to boric acid.

[0488] R1 represents halogen, lower alkyl, lower alkenyl, lower alkynyl, carbonyl, thiocarbonyl, amino, acylamino, acylamino, cyano, nitro, azide, sulfate, sulfonate, sulfonylamino, -CF3, -(CH2)m-R3, -(CH2)mOH, -(CH2)mO-lower alkyl, -(CH2)mO-lower alkenyl, -(CH2)nO-(CH2)m-R3, -(CH2)m-SH, -(CH2)mS-lower alkyl, -(CH2)mS-lower alkenyl or -(CH2)nS-(CH2)m-R3;

[0489] R2 indicates hydrogen, lower alkyl, lower alkynyl, -(CH2)m-R3, -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl or -C(=O)-(CH2)m-R3 each time it appears;

[0490] R3 indicates hydrogen or a substituted or unsubstituted lower alkyl, lower alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocyclic ring each time it appears;

[0491] R4 represents hydrogen, lower alkyl, lower alkenyl, lower alkynyl, -(CH2)m-R3, -(CH2)n-OH, -(CH2)nO-lower alkyl, -(CH2)nO-alkenyl, -(CH2)nO-alkynyl, -(CH2)nO-(CH2)m-R7, -(CH2)n-SH, -(CH2)nS-lower alkyl, -(CH2)nS-lower alkenyl, -(CH2)nS-lower alkynyl, -(CH2)nS-(CH2)m-R3, -C(O)C(O)NH2 or -C(O)C(O)OR8;

[0492] R5 represents O or S;

[0493] R6 represents N3, SH, NH2, NO2, or OR8;

[0494] R7 represents hydrogen, a lower alkyl group, an amine, OR8 or a pharmaceutically acceptable salt, or R5 and R6 together with the phosphorus atom to which they are attached to form a heterocycle having 5 to 8 atoms in the ring structure.

[0495] R8 represents hydrogen, substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl or heterocyclic groups;

[0496] R9 and R10 are each independently absent or represent one to three substitutions of the ring A or ring Z to which they are connected, and each can be independently halogen, lower alkyl, lower alkenyl, lower alkynyl, carbonyl (such as carboxyl, ester, formate or ketone), thiocarbonyl (such as thioester, thioacetate or thioformate), amino, acylamino, acylamino, cyano, isocyano, cyanothio, isocyanothio, cyanoxy, nitro, azide, sulfate, sulfonate, sulfonylamino, -(CH2)m-R7, -(CH2)m-OH, -(CH2)mO-lower alkyl, -(CH2)mO-lower alkenyl, -(CH2)nO-(CH2)m-R3, -(CH2)m-SH, -(CH2)mS-lower alkyl, -(CH2)mS-lower alkenyl, -(CH2)nS-(CH2)m-R3;

[0497] n is 0, 1, 2, or 3; and

[0498] m can be 0, 1, 2, or 3.

[0499] In some preferred embodiments, the immune-DASH inhibitor is a borate inhibitor of DASH enzymes DPP8 and DPP9 (and optionally also DPP-4 and / or FAP).

[0500] In some preferred embodiments, the immuno-DASH inhibitor is a dipeptide borate inhibitor of the DASH enzymes DPP8 and DPP9 (and optionally also DPP-4 and / or FAP). In some preferred embodiments, the immuno-DASH inhibitor dipeptide borate has a proline or proline analog at the P1 position. The immuno-DASH inhibitor can mediate tumor regression through an immune-mediated mechanism. The immuno-DASH inhibitor induces pyroptosis of macrophages and has activities such as immunogenic regulation, directly or indirectly, sensitizing tumor cells to antigen-specific CTL killing, altering immune cell subsets and functions, accelerating T cell initiation via regulation of dendritic cell transport, and inducing universal T cell-mediated antitumor activity.

[0501] In some implementations, the combination of a topical immune-DASH inhibitor and a PD-1 inhibitor may be administered as part of a therapy involving one or more other chemotherapeutic agents, immuno-oncology agents, or radiation. It may also be used as part of therapies including tumor vaccines, adoptive cell therapy, gene therapy, oncolytic virus therapy, and the like.

[0502] In some embodiments, the immune-DASH inhibitor of the method of the present invention is represented by Formula I or a drug salt thereof:

[0503]

[0504] in

[0505] Ring A represents a 3-10 element ring structure;

[0506] The Z-ring represents a 4-10 membered heterocycle including N and Cα carbons;

[0507] W represents a functional group such as -CN, -CH=NR4, or that reacts with the active site residues of the target.

[0508]

[0509] X is either O or S;

[0510] X1 represents halogen;

[0511] Y1 and Y2 are OH independently, or together with the boron atom attached to them, they represent groups that can be hydrolyzed to boric acid, or together with the boron atom attached to them, they form a 5-8 membered ring that can be hydrolyzed to boric acid.

[0512] R1 represents halogen, lower alkyl, lower alkenyl, lower alkynyl, carbonyl, thiocarbonyl, amino, acylamino, acylamino, cyano, nitro, azide, sulfate, sulfonate, sulfonylamino, -CF3, -(CH2)m-R3, -(CH2)mOH, -(CH2)mO-lower alkyl, -(CH2)mO-lower alkenyl, -(CH2)nO-(CH2)m-R3, -(CH2)m-SH, -(CH2)mS-lower alkyl, -(CH2)mS-lower alkenyl or -(CH2)nS-(CH2)m-R3;

[0513] R2 indicates hydrogen, lower alkyl, lower alkynyl, -(CH2)m-R3, -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-alkynyl or -C(=O)-(CH2)m-R3 each time it appears;

[0514] R3 indicates hydrogen or a substituted or unsubstituted lower alkyl, lower alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocyclic ring each time it appears;

[0515] R4 represents hydrogen, lower alkyl, lower alkenyl, lower alkynyl, -(CH2)m-R3, -(CH2)n-OH, -(CH2)nO-lower alkyl, -(CH2)nO-alkenyl, -(CH2)nO-alkynyl, -(CH2)nO-(CH2)m-R7, -(CH2)n-SH, -(CH2)nS-lower alkyl, -(CH2)nS-lower alkenyl, -(CH2)nS-lower alkynyl, -(CH2)nS-(CH2)m-R3, -C(O)C(O)NH2 or -C(O)C(O)OR8;

[0516] R5 represents O or S;

[0517] R6 represents N3, SH, NH2, NO2, or OR8;

[0518] R7 represents hydrogen, a lower alkyl group, an amine, OR8 or a pharmaceutically acceptable salt, or R5 and R6 together with the phosphorus atom to which they are attached to form a heterocycle having 5 to 8 atoms in the ring structure.

[0519] R8 represents hydrogen, substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl or heterocyclic groups;

[0520] R9 and R10 are each independently absent or represent one, two, or three substitutions of the ring A or ring Z to which they are linked, and can each be independently halogen, lower alkyl, lower alkenyl, lower alkynyl, carbonyl (such as carboxyl, ester, formate, or ketone), thiocarbonyl (such as thioester, thioacetate, or thiocarbamate), amino, acylamino, amide, cyano, isocyano, cyanothio, isocyanothio, cyanoxy, nitro, azide, sulfate, sulfonate, sulfonylamino, lower Alkyl-C(O)OH, -O-lower alkyl-C(O)OH, -guanidinyl; -(CH2)m-R7, -(CH2)m-OH, -(CH2)mO-lower alkyl, -(CH2)mO-lower alkenyl, -(CH2)nO-(CH2)m-R3, -(CH2)m-SH, -(CH2)mS-lower alkyl, -(CH2)mS-lower alkenyl, -(CH2)nS-(CH2)m-R3;

[0521] n is 0, 1, 2, or 3; and

[0522] m can be 0, 1, 2, or 3.

[0523] In some implementations, the immune-DASH inhibitor of Formula I is represented by Formula Ia, or its drug salt:

[0524]

[0525] Among them, X, W, Z, R 1 R 2 R 9 and R 10 As defined above for Equation I, where p is 1, 2, or 3.

[0526] In some preferred embodiments of Ia: R 1 It is a lower alkyl group; R 9 It is absent or, each time it appears independently, is a lower alkyl group, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl-C(O)OH, -guanidinyl; X is O; each R 2 For hydrogen, R 10 The absence of or representation of a single substitution of -OH, -NH2, -CN or -N3; ​​and W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0527] In some implementations, the immune-DASH inhibitor of Formula I is represented by Formula Ib, or its drug salt:

[0528]

[0529] Among them, X, W, and R 1 R2 R 9 and R 10 As defined above for Equation I, where p is 1, 2, or 3.

[0530] In some preferred embodiments of Ib: R 1 It is a lower alkyl group; R 9 It is absent or, each time it appears independently, is a lower alkyl group, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl-C(O)OH, -guanidinyl; X is O; each R 2 For hydrogen, R 10 The absence of or representation of a single substitution of -OH, -NH2, -CN or -N3; ​​and W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0531] In some implementations, the immune-DASH inhibitor of Formula I is represented by Formula Ic, or its drug salt:

[0532]

[0533] Among them, X, W, and R 1 R 2 R 9 and R 10 As defined above for Equation I, where p is 1, 2, or 3.

[0534] In some preferred embodiments of Ic: R 1 It is a lower alkyl group; R 9 It is absent or, each time it appears independently, is a lower alkyl group, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl-C(O)OH, -guanidinyl; X is O; each R 2 For hydrogen, R 10 The absence of or representation of a single substitution of -OH, -NH2, -CN or -N3; ​​and W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0535] In some implementations, immune-DASH inhibitors are represented by the following:

[0536]

[0537] Another aspect of the present invention relates to an immune-DASH inhibitor represented by Formula II or a pharmaceutical salt thereof:

[0538]

[0539] in

[0540] Ring A and each occurrence of R 1a Together, they represent a 7-12 elemental multi-ring structure;

[0541] The Z-ring represents a 4-10 membered heterocycle including N and Cα carbons;

[0542] W represents -CN, -CH=NR 4 Functional groups that react with the active site residues of the target, or

[0543]

[0544] X is either O or S;

[0545] X 1 Indicates halogen;

[0546] Y is either C or N;

[0547] Y 1 and Y 2 It can be represented independently as OH, or together with the boron atom it is attached to, as a group that can be hydrolyzed to boric acid, or together with the boron atom it is attached to, as a 5-8 membered ring that can be hydrolyzed to boric acid;

[0548] R1a represents a lower alkyl group, -(CH2)m-, -(CH2)mO-(CH2)m-; -(CH2)mN-(CH2)m-; or -(CH2)mS-(CH2)m-;

[0549] R 2 Each occurrence indicates hydrogen, lower alkyl, lower alkynyl, or -(CH2)mR. 3 -C(=O)-alkyl, -C(=O)-alkenyl, -C(=O)-ynyl or -C(=O)-(CH2)mR 3 ;

[0550] R 3 Each occurrence indicates hydrogen or a substituted or unsubstituted lower alkyl, lower alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocyclic compound;

[0551] R 4 This indicates hydrogen, lower alkyl, lower alkenyl, lower alkynyl, or -(CH2). m -R 3 -(CH2) n -OH, -(CH2) n -O-lower alkyl group, -(CH2) n -O-alkenyl, -(CH2) n -O-acetylinyl group, -(CH2) n -O-(CH2) m -R 7 -(CH2)n -SH, -(CH2) n -S-lower alkyl group, -(CH2) n -S-lower alkenyl groups, -(CH2) n -S-lower alkyne group, -(CH2) n -S-(CH2) m -R 3 -C(O)C(O)NH2 or -C(O)C(O)OR 8 ;

[0552] R 5 Indicates O or S;

[0553] R 6 Indicates N3, SH, NH2, NO2, or OR 8 ;

[0554] R 7 Indicates hydrogen, lower alkyl groups, amines, OR 8 Or a pharmaceutically acceptable salt, or R 5 and R 6 Together with the phosphorus atoms it is attached to, it forms a heterocycle with 5 to 8 atoms in the ring structure;

[0555] R 8 Indicates hydrogen, substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl or heterocyclic groups;

[0556] R 9 and R 10 Each of the following groups of elements independently lacks or represents one, two, or three substitutions of the ring A or ring Z to which it is linked, and each can independently be a halogen, lower alkyl, lower alkenyl, lower alkynyl, carbonyl (such as carboxyl, ester, formate, or ketone), thiocarbonyl (such as thioester, thioacetate, or thiocarbamate), amino, acylamino, amide, cyano, isocyano, cyanothio, isocyanothio, cyanoxy, nitro, azide, sulfate, sulfonate, sulfonylamino, lower alkyl -C(O)OH, -O-lower alkyl -C(O)OH, -guanidinyl; -(CH2) m -R 7 -(CH2) m -OH, -(CH2) m -O-lower alkyl group, -(CH2) m -O-lower alkenyl group, -(CH2) n -O-(CH2)mR 3 -(CH2) m -SH, -(CH2) m -S-lower alkyl group, -(CH2) m -S-lower alkenyl groups, -(CH2)n -S-(CH2) m -R 3 ;

[0557] n is 0, 1, 2, or 3;

[0558] m is 0, 1, 2, or 3; and

[0559] p is 1, 2, or 3.

[0560] In some implementations, the immune-DASH inhibitor of formula II is represented by formula IIa, or its drug salt:

[0561]

[0562] Among them, X, W, Z, R 2 R 9 and R 10 As defined above for Equation II.

[0563] In certain preferred embodiments of IIa: R 9 Each occurrence is independently of lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl-C(O)OH, -guanidinyl; X is O; each R 2 For hydrogen, R 10 The absence of or representation of a single substitution of -OH, -NH2, -CN or -N3; ​​and W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0564] In some implementations, the immune-DASH inhibitor of formula II is represented by formula IIb, or its drug salt:

[0565]

[0566] Among them, X, W, and R 2 R 9 and R 10 As defined above for Equation II.

[0567] In certain preferred embodiments of IIb: R 9 Each occurrence is independently of lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl-C(O)OH, -guanidinyl; X is O; each R 2 For hydrogen, R 10 The absence of or representation of a single substitution of -OH, -NH2, -CN or -N3; ​​and W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0568] In some implementations, the immune-DASH inhibitor of formula II is represented by formula IIc, or its drug salt:

[0569]

[0570] Among them, X, W, and R 2 R 9 and R 10 As defined above for Equation II.

[0571] In certain preferred embodiments of IIc: R 9 Each occurrence is independently of lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl-C(O)OH, -guanidinyl; X is O; each R 2 For hydrogen, R 10 The absence of or representation of a single substitution of -OH, -NH2, -CN or -N3; ​​and W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0572] In some implementations, the immune-DASH inhibitor of formula II is represented by formula IId, or its drug salt:

[0573]

[0574] Among them, X, W, and R 2 R 9 and R 10 As defined above for Equation II.

[0575] In certain preferred embodiments of IId: R 9 Each occurrence is independently of lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl-C(O)OH, -guanidinyl; X is O; each R 2 For hydrogen, R 10 The absence of or representation of a single substitution of -OH, -NH2, -CN or -N3; ​​and W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0576] In some implementations, the immune-DASH inhibitor of formula II is represented by formula IIe, or its drug salt:

[0577]

[0578] Among them, X, W, Z, R 2 R 9 and R 10 As defined above for Equation II.

[0579] In certain preferred embodiments of IIe: R 9 Each occurrence is independently of lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl-C(O)OH, -guanidinyl; X is O; each R 2 For hydrogen, R 10 The absence of or representation of a single substitution of -OH, -NH2, -CN or -N3; ​​Z is a pyrrolidine or piperidine ring (more preferably a pyrrolidine ring); and W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0580] In some implementations, the immune-DASH inhibitor is one of the following:

[0581]

[0582] (ii) Exemplary STING agonists

[0583] Non-limiting examples of STING agonists include agonists represented by one of the following general formulas:

[0584]

[0585] in

[0586] X1 and X2 are independently O or S and preferably the same (O, O or S, S);

[0587] X3 and X4 are purines, such as guanine or guanine analogs or pyrimidines, and the wavy line indicates a covalently linked site with L1, or, in the case of a straight bond with L1, a covalently linked site with a substrate recognition sequence.

[0588] R1 and R2 are independently H, hydroxyl, halogen (preferably F or Cl), or optionally substituted straight-chain alkyl having 1 to 18 carbons and 0 to 3 heteroatoms, optionally substituted alkenyl having 1 to 9 carbons, optionally substituted alkynyl having 1 to 9 carbons, or optionally substituted aryl, wherein one or more substitutions, when present, can be independently selected from the group consisting of: straight-chain or branched C 1-6 Alkyl, benzyl, halogen, trihalomethyl, C 1-6 Alkyl group, -NO2, -NH2, -OH, =O, -COOR', or -OR', where R1 and R2 are not H.

[0589] R' is H or a lower alkyl group, -CH2OH or -CONH2.

[0590] In some implementations, the STING agonist is represented by one of the following formulas:

[0591]

[0592] In the above STING agonist structures, X3 and X4 can each independently be, for example, 9-purine, 9-adenine, 9-guanine, 9-hypoxanthine, 9-xanthine, 9-uric acid, or 9-isoguanine, provided that one of X3 or X4 includes if L 2 For triggered self-degrading connectors, it is similar to L 2 Shared functional groups of straight keys, or if L 2 If it is a direct bond, it shares the functional groups of the direct bond with DM.

[0593] X3 and X4 can be the same or different.

[0594] In some embodiments, the STING agonist may be provided as a predominant Rp,Rp or Rp,Sp stereoisomer.

[0595] Exemplary STING agonists include:

[0596]

[0597]

[0598]

[0599]

[0600] In some implementations, the STING agonist is represented by one of the following structures:

[0601]

[0602] Another STING agonist that can be used as the pharmaceutical portion in the binder conjugate of this invention is

[0603]

[0604] By way of example only, other exemplary STING agonist teachings that can be readily applied as the pharmaceutical part of the conjugates of the present invention are in PCT Publications WO2017123669A1 and WO2015077354A1 and U.S. Patent Publication US20150056224A1 (each of which is incorporated herein by reference).

[0605] Those skilled in the art will also understand that, especially when using triggered self-degrading linkers, STING agonists can couple with linkers via functional groups other than amines as described above, such as via free hydroxyl groups.

[0606] (iii) Exemplary TLR agonists

[0607] Examples of “Toll-like receptor (TLR) agonists” include (but are not limited to) TLR1 / 2 agonists, TLR2 agonists, TLR3 agonists (e.g., PolyI:C), TLR4 agonists (e.g., S-type lipopolysaccharide, paclitaxel, lipid A, and monophospholipid A), TLR5 agonists (e.g., flagellin), TLR6 / 2 agonists (e.g., MALP-2), TLR7 agonists, TLR7 / 8 agonists (e.g., gademoti, imiquimoti, loxolibin, and resimimoti (R848)), TLR7 / 9 agonists (e.g., hydroxychloroquine sulfate), TLR8 agonists (e.g., motomoti (VTX-2337)), TLR9 agonists (e.g., CpG-ODN), and TLR11 agonists (e.g., repressor protein).

[0608] Exemplary TRL agonists that can be used as the pharmaceutical portion in the binding conjugates of the present invention include S-27609, CL307, UC-IV150, imiquimod, gademod, resimimod, motomod, VTS-1463GS-9620, GSK2245035, TMX-101, TMX-201, TMX-202, essatoribine, AZD8848, MEDI9197, 3M-051, 3M-852, 3M-052, 3M-854A, S-34240, KU34B, or CL663, or their analogues, where appropriate, having suitable functional groups for direct binding and release from the substrate recognition sequence or by binding to a triggered self-degrading linker.

[0609] Exemplary TRL agonists, particularly TRL7 agonists, TRL8 agonists, and TRL7 / 8 agonists, include:

[0610]

[0611]

[0612] In some embodiments, the pharmaceutical component is a TRL7 / 8 agonist represented by the following general formula:

[0613]

[0614] in

[0615] X is CH2, O, S or N, preferably CH2, O or N, and more preferably CH2 or O;

[0616] n is 0 (N to 0 are direct keys) or an integer from 1 to 5, preferably 1 or 2;

[0617] z is an integer from 1 to 5;

[0618] m is an integer from 1 to 20, preferably from 1 to 16;

[0619] p is 0 (the ring to X is a direct bond) or an integer from 1 to 5, preferably 1 or 2; and

[0620] q is an integer from 1 to 5, preferably 1 or 2.

[0621] For example, TRL agonists are TRL7 / 8 agonists, such as one of the following:

[0622]

[0623] Publications WO2008135791 and WO2016141092 also describe a class of imidazoquinoline compounds that have immunomodulatory properties acting via TLR7.

[0624] Other exemplary TLR agonists readily applicable as the pharmaceutical part of the binding conjugates of the present invention are disclosed, for example, in the following literature: Yoo et al., “Structure-activity relationships in Toll-like receptor 7 agonistic 1H-imidazo[4,5-c]pyridines”, Org. Biomol. Chem., 2013, 11, 6526-6545; Fletcher et al., “Masked oral prodrugs of Toll-like receptor 7 agonists: a new approach for the treatment of infectious disease”, 2006 Current opinion in investigational drugs (London, England). 7.702-708; and Pryde et al., “The discovery of a novel prototype small molecule TLR7 agonist for the treatment of hepatitis C virus infection”, Med. Chem. Commun., 2011, 2, 185-189.

[0625] Those skilled in the art will also understand that, especially when using triggered self-degrading linkers, TRL agonists can couple with the linker via functional groups other than the amines described above, such as via free hydroxyl groups.

[0626] (iv) Exemplary RIG-1 agonists

[0627] The conjugate of any of the foregoing embodiments, wherein the immunostimulatory agonist is a RIG-I agonist, wherein the RIG-I agonist is KIN700, KIN1148, KIN600, KIN500, KIN100, KIN101, KIN400, KIN2000 or SB-9200.

[0628] (v) Exemplary anthracycline

[0629] In some embodiments, the pharmaceutical component is anthracycline or a derivative thereof, preferably doxorubicin or other analogues capable of inducing immunogenic cell death in tumor cells.

[0630] Anthracyclines and their analogues particularly include (but are not limited to) doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, valrubicin, arubicin, mitoxantrone, actinomycin, bleomycin, plicamycin, and mitomycin. For example, the anthracycline moiety can be represented by the following formula:

[0631]

[0632] in

[0633] R c It represents (C1-C6)alkyl, (C1-C6)hydroxyalkyl or (C1-C6)alkylalkyl, especially methyl, hydroxymethyl, diethoxyacetoxymethyl or butyryloxymethyl;

[0634] R d It represents hydrogen, hydroxyl, or (C1-C6)alkoxy, especially methoxy;

[0635] R e and R f One of them represents a hydrogen atom; and the other represents a hydrogen atom or a hydroxyl group or a tetrahydropyran-2-yloxy group (OTHP).

[0636] (vi) Exemplary proteasome inhibitors

[0637] In some embodiments, the pharmaceutical component is a proteasome inhibitor. Exemplary proteasome inhibitors include...

[0638]

[0639]

[0640] d. Cell-binding portion

[0641] In some embodiments, the diseased tissue is a tumor. In some embodiments, the cell-binding portion of the binder-drug conjugate is selectively configured to bind to cell surface proteins on tumor cells. In other embodiments, the cell-binding portion of the binder-drug conjugate is selectively configured to bind to cell surface proteins on macrophages, monocyte-derived suppressor cells (MDSCs), dendritic cells, fibroblasts, T cells, NK cells, mast cells, granulocytes, eosinophils, and B cells.

[0642] In some embodiments, the cell-binding portion of the binder-drug conjugate is selected such that when the binder-drug conjugate binds to a surface feature on a target cell, it has an internalization half-life of at least 6 hours, more preferably at least 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 75 hours, or even 100 hours.

[0643] In some embodiments, the cell-binding portion of the binder-drug conjugate binds to cell surface proteins selectively expressed or upregulated by target cells in diseased tissue relative to normal cells from healthy tissue. For example, the protein is detectable on the surface of target cells at levels at 2 times higher than those from normal cells from the tissue, and even more preferably at levels at least 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, or even 1000 times higher than those from normal cells from the tissue.

[0644] In some embodiments, the cell-binding portion of the binder-drug conjugate is selectively bound to cell surface proteins selectively expressed or upregulated by target cells in diseased tissues relative to cells from other tissues, particularly cells from critical organs. For example, the protein is detectable on the surface of target cells at levels 2 times higher than those from cells from other tissues, and even more preferably at levels at least 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, or even 1000 times higher than those from cells from other tissues.

[0645] In some embodiments, the cell-binding portion of the binder-drug conjugate is selected to bind to a checkpoint protein, and the cell-binding portion is preferably a checkpoint antagonist. Examples of checkpoint proteins include those selected from the group consisting of: CTLA-4, PD-1, LAG-3, BTLA, KIR, TIM-3, PD-L1, PD-L2, B7-H3, B7-H4, HVEM, GAL9, CD160, VISTA, BTNL2, TIGIT, PVR, BTN1A1, BTN2A2, BTN3A2, and CSF-1R, more preferably CTLA-4, PD-1, LAG-3, TIM-3, BTLA, VISTA, HVEM, TIGIT, PVR, PD-L1, and CD160.

[0646] In some embodiments, the cell-binding portion of the binder-drug conjugate is selected to bind to a co-stimulatory receptor, and the cell-binding portion is a co-stimulatory receptor agonist. Examples include co-stimulatory receptors or ligands with surface features selected from the group consisting of: 4-1BB, 4-1BB-L, OX40, OX40-L, GITR, CD28, CD40, CD40-L, ICOS, ICOS-L, LIGHT, and CD27, more preferably 4-1BB, OX40, GITR, CD40, and ICOS.

[0647] In some implementations, the cell-binding portion is an antibody, such as a humanized antibody, a human antibody, or a chimeric antibody, or includes an antigen-binding portion that binds to cell surface features, such as Fab, F(ab)2, F(ab'), F(ab')2, F(ab')3, Fd, Fv, disulfide-linked Fv, dAb or sdAb (or nanobodies), CDR, scFv, (scFv)2, di-scFv, bi-scFv, tascFv (tandem scFv), AVIBODY (e.g., bifunctional, trifunctional, tetrafunctional antibodies), T-cell conjugating molecules (BiTE), scFv-Fc, Fcab, mAb2, small modular immunopharmaceuticals (SMIPs), Genmab / monoantibody or bispecific antibody, V-NAR domain, IgNAR, microantibody, IgGACH2, DVD-Ig, antibody precursor, intracellular antibody, or multispecific antibody.

[0648] In other embodiments, the cell-binding portion is a non-antibody scaffold selected from the group consisting of: affinity antibodies, affinity molecules, afenine, anticarrier proteins, atrimer, high-affinity multimers, DARPin, FN3 scaffolds (e.g., adenettin and sintine), fenomomovir, Kunitz domains, nanofitin, pronectin, OBodies, trifunctional antibodies, high-affinity multimers, bicyclic peptides, and Cys knots.

[0649] (i) Binding to the affinity of PD-L1

[0650] In some embodiments, the cell-binding portion is an affinity for PD-L1. The affinity is a stefin A-based scaffold, meaning it has a sequence derived from stefin A, such as mammalian stefin A, and more preferably human stefin A. One aspect of this application provides an affinity for PD-L1 (also referred to as an "anti-PD-L1 affinity") comprising one or more solvent-accessible loops from wild-type stefin A protein having the ability to bind PD-L1 (preferably selectively and preferably in 10...). -6 The affinity of the affinity of the amino acid sequence of the affinity of M or lower Kd).

[0651] In some implementations, the anti-PD-L1 affinity is derived from a wild-type human stefin A protein with a main chain sequence in which loop 2 [is referred to as (Xaa)]. n ] and ring 4 [called (Xaa)] m One or two of the sequences in the sequence are derived from the substitution ring sequence (Xaa). n and(Xaa) m Permutation, to have general formula (i)

[0652] FR1-(Xaa) n -FR2-(Xaa) m -FR3 (I)

[0653] in

[0654] FR1 is a polypeptide sequence represented by MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID No. 1) or a polypeptide sequence having at least 70% homology with it;

[0655] FR2 is a polypeptide sequence represented by GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID No. 2) or a polypeptide sequence having at least 70% homology with it;

[0656] FR3 is a polypeptide sequence represented by EDLVLTGYQV DKNKDDELTG F (SEQ ID No. 3) or a polypeptide sequence having at least 70% homology with it; and

[0657] Each occurrence of Xaa represents an amino acid residue, and n and m each represent an integer from 3 to 20.

[0658] In some embodiments, FR1 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% homology with SEQ ID No. 1. In some embodiments, FR1 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% homology with SEQ ID No. 1; in some embodiments, FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% homology with SEQ ID No. 2. In some embodiments, FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% homology with SEQ ID No. 2; in some embodiments, FR3 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% homology with SEQ ID No. 3. In some embodiments, FR3 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% homology with SEQ ID No. 3.

[0659] For embodiments in which at least one drug-conjugate moiety is linked to the affinity sequence via a thiol side chain of a cysteine ​​residue introduced into the affinity sequence, the cysteine ​​will preferably be provided in a portion of the affinity sequence region corresponding to FR1, FR2, and / or FR3, and more preferably, will replace the side chain in the affinity sequence with a solvent-accessible amino acid residue that is independent of hydrogen bonding of other parts of the affinity sequence. Generally, the cysteine ​​will not introduce a ring (Xaa). n Or (Xaa) m middle.

[0660] In some embodiments, the anti-PD-L1 affinity has the amino acid sequence represented in the following general formula (SEQ ID No. 4):

[0661] MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(Xaa) n -Xaa2-TNYYIKVRAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa) m -Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF

[0662] in

[0663] Each occurrence of Xaa is an individual amino acid residue; n and m are each an independent integer from 3 to 20; Xaa1 is Gly, Ala, Val, Arg, Lys, Asp, or Glu, more preferably Gly, Ala, Arg, or Lys, and even more preferably Gly or Arg; Xaa2 is Gly, Ala, Val, Ser, or Thr, more preferably Gly or Ser; Xaa3 is Arg, Lys, Asn, Gln, Ser, Thr, more preferably Arg, Lys, Asn, or Gln, and even more preferably Lys or Asn; Xaa4 is Gl y, Ala, Val, Ser or Thr, more preferably Gly or Ser; Xaa5 is Ala, Val, Ile, Leu, Gly or Pro, more preferably Ile, Leu or Pro, and even more preferably Leu or Pro; Xaa6 is Gly, Ala, Val, Asp or Glu, more preferably Ala, Val, Asp or Glu, and even more preferably Ala or Glu; and Xaa7 is Ala, Val, Ile, Leu, Arg or Lys, more preferably Ile, Leu or Arg, and even more preferably Leu or Arg.

[0664] For those embodiments in which at least one drug-conjugate moiety is linked to the affinity sequence via a thiol side chain of a cysteine ​​residue introduced into the affinity sequence, the cysteine ​​residue will preferably be provided in the circumcyclic sequence (Xaa). n Or (Xaa) m The affinity sequence portion outside the sequence. Therefore, SEQ ID No. 4 may include 1 to 5 cysteine ​​residues replacing amino acid residues at the changed positions in the sequence.

[0665] For example, the anti-PD-L1 affinity may have the amino acid sequence represented in the following general formula (SEQ ID No. 5):

[0666] MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(Xaa) n -STNYYIKVRAGDNKYMHLKVFNGP-(Xaa) m -ADRVLTGYQVDKNKDDELTGF

[0667] Each occurrence of Xaa represents an individual amino acid residue; n and m are each an independent integer from 3 to 20.

[0668] In some implementations, n is 3 to 15, 3 to 12, 3 to 9, 3 to 7, 5 to 7, 5 to 9, 5 to 12, 5 to 15, 7 to 12, or 7 to 9.

[0669] In some implementations, m is 3 to 15, 3 to 12, 3 to 9, 3 to 7, 5 to 7, 5 to 9, 5 to 12, 5 to 15, 7 to 12, or 7 to 9.

[0670] In some embodiments, Xaa is an amino acid each time it appears, which can be added to the polypeptide by recombinant expression in prokaryotic or eukaryotic cells, and even more preferably one of 20 naturally occurring amino acids.

[0671] For those embodiments in which at least one drug-conjugate moiety is linked to the affinity sequence via a thiol side chain of a cysteine ​​residue introduced into the affinity sequence, the cysteine ​​residue will preferably be provided in the circumcyclic sequence (Xaa). n Or (Xaa) m The affinity sequence portion outside the sequence. Therefore, SEQ ID No. 5 may include 1 to 5 cysteine ​​residues replacing amino acid residues at the modified positions in the sequence.

[0672] In some implementations of the above sequence and formula, (Xaa) n The amino acid sequence is represented by general formula (II):

[0673] -aa1-aa2-aa3-Gly-Pro-aa4-aa5-Trp-aa6- (II)

[0674] in

[0675] aa1 represents an amino acid residue with a basic side chain, more preferably Lys, Arg, or His, and even more preferably Lys or Arg;

[0676] aa2 represents an amino acid residue, preferably having a neutral polar or nonpolar side chain or a charged (acidic or basic) side chain, more preferably an amino acid residue with a small aliphatic side chain, a neutral polar side chain or a basic or acidic side chain, even more preferably Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg or His, and even more preferably Ala, Gln, Asp or Glu;

[0677] aa3 represents an amino acid residue having an aromatic or basic side chain, preferably Phe, Tyr, Trp, Lys, Arg or His, more preferably Phe, Tyr, Trp and even more preferably His or Tyr, Trp or His.

[0678] aa4 indicates an amino acid residue having a neutral polar or nonpolar side chain or a charged (acidic or basic) side chain, preferably a neutral polar side chain or a charged (acidic or basic) side chain; more preferably Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg or His, and even more preferably Gln, Lys, Arg, His, Asp or Glu;

[0679] aa5 indicates an amino acid residue having a neutral polar or charged (acidic or basic) or small aliphatic or aromatic side chain, preferably a neutral polar or charged side chain; more preferably Ser, Thr, Asn, Gln, Asp, Glu, Arg, or His, and even more preferably Ser, Asn, Gln, Asp, Glu, or Arg; and

[0680] aa6 represents an amino acid residue having an aromatic or acidic side chain, preferably Phe, Tyr, Trp, Asp, or Glu; more preferably Trp or Asp; and even more preferably Trp.

[0681] In some implementations of the above sequence and formula, (Xaa) n The amino acid sequence is represented by general formula (III):

[0682] -aa1-aa2-aa3-Phe-Pro-aa4-aa5-Phe-Trp- (III)

[0683] in

[0684] aa1 represents an amino acid residue having a basic side chain or an aromatic side chain, preferably Lys, Arg, His, Ser, Thr, Asn or Gln, more preferably Lys, Arg, His, Asn or Gln, and even more preferably Lys or Asn.

[0685] aa2 represents an amino acid residue, preferably having a neutral polar or nonpolar side chain or a charged (acidic or basic) side chain, more preferably an amino acid residue with a small aliphatic side chain, a neutral polar side chain or a basic or acidic side chain, even more preferably Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg or His, and even more preferably Ala, Gln, Asp or Glu;

[0686] aa3 represents an amino acid residue having an aromatic or basic side chain, preferably Phe, Tyr, Trp, Lys, Arg or His, more preferably Phe, Tyr, Trp or His, and even more preferably Tyr, Trp or His.

[0687] aa4 indicates an amino acid residue having a neutral polar or nonpolar side chain or a charged (acidic or basic) side chain, preferably a neutral polar side chain or a charged (acidic or basic) side chain; more preferably Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg, or His, and even more preferably Gln, Lys, Arg, His, Asp, or Glu; and

[0688] aa5 indicates an amino acid residue having a neutral polar or charged (acidic or basic) or small aliphatic or aromatic side chain, preferably a neutral polar or charged side chain; more preferably Ser, Thr, Asn, Gln, Asp, Glu, Arg or His, and even more preferably Ser, Asn, Gln, Asp, Glu or Arg.

[0689] In some implementations of the above sequence and formula, (Xaa) n The amino acid sequence is selected from SEQ ID No. 6 to 40, or has at least 80%, 85%, 90%, 95%, or even 98% homology with the sequence selected from SEQ ID No. 6 to 40. In some embodiments, (Xaa) n An amino acid sequence having at least 80%, 85%, 90%, 95%, or even 98% identity with a sequence selected from SEQ ID No. 6 to 40.

[0690]

[0691]

[0692] In some implementations of the above sequence and formula, (Xaa) m The amino acid sequence is represented by general formula (IV):

[0693] -aa7-aa8-aa9-aa10-aa11-aa12-aa13-aa14-aa15- (IV)

[0694] in

[0695] aa7 represents an amino acid residue having a neutral polar or nonpolar side chain or an acidic side chain; preferably Gly, Ala, Val, Pro, Trp, Gln, Ser, Asp or Glu and even more preferably Gly, Ala, Trp, Gln, Ser, Asp or Glu.

[0696] aa8 represents an amino acid residue, preferably having a neutral polar or nonpolar side chain, a charged (acidic or basic) side chain, or an aromatic side chain, more preferably an amino acid residue with a charged (acidic or basic) side chain, more preferably Asp, Glu, Lys, Arg, His, Gln, Ser, Thr, Asn, Ala, Val, Pro, Gly, Tyr, or Phe, and even more preferably Asp, Glu, Lys, Arg, His, or Gln;

[0697] aa9 represents an amino acid residue, preferably having a neutral polar or nonpolar side chain, or a charged (acidic or basic) side chain, or an aromatic side chain, more preferably an amino acid residue with a neutral polar side chain or an acidic side chain, more preferably Gln, Ser, Thr, Asn, Asp, Glu, Arg, Lys, Gly, Leu, Pro, or Tyr, and even more preferably Gln, Thr, or Asp;

[0698] aa10 represents an amino acid residue, preferably having a neutral polar or nonpolar side chain, a charged (acidic or basic) side chain, or an aromatic side chain, more preferably an amino acid residue with a neutral polar side chain, a basic side chain, or an acidic side chain, more preferably Asp, Glu, Arg, His, Lys, Ser, Gln, Asn, Ala, Leu, Tyr, Trp, Pro, or Gly, and even more preferably Asp, Glu, His, Gln, Asn, Leu, Trp, or Gly;

[0699] aa11 represents an amino acid residue, preferably having a neutral polar side chain or a charged (acidic or basic) side chain or a nonpolar aliphatic side chain or an aromatic side chain, more preferably an amino acid residue with a neutral polar side chain or a basic or acidic side chain, more preferably Asp, Glu, Ser, Thr, Gln, Arg, Lys, His, Val, Ile, Tyr or Gly and even more preferably Asp, Glu, Ser, Thr, Gln, Lys or His;

[0700] aa12 represents an amino acid residue, preferably an amino acid residue having a neutral polar side chain or a charged (acidic or basic) side chain or a nonpolar aliphatic side chain or an aromatic side chain, more preferably an acidic side chain, more preferably Asp, Glu, Ser, Thr, Gln, Asn, Lys, Arg, Val, Leu, Ile, Trp, Tyr, Phe or Gly and even more preferably Asp, Glu, Ser, Tyr, Trp, Arg or Lys;

[0701] aa13 represents an amino acid residue, preferably an amino acid residue having a neutral polar side chain, a charged (acidic or basic) side chain, a nonpolar aliphatic side chain, or an aromatic side chain, more preferably an acidic side chain, and even more preferably Ser, Thr, Gln, Asn, Val, Ile, Leu, Gly, Pro, Asp, Glu, His, Arg, Trp, Tyr, or Phe, and even more preferably Ser, Thr, Gln, Asn, Val, Ile, Leu, Gly, Asp, or Glu;

[0702] aa14 represents an amino acid residue, preferably an amino acid residue having a neutral polar side chain or a charged (acidic or basic) side chain; more preferably Ala, Ile, Trp, Pro, Asp, Glu, Arg, Lys, His, Ser, Thr, Gln, or Asn, and even more preferably Ala, Pro, Asp, Glu, Arg, Lys, Ser, Gln, or Asn; and

[0703] aa15 represents an amino acid residue, preferably an amino acid residue having a neutral polar or neutral nonpolar side chain or a charged (acidic or basic) side chain, more preferably His, Arg, Lys, Asp, Ser, Thr, Gln, Asn, Ala, Val, Leu, Gly, or Phe, and even more preferably His, Arg, Lys, Asp, Ser, Thr, Gln, or Asn.

[0704] In some implementations of the above sequence and formula, (Xaa) m The amino acid sequence is selected from SEQ ID No. 41 to 75 or has at least 80%, 85%, 90%, 95%, or even 98% homology with the sequences selected from SEQ ID No. 41 to 75. In some embodiments, (Xaa) m An amino acid sequence having at least 80%, 85%, 90%, 95%, or even 98% identity with a sequence selected from SEQ ID No. 41 to 75.

[0705]

[0706]

[0707] In some embodiments, the anti-PD-L1 affinity has an amino acid sequence selected from SEQ ID No. 76 to 84, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or even 98% homology with a sequence selected from SEQ ID No. 76 to 84. In some embodiments, the anti-PD-L1 affinity has an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or even 98% identity with a sequence selected from SEQ ID No. 76 to 84.

[0708] For those embodiments in which at least one drug-conjugate moiety is linked to the affinity sequence via a thiol side chain of a cysteine ​​residue introduced into the affinity sequence, the cysteine ​​residue will preferably be provided in the circumcyclic sequence (Xaa). n Or (Xaa) m The affinity sequence portion outside the ring. Therefore, by including at least 1 to 5 cysteine ​​residues in place of amino acid residues at varying positions in the sequence, but preferably not in the ring 2 or ring 4 sequence, the anti-PD-L1 affinity will have a sequence varying between SEQ ID No. 76 to 84.

[0709]

[0710]

[0711] In some embodiments, the anti-PD-L1 affinity has an amino acid sequence encoded by a nucleic acid having a coding sequence corresponding to nucleotides 1-336 of one of SEQ ID Nos. 85 to 92, or may be encoded by a nucleic acid having a coding sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or even 98% identity with nucleotides 1-336 of one of SEQ ID Nos. 85 to 92, or may be encoded by a nucleic acid having a coding sequence having hybridized with nucleotides 1-336 of one of SEQ ID Nos. 85 to 92 under stringent conditions (such as at 45°C in the presence of 6X sodium chloride / sodium citrate (SSC), followed by washing at 65°C in 0.2X SSC).

[0712] For those embodiments in which at least one drug-conjugate moiety is linked to the affinity sequence via a thiol side chain of a cysteine ​​residue introduced into the affinity sequence, the cysteine ​​residue will preferably be provided in the circumcyclic sequence (Xaa). n Or (Xaa) mThe anti-PD-L1 affinity will have a sequence that varies between the amino acid sequences encoded by SEQ ID No. 85 to 92, by including at least 1 to 5 cysteine ​​residues in place of amino acid residues at the varied positions in the sequence, but preferably not in the loop 2 or loop 4 sequences.

[0713]

[0714]

[0715] In addition, minor modifications may include, in addition to the ring 2 and ring 4 inserts described above, minor deletions or additions to the stefin A or stefin A-derived sequences disclosed herein, such as the addition or deletion of up to 10 amino acids compared to the stefin A or stefin A-derived affinity polypeptide.

[0716] In some embodiments, the affinity peptide binding to PD-L1 is a monomer with a dissociation constant (KL) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. D () Combined with human PD-L1.

[0717] In some embodiments, the affinity peptide moiety that binds to PD-L1 is in monomeric form, such as approximately 10 as measured by Biacore. -3 s -1 (That is, the unit is 1 / second) or slower, about 10 -4 s -1 Or slower or even about 10 -5 s -1 Or a slower dissociation rate constant (K) off () Combined with human PD-L1.

[0718] In some embodiments, the affinity peptide moiety that binds to PD-L1 is a monomeric form at least about 10, as measured by Biacore. 3 M -1 s -1 Or faster, at least about 10 4 M -1 s -1 Or faster, at least about 10 5 M -1 s -1 Or faster or even at least about 10 6 M - 1 s -1 Or a faster association constant (K) on () Combined with human PD-L1.

[0719] In some embodiments, in a competitive binding assay with human PD-1, the affinity polypeptide moiety that binds to PD-L1 is a monomer that binds to human PD-1 at an IC50 of 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less.

[0720] Fusion Proteins - Review

[0721] In some embodiments, the affinity peptide may also include additional insertions, substitutions, or deletions that modulate the bioactivity of the affinity peptide. For example, addition, substitution, or deletion can modulate one or more properties or activities of the modified affinity peptide. For example, addition, substitution, or deletion can modulate the affinity of the affinity peptide for binding to and inhibiting PD-1, modulate the circulating half-life, modulate the therapeutic half-life, modulate the stability of the affinity peptide, modulate protease cleavage, modulate the dosage, modulate the release or bioavailability, promote purification, reduce deamidation, improve shelf life, or modify or alter a particular route of administration. Similarly, the affinity peptide may include a protease cleavage sequence, a reactive group, an antibody-binding domain (including (but not limited to) FLAG or poly-His), or other affinity-based sequences (including (but not limited to) FLAG, poly-His, GST, etc.) or linker molecules (including (but not limited to) biotin) that improve the detection, purification, or other properties of the peptide.

[0722] In some cases, these additional sequences are added to one and / or the other end of the affinity polypeptide in the form of a fusion protein. Therefore, in certain aspects of the invention, the binder-drug conjugate is a fusion protein (“fusion domain” herein) having at least one affinity polypeptide sequence and one or more heterologous polypeptide sequences. The fusion domain may be selected to confer desired properties, such as, by way of example only, self-secretion from cells or retention on the cell surface (i.e., for the encoded affinity), acting as a substrate for post-translational modifications or other recognition sequences, establishing polymeric structures aggregated via protein-protein interactions, altering (typically prolonging) serum half-life, or altering tissue localization or tissue exclusion and other ADME properties.

[0723] For example, some fusion domains are particularly useful for the isolation and / or purification of fusion proteins, such as by affinity chromatography. By way of illustration only, well-known examples of such fusion domains that facilitate expression or purification include affinity tags such as polyhistidine (i.e., the His6 tag), Strep II tags, streptavidin-binding peptide (SBP) tags, calmodulin-binding peptide (CBP) tags, glutathione S-transferase (GST), maltose-binding protein (MBP), S-tags, HA tags, c-Myc tags, thioredoxin, protein A, and protein G.

[0724] In order for the affinity peptide to be secreted when prepared in a recombinant manner, it typically contains a signal sequence that guides the protein's transport into the lumen of the endoplasmic reticulum and ultimately to secretion (or, if it is a transmembrane domain or other cell surface-retained signal, to the cell surface). The signal sequence (also called the signal peptide or leader sequence) is located at the N-terminus of the nascent polypeptide. It directs the polypeptide to the endoplasmic reticulum and sorts the protein to its destination, such as into the interior space of the organelle, into the inner membrane, into the outer membrane, or to the extracellular space via secretion. After the protein is transported into the endoplasmic reticulum, most of the signal sequence is cleaved from the protein by signal peptidase. The cleavage of the signal sequence from the polypeptide typically occurs at specific sites in the amino acid sequence and depends on the amino acid residues within the signal sequence.

[0725] In some embodiments, the signal peptide is about 5 to about 40 amino acids in length (such as about 5 to about 7, about 7 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, or about 25 to about 30, about 30 to about 35, or about 35 to about 40 amino acids in length).

[0726] In some embodiments, the signal peptide is a natural signal peptide derived from a human protein. In other embodiments, the signal peptide is a non-natural signal peptide. For example, in some embodiments, the non-natural signal peptide is a mutant natural signal peptide derived from a corresponding natural human secreted protein, and may include one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitutions, insertions, or deletions.

[0727] In some embodiments, the signal peptide is a signal peptide from a non-IgSF protein family or a mutant thereof, such as a signal peptide from immunoglobulins (such as the IgG heavy chain or IgG-κ light chain), cytokines (such as interleukin-2 (IL-2) or CD33), serum albumin proteins (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, luciferase, trypsinogen (e.g., chymotrypsinogen or trypsinogen), or other signal peptides capable of efficiently secreting proteins from the cell. Exemplary signal peptides include (but are not limited to):

[0728] Natural protein signal sequences

[0729]

[0730] Thematic fusion proteins may also include one or more isolated heterologous protein sequences or domains, i.e., linkers that separate the cell-binding moiety in cases where the drug-binding conjugate includes more than one cell-binding moiety. As used herein, the term "linker" refers to a linker amino acid sequence inserted between a first polypeptide (e.g., an affinity peptide) and a second polypeptide (e.g., a second affinity peptide, an Fc region, a receptor trap, albumin, etc.). Empirical linkers designed by researchers are generally classified into three categories based on their structure: flexible linkers, rigid linkers, and in vivo cleavable linkers. In addition to their basic functions of linking functional domains together (as in flexible and rigid linkers) or releasing free functional domains in vivo (as in in vivo cleavable linkers), linkers offer many other advantages for the production of fusion proteins, such as improved biological activity, increased expression levels, and achieving a desirable pharmacokinetic profile. Linkers should not adversely affect the expression, secretion, or biological activity of the fusion protein. Linkers should not be antigenic and should not trigger an immune response.

[0731] Suitable linkers are known to those skilled in the art and typically comprise a mixture of glycine and serine residues, and generally include non-sterically hindered amino acids. Other amino acids that may be incorporated into a usable linker include threonine and alanine residues. The length of the linker can be within a range, for example, 1-50 amino acids, 1-22 amino acids, 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the linker may include a cleavage site. In some embodiments, the linker may include an enzyme cleavage site, allowing the second polypeptide to be separated from the first polypeptide.

[0732] In some preferred embodiments, the linker may be characterized as flexible. Flexible linkers are generally suitable when the joined domains require some degree of mobility or interaction. They are typically composed of small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. See, for example, Argos P. (1990) “An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion” J Mol Biol. 211:943-958. The small size of these amino acids provides flexibility and enables the mobility of the linked functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, and thus reduce unfavorable interactions between the linker and the protein moieties. The most commonly used flexible linkers have sequences consisting primarily of extensions of Gly and Ser residues (“GS” linkers). The most widely used example of a flexible linker has a (Gly-Gly-Gly-Gly-Ser)n sequence. By adjusting the copy number "n", the length of this GS linker can be optimized to achieve proper separation of functional domains or maintain necessary interdomain interactions. Besides the GS linker, many other flexible linkers have been designed for recombinant fusion proteins. Although these flexible linkers are also rich in small or polar amino acids, such as Gly and Ser, they may contain additional amino acids such as Thr and Ala to maintain flexibility, and polar amino acids such as Lys and Glu to improve solubility.

[0733] In some preferred embodiments, the linker may be rigid. While flexible linkers have the advantage of passively connecting functional domains and enabling some degree of movement, their lack of rigidity can limit expression levels or biological activity in certain fusion protein embodiments. The ineffectiveness of flexible linkers in these cases is attributed to inefficient separation of protein domains or insufficient reduction of interference between them. In these situations, rigid linkers have been successfully used to maintain a fixed distance between domains and preserve their independent function.

[0734] Many natural linkers exhibit α-helical structures. α-Helical structures are rigid and stable, with intrasegmental hydrogen bonds and a tightly packed backbone. Therefore, rigid α-helical linkers can act as rigid spacers between protein domains. (George et al. (2002), “An analysis of protein domain linkers: their classification and role in protein folding”, Protein Eng. 15(11):871-9). Generally, rigid linkers exhibit relatively rigid structures by employing α-helical structures or by containing multiple Pro residues. In many cases, they are more efficient at separating functional domains than flexible linkers. The length of the linker can be easily tuned by changing the copy number to achieve the optimal distance between domains. Therefore, rigid linkers are chosen when the spatial separation of domains is important for maintaining the stability or biological activity of the fusion protein. In this regard, linkers forming α-helices with the (EAAAK)n sequence have been used to construct many recombinant fusion proteins. Another type of rigid linker has a Pro-rich sequence, (XP)n, where X represents any amino acid, preferably Ala, Lys, or Glu.

[0735] For illustrative purposes only, an exemplary connector includes:

[0736]

[0737]

[0738] Other linkers that can be used in the subject fusion protein include (but are not limited to) SerGly, GGSG, GSGS, GGGS, S(GGS)n (where n is 1-7), GRA, poly(Gly), poly(Ala), GGGSGGG, ESGGGGVT, LESGGGGVT, GRAQVT, WRAQVT, and ARGRAQVT. The hinge region of the Fc fusion protein described below can also be considered as a linker.

[0739] Other modifications that may be made to the affinity polypeptide sequence itself or to the side-connected polypeptide portion provided as part of a fusion protein are one or more sequences that serve as sites for post-translational modifications performed by an enzyme. These modifications may include (but are not limited to) glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitic acid addition, phosphorylation, glycolipid bond modification, etc.

[0740] Engineering modification PK and ADME features

[0741] In some embodiments, the binder-drug conjugate may not have an optimal half-life and / or PK profile for the route of administration, such as parenteral therapeutic administration. The term "half-life" refers to the amount of time it takes for a substance (such as the binder-drug conjugate of the present invention) to lose half of its pharmacological or physiological activity or concentration. Biological half-life can be affected by the elimination, secretion, degradation (e.g., enzymatic), or absorption and concentration of a substance in certain organs or tissues of the body. In some embodiments, biological half-life can be assessed by determining the time required for a substance to reach half its steady-state plasma concentration ("plasma half-life"). To overcome this drawback, several common strategies for extending half-life, used in the case of other protein therapeutics, exist, including incorporating a half-life extension portion as part of the binder-drug conjugate.

[0742] The term “half-life extended portion” refers to a pharmaceutically acceptable portion, domain, or molecule of an affinity peptide, optionally covalently linked (“conjugated” or “fused”) to the affinity peptide via a non-naturally encoded amino acid, directly or via a linker, to form the binder-drug conjugate described herein. This conjugate prevents or slows in vivo proteolytic degradation or other activity-reducing modifications of the affinity peptide, prolongs its half-life, and / or improves or alters other pharmacokinetic or physiological properties, including (but not limited to) improved absorption, reduced toxicity, improved solubility, reduced protein aggregation, improved bioactivity and / or target selectivity, improved manufacturability, and / or reduced immunogenicity of the modified affinity peptide compared to a comparative substance, such as the unconjugated form of the modified affinity peptide. The term "half-life extended portion" includes non-protein half-life extended portions, such as water-soluble polymers, such as polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), lipids, branched or unbranched acyl groups, branched or unbranched C8-C30 acyl groups, branched or unbranched alkyl groups, and branched or unbranched C8-C30 alkyl groups; and protein half-life extended portions, such as serum albumin, transferrin, fibronectin (e.g., albumin-bound or pharmacokineticly extended (PKE) fibronectin), Fc domains and unstructured polypeptides, such as XTEN and PAS polypeptides (e.g., conformationally disordered polypeptide sequences composed of amino acids Pro, Ala, and / or Ser), and fragments of any of the foregoing. Examination of the crystal structure of the affinity and its interaction with its target (such as the anti-PD-L1 affinity complex with PD-1 shown in the figure) can indicate which amino acid residues have side chains that are fully or partially solvent-accessible.

[0743] In some embodiments, the half-life extension portion prolongs the half-life of the resulting binder-drug conjugate circulating in mammalian serum compared to the half-life of a protein not conjugated (e.g., relative to a single affinity peptide). In some embodiments, the half-life extension is greater than or equal to about 1.2, 1.5, 2.0, 3.0, 4.0, 5.0, or 6.0 times. In some embodiments, the half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week after in vivo administration compared to a protein without the half-life extension portion.

[0744] As a further example, the half-life-extended portion of the binder-drug conjugate of the present invention may include the following:

[0745] • Gene fusions of pharmacological affinity sequences with native long-half-life proteins or protein domains (e.g., Fc fusions, transferrin [Tf] fusions, or albumin fusions). See, for example, Beck et al. (2011) "Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies". MAbs. 3:1-2; Czajkowsky et al. (2012) "Fc-fusion proteins: new developments and future perspectives". EMBO Mol Med. 4: 1015-28; Huang et al. (2009) "Receptor-Fcfusion therapeutics, traps, and Mimetibody technology" Curr OpinBiotechnol. 2009; 20: 692-9; Keefe et al. (2013) "Transferrin fusion proteintherapies: acetylcholine receptor-transferrin fusion protein as a model". Edited by Schmidt S, Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; pp. 345-56; Weimer et al. (2013) “Recombinant albumin fusion proteins". Edited by Schmidt S. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013. Pages 297-323; Walker et al. (2013) "Albumin-binding fusion proteins in the development of novel long-acting therapeutics". Edited by Schmidt S, Fusion protein technologies for biopharmaceuticals: applications andchallenges. Hoboken: Wiley; 2013. pp. 325-43.

[0746] • Gene fusions of pharmacological affinity sequences with inert peptides, such as XTEN (also known as recombinant PEG or “rPEG”), high amino acid polymers (HAP; HAP-modified), proline-alanine-serine polymers (PAS; PAS-modified), or elastin-like peptides (ELP; ELP-modified). See, for example, Schellenberger et al. (2009) "A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner". Nat Biotechnol. 2009; 27:1186-90; Schlapschy et al., Fusion of a recombinantantibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life. Protein Eng Des Sel. 2007; 20: 273-84; Schlapschy (2013) PASylation: a biological alternative to PEGylation for extending the plasma halflife of pharmaceutically active proteins. Protein EngDes Sel. 26: 489-501. Floss et al. (2012) "Elastin-like polypeptides revolutionizerecombinant protein expression and their biomedical application". TrendsBiotechnol. 28: 37-45. Floss et al. "ELP-fusion technology for biopharmaceuticals". Edited by Schmidt S, Fusion protein technologies for biopharmaceuticals: application and challenges. Hoboken: Wiley; 2013. Pages 372-98.

[0747] • Increase the hydrodynamic radius by chemically conjugating pharmacologically active peptides or proteins with repeating chemical moieties, such as PEG (PEGylation) or hyaluronic acid. See, for example, Caliceti et al. (2003) "Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates" AdvDrug Delivery Rev. 55:1261-77; Jevsevar et al. (2010) PEGylation of therapeuticproteins. Biotechnol J 5:113-28; Kontermann (2009) "Strategies to extend Plasmahalf-lives of recombinant antibodies" BioDrugs. 23:93-109; Kang et al. (2009) "Emerging PEGylated drugs" Expert Opin Emerg Drugs. 14:363-80; and Mero et al. (2013) "Conjugation of hyaluronan to proteins" Carb Polymers. 92:2163-70.

[0748] • By significantly increasing the negative charge of the fused pharmacologically active peptide or protein through polysialylation; or alternatively, (b) fusing a negatively charged, highly sialylated peptide (e.g., a carboxyl-terminal peptide [CTP; human chorionic gonadotropin (CG) b-chain]) with a biological drug candidate, known to extend the half-life of natural proteins (such as human CG b-subunits). See, for example, Gregoriadis et al. (2005) "Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids" Int J Pharm. 2005;300:125-30; Duijkers et al. "Single dose pharmacokinetics and effects on follicular growth and serumhormones of a long-acting recombinant FSH preparation (FSHCTP) in healthy pituitary-suppressed" females" (2002) Hum Reprod. 17:1987-93; and Fares et al. "Design of a longacting follitropin agonist by fusing the C-terminal sequence of thechorionic gonadotropin beta subunit to the follitropin beta subunit" (1992) Proc Natl Acad Sci USA.89:4304-8.35; and Fares "Half-life extension through O-glycosylation”.

[0749] • It can non-covalently bind to common long-half-life proteins, such as HSA, human IgG, transferrin, or fibronectin, via a peptide or protein-binding domain. See, for example, Andersen et al. (2011) "Extending half-life by indirect targeting of the neonatal Fc receptor (FcRn) using a minimal albumin binding domain" J Biol Chem. 286:5234-41; O'Connor-Semmes et al. (2014) "GSK2374697, a novel albumin-binding domain antibody (albudAb), extends systemic exposure of extendin-4: first study in humans-PK / PD and safety" Clin Pharmacol Ther. 2014;96:704-12; Sockolosky et al. (2014) "Fusion of ashort peptide that binds immunoglobulin G to a recombinant protein substantially increases its plasma half-life in mice" PLoS One. 2014;9:e102566.

[0750] Classic gene fusions with long-lived serum proteins offer alternative methods for extending half-life, distinct from chemical conjugations with PEG or lipids. Traditionally, two main proteins are used as fusion partners: antibody Fc domains and human serum albumin (HSA). Fc fusions involve the fusion of the extracellular domain of a peptide, protein, or receptor with the Fc moiety of an antibody. Both Fc and albumin fusions extend half-life not only by increasing the size of the peptide drug but also by utilizing the body's natural recycling mechanism: the nascent Fc receptor FcRn. The pH-dependent binding of these proteins to FcRn prevents degradation of the fusion protein in the endosome. Fusions based on these proteins can have half-lives ranging from 3 to 16 days, significantly longer than typical PEGylated or lipotropic peptides. Fusion with antibody Fc domains can improve the solubility and stability of peptide or protein drugs. An example of a peptide Fc fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials. Human serum albumin (the same protein used by fatty acid acylated peptides) is another popular fusion partner. Albiglutide is a GLP-1 receptor agonist based on this platform. The main difference between Fc and albumin is the dimerization property of Fc relative to the monomeric structure of HSA, which causes the fused peptide to present as a dimer or monomer, depending on the choice of fusion partner. The dimerization property of the affinity-Fc fusion can produce an affinity effect if the affinity target (such as PD-L1 on tumor cells) is sufficiently close together or is itself a dimer. This may be desirable or target-independent.

[0751] Fc Fusion

[0752] In some embodiments, the affinity polypeptide may be part of a fusion protein having an immunoglobulin Fc domain (“Fc domain”) or a fragment or variant thereof (such as a functional Fc region). In this case, the Fc fusion (“Fc-fusion”), such as a binder-drug conjugate produced in the form of an affinity-Fc fusion protein, is a polypeptide comprising one or more affinity sequences covalently linked (directly or indirectly) to the Fc region of an immunoglobulin via a peptide backbone. The Fc-fusion may comprise, for example, the Fc region of an antibody (which contributes to effector function and pharmacokinetics) and an affinity sequence as part of the same polypeptide. The immunoglobulin Fc region may also be indirectly linked to one or more affinity sequences. Various linkers are known in the art and may optionally be used to link the Fc to a polypeptide including the affinity sequence to produce the Fc-fusion. In some embodiments, the Fc-fusion may dimerize to form a homodimer, or use different Fc domains to form a heterodimer.

[0753] Several reasons exist for selecting the Fc region of human antibodies to generate subject-drug conjugates in the form of affinity fusion proteins. The basic principles are the generation of stable proteins large enough to exhibit a pharmacokinetic profile similar to that of antibodies; and the utilization of properties conferred by the Fc region. This includes the rescue of nascent FcRn receptor pathways: FcRn-mediated fusion proteins are recirculated to the cell surface after endoplasmosis, avoiding lysosomal degradation and re-release into the bloodstream, thus contributing to prolonged serum half-life. Another significant benefit is the binding of the Fc domain to protein A, which simplifies downstream processing during conjugate generation and allows for the production of highly purified formulations of the conjugate.

[0754] Therefore, the Fc domain will include the antibody constant region except for the first constant region immunoglobulin domain. Thus, the Fc domain refers to the last two constant regions of the immunoglobulin domains of IgA, IgD, and IgG, and the last three constant regions of the immunoglobulin domains of IgE and IgM, and the flexible hinges at the N-terminus of these domains. For IgA and IgM, Fc may include the J-chain. For IgG, Fc contains the immunoglobulin domains Cγ2 and Cγ3, and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc domain can vary, the human IgG heavy chain Fc region is generally defined to contain residues C226 or P230 to its carboxyl terminus, where the numbering is based on the EU index as described in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, NIH, Bethesda, Md. (1991)). Fc may refer to the region alone or in the case of a complete antibody, antibody fragment, or Fc fusion protein. Polymorphism has been observed at many different Fc locations, including as an Fc structural domain, as used in this paper.

[0755] In some embodiments, as used herein, "functional Fc region" refers to an Fc domain or fragment thereof that maintains the ability to bind FcRn. The functional Fc region binds to FcRn but does not have effector function. The ability of an Fc region or fragment thereof to bind to FcRn can be determined by standard binding assays known in the art. Exemplary "effective functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. These effector functions can be assessed using various assays known in the art for evaluating antibody effector functions.

[0756] In one exemplary implementation, the Fc domain is derived from the IgG1 subclass; however, other subclasses (e.g., IgG2, IgG3, and IgG4) may also be used. An exemplary sequence of the human IgG1 immunoglobulin Fc domain that can be used is as follows:

[0757]

[0758] In some embodiments, the Fc region for the fusion protein may comprise a hinge region of the Fc molecule. An exemplary hinge region comprises core hinge residues (i.e., DKTHTCPPCPAPELLG) spanning positions 1-16 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In some embodiments, the fusion protein containing the affinity protein may be polymeric (e.g., a dimer) due in part to cysteine ​​residues at positions 6 and 9 within the hinge region of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In other embodiments, the hinge region, as used herein, may also comprise residues of the core hinge sequence derived from the CH1 and CH2 regions and flanked by the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In other embodiments, the hinge sequence may comprise or consist of GSTHTCPPCPAPELLG or EPKSCDKTHTCPPCPAPELLG or thereof.

[0759] In some embodiments, the hinge sequence may include one or more substitutions that impart desired pharmacokinetic, biophysical, and / or biological properties. Some exemplary hinge sequences include:

[0760] EPKSCDKTHTCPPCPAPELLGGPS;

[0761] EPKSSDKTHTCPPCPAPELLGGPS;

[0762] EPKSSDKTHTCPPCPAPELLGGSS;

[0763] EPKSSGSTHTCPPCPAPELLGGSS;

[0764] DKTHTCPPCPAPELLGGPS; and

[0765] DKTHTCPPCPAPELLGGSS.

[0766] In one embodiment, residue P at position 18 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above may be replaced with S to remove Fc effector function; an example of this replacement is in the hinge having sequences EPKSSDKTHTCPPCPAPELLGGSS, EPKS SGSTHTCPPCPAPELLGGSS, and DKTHTCPPCPAPELLGGSS. In another embodiment, residue DK at positions 1-2 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above may be replaced with GS to remove potential cleavage sites; an example of this replacement is in the sequence EPKSSGSTHTCPPCPAPELLGGSS. In yet another embodiment, residue C at position 103 of the heavy chain constant region (i.e., domains CH1-CH3) of human IgG1 may be replaced with S to prevent the formation of inappropriate cysteine ​​bonds in the absence of light chains; an example of this replacement is in EPKSSDKTHTCPPCPAPELLGGSS, EPKS SDKTHTCPPCPAPELLGGSS, and EPKSSGSTHTCPPCPAPELLGGSS.

[0767] In some embodiments, the Fc region is a mammalian Fc, such as a human Fc, including Fc domains derived from IgG1, IgG2, IgG3, or IgG4. The Fc region may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the natural Fc region and / or the Fc region of the parental polypeptide. In some embodiments, the Fc region may have at least about 90% sequence identity with the natural Fc region and / or the Fc region of the parental polypeptide.

[0768] In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NO:93, or an Fc sequence derived from the examples provided in SEQ ID Nos.94-106. It should be understood that the C-terminal lysine of the Fc domain is an optional component of the fusion protein comprising the Fc domain. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NO:93-106, but the C-terminal lysine is omitted. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO:93. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO:93, but the C-terminal lysine is omitted.

[0769]

[0770]

[0771]

[0772] Exemplary Fc fusions of the PD-L1 affinity peptide and Fc are provided in the embodiments and figures, illustrating that the affinity sequence can be positioned at the N-terminus or C-terminus of the Fc domain, and can be directly linked, or the fusion protein can have other polypeptide sequences intermediate between the Fc domain and the affinity peptide sequence. In the illustrated embodiments, an unstructured (flexible) linker (Gly4Ser) is used. n Used in conjunction with the PD-L1 affinity "251" (SEQ ID No. 84) and the Fc domain of human IgG1 (SEQ ID No. 93), wherein the hinge region is EPKSCDKTHTCPPCPAPELLG. All constructs include the CD33 secretion signal sequence MPLLLLLPLLWAGALA, derived from the cleavage of the protein's mature form.

[0773]

[0774]

[0775] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secretory Ig that binds to the Fc receptor (FcR) present on certain cytotoxic cells (such as natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to target cells carrying antigens and then kill the target cells with cytotoxins.

[0776] In some embodiments, the fusion protein includes an Fc domain sequence that renders the resulting binder-drug conjugate non-functional (or with reduced) ADCC and / or complement activation or effector function. For example, the Fc domain may contain a naturally disabling constant region of the IgG2 or IgG4 isotype or a mutant IgG1 constant region. Examples of suitable modifications are described in EP0307434. One example includes substitutions of alanine residues at positions 235 and 237 (EU index number).

[0777] In other embodiments, the fusion protein includes, for example, an Fc domain from human IgG1 or IgG3, such that the resulting binder-drug conjugate will retain some or all of its Fc functionality, for example, an Fc domain sequence capable of having one or both of ADCC and CDC activities. The level of effector function can be varied according to known techniques, such as mutations in the CH2 domain, where the IgG1 CH2 domain has one or more mutations selected from positions 239, 332, and 330, for example, mutations selected from S239D, I332E, and A330L, resulting in enhanced effector function of the antibody, and / or, for example, altering the glycosylation profile of the antigen-binding protein of the present invention to reduce fucosylation in the Fc region.

[0778] albumin fusion

[0779] In other embodiments, the binder-drug conjugate is a fusion protein comprising an albumin sequence or albumin fragment in addition to at least one affinity sequence. In other embodiments, the binder-drug conjugate is conjugated to the albumin sequence or albumin fragment via chemical bonds, rather than being incorporated into a polypeptide sequence including the affinity. In some embodiments, the albumin, albumin variant, or albumin fragment is human serum albumin (HSA), human serum albumin variant, or human serum albumin fragment. Albumin serum proteins equivalent to HSA have been found in, for example, cynomolgus monkeys, cattle, dogs, rabbits, and rats. In non-human species, bovine serum albumin (BSA) is structurally most similar to HSA. See, for example, Kosa et al., (2007) JPharm Sci. 96(11):3117-24. This invention covers albumins from non-human species, including (but not limited to) the use of albumin sequences derived from cynomolgus monkey serum albumin or bovine serum albumin.

[0780] Mature human serum albumin (HSA), a 585-amino acid polypeptide (approximately 67 kDa) with a serum half-life of approximately 20 days, is primarily responsible for maintaining colloid osmotic pressure, blood pH, and the transport and distribution of numerous endogenous and exogenous ligands. The protein has three structurally homologous domains (domains I, II, and III), is almost entirely in an α-helical conformation, and is highly stable through 17 disulfide bonds. In some preferred embodiments, the binder-drug conjugate may be an albumin fusion protein comprising one or more affinity polypeptide sequences and a sequence of mature human serum albumin (SEQ ID No. 111) or a variant or fragment thereof that maintains the PK and / or biodistribution properties of mature albumin to the extent required in the fusion protein.

[0781]

[0782]

[0783] Albumin sequences can be separated from affinity peptide sequences or other side-connecting sequences in binder-drug conjugates using adapter sequences as described above.

[0784] Unless otherwise indicated, references to “albumin” or “mature albumin” in this document refer to HSA. However, it should be noted that the full-length HSA has an 18-amino acid signal peptide (MKWVTFISLLFLFSSAYS) followed by a 6-amino acid pre-domain (RGVFRR); this 24-amino acid peptide may be referred to as the pre-pre-domain. Affinity-HSA fusion proteins can be expressed and secreted using the HSA pre-pre-domain in the recombinant protein-coding sequence. Alternatively, affinity-HSA fusions can be expressed and secreted via other secretion signal sequences, such as those described above.

[0785] In an alternative embodiment, instead of being provided as part of a fusion protein having an affinity peptide, the serum albumin peptide may be covalently coupled to the affinity peptide via bonds other than the main chain amide bond, such as through chemical conjugation between the amino acid side chains of each of the albumin peptides and the affinity peptide.

[0786] Albumin-binding domain

[0787] In some embodiments, the binder-drug conjugate may include a serum-binding portion—as part of a fusion protein (if also a polypeptide) having an affinity polypeptide sequence—or via site chemical conjugation other than as part of an adjacent polypeptide chain.

[0788] In some embodiments, the serum-binding polypeptide is the albumin-binding moiety. Albumin contains multiple hydrophobic binding pockets and naturally acts as a transporter for a variety of different ligands (such as fatty acids and steroids) and different drugs. Furthermore, albumin has a negatively charged surface, giving it high water solubility.

[0789] As used herein, the term "albumin-binding moiety" refers to any chemical group capable of binding to albumin, i.e., having albumin-binding affinity. Albumin binds to endogenous ligands, such as fatty acids; however, it also interacts with exogenous ligands, such as warfarin, penicillin, and diazepam. Because the binding of these drugs to albumin is reversible, albumin-drug complexes act as drug reservoirs that can enhance drug biodistribution and bioavailability. Incorporating components that mimic endogenous albumin-binding ligands, such as fatty acids, can enhance albumin association and improve drug efficacy.

[0790] In some implementations, lipolysis, a chemical modification method to enhance protein half-life, can be employed in the generation of the subject binder-drug conjugate, involving the covalent binding of fatty acids to peptide side chains. Initially conceived and developed as a method for prolonging the half-life of insulin, lipolysis shares the same basic half-life extension mechanism as polyethylene glycol (PEG) conversion: increasing the hydrodynamic radius to reduce renal filtration. However, the lipid moiety itself is relatively small, and its effect is indirectly mediated via the nonvalent binding of the lipid moiety to circulating albumin. One consequence of lipolysis is its reduced water solubility of the peptide, but this effect can be modulated, for example, by engineering the linker between the peptide and fatty acid using glutamate or microPEG within the linker. Linker engineering and changes in the lipid moiety can affect autoaggregation, which can prolong half-life (independent of albumin) by delaying biodistribution. See, for example, Jonassen et al. (2012) Pharm Res. 29(8):2104-14.

[0791] Other examples of albumin-binding moieties used to generate certain binder-drug conjugates include albumin-binding (PKE2) fibronectin (see WO2011140086, “Serum Albumin Binding Molecules”; WO2015143199, “Serum albumin-binding Fibronectin Type III Domains”; and WO2017053617, “Fast-off rate serum albumin binding fibronectin type iii domains”), albumin-binding domain 3 (ABD3) of protein G from Streptococcus strain G148, and albumin-binding nanobody moieties of albumin-binding domain antibodies GSK2374697 (“AlbudAb”) or ATN-103 (Ozoralizumab).

[0792] Polyethylene glycolation, XTEN, PAS and other polymers

[0793] Various macromolecular polymers and other molecules can be linked to the affinity-containing peptides of the present invention to modulate the biological properties of the resulting binder-drug conjugates and / or impart novel biological properties to the binder-drug conjugates. These macromolecular polymers can be linked to affinity-containing peptides via naturally encoded amino acids, non-naturally encoded amino acids, or any functional substituents of natural or non-natural amino acids, or any substituents or functional groups added to natural or non-natural amino acids. The molecular weight of the polymers can be in a wide range, including (but not limited to) about 100 Da and about 100,000 Da or greater. The molecular weight of the polymer can be between about 100 Da and about 100,000 Da, including (but not limited to) 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30, The molecular weights of the polymers are approximately 100 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da.

[0794] For the purposes described, various methods have been developed, including polyethylene glycolation, polysialylation, hydroxyethyl starchization, glycosylation, or fusion with flexible and hydrophilic amino acid chains (500 to 600 amino acids) to produce recombinant PEG analogs (see Chapman, (2002) Adv Drug Deliv Rev. 54. 531-545; Schlapschy et al., (2007) Prot Eng DesSel. 20, 273-283; Contermann (2011) Curr Op Biotechnol. 22, 868-876; Jevsevar et al., (2012) Methods Mol Biol. 901, 233-246).

[0795] Examples of polymers include (but are not limited to) polyalkyl ethers and their alkoxy-terminated analogs (e.g., polyoxyethylene glycol, polyoxyethylene / propylene glycol and their methoxy or ethoxy-terminated analogs, especially polyoxyethylene glycol, the latter also known as polyethylene glycol or PEG); discrete PEG (dPEG); polyvinylpyrrolidone; polyvinylalkyl ethers; polyoxazolines, polyalkyloxazolines and polyhydroxyalkyloxazolines; polyacrylamide, polyalkylacrylamide and polyhydroxyalkylacrylamide (e.g., polyhydroxypropyl methacrylamide and its derivatives); polyhydroxyalkyl acrylates; polysialic acid and its analogs; hydrophilic peptide sequences; polysaccharides and their derivatives, including dextran and dextran derivatives, such as carboxyl groups. Methyl dextran, sulfated dextran, amino dextran; cellulose and its derivatives, such as carboxymethyl cellulose, hydroxyalkyl cellulose; chitin and its derivatives, such as chitosan, succinylated chitosan, carboxymethyl chitin, carboxymethyl chitosan; hyaluronic acid and its derivatives; starch; alginate; chondroitin sulfate; albumin; pullulan and carboxymethyl pullulan; polyamino acids and their derivatives, such as polyglutamic acid, polylysine, polyaspartic acid, polyasparagine; maleic anhydride copolymers, such as styrene-maleic anhydride copolymers, divinylethyl ether-maleic anhydride copolymers; polyvinyl alcohol; copolymers thereof; trimers thereof; mixtures thereof; and derivatives thereof.

[0796] The selected polymer may be water-soluble so that the bound drug conjugate does not precipitate in aqueous environments (such as physiological environments). The water-soluble polymer may be in any structural form, including (but not limited to) linear, forked, or branched forms. Typically, the water-soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water-soluble polymers may also be used. For example, PEG is used to describe certain embodiments of the invention. For the therapeutic use of the bound drug conjugate, the polymer may be pharmaceutically acceptable.

[0797] The widely used term "PEG" encompasses any polyethylene glycol molecule, regardless of size or modification at one end of the PEG, and can be represented as a polypeptide linked to an affinity peptide, as follows:

[0798] XO-(CH2CH2O) n -CH2CH2-

[0799] or

[0800] XO-(CH2CH2O) n -

[0801] Wherein n is from 2 to 10,000, and X is H or a terminal modification, including (but not limited to) C1-4 alkyl groups, protecting groups, or terminal functional groups. In some cases, the PEG used in the polypeptides of the present invention is terminated at an end having a hydroxyl or methoxy group, i.e., X is H or CH3 (“methoxyPEG”).

[0802] It should be noted that the other end of the PEG, indicated by a terminal "-" in the above formula, can be linked to a polypeptide containing an affinity group via a naturally occurring or non-naturally encoded amino acid. For example, the linkage can occur via an amide, carbamate, or urea bond to an amino group of the polypeptide (including, but not limited to, the ε-amine of lysine or the N-terminus). Alternatively, the polymer can be linked to a thiol group (including, but not limited to, the thiol group of cysteine) via a maleimide bond, which requires the residues in the affinity sequence to be changed to cysteine ​​when linked to the affinity polypeptide sequence itself.

[0803] The number of water-soluble polymers linked to the affinity peptide (i.e., the degree of PEGylation or glycosylation) can be adjusted to provide altered (including, but not limited to, increased or decreased) pharmacological, pharmacokinetic, or pharmacodynamic characteristics, such as in vivo half-life, in the resulting binder-drug conjugate. In some embodiments, the half-life of the resulting binder-drug conjugate is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or at least about 100-fold compared to the unmodified peptide.

[0804] Another variation of polymer systems that can be used to improve the PK or other biological properties of the resulting binder-drug conjugates is the use of functional analogues of PEG, particularly unstructured hydrophilic amino acid polymers as part of a fusion protein with an affinity peptide sequence. The inherent biodegradability of peptide platforms makes them attractive as potentially more benign alternatives to PEG. Another advantage is the precise molecular structure of recombinant molecules compared to the polydispersity of PEG. Unlike HSA and Fc peptide fusions, where the three-dimensional folding of the fusion partner needs to be maintained, recombinant fusions with unstructured partners can, in many cases, undergo higher temperatures or stringent conditions, such as HPLC purification.

[0805] A more advanced polypeptide in this class is called XTEN (Amunix) and is 864 amino acids long, containing six amino acids (A, E, G, P, S, and T). See Schellenberger et al., “A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner”, 2009 Nat Biotechnol. 27(12): 1186-90. Due to the biodegradable nature of the polymer, this is much larger than the commonly used 40 kDa PEG and imparts a greater degree of half-life extension. Fusions of XTEN with polypeptides containing affinity peptides should result in a 60 to 130-fold increase in the half-life of the final binder-drug conjugate compared to the unmodified polypeptide.

[0806] The second polymer based on similar conceptual considerations is PAS (XL-Protein GmbH). Schlapschy et al., “PASYlation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins”, 2013, Protein Eng Des Sel. 26(8): 489-501. Random coil polymers are composed of even more restricted sets of only three small, uncharged amino acids (proline, alanine, and serine). Similar to Fc, HAS, and XTEN, PAS modification can be genetically encoded along with the affinity polypeptide sequence to produce an inline fusion protein upon expression.

[0807] Multispecific fusion protein

[0808] In some embodiments, the binder-drug conjugate is a multispecific polypeptide comprising, for example, a first anti-PD-L1 affinity polypeptide and at least one additional binding domain. For illustration, the additional binding domain may be a polypeptide sequence selected from: a second affinity polypeptide sequence (which may be the same as or different from the first affinity polypeptide sequence), an antibody or a fragment thereof or other antigen-binding polypeptide, a ligand-binding portion of a receptor (such as a receptor trapping polypeptide), a receptor-binding ligand (such as a cytokine, growth factor, etc.), an engineered T-cell receptor, an enzyme or a catalytic fragment thereof, or other polypeptide sequences endowed with such properties.

[0809] In some embodiments, the binder-drug conjugate includes one or more additional affinity polypeptide sequences that also target PD-L1. The additional anti-PD-L1 affinity (or a mixture thereof) may be the same as or different from the first anti-PD-L1 affinity polypeptide to produce a multispecific affinity fusion protein. The binder-drug conjugate may bind to the same or overlapping sites on PD-L1, or it may bind to two different sites, such that the binder-drug conjugate can simultaneously bind to two sites (double complementary sites) or more than two sites (multiple complementary sites) on the same PD-L1 protein.

[0810] In some embodiments, the binder-drug conjugate includes one or more antigen-binding sites from the antibody. The resulting binder-drug conjugate may be a single chain comprising both an anti-PD-L1 affinity and an antigen-binding site (as in the case of scFV), or may be a polyprotein complex, such as in which the sequence of the anti-PD-L1 antibody is further fused with an antibody assembled with heavy and / or light chains. An exemplary affinity / antibody fusion of the aforementioned format is... Figure 11 The ipulimab-AVA04-141 bispecific antibody shown in A is bivalent for both CTLA-4 and PD-L1. Another one is... Figure 13 The bevacizumab-AVA04-251 bispecific antibody shown in A is bivalent for each of VEGF-A and PD-L1.

[0811] In the case of the described iplemab-AVA04-141 bispecific antibody, the anti-PD-L1 affinity peptide is provided as an inline fusion compound at the C-terminus of the heavy chain of the anti-CTLA-4 antibody, wherein the heavy chain (including the removable secretion signal sequence MPLLLLLPLLWAGALA and the Gly4-Ser repeat linker) has an affinity fusion sequence:

[0812]

[0813] Furthermore, the light chain (including the removable secretion signal sequence MPLLLLLPLLWAGALA) possesses the sequence of the natural iplemab antibody:

[0814]

[0815] Similarly, in the case of the described bevacizumab-AVA04-251 bispecific antibody, the anti-PD-L1 affinity peptide is provided as an inline fusion compound at the C-terminus of the heavy chain of the anti-VEGF-A antibody, wherein the heavy chain (including the removable secretion signal sequence MPLLLLLPLLWAGALA and the flexible Gly4-Ser repeat linker) has an affinity fusion sequence:

[0816]

[0817] Furthermore, the light chain (including the removable secretion signal sequence MPLLLLLPLLWAGALA) possesses the sequence of the natural bevacizumab antibody:

[0818]

[0819] To further illustrate the flexibility in formatting the affinity of the present invention, a bispecific antibody, bevacizumab-AVA04-251, is also produced in the following form, wherein the light chain is the same as above, but the heavy chain includes a rigid linker between the antibody heavy chain and the anti-PD-L1 affinity, wherein the heavy chain (including the removable secretion signal sequence MPLLLLLPLLWAGALA and the rigid A(EAAAK)3 linker) has an affinity fusion sequence:

[0820]

[0821] As will be obvious to those skilled in the art and Figure 17 As shown, the anti-PD-L1 affinity polypeptide sequence can be added to either the N-terminus or C-terminus of the antibody heavy or light chain, or a combination / arrangement thereof. Furthermore, as... Figure 9 As shown, in the case of multi-affinity, any given antibody chain may include more than one affinity sequence.

[0822] In some embodiments of multispecific binder-drug conjugates comprising full-length immunoglobulins, the fusion of the affinity polypeptide sequence with the antibody preserves the Fc function of the Fc region of the immunoglobulin. For example, in some embodiments, the binder-drug conjugate is capable of binding to the Fc receptor on Fc receptor-positive cells via its Fc moiety. In some other embodiments, the binder-drug conjugate can activate Fc receptor-positive cells by binding to them, thereby initiating or increasing the expression of cytokines and / or co-stimulatory antigens. Furthermore, the binder-drug conjugate can transfer a second activation signal required for the physiological activation of T cells to T cells, at least via co-stimulatory antigens and / or cytokines.

[0823] In some embodiments, the binder-drug conjugate may possess antibody-dependent cytotoxicity (ADCC) function due to the binding of its Fc portion to other cells expressing Fc receptors present on the surface of effector cells from the immune system, such as immune cells, hepatocytes, and endothelial cells. ADCC is a cell-mediated immune defense mechanism in which effector cells of the immune system effectively dissolve membrane surface antigens on target cells that have been bound by antibodies, thus triggering tumor cell death via ADCC. In some other embodiments, the binder-drug conjugate is capable of exhibiting ADCC function.

[0824] As mentioned above, in addition to Fc-mediated cytotoxicity, the Fc moiety can help maintain serum levels of the binder-drug conjugate, which is crucial for its stability and persistence in vivo. For example, when the Fc moiety binds to Fc receptors on endothelial cells and phagocytes, the binder-drug conjugate can be internalized and recirculated back into the bloodstream, enhancing its half-life in vivo.

[0825] For illustrative purposes only, exemplary targets of additional affinity peptides include (but are not limited to) another immune checkpoint protein and immune co-stimulatory receptors (especially in cases where one or more additional affinity peptides can activate co-stimulatory receptors), receptors, cytokines, growth factors, or tumor-associated antigens.

[0826] In cases where the binder-drug conjugate is an affinity / antibody fusion protein, the immunoglobulin portion of the immunoglobulin may be a monoclonal antibody against CD20, CD30, CD33, CD38, CD52, VEGF, VEGF receptor, EGFR, or Her2 / neu. A few exemplary examples of such immunoglobulins include antibodies contained in any of the following: trastuzumab, panitumumab, cetuximab, obinutuzumab, rituximab, pertuzumab, alemtuzumab, bevacizumab, tositumomab, ibritumomab, ofatumumab, brentuximab, and gemtuzumab.

[0827] In some embodiments, the anti-PD-L1 affinity peptide is part of a binder-drug conjugate comprising one or more binding domains that inhibit immune checkpoint molecules expressed on T cells, including (but not limited to) PD-1, PD-L2, CTLA-4, NKG2A, KIR, LAG-3, TIM-3, CD96, VISTA, or TIGIT.

[0828] In some embodiments, the anti-...

Claims

1. A binder-drug conjugate, represented by any one of the following formulas: in CBM is a PD-L1-binding affinity polypeptide having an amino acid sequence represented by the following general formula (I): FR1-(Three) n -FR2-(No) m -FR3 (SEQ ID NO: 121) (I) in FR1 is a polypeptide sequence represented by MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID No. 1) or a polypeptide sequence having at least 95% homology with it; FR2 is a polypeptide sequence represented by GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID No. 2) or a polypeptide sequence having at least 95% homology with it; FR3 is a polypeptide sequence represented by EDLVLTGYQV DKNKDDELTG F (SEQ ID No. 3) or a polypeptide sequence having at least 95% homology with it; and Each occurrence of Xaa is individually an amino acid sequence that has at least 95% homology with any one of SEQ ID Nos. 6 to 75; n and m are each independent integers from 3 to 20; L 1 It is a polyether, N-succinimide-4-(2-pyridylthio)valerate, N-succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate, or N-succinimide-(4-iodo-acetyl)aminobenzoate. p represents an integer selected from 1 to 20; SRS has the characteristics of The structure represented; in R 2 Indicates H or (C1-C6) alkyl; R 3 Indicates H or (C1-C6) alkyl; R 4 It does not exist or represents (C1-C6) alkyl, -OH, -NH2 or halogen; X represents O or S; If L 2 For a triggered self-degrading connector, NH represents L 2 A portion of the amine, or if L 2 If it is a straight bond, then NH represents an amine that is part of DM; L 2 This refers to a triggered self-degrading linker or straight bond that can be cleaved by enzymes present outside the cells in diseased tissues; DM indicates the drug component; m represents an integer from 1 to 6; and n represents an integer from 1 to 500.

2. The binder-drug conjugate of claim 1, wherein n is an integer from 1 to 100.

3. The binder-drug conjugate as claimed in claim 1, wherein n is an integer from 1 to 5.

4. The binder-drug conjugate of claim 1, wherein R 2 For H.

5. The binder-drug conjugate of claim 1, wherein R 3 It can be methyl, ethyl, propyl or isopropyl.

6. The binder-drug conjugate of claim 1, wherein R 3 It is a methyl group.

7. The binder-drug conjugate of claim 1, wherein the diseased tissue is a tumor.

8. The binder-drug conjugate of claim 1, wherein the PD-L1 affinity polypeptide has an amino acid sequence represented by the following general formula: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEKTNETYGKLEAVQYKTQVLA-(Xaa) n -stone2-TNYYKVRAGDNKYMHLKVF-stone3-stone4-stone5-(stone) m -Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF(SEQ ID No. 4) in Each occurrence of Xaa is individually an amino acid sequence that has at least 95% homology with any one of SEQ ID Nos. 6 to 75; n and m are each an independent integer selected from 3 to 20; Xaa1 can be Gly, Ala, Val, Arg, Lys, Asp, or Glu; Xaa2 is Gly, Ala, Val, Ser, or Thr; Xaa3 can be Arg, Lys, Asn, Gln, Ser, or Thr; Xaa4 is Gly, Ala, Val, Ser, or Thr; Xaa5 can be Ala, Val, Ile, Leu, Gly, or Pro; Xaa6 is Gly, Ala, Val, Asp, or Glu; and Xaa7 can be Ala, Val, Ile, Leu, Arg, or Lys.

9. The binder-drug conjugate of claim 1, wherein the PD-L1 affinity polypeptide has an amino acid sequence represented by the following general formula: MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(Xaa) n -STNYYIKVRAGDNKYMHLKVFNGP-(Xaa) m -ADRVLTGYQVDKNKDDELTGF (SEQ ID No. 5) in Each occurrence of Xaa is individually an amino acid sequence with at least 95% homology to any one of SEQ ID Nos. 6 to 75; and n and m are each an independent integer selected from 3 to 20.

10. The binder-drug conjugate of claim 1, wherein L 1 It is N-succinimide-4-(2-pyridylthio)valerate, N-succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate or N-succinimide-(4-iodo-acetyl)aminobenzoate.

11. The binder-drug conjugate of claim 1, wherein L 1 It has a structure represented by the following formula: 。 12. The binder-drug conjugate of claim 1, wherein L 1 It is a polyether.

13. The binder-drug conjugate of claim 12, wherein L 1 It is poly(ethylene glycol).

14. The binder-drug conjugate of claim 1, wherein: CBM contains thiols; L 1 Depend on express; and p represents an integer from 1 to 100.

15. The binder-drug conjugate of claim 14, wherein p is 6 to 50.

16. The binder-drug conjugate of claim 14, wherein p is 6 to 12.

17. The binder-drug conjugate of claim 1, wherein: CBM contains thiols; L 1 Depend on express; and p is an integer from 1 to 20.

18. The binder-drug conjugate of claim 17, wherein p is 1 to 4.

19. The binder-drug conjugate of claim 1, wherein L 2 Choose from the following groups: -NH-(CH2)4-C(=O)-, -NH-(CH2)3-C(=O)-, p-aminobenzyloxycarbonyl (PABC) and 2,4-bis(hydroxymethyl)aniline.

20. The binder-drug conjugate of claim 1, wherein the drug portion is an immunomodulator.

21. The binder-drug conjugate of claim 1, wherein the drug portion is an immune activator.

22. The binder-drug conjugate of claim 21, wherein the immune activator is a STING agonist.

23. The binder-drug conjugate of claim 21, wherein the immune activator is a RIG-1 agonist.

24. The binder-drug conjugate of claim 21, wherein the immune activator is a Toll-like receptor (TLR) agonist.

25. The binder-drug conjugate of claim 24, wherein the TLR agonist is selected from the group consisting of TLR1 / 2 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 / 2 agonists, TLR7 agonists, TLR7 / 8 agonists, TLR7 / 9 agonists, TLR8 agonists, TLR9 agonists, and TLR11 agonists.

26. The binder-drug conjugate of claim 24, wherein the TLR agonist is selected from the group consisting of TLR3 agonists, TLR7 agonists, TLR7 / 8 agonists and TLR9 agonists.

27. The binder-drug conjugate of claim 20, wherein the immunomodulator is an immuno-DASH inhibitor that inhibits the enzymatic activity of DPP8 and DPP9 and induces pyroptosis in macrophages.

28. The binder-drug conjugate of claim 27, wherein the immunomodulator has a molecular weight of less than 5,000 amu.

29. The binder-drug conjugate of claim 27, wherein the immunomodulator has a molecular weight of less than 2,500 amu.

30. A binder-drug conjugate comprising a polypeptide including an affinity sequence of a PD-L1 receptor on target cells in a tumor and having one or more drug-conjugate moieties linked thereto, said drug-conjugate moieties being represented by the following formula: in L 1 Indicates a spacer base or a straight bond; SRS has the characteristics of The structure represented; L 2 Indicates a trigger-activated self-degrading connector or a straight key; R 2 Indicates H or (C1-C6) alkyl; R 3 Indicates H or (C1-C6) alkyl; R 4 It does not exist or represents (C1-C6) alkyl, -OH, -NH2 or halogen; X represents O or S; If L 2 For a triggered self-degrading connector, NH represents L 2 A portion of the amine, or if L 2 If it is a straight bond, then NH represents an amine that is part of DM; DM represents the drug component; n represents an integer selected from 1 to 500; and The affinity polypeptide has an amino acid sequence represented by the following general formula (I): FR1-(Three) n1 -FR2-(No) m1 -FR3 (I) in FR1 is a polypeptide sequence represented by MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID No. 1) or a polypeptide sequence having at least 95% homology with it; FR2 is a polypeptide sequence represented by GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID No. 2) or a polypeptide sequence having at least 95% homology with it; FR3 is a polypeptide sequence represented by EDLVLTGYQV DKNKDDELTG F (SEQ ID No. 3) or a polypeptide sequence having at least 95% homology with it; and Each time Xaa appears, it is an individual amino acid residue; and n1 and m1 are each an independent integer selected from 3 to 20.

31. The binder-drug conjugate of claim 30, wherein n is an integer selected from 1 to 100.

32. The binder-drug conjugate of claim 30, wherein n is an integer selected from 1 to 5.

33. The binder-drug conjugate of claim 30, wherein R 2 For H.

34. The binder-drug conjugate of claim 30, wherein R 3 It can be methyl, ethyl, propyl or isopropyl.

35. The binder-drug conjugate of claim 30, wherein R 3 It is a methyl group.

36. The binder-drug conjugate of claim 30, wherein the affinity polypeptide has an amino acid sequence represented by the following general formula: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEKTNETYGKLEAVQYKTQVLA-(Xaa) n1 -stone2-TNYYKVRAGDNKYMHLKVF-stone3-stone4-stone5-(stone) m1 -Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF(SEQ ID No. 4) in Each time Xaa appears, it is an individual amino acid residue; n1 and m1 are each an independent integer selected from 3 to 20; Xaa1 can be Gly, Ala, Val, Arg, Lys, Asp, or Glu; Xaa2 is Gly, Ala, Val, Ser, or Thr; Xaa3 can be Arg, Lys, Asn, Gln, Ser, or Thr; Xaa4 is Gly, Ala, Val, Ser, or Thr; Xaa5 can be Ala, Val, Ile, Leu, Gly, or Pro; Xaa6 is Gly, Ala, Val, Asp, or Glu; and Xaa7 can be Ala, Val, Ile, Leu, Arg, or Lys.

37. The binder-drug conjugate of claim 30, wherein the affinity polypeptide has an amino acid sequence represented by the following general formula: MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(Xaa) n1 -STNYYIKVRAGDNKYMHLKVFNGP-(Xaa) m1 -ADRVLTGYQVDKNKDDELTGF (SEQ ID No. 5) in Each time Xaa appears, it is an individual amino acid residue; and n1 and m1 are each an independent integer selected from 3 to 20.

38. The binder-drug conjugate of claim 30, wherein the affinity polypeptide has an amino acid sequence selected from SEQ ID No. 76 to 84, or an amino acid sequence having at least 95% homology thereto.

39. The binder-drug conjugate of claim 30, wherein at least one drug-conjugate moiety is linked to the affinity sequence via a thiol side chain of a cysteine ​​residue in the affinity sequence.

40. The binder-drug conjugate of claim 30, wherein at least one drug-drug conjugate portion is linked to the affinity sequence via a thiol side chain of a cysteine ​​residue in a portion of the affinity sequence region corresponding to FR1, FR2 and / or FR3.

41. The binder-drug conjugate of claim 30, wherein the affinity is a portion of a fusion protein comprising a polypeptide portion selected from the group consisting of: an Fc domain or a portion thereof, albumin or a portion thereof, a polypeptide portion binding albumin, transferrin or a portion thereof, a polypeptide portion binding transferrin, fibronectin or a portion thereof, or a polypeptide portion binding fibronectin.

42. The binder-drug conjugate of claim 30, wherein L 1 It is N-succinimide-4-(2-pyridylthio)valerate, N-succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate or N-succinimide-(4-iodo-acetyl)aminobenzoate.

43. The binder-drug conjugate of claim 30, wherein L 1 It is a polyether.

44. The binder-drug conjugate of claim 43, wherein L 1 It is poly(ethylene glycol).

45. The binder-drug conjugate of claim 30, wherein: The affinity group comprises a thiol; L 1 Depend on It means; and p represents an integer selected from 1 to 100.

46. ​​The binder-drug conjugate of claim 45, wherein p is 6 to 50.

47. The binder-drug conjugate of claim 45, wherein p is 6 to 12.

48. The binder-drug conjugate of claim 30, wherein: The affinity group comprises a thiol; L 1 Depend on It means; and p is an integer selected from 1 to 20.

49. The binder-drug conjugate of claim 48, wherein p is 1 to 4.

50. The binder-drug conjugate of claim 30, wherein L 2 Choose from the following groups: -NH-(CH2)4-C(=O)-, -NH-(CH2)3-C(=O)-, p-aminobenzyloxycarbonyl (PABC) and 2,4-bis(hydroxymethyl)aniline.

51. The binder-drug conjugate of claim 30, wherein the drug portion is an immunomodulator.

52. The binder-drug conjugate of claim 30, wherein the drug portion is an immune activator.

53. The binder-drug conjugate of claim 52, wherein the immunomodulator is a STING agonist.

54. The binder-drug conjugate of claim 52, wherein the immunomodulator is a RIG-1 agonist.

55. The binder-drug conjugate of claim 52, wherein the immunomodulator is a Toll-like receptor (TLR) agonist.

56. The binder-drug conjugate of claim 55, wherein the TLR agonist is selected from the group consisting of TLR1 / 2 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 / 2 agonists, TLR7 agonists, TLR7 / 8 agonists, TLR7 / 9 agonists, TLR8 agonists, TLR9 agonists, and TLR11 agonists.

57. The binder-drug conjugate of claim 55, wherein the TLR agonist is selected from the group consisting of TLR3 agonists, TLR7 agonists, TLR7 / 8 agonists and TLR9 agonists.

58. The binder-drug conjugate of claim 51, wherein the immunomodulator is an immuno-DASH inhibitor that inhibits the enzymatic activity of DPP8 and DPP9 and induces pyroptosis in macrophages.

59. The binder-drug conjugate of claim 51, wherein the immunomodulator has a molecular weight of less than 5,000 amu.

60. The binder-drug conjugate of claim 51, wherein the immunomodulator has a molecular weight of less than 2,500 amu.

61. Use of the binder-drug conjugate according to any one of claims 1-60 in the manufacture of a medicament for treating cancer, wherein the cancer is acute myeloid leukemia (AML).

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