Tumor microenvironment-activated drug-binder conjugates, and uses related thereto

Abstract

To provide a new system for co-delivery of two classes of therapeutic agents.SOLUTION: Disclosed are binder-drug conjugates that are activated extracellular, with both the binder and the free drug moiety have pharmacological activity.SELECTED DRAWING: None

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 680,300, filed on June 4, 2018.

Background Art

[0002] The PD - 1 - PD - L1 interaction is known to cause 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 affected by complex immune regulatory networks. In most advanced cancers, except for Hodgkin lymphoma (which has high PD - L1 / L2 expression) and melanoma (which has a high amount of tumor gene mutations), the objective response rate by anti - PD - 1 / PD - L1 monotherapy is only about 20%, and immune - related toxicities and rapid progression can occur in a small subset of patients during PD - 1 / PD - L1 inhibition therapy. The fact that the lack of efficacy in up to 80% of patients is not necessarily associated with negative expression of PD - 1 and PD - L1 suggests that the role of PD - 1 / PD - L1 in immunosuppression and the mechanism of action of antibodies remains poorly defined. Similarly, similar limitations have been observed in CTLA - 4 and other checkpoint pathways. Therefore, important synergistic immune regulatory mechanisms within or outside the scope of the PD - 1 / PD - L1, CTLA - 4, and other checkpoint networks need to be targeted to increase the efficacy of immune checkpoint inhibition therapy and, in some cases, reduce toxicity.

[0003] In this regard, drugs that induce innate immune responses, such as agonists of STING, RIG - I, and TLR, are thought to have the potential to enhance the efficacy of immune - oncology checkpoint inhibitors. However, dose - limiting toxicities are products of systemic innate immune activation, and the maximum tolerated dose does not achieve the therapeutic dose in many patients, so these types of agents are often too toxic for systemic use.

[0004] The present invention is based on a new system for the co-delivery, in a form that addresses the problem of systemic toxicity of any of the components, particularly the innate immune inducer, while keeping it in a pharmacologically inactive form until released by proteases in the tumor microenvironment, of two classes of therapeutic agents - an innate immune inducer that causes local inflammatory events in tumors that induce a strong immune response, and one or more checkpoint inhibitors or co-stimulatory agonists that promote or maintain an adaptive immune response. More simply put, one agent induces an anti-tumor immune response, and the other agent ensures that it functions effectively when it reaches the tumor. The checkpoint inhibitor or co-stimulatory agonist in combination with TME enzyme release helps to place the drug within the tumor and improve the therapeutic index compared to using the drugs of each component individually.

Summary of the Invention

Means for Solving the Problems

[0005] One aspect of the present invention relates to a binder-drug conjugate comprising: (i) a cell-binding moiety that binds to a cell surface feature on target cells in the tissue of a disease state, wherein the cell surface feature undergoes slow internalization upon binding of the binder-drug conjugate; (ii) a drug moiety that has a pharmacological effect on bystander cells in proximity to the target cells and has an EC50 for a pharmacological effect that is at least 2-fold attenuated when part of the binder-drug conjugate compared to the free drug moiety released from the binder-drug conjugate; and (iii) a linker moiety that covalently attaches the polypeptide binder moiety to the drug moiety and contains a substrate recognition sequence that is cleavable by an enzyme that is extracellularly present in the diseased tissue, such that in the presence of the enzyme, the linker moiety is cleaved to release the free drug moiety.

[0006] In certain embodiments, the drug moiety has an EC50 for a pharmacological effect that is at least 5-fold attenuated, more preferably at least 10, 20, 30, 40, 50, 75, 100, 250, 500, or even 1000-fold attenuated, when it is part of the binder-drug conjugate as compared to the free drug moiety released from the binder-drug conjugate.

[0007] In certain embodiments, the diseased tissue is a tumor. In certain embodiments, the target cells are tumor cells. In certain embodiments, the target cells are macrophages, monocyte-derived suppressor cells (MDSC), dendritic cells, fibroblasts, T cells, NK cells, mast cells, granulocytes, eosinophils, and B cells.

[0008] In certain embodiments, when the binder-drug conjugate binds to a surface feature on the target cell, it has an internalization half-life of at least 6 hours, more preferably at least 10, 12, 14, 16, 18, 20, 24, 36, 48, 60, 75, or even 100 hours.

[0009] In certain embodiments, the cell surface feature is a protein that is selectively expressed or upregulated by the target cells in the diseased tissue as compared to normal cells from the healthy state of the tissue. For example, the protein is detectable on the surface of the target cells at a level that is 2-fold higher than that of normal cells from the tissue, and even more preferably at a level that is at least 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, or even 1000-fold higher than that of normal cells from the tissue.

[0010] In certain embodiments, the cell surface feature is a protein that is selectively expressed or upregulated by the target cells in the diseased tissue as compared to cells from other tissues, particularly cells from vital organs. For example, the protein is detectable on the surface of the target cells at a level that is 2-fold higher than that of cells from the tissue, and even more preferably at a level that is at least 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, or even 1000-fold higher than that of cells from other tissues.

[0011] In certain embodiments, the cell surface feature is a checkpoint protein and the binder moiety is an antagonist of that checkpoint. Examples of checkpoint proteins include 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, CSF-1R, and more preferably those selected from the group consisting of CTLA-4, PD-1, LAG-3, TIM-3, BTLA, VISTA, HVEM, TIGIT, PVR, PD-L1, and CD160.

[0012] In certain embodiments, the cell surface feature is a co-stimulatory receptor and the binder moiety is a co-stimulatory agonist of the receptor. Examples include 4-1BB, 4-1BB-L, OX40, OX40-L, GITR, CD28, CD40, CD40-L, ICOS, ICOS-L, LIGHT, and CD27, and more preferably surface features that are co-stimulatory receptors or ligands selected from the group consisting of 4-1BB, OX40, GITR, CD40, and ICOS.

[0013] In certain embodiments, the cell-binding moiety is an antibody such as, for example, a humanized antibody, a human antibody, or a chimeric antibody, or its antigen-binding portion that binds to a cell surface feature, such as Fab, F(ab)2, F(ab’), F(ab’)2, F(ab’)3, Fd, Fv, disulfide-bonded Fv, dAb, or sdAb (or nanobody), CDR, scFv, (scFv)2, diabody-scFv, bis-scFv, tascFv (tandem scFv), AVIBODY (e.g., diabody, triabody, tetrabody), T cell engager (BiTE), scFv-Fc, Fcab, mAb2, small modular immunopharmaceutical (SMIP), Genmab / unibody or duo-body, V-NAR domain, IgNAR, minibody, IgGACH2, DVD-Ig, probody, intrabody, or multispecific antibody.

[0014] In other embodiments, the binder moiety is a non-antibody scaffold such as selected from the group consisting of an affibody, an affimer, an affilin, an anticalin, an atrimer, an abimer, DARPins, FN3 scaffolds (e.g.,adnectin and centyrin), finomers, knotted domains, nanofitins, propeptins, OBodys, tribodies, abimers, bicyclic peptides, and Cys-knots.

[0015] In certain embodiments, the linker moiety comprises 2, 3 or even 4 substrate recognition sequences that are cleavable by the same or different enzymes present in diseased tissue (at least one of which is extracellular), such that in the simultaneous or sequential presence of various enzymes, the linker moiety can be completely cleaved to liberate the free drug moiety. For example, a linker having two different substrate recognition sequences can be engineered to require cleavage by both MMP and FAPα. In a preferred embodiment, cleavage by one of the two enzymes is required to have occurred first by the other enzyme - i.e., by creating a linker that is a poor substrate for FAPα when intact and is improved as a substrate for cleavage by FAPα after MMP cleavage has occurred, MMP cleavage can be required before FAPα cleavage.

[0016] Further to explain, the binder-drug conjugate can be represented by one of the formulas,

Chemical formula

[0017] In certain embodiments, L 1 is a hydrocarbon (linear or cyclic) such as 6-maleimidocaproyl, maleimidopropanoyl, and maleimidomethylcyclohexane-1-carboxylate, or L 1 is N-succinimidyl 4-(2-pyridylthio)pentanoate, N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, N-succinimidyl (4-iodo-acetyl)aminobenzoate.

[0018] In certain embodiments, L 1 is a polyether such as poly(ethylene glycol) or other hydrophilic linker. For example, when the CBM contains a thiol (e.g., a cysteine residue), L 1 can be poly(ethylene glycol) bonded to the thiol group via a maleimide moiety as represented by the following formula, [Chemical formula] wherein p represents an integer from 1 to 100, preferably from 6 to 50, more preferably from 6 to 12.

[0019] In other embodiments, when the CBM contains a thiol and L 1 is a hydrocarbon moiety bonded to the thiol group via a maleimide moiety, L 1 can be represented by the formula, [Chemical formula] wherein p represents an integer from 1 to 20, preferably from 1 to 4.

[0020] In certain embodiments of the binder-drug conjugates of the present invention, the substrate recognition sequence is cleaved by extracellular proteases, preferably proteases having protease activity located in the extracellular domain of the target tissue - i.e., serine proteases, metalloproteases or cysteine proteases as cell surface proteases or secreted / free proteases.

[0021] In certain embodiments, the protease is present extracellularly in the diseased state of the patient's tissue at a level that is at least 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, or even more than 1000-fold higher than that present extracellularly in the healthy state of the patient's tissue.

[0022] In certain embodiments, the protease is present extracellularly in the diseased state of the patient's tissue at a level that is at least 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, or even more than 1000-fold higher than that present in other tissues of the patient.

[0023] In certain embodiments, the protease is a matrix metalloproteinase. The matrix metalloproteinase can be a membrane-bound matrix metalloproteinase (e.g., MMP14-17 and MMP24-25) or a secreted matrix metalloproteinase (e.g., MMP1-13 and MMP18-23 and MMP26-28, etc.). In certain embodiments, the metalloproteinase is MMP1, MMP2, MMP3, MMP4, MMP9, MMP11, MMP13, MMP14, MMP17 or MMP19, and more preferably, MMP2, MMP9 or MMP14.

[0024] In certain embodiments, the protease is an A disintegrin and metalloproteinase (ADAM), or an A disintegrin or metalloproteinase with thrombospondin motif (ADAMTS).

[0025] In certain embodiments, the protease is legumain, matriptase (MT-SP1), neutrophil elastase, TMPRSS, thrombin, u-type plasminogen activator (also called uPA, urokinase), PSMA or CD10 (CALLA).

[0026] In certain embodiments, the protease is a post-proline cleaving protease such as, for example, fibroblast activation protein alpha (FAPα).

[0027] In certain embodiments of the subject binder-drug conjugate, the substrate recognition sequence is cleaved by fibroblast activation protein alpha (FAPα) and is represented by the following formula:

Chemical formula

[0028] In certain embodiments, R 2 is H, R 3 is methyl, R 4 is absent, and X is O.

[0029] In certain embodiments, L 2is a self-cleaving linker 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 certain embodiments, p-aminobenzyloxycarbonyl (PABC) satisfies the P'1 specificity requirement of FAPα, so especially when the target recognition sequence is cleaved by FAPα, L 2 is p-aminobenzyloxycarbonyl (PABC).

[0030] In certain embodiments, the free drug moiety interacts with an intracellular target, and the pharmacological effect of the drug moiety depends on the free drug moiety being cell permeable, i.e., able to interact with its intracellular target, whereas when part of the binder-drug conjugate, the drug moiety is substantially cell impermeable. For example, the accumulation rate of the binder-drug conjugate in cells is less than 50% of the accumulation rate of the free drug moiety, more preferably less than 25%, 10%, 5%, 1%, or 0.1% of the accumulation rate of the free drug moiety. For example, the EC50 for the pharmacological effect of the free drug moiety is at least one-half (more potent) of the binder-drug conjugate, more preferably at least 5, 10, 20, 30, 40, 50, 100, 250, 500, or even 1 / 1000 of the binder-drug conjugate.

[0031] In certain embodiments, the free drug moiety interacts with an extracellular target, and the pharmacological effect of the drug moiety is substantially attenuated when covalently linked to L 1 For example, the EC50 for the pharmacological effect of the free drug moiety is at least one-half (more potent) of the binder-drug conjugate, more preferably at least 5, 10, 20, 30, 40, 50, 100, 250, 500, or even 1 / 1000 of the binder-drug conjugate.

[0032] In certain embodiments, the binder-drug conjugate, when systemically delivered, has a therapeutic index that is at least 2-fold greater, and even more preferably at least 5, 10, 20, 30, 40, 50, 100, 250, 500, or even 1000-fold greater than the systemic delivery of the free drug moiety.

[0033] In certain embodiments, the free drug moiety is an immunomodulatory agent, which includes drug moieties that act as immunostimulants and / or inducers of innate immune pathway responses. In certain embodiments, the free drug moiety induces the production of IFN-α. In certain embodiments, the free drug moiety induces the production of pro-inflammatory cytokines. In certain embodiments, the free drug moiety induces the production of IL-1β. In certain embodiments, the free drug moiety induces the production of IL-18.

[0034] In certain embodiments, the free drug moiety promotes the proliferation and survival of effector cells including NK, γδT, and CD8+ T cells.

[0035] In certain embodiments, the free drug moiety is an ImmunoDASH inhibitor that inhibits the enzymatic activity of DPP8 and DPP9 and induces macrophage pyroptosis in vitro and / or in vivo.

[0036] In certain embodiments, the free drug moiety is a molecule of a damage-associated molecular pattern. In certain embodiments, the free drug moiety is a molecule of a pathogen-associated molecular pattern.

[0037] In certain embodiments, the free drug moiety is a STING agonist.

[0038] In certain embodiments, the free drug moiety is a RIG-1 agonist.

[0039] In certain embodiments, the free drug moiety is a toll-like receptor (TLR) agonist selected from the group consisting of TLR1 / 2 agonist, TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR6 / 2 agonist, TLR7 agonist, TLR7 / 8 agonist, TLR7 / 9 agonist, TLR8 agonist, TLR9 agonist, and TLR11 agonist, preferably TLR3 agonist, TLR7 agonist, TLR7 / 8 agonist, and TLR9 agonist.

[0040] In certain embodiments, the free drug moiety is a cyclic dinucleotide.

[0041] In certain embodiments, the free drug moiety is ADU-S100.

[0042] In certain embodiments, the free drug moiety is a RIG-I agonist, and the RIG-I agonist is KIN700, KIN1148, KIN600, KIN500, KIN100, KIN101, KIN400, KIN2000, or SB-9200.

[0043] In certain embodiments, the free drug moiety 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.

[0044] In certain embodiments, the free drug moiety is cytotoxic to cancer-associated fibroblasts (CAFs).

[0045] In certain embodiments, the free drug moiety polarizes the tumor-associated macrophage population towards M1 macrophages and / or inhibits the immunosuppressive activity of M2 macrophages.

[0046] In certain embodiments, the free drug moiety accelerates T cell priming and / or dendritic cell trafficking.

[0047] In certain embodiments, the free drug moiety inhibits or depletes Treg cells, for example, by blocking immunosuppressive function or migration to lymph nodes and / or the tumor microenvironment.

[0048] In certain embodiments, the therapeutic index (TI) of the conjugate-drug conjugate is at least 5-fold greater, more preferably at least 10, 20, 30, 40, 50, 75-fold, or even 100-fold greater than the therapeutic index of the free drug moiety when administered systemically.

[0049] In certain embodiments, the free drug moiety is a small molecule inhibitor, i.e., a small molecule inhibitor having a molecular weight of less than 5000 amu, preferably less than 2500 amu, and even more preferably less than 1500 amu.

[0050] Another aspect of the invention provides a conjugate-drug conjugate comprising one or more small domain-binding polypeptide sequences (e.g., antibody fragments or non-antibody scaffolds), preferably one or more affimer sequences, that bind to and have attached thereto one or more drug conjugate moieties on the cell surface proteins on cells within the tumor, wherein the drug conjugate moiety is represented by the formula

Chemical formula

[0051] When the binder-drug conjugate binds to the surface characteristics of the target cell, it has an internal migration half-life of at least 6 hours, more preferably at least 10, 12, 14, 16, 18, 20, 24, 36, 48, 60, 75, or even 100 hours.

[0052] In certain embodiments, at least one of the small domain binding polypeptide sequences is a PD-L1 binding moiety.

[0053] In certain embodiments, the polypeptide of the binder-drug conjugate has a Kd of 1×10 -6 M or less (and in embodiments where the polypeptide is multivalent with a valence of two or more for binding to PD-L1, 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 less) and binds to PD-L1, inhibiting the interaction of PD-L1 with PD-1 to which the polypeptide binds.

[0054] Another aspect of the invention provides a multispecific binder-drug conjugate comprising: (i) a polypeptide comprising two or more different binding domain polypeptide sequences that selectively bind to two different cell surface proteins on two different cell types within a tumor, and (ii) one or more drug conjugate moieties attached to the polypeptide, the drug conjugate moiety being represented by the formula

Chemical formula

[0055] When the multi-specific binding agent-drug conjugate binds to any of the surface proteins, it has an internalization half-life of at least 6 hours, more preferably at least 10, 12, 14, 16, 18, 20, 24, 36, 48, 60, 75 hours, or 100 hours.

[0056] In certain embodiments of the multi-specific binding agent-drug conjugate, the polypeptide comprises a first binding domain polypeptide sequence that selectively binds to a tumor cell antigen and a second binding domain polypeptide sequence that selectively binds to a cell selected from the group consisting of macrophages, monocyte-derived suppressor cells (MDSC), dendritic cells, fibroblasts, NK cells, mast cells, granulocytes, eosinophils, and B cells.

[0057] In certain embodiments of the multi-specific binding agent-drug conjugate, the polypeptide is a checkpoint inhibitor or co-stimulatory agonist and comprises a first binding domain polypeptide sequence that binds to a checkpoint protein or co-stimulatory receptor protein expressed on tumor-infiltrating lymphocytes (e.g., 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 co-stimulatory proteins) and a second binding domain polypeptide sequence that is a checkpoint inhibitor that binds to a checkpoint (such as PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, CD155, HVEM, or galectin-9) expressed on tumor cells.

[0058] Yet another aspect of the invention relates to a combination of a PD-L1 inhibitor / natural immunity stimulant comprising a PD-L1 binding polypeptide conjugated to a drug moiety that is an inducer of sterile natural immune responses (an ImmunoDASH inhibitor, a STING agonist, a TLR7 / 8 agonist, or a RIG-1 agonist), wherein the PD-L1 binding polypeptide causes accumulation of the PD-L1 inhibitor / natural immunity stimulant within the tumor as compared to other tissues of the patient, and the drug moiety is selectively released from the PD-L1 binding polypeptide within the tumor microenvironment as compared to other tissues of the patient.

[0059] In certain embodiments of the drug conjugates of the invention, the molecule has a Kd of 1×10 -6 M or less (and more preferably a Kd of 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M or less) for binding to PD-L1 and comprises a PD-L1 binding moiety that is an Affimer polypeptide sequence that inhibits the interaction of the PD-L1 to which it binds with PD-1.

[0060] In certain embodiments, the PD-L1 binding Affimer polypeptide binds human PD-L1 and blocks the interaction with human PD-1. In certain embodiments, the PD-L1 binding Affimer polypeptide has a Kd of 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, or even 1×10 -10 M or less for binding to PD-L1. In certain embodiments, the PD-L1 binding Affimer polypeptide has a K -3 off -1 of 10 -4 s -1 or less, 10 -5 s -1 or less, or even 10 off s 3 or less for binding to PD-L1. In certain embodiments, the PD-L1 binding Affimer polypeptide has an on rate of 10 -1 s -1 or faster, 104 M -1 s -1 Faster than, 10 5 M -1 s -1 Faster than, or even 10 6 M -1 s -1 Faster than K on binds to PD-L1. In certain embodiments, the PD-L1 binding affimer polypeptide binds to PD-L1 with 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 in a competitive binding assay with human PD-1.

[0061] In certain embodiments, the PD-L1 binding affimer polypeptide 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 certain 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.

[0062] In certain embodiments, the PD-L1 binding affimer polypeptide has an amino acid sequence represented by the general formula (I), FR1-(Xaa) n -FR2-(Xaa) m -FR3(I) wherein, 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 thereto; FR2 is a polypeptide sequence represented by GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2), or a polypeptide sequence having at least 70% homology thereto; FR3 is a polypeptide sequence represented by EDLVLTGYQV DKNKDDELTG F (SEQ ID NO: 3), or a polypeptide sequence having at least 70% homology thereto; and, Xaa is an amino acid residue that is individual for each occurrence; and, n and m are each independently an integer from 3 to 20.

[0063] For certain embodiments, FR1 may be a polypeptide sequence having at least 80%, 85%, 90%, 95% or 98% homology with SEQ ID NO: 1. For certain embodiments, FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or 98% homology with SEQ ID NO: 2. For certain embodiments, FR3 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or 98% homology with SEQ ID NO: 2.

[0064] For those embodiments in which at least one drug conjugate moiety is attached to the Affimer sequence via the thiol side chain of a cysteine introduced into the Affimer sequence, the cysteine is preferably provided as part of the Affimer sequence region corresponding to FR1, FR2 and / or FR3, and more preferably, a substitution to an amino acid residue in the Affimer is provided such that its side chain is solvent accessible and not involved in hydrogen bonding with other parts of the Affimer. Generally, cysteine will not be introduced into loop (Xaa) n or (Xaa) m will not be introduced.

[0065] In certain embodiments, the PD-L1 binding Affimer polypeptide has an amino acid sequence represented by the general formula: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(Xaa) n -Xaa2-TNYYIKVRAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa) m -Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4) wherein, Xaa is an amino acid residue that is individual for each occurrence; n and m are each independently an integer from 3 to 20; Xaa1 is Gly, Ala, Val, Arg, Lys, Asp, or Glu; Xaa2 is Gly, Ala, Val, Ser or Thr; Xaa3 is Arg, Lys, Asn, Gln, Ser, or Thr; Xaa4 is Gly, Ala, Val, Ser, or Thr; Xaa5 is Ala, Val, Ile, Leu, Gly or Pro; Xaa6 is Gly, Ala, Val, Asp, or Glu; and Xaa7 is Ala, Val, Ile, Leu, Arg, or Lys.

[0066] For certain embodiments, Xaa1 is Gly, Ala, Arg or Lys, and more preferably Gly or Arg at a further higher level. For certain embodiments, Xaa2 is Gly or Ser. For certain embodiments, Xaa3 is Arg, Lys, Asn or Gln, more preferably Lys or Asn. For certain embodiments, Xaa4 is Gly or Ser. For certain embodiments, Xaa5 is Ala, Val, Ile, Leu, Gly or Pro, more preferably Ile, Leu or Pro, even more preferably Leu or Pro. For certain embodiments, Xaa6 is Ala, Val, Asp or Glu, even more preferably Ala or Glu. For certain embodiments, Xaa7 is Ile, Leu or Arg, more preferably Leu or Arg.

[0067] For those embodiments in which at least one drug conjugate moiety is attached to the affimer sequence via the thiol side chain of a cysteine introduced into the affimer sequence, the cysteine is preferably in a loop sequence (Xaa) n or (Xaa) mIt will be provided in a part of the affimer sequence that is not accompanied. Therefore, SEQ ID NO: 4 may contain 1 to 5 cysteines instead of amino acid residues at various positions of the sequence.

[0068] In certain embodiments, the PD-L1 binding affimer polypeptide has an amino acid sequence represented by the general formula: MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(Xaa) n -STNYYIKVRAGDNKYMHLKVFNGP-(Xaa) m -ADRVLTGYQVDKNKDDELTGF (SEQ ID NO: 5) Wherein Xaa is an amino acid residue individually for each occurrence; and n and m are each independently an integer from 3 to 20.

[0069] For those embodiments in which at least one drug conjugate moiety is added to the affimer sequence via the thiol side chain of cysteine introduced into the affimer sequence, the cysteine is preferably in the loop sequence (Xaa) n Or (Xaa) m It will be provided in a part of the affimer sequence other than that provided. Therefore, SEQ ID NO: 5 may contain 1 to 5 cysteines instead of amino acid residues at various positions of the sequence.

[0070] In a specific embodiment of the above sequence, (Xaa) n ("Loop 2") is an amino acid sequence represented by the general formula (II), -aa1-aa2-aa3-Gly-Pro-aa4-aa5-Trp-aa6- (II) Wherein, aa1 represents an amino acid residue having a basic side chain; aa2 represents an amino acid residue, preferably a neutral polar or non-polar side chain or a charged (acidic or basic) side chain, more preferably an amino acid residue having a small aliphatic side chain, a neutral polar side chain or a basic or acidic side chain; aa3 represents an amino acid residue having an aromatic or basic side chain; aa4 represents 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; aa5 represents an amino acid residue having a neutral polar or charged (acidic or basic) or small aliphatic or aromatic side chain; preferably a neutral polar side chain or a charged side chain; and aa6 represents an amino acid residue having an aromatic or acidic side chain.

[0071] For certain embodiments, aa1 represents Lys, Arg or His, more preferably Lys or Arg. For certain embodiments, aa2 represents Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg or His, more preferably Ala, Gln, Asp or Glu. For certain embodiments, aa3 represents Phe, Tyr, Trp, Lys, Arg or His, preferably Phe, Tyr, Trp, more preferably His or Tyr, Trp or His. For certain embodiments, aa4 represents Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg or His, more preferably Gln, Lys, Arg, His, Asp or Glu. For certain embodiments, aa5 represents Ser, Thr, Asn, Gln, Asp, Glu, Arg or His, more preferably Ser, Asn, Gln, Asp, Glu or Arg. For certain embodiments, aa6 represents Phe, Tyr, Trp, Asp or Glu, preferably Trp or Asp, more preferably Trp.

[0072] In certain other embodiments of the above sequences, (Xaa) n ("Loop 2") is an amino acid sequence represented by the general formula (III), -aa1-aa2-aa3-Phe-Pro-aa4-aa5-Phe-Trp- (III) wherein, aa1 represents an amino acid residue having a basic side chain or an aromatic side chain; 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 having a small aliphatic side chain, a neutral polar side chain or a basic or acidic side chain; 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, even more preferably Tyr, Trp or His; aa4 represents a neutral polar side chain or a nonpolar side chain or a charged (acidic or basic) side chain, preferably an amino acid residue having 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, even more preferably Gln, Lys, Arg, His, Asp or Glu; and aa5 represents a neutral polar side chain or a charged (acidic or basic) side chain or a small aliphatic or aromatic side chain; preferably an amino acid residue having a neutral polar side chain or a charged side chain, more preferably Ser, Thr, Asn, Gln, Asp, Glu, Arg or His, even more preferably Ser, Asn, Gln, Asp, Glu or Arg.

[0073] For certain 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 certain embodiments, aa2 represents Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg or His, and even more preferably Ala, Gln, Asp or Glu. For certain 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 certain 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 certain embodiments, aa5 represents Ser, Thr, Asn, Gln, Asp, Glu, Arg or His, and even more preferably Ser, Asn, Gln, Asp, Glu or Arg.

[0074] In certain embodiments of the above sequences, (Xaa) n ("Loop 2") is an amino acid sequence selected from amino acid residues 6 to 40 of SEQ ID NO: 6, or an amino acid sequence having at least 80% homology thereto, more preferably an amino acid sequence having at least 85%, 90%, 95% or even 98% homology thereto.

[0075] In certain embodiments of the above sequences, (Xaa) n ("Loop 2") is an amino acid sequence selected from amino acid residues 6 to 40 of SEQ ID NO: 6, or an amino acid sequence having at least 80% identity thereto, more preferably an amino acid sequence having at least 85%, 90%, 95% or even 98% identity thereto.

[0076] In certain embodiments of the above sequences, (Xaa) m ("Loop 4") is an amino acid sequence represented by the general formula (IV), -aa7-aa8-aa9-aa10-aa11-aa12-aa13-aa14-aa15-(IV) In the formula, aa7 represents an amino acid residue having a neutral polar side chain, a nonpolar side chain, or an acidic side chain; aa8 represents an amino acid residue, preferably an amino acid residue having a neutral polar side chain, a nonpolar side chain, a charged (acidic or basic) side chain, or an aromatic side chain, more preferably a charged (acidic or basic) side chain; aa9 represents an amino acid residue, preferably an amino acid residue having a neutral polar side chain, a nonpolar side chain, a charged (acidic or basic) side chain, or an aromatic side chain, more preferably a neutral polar side chain or an acidic side chain; aa10 represents an amino acid residue, preferably an amino acid residue having a neutral polar side chain, a nonpolar side chain, a charged (acidic or basic) side chain, or an aromatic side chain, more preferably a neutral polar side chain or a basic or acidic side chain; 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 a neutral polar side chain or a basic or acidic side chain; 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, more preferably an acidic side chain; 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; 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; and aa15 represents an amino acid residue, preferably an amino acid residue having a neutral polar side chain, a neutral nonpolar side chain, or a charged (acidic or basic) side chain.

[0077] For certain embodiments, aa7 represents Gly, Ala, Val, Pro, Trp, Gln, Ser, Asp or Glu, even more preferably Gly, Ala, Trp, Gln, Ser, Asp or Glu. For certain embodiments, aa8 represents Asp, Glu, Lys, Arg, His, Gln, Ser, Thr, Asn, Ala, Val, Pro, Gly, Tyr or Phe, even more preferably Asp, Glu, Lys, Arg, His or Gln. For certain embodiments, aa9 represents Gln, Ser, Thr, Asn, Asp, Glu, Arg, Lys, Gly, Leu, Pro or Tyr, even more preferably Gln, Thr or Asp. For certain embodiments, aa10 represents Asp, Glu, Arg, His, Lys, Ser, Gln, Asn, Ala, Leu, Tyr, Trp, Pro or Gly, even more preferably Asp, Glu, His, Gln, Asn, Leu, Trp or Gly. For certain embodiments, aa11 represents Asp, Glu, Ser, Thr, Gln, Arg, Lys, His, Val, Ile, Tyr or Gly, even more preferably Asp, Glu, Ser, Thr, Gln, Lys or His. For certain embodiments, aa12 represents Asp, Glu, Ser, Thr, Gln, Asn, Lys, Arg, Val, Leu, Ile, Trp, Tyr, Phe or Gly, even more preferably Asp, Glu, Ser, Tyr, Trp, Arg or Lys. For certain embodiments, aa13 represents Ser, Thr, Gln, Asn, Val, Ile, Leu, Gly, Pro, Asp, Glu, His, Arg, Trp, Tyr or Phe, even more preferably Ser, Thr, Gln, Asn, Val, Ile, Leu, Gly, Asp or Glu. For certain embodiments, aa14 represents Ala, Ile, Trp, Pro, Asp, Glu, Arg, Lys, His, Ser, Thr, Gln or Asn, even more preferably Ala, Pro, Asp, Glu, Arg, Lys, Ser, Gln or Asn.For certain embodiments, aa15 represents His, Arg, Lys, Asp, Ser, Thr, Gln, Asn, Ala, Val, Leu, Gly or Phe, and more preferably His, Arg, Lys, Asp, Ser, Thr, Gln or Asn.

[0078] In certain embodiments of the above sequence, (Xaa) n ("Loop 4") is an amino acid sequence selected from SEQ ID NOs: 41 to 75, or an amino acid sequence having at least 80% homology thereto, more preferably an amino acid sequence having at least 85%, 90%, 95% or 98% homology thereto.

[0079] In certain embodiments of the above sequence, (Xaa) n ("Loop 4") is an amino acid sequence selected from SEQ ID NOs: 41 to 75, or an amino acid sequence having at least 80% identity thereto, more preferably an amino acid sequence having at least 85%, 90%, 95% or even 98% identity thereto.

[0080] In certain embodiments, the PD-L1 binding affimer polypeptide has an amino acid sequence selected from SEQ ID NOs: 76 to 84, or an amino acid sequence having at least 70% homology thereto, more preferably an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or even 98% homology thereto.

[0081] In certain embodiments, the PD-L1 binding affimer polypeptide has an amino acid sequence selected from SEQ ID NOs: 76 to 84, or an amino acid sequence having at least 70% identity thereto, more preferably an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or even 98% identity thereto.

[0082] In certain embodiments, the PD-L1 binding affimer polypeptide has an amino acid sequence encoded by a coding sequence corresponding to nucleotides 1 to 336 of any one of SEQ ID NOs: 85 to 92, or a coding sequence that is at least 70% identical thereto, more preferably at least 75%, 80%, 85%, 90%, 95% or even 98% identical.

[0083] In certain embodiments, the PD-L1 binding affimer polypeptide has an amino acid sequence encoded by a nucleic acid having a coding sequence that hybridizes to any one of SEQ ID NOs: 85 to 92 under stringent conditions of washing in 6× sodium chloride / sodium citrate (SSC) at 45° C. followed by 0.2× SSC at 65° C.

[0084] In certain embodiments, the PD-L1 binding affimers described herein bind PD-L1 in a manner that competes with PD-L1 binding by the anti-PD-L1 antibodies atezolizumab, avelumab and / or durvalumab.

[0085] In certain embodiments, the PD-L1 binding affimer polypeptide forms a crystal structure with PD-L1 that includes a contact surface that includes at least 10 residues of PD-L1 selected from Ile-54, Tyr-56, Glu-58, Glu-60, Asp-61, Lys-62, Asn-63, Gln66, 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.

[0086] In certain embodiments, the PD-L1 binding affimer polypeptide that binds to PD-L1 (a) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (b) increases the production of interferon-γ in an MLR assay; and / or (c) increases the secretion of interleukin-2 (IL-2) in an MLR assay.

[0087] In certain embodiments, the binder-drug conjugate of the present invention, in addition to the PD-L1 binding affimer polypeptide or other target binding moiety, for purposes of illustration, a secretion signal sequence, a peptide linker sequence, an affinity tag, a transmembrane domain, a cell surface retention sequence, a substrate recognition sequence for post-translational modification, a multimerization domain for creating a multimeric structure of a protein that aggregates via protein-protein interaction, a half-life extending polypeptide moiety, a polypeptide sequence for altering the tissue localization and antigen binding site of an antibody, and one or more additional amino acid sequences selected from the group consisting of one or more additional affimer polypeptide sequences that bind to other different targets, may be a fusion protein comprising.

[0088] In certain embodiments, the fusion protein comprises a half-life extending polypeptide moiety selected from the group consisting of an Fc domain or a portion thereof, an albumin protein or a portion thereof, an albumin binding polypeptide moiety, a transferrin or a portion thereof, a transferrin binding polypeptide moiety, a fibronectin or a portion thereof, or a fibronectin binding polypeptide moiety.

[0089] When the fusion protein comprises an Fc domain or a portion thereof, in certain embodiments, it is an Fc domain that retains FcN binding.

[0090] When the fusion protein comprises an Fc domain or a portion thereof, in certain embodiments, the Fc domain or a portion thereof is derived from IgA, IgD, IgE, IgG, and IgM, or a subclass (isotype) thereof such as, for example, IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

[0091] In certain embodiments, the fusion protein has the amino acid sequence of SEQ ID NO: 108 or 109, or a sequence having at least 70% homology thereto, even more preferably at least 75%, 80%, 85%, 90%, 95% or even 98% homology thereto.

[0092] When the fusion protein contains an Fc domain or a part thereof, in certain embodiments, the Fc domain or a part thereof retains effector functions selected from C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of the B cell receptor, or combinations thereof.

[0093] In certain embodiments, when the fusion protein contains a half-life extending polypeptide moiety, that moiety increases the serum half-life of the protein by at least 5-fold, more preferably 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 500-fold, or even 1000-fold compared to the case where the protein is absent.

[0094] In certain embodiments, the fusion protein of the present invention is provided as a pharmaceutical formulation suitable for therapeutic use in human patients, further comprising one or more pharmaceutically acceptable excipients, buffers, salts, etc.

[0095] Yet another aspect of the present invention relates to a pharmaceutical formulation suitable for therapeutic use in human patients, comprising (i) a binder-drug conjugate or a combination of a PD-L1 inhibitor / natural immune stimulant described herein, and (ii) one or more pharmaceutically acceptable excipients, buffers, salts, etc.

[0096] In certain embodiments of the drug conjugate of the present invention, the free drug moiety is an immunodash inhibitor. In certain embodiments, the immunodash inhibitor has an in vitro intracellular IC50 of less than 200 nM in human macrophages for DPP8 and DPP9 inhibition. In certain embodiments, the in vitro cell-free IC50 for inhibition of 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 certain embodiments, the EnPlex IC50 for inhibition of 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 picomole), or even 0.0001 nM (100 femtomoles). In certain embodiments, the Ki for inhibition of 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 picomole), or even 0.0001 nM (100 femtomoles).

[0097] In certain embodiments, the subject immunodash inhibitor also inhibits fibroblast activation protein (FAP) within the concentration range of a drug that is an effective anti-tumor agent. For example, the immunodash inhibitor can have a Ki for FAP inhibition of less than 100 nM, 10 nM, 1.0 nM, 0.1 nM, 0.01 nM, 0.001 nM (1 picomole) or even 0.0001 nM (100 femtomoles).

[0098] In certain embodiments, the subject immunodash inhibitor inhibits human fibroblast activation protein (FAP) with an IC50 that is at least 2-fold higher, more preferably at least 3, 4, 5, 10, 20, 30, 40, 50 or even 100-fold higher than the IC50 for induction of pyroptosis in human macrophages - i.e., immunodash is a more potent inducer of pyroptosis than FAP inhibition.

[0099] In certain embodiments, the immunoDASH inhibitor exhibits slow binding inhibition kinetics. In certain embodiments, the immunoDASH inhibitor has an off-rate of less than 1×10-4 / sec, preferably less than 5×10-5 / sec, 3×10-5 / sec or even less than 1×10-5 / sec for its interaction with DPP4.

[0100] In certain embodiments, the immunoDASH inhibitor is administered to a patient as a binder-drug conjugate in an amount sufficient to cause a decrease in the number of tumor-associated macrophages.

[0101] In certain embodiments, the immunoDASH inhibitor is administered to a patient as a binder-drug conjugate in an amount sufficient to reduce monocytic myeloid-derived suppressor cells within the tumor.

[0102] In certain embodiments, the immunoDASH inhibitor is administered to a 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.

[0103] In certain embodiments, the immunoDASH inhibitor is administered to a patient as a binder-drug conjugate in a therapeutically effective amount that results in complete tumor regression, where the therapeutically effective amount is less than the maximum tolerated dose of the binder-drug conjugate.

[0104] In certain embodiments, the immunoDASH inhibitor is administered to a patient as a binder-drug conjugate alone or in combination with a PGE2 inhibitor such as a cPLA-2 inhibitor.

[0105] In certain embodiments, the immunoDASH inhibitor is administered to a patient as a binder-drug conjugate alone or in combination with a DPP4 inhibitor such as sitagliptin, vildagliptin, saxagliptin, linagliptin, and alogliptin. BRIEF DESCRIPTION OF THE DRAWINGS

[0106]

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BRIEF DESCRIPTION OF THE INVENTION

[0107] I. General One aspect of the invention relates to a binder-drug conjugate comprising the following: (i) a cell-binding moiety that binds to a cell surface feature on target cells in diseased tissue, wherein the cell surface feature undergoes slow internalization upon binding of the binder-drug conjugate; (ii) a drug moiety that has a pharmacological effect on bystander cells in proximity to the target cells, having an EC50 for a pharmacological effect that is at least 2-fold attenuated when part of the binder-drug conjugate compared to the free drug moiety released from the binder-drug conjugate; and (iii) a linker moiety that covalently attaches the polypeptide binder moiety to the drug moiety, comprising a substrate recognition sequence cleavable by an enzyme present extracellularly in diseased tissue, such that in the presence of the enzyme, the linker moiety is cleaved and the free drug moiety can be released.

[0108] II. Definitions To facilitate understanding of the invention, a number of terms and phrases are defined below.

[0109] a. Affimer The term "Stefin polypeptide" refers to a subgroup of proteins of the cystatin superfamily, a family that includes proteins containing multiple cystatin-like sequences.

[0110] The Stefin subgroup of the cystatin family are relatively small (about 100 amino acids) single domain proteins. They have not been subject to known post-translational modifications and lack disulfide bonds, suggesting that they can fold in the same way in a wide range of extracellular and intracellular environments. Stefin A itself is a 98 amino acid monomeric, single-chain, single domain protein. The structure of Stefin A has been analyzed, facilitating rational mutations to the Affimer scaffold of Stefin A. The only known biological activity of cystatin is the inhibition of cathepsin activity, which has allowed us to thoroughly investigate the residual biological activity of our engineered proteins.

[0111] The term "affimer" (or "affimer scaffold" or "affimer polypeptide") refers to a small and very stable protein that is a recombinantly engineered variant of a stefin polypeptide. Affimer proteins, like monoclonal antibodies, exhibit two peptide loops and an N-terminal sequence that are all randomized and can bind to a desired target protein with high affinity and specificity. The stabilization of the two peptides by the stefin protein scaffold constrains the possible conformational structures that the peptides can adopt, enhancing the binding affinity and specificity compared to a library of free peptides. These engineered non-antibody binding proteins are designed to mimic the molecular recognition properties of monoclonal antibodies in various applications. Modifications can be made to other parts of the stefin polypeptide sequence, such modifications improving the properties of these affinity reagents, such as enhancing stability, and rendering them robust over ranges such as temperature and pH. Preferably, an affimer comprises a sequence derived from stefin A and shares substantial identity with a stefin A wild-type sequence such as human stefin A. It will be apparent to those skilled in the art that modifications may be made to the scaffold sequence without departing from the present invention. In particular, the affimer scaffold can be, for example, at least 25%, 35%, 45%, 55% or 60% identical, 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 the sequence corresponding to human stefin A, provided that sequence variations do not adversely affect the ability of the scaffold to bind to a desired target (e.g., PD-L1), and can have an amino acid sequence that does not retain or generate a biological function such as that which wild-type stefin A possesses but which has been rendered ineffective by the mutational changes described herein.

[0112] "Binding agent-drug conjugate" refers to a polypeptide that comprises an affimer polypeptide sequence and has any other optional modifications (e.g., conjugation, post-translational modifications, etc.) for representing a therapeutically active protein for delivery to a patient.

[0113] "Programmed death ligand 1", also known as "PD-L1", "cluster of differentiation 274", "CD274", "B7 homolog 1" or "B7-H1", refers to the protein encoded by the CD274 gene in humans. Human PD-L1 is a 40 kDa type I transmembrane protein that plays a major role in suppressing the immune system under various circumstances. A representative human PD-L1 sequence is provided by the UniProtKB primary accession number Q9NZQ7 and will include other human isoforms. PD-L1 binds to the receptor PD-1 found on activated T cells, B cells, and myeloid cells and regulates activation or inhibition. PD-L1 also has a significant affinity for the costimulatory molecule CD80 (B7-1). Engagement of PD-L1 with its receptor PD-1 ( "programmed death ligand 1" or "CD279") on T cells delivers a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation. In this regard, PD-L1 is considered a checkpoint, and its upregulated expression in tumors contributes to the inhibition of T cell-mediated antitumor responses. While PD-L1 is generally used with reference to PD-L1 from various mammalian species, it will be understood throughout the application that references to PD-L1 include human PD-L1 and preferably refer to human PD-L1 itself.

[0114] "PD-L1 binding agent-drug conjugate" refers to a binding agent-drug conjugate having at least one affimer polypeptide that binds to PD-L1, particularly human PD-L1, with a dissociation constant (Kd) of at least 10 -6 M.

[0115] b. Polypeptide The terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also include amino acid polymers that are naturally or artificially modified, such as by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the scope of the definition are, for example, one or more analogs of amino acids (including, for example, non-natural amino acids, etc.) and polypeptides containing other modifications known in the art.

[0116] The terms "amino acid residue" and "amino acid" are used interchangeably and, in the context of a polypeptide, mean an amino acid that is involved in another peptide bond of the polypeptide. In general, the abbreviations used herein to specify amino acids are based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry, (1972), 11:1726-1732). For example, Met, Ile, Leu, Ala, and Gly each represent the "residues" of methionine, isoleucine, leucine, alanine, and glycine, respectively. A residue means a radical derived from the corresponding amino acid by removing the OH portion of the carboxyl group and the H portion of the -amino group. The term "amino acid side chain" is the portion of an amino acid excluding the -CH(NH2)COOH portion, as defined by K.D. Kopple, "Peptides and Amino Acids", W.A. Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33.

[0117] In most cases, the amino acids used in the application of the present invention are naturally occurring amino acids found in proteins, or naturally occurring catabolites or metabolites of such amino acids containing amino groups and carboxyl groups. Particularly preferred amino acid side chains include the 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, and those of amino acids and amino acid analogs identified as components of peptidoglycan bacterial cell walls.

[0118] Amino acid residues having a "basic side chain" include Arg, Lys, and His. Amino acid residues having an "acidic side chain" include Glu and Asp. Amino acid residues having a "neutral polar side chain" include Ser, Thr, Asn, Gln, Cys, and Tyr. Amino acid residues having a "neutral nonpolar side chain" include Gly, Ala, Val, Ile, Leu, Met, Pro, Trp, and Phe. Amino acid residues having a "nonpolar aliphatic side chain" include Gly, Ala, Val, Ile, and Leu. Amino acid residues having a "hydrophobic side chain" include Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp. Amino acid residues having a "small hydrophobic side chain" include Ala and Val. Amino acid residues having an "aromatic side chain" include Tyr, Trp, and Phe.

[0119] The term amino acid residue further includes analogs, derivatives and homologs of any particular amino acid referred to herein. For example, the subject Affimers (especially when produced by chemical synthesis) can include amino acid analogs such as, for example, cyanalanine, canavanine, genisteic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diminopimelic acid, ornithine, or diaminobutyric acid. Other naturally occurring metabolites or precursors of amino acids having side chains suitable herein will be recognized by those skilled in the art and are included within the scope of the present invention.

[0120] Also included are the (D) and (L) stereoisomers of such amino acids when the structure of the amino acid admits of stereoisomers. The configuration of amino acids and amino acid residues herein is indicated by the appropriate symbol (D), (L) or (DL), and further, when no configuration is specified, the amino acid or residue can have the configuration (D), (L) or (DL). It will be mentioned that some structures of the compounds of the present invention contain asymmetric carbon atoms. Thus, it should be understood that isomers resulting from such asymmetry are included within the scope of the present invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereocontrolled synthesis. For the purposes of the present application, unless expressly stated to the contrary, the specified amino acids should be construed to include both the (D) and (L) stereoisomers.

[0121] The terms "identical" or "percent identity" in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that, when compared and aligned for maximum correspondence (introducing gaps if necessary), are the same or have a specified percentage of the same nucleotide or amino acid residues, without considering conservative amino acid substitutions as part of sequence identity. Percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain sequence comparisons of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical if, when compared and aligned for maximum correspondence as measured using a sequence comparison algorithm 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 over a region of an amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40 - 60 residues, at least about 60 - 80 residues in length, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60 - 80 residues, such as at least about 80 - 100 residues, and in some embodiments, the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or antibody. In some embodiments, identity exists over a region of a nucleotide sequence that is at least about 10 bases, at least about 20 bases, at least about 40 - 60 bases, at least about 60 - 80 bases in length, or any integer value therebetween.In some embodiments, the identity exists over a region longer than 60 - 80 bases, such as at least about 80 - 1000 bases or more, and in some embodiments, the sequences are substantially identical over the full length of the sequences being compared, such as the nucleotide sequences encoding the protein of interest.

[0122] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains, 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), are generally defined in the art. For example, the substitution of tyrosine with phenylalanine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and / or antibodies of the present invention do not inhibit binding to the target binding site of the polypeptide, soluble protein, or antibody containing the amino acid sequence. Methods for identifying conservative substitutions of amino acids that do not eliminate binding are well known in the art.

[0123] A polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is "isolated" is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in their natural form. In some embodiments, the isolated polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0124] As used herein, the term "substantially pure" refers to a substance 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%.

[0125] As used herein, the term "fusion protein" or "fusion polypeptide" refers to a hybrid protein expressed by a nucleic acid molecule containing the nucleotide sequences of at least two genes.

[0126] As used herein, the term "linker" or "linker region" refers to a linker inserted between a first polypeptide (e.g., a copy of an affimer) and a second polypeptide (e.g., another affimer, Fc domain, ligand-binding domain, etc.). In some embodiments, the linker portion is a peptide linker. The linker must not adversely affect the expression, secretion, or biological activity of the polypeptide. Preferably, the linker is not antigenic and does not induce an immune response.

[0127] "Affimer-antibody fusion" refers to a fusion protein comprising an affimer polypeptide portion and the variable region of an antibody. Affimer-antibody fusions include, for example, full-length antibodies having one or more affimer polypeptide sequences added to one or more C-termini and / or N-termini of their VH and / or VL chains, i.e., at least one chain of the constructed antibody is a fusion protein with an affimer polypeptide. Affimer-antibody fusions also include embodiments in which one or more affimer polypeptide sequences are provided as part of a fusion protein with the antigen-binding site or variable region of an antibody fragment.

[0128] 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 a combination of any of the foregoing, via at least one antigen recognition site, where the antigen recognition site is typically within the variable region of the immunoglobulin molecule. As used herein, the term includes intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (e.g., Fab, Fab’, F(ab’)2, and Fv fragments), single-chain Fv (scFv) antibodies provided that such 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 that include the antigen-binding site of an antibody (formatted to include an Fc or other FcγRIII binding domain), and any other modified immunoglobulin molecule that includes an antigen-binding site so long as the antibody exhibits the desired biological activity.

[0129] Antibodies can be of any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the uniqueness of the heavy-chain constant domains called alpha, delta, epsilon, gamma, and mu.

[0130] The term "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. Generally, the variable regions of the heavy and light chains each consist of four framework regions (FRs) and three complementarity-determining regions (CDRs), also known as "hypervariable regions". The CDRs within each chain are held together in close proximity by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) a method based on the variability of sequences between species (i.e., Kabat, et al. Sequences of Proteins of Immunological Interest, 5th ed., 1991, National Institutes of Health, Bethesda, Md.), and (2) a method based on crystallographic studies of antigen-antibody complexes (Al Lazikani, et al., 1997, J. Mol. Biol., 273:927-948). In addition, combinations of these two methods may be used in the art to determine CDRs.

[0131] As used herein, the term "humanized antibody" refers to a form of a non-human antibody that is a particular immunoglobulin chain, chimeric immunoglobulin, or fragment thereof that contains minimal non-human (e.g., mouse) sequences. Typically, a humanized antibody is a human immunoglobulin in which the residues of the CDRs are replaced by residues derived from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) having the desired specificity, affinity, and / or binding function. In some cases, residues of the Fv framework region of the human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species. A humanized antibody can be further modified by additional residue substitutions in the Fv framework region and / or within the replaced non-human residues to improve and optimize the specificity, affinity, and / or binding function of the antibody. A humanized antibody may include a variable domain that contains all or substantially all of the CDRs corresponding to a non-human immunoglobulin, whereas all or substantially all of the FR regions are those of a human immunoglobulin sequence. In some embodiments, the variable domain includes a framework region of a human immunoglobulin sequence. In some embodiments, the variable domain includes a framework region of a human immunoglobulin consensus sequence. A humanized antibody can also include at least a portion (Fc) of an immunoglobulin constant region or constant domain, typically at least a portion of a human immunoglobulin. A humanized antibody is generally considered to be distinct from a chimeric antibody.

[0132] The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen that can be recognized and specifically bound by a particular antibody, a particular affimer, or other particular binding domain. When the antigen is a polypeptide, an epitope can be formed from both adjacent amino acids and non-adjacent amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed from adjacent amino acids (also called linear epitopes) are usually retained upon protein denaturation, whereas epitopes formed by tertiary folding (also called conformational epitopes) are usually lost upon protein denaturation. Epitopes usually contain at least 3, more usually at least 5, 6, 7 or 8 - 10 amino acids in a unique spatial structure.

[0133] As used herein, the terms "specifically binds to" or "is specific for" refer to a measurable and reproducible interaction such as binding between a target and an affimer, antibody or other binding partner that determines the presence of the target in the presence of a heterogeneous population of molecules including biomolecules. For example, an affimer that specifically binds to a certain target is an affimer that binds to this target with higher affinity, binding strength (if formed as a multimer), more readily, and / or for a longer duration than it binds to other targets.

[0134] c. Checkpoint inhibitors, co-stimulatory agonists and chemotherapeutic agents "Checkpoint molecule" refers to a protein expressed by tissues and / or immune cells that decreases the effectiveness of an immune response depending on the expression level of the checkpoint molecule. When these proteins are blocked, the "brake" of the immune system is released, and for example, T cells can 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.

[0135] "Checkpoint inhibitor" refers to a drug entity that reverses immunosuppressive signaling from checkpoint molecules.

[0136] "Costimulatory molecule" refers to an immune cell that specifically binds to a costimulatory ligand and thereby mediates costimulation, such as but not limited to proliferation, of T cell cognate binding partners. A costimulatory molecule is a cell surface molecule other than an antigen receptor or ligand that promotes an effective immune response. Costimulatory molecules include, but are not limited to, MHC I molecules, BTLA receptors and toll ligands, and OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Examples of costimulatory molecules include, but are not limited to, CD5, 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), PSGL1, 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 ligand.

[0137] "Co-stimulatory agonist" refers to a drug entity that activates (stimulates) co-stimulatory molecules such as co-stimulatory ligands to generate immune-stimulatory signals or otherwise enhance the potency or efficacy of an immune response.

[0138] "Chemotherapeutic agents" are compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylene imines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bratasin and bratasinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analogs topotecan (HYCAMTIN), CPT-11 (irinotecan, CAMPTOSAR), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; pemetrexed; calistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllinic acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); erythrobins; pancratistatin; TLK-286; CDP323, an oral alpha-4 integrin inhibitor; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics (e.g., calicheamicin, especially antibiotics such as calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Nicolaou, et al., Angew.Chem Intl.Ed.Engl., 33:183-186 (1994)); dynemicin including dynemicin A; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), actinomycins, aclacinomycins, authramycin, azaserine, bleomycin, cactinomycin, carabicin, calminomycin, cardifilin, chromomycinis, daunorubicin, doxorubicin (ADRIAMYCIN, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL), and including deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodomycin, streptozocin, streptonigrin, tubercidin, ubenimex, dinostatin, zorubicin; antibiotics; metabolic antagonists such as methotrexate, gemcitabine (GEMZAR), tegafur (UFTORAL), capecitabine (XELODA), epothilone, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyruridine, doxifluridine, enocitabine, floxuridine, and imatinib (2-phenylaminopyrimidine derivative), and other c-Kit inhibitors; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;Demecolcine; Diacontin; Elfornithine; Elliptinium acetate; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Razoxane; Rizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triacontin; 2,2’,2”-Trichlorotriethylamine; Trichothecenes (especially, T-2 toxin, verracurin A, roridin A, and anguidine); Urethane; Vindesine (ELDISINE, FILDESIN); Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside (“Ara-C”); Thiotepa; Taxoids, for example, paclitaxel (TAXOL), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE), and docetaxel (TAXOTERE); Chlorambucil; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as cisplatin and carboplatin; Vinblastine (VELBAN); Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine (ONCOVIN); Oxaliplatin; Leucovovin; Vinorelbine (NAVELBINE); Novantrone; Edatrexate; Daunomycin; Aminopterin; Ibandronate; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids such as retinoic acid; Any pharmaceutically acceptable salt, acid, or derivative of any of the above;And abbreviations of combination therapies of CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisone, and abbreviations of treatment regimens using oxaliplatin (ELOXATIN (trademark)) combined with FOLFOX, 5-FU, and leucovorin, such as the above two or more combinations are exemplified.;

[0139] Also included in this definition are antihormonal agents that act to modulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth and that are often in the form of systemic or whole-body treatment. They may be the hormones themselves. Examples include, for example, antiestrogens and selective estrogen receptor modulators (SERMs) such as tamoxifen (including NOLVADEX tamoxifen), raloxifene (EVISTA), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); antiprogesterone drugs; estrogen receptor downregulators (ERDs); estrogen receptor antagonists such as fulvestrant (FASLODEX); agents that function to suppress or arrest ovarian activity, such as luteinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate (LUPRON and ELIGARD), goserelin acetate, buserelin acetate, and tripterelin; antiandrogen drugs such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase that 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), etc.In addition, such a definition of a chemotherapeutic agent 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-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in the growth of adherent cells, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE vaccine and gene therapy vaccines, for example, ALLOVECTIN vaccine, LEUVECTIN vaccine, and VAXID vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN); anti-estrogen drugs 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; arinotecan; rmRH (e.g., ABARELIX); lapatinib and lapatinib ditosylate (a small molecule dual tyrosine kinase inhibitor of ErbB-2 and EGFR also known as GW572016); 17AAG (a geldanamycin derivative that is a heat shock protein (Hsp) 90 poison), and pharmaceutically acceptable salts, acids, or derivatives of any of the above are included.

[0140] As used herein, the term "cytokine" generically refers to a protein released by one cell population that acts as an intercellular mediator on another cell or has an autocrine effect on the cell producing the protein. Examples of such cytokines include lymphokines, monokines; interleukins ("IL"), 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 - IL-29 (e.g., IL-23), IL-31; tumor necrosis factors such as TNF-α or TNF-β, TGF-β1 - 3; and other polypeptide factors including leukemia inhibitory factor ("LIF"), ciliary neurotrophic factor ("CNTF"), CNTF-like cytokine ("CLC"), cardiotrophin ("CT"), and kit ligand ("KL").

[0141] As used herein, the term "chemokine" refers to a soluble factor (e.g., cytokine) having the ability to selectively induce the chemotaxis and activation of leukocytes. They also cause processes such as angiogenesis, inflammation, wound healing, and tumorigenesis. Exemplary chemokines include IL-8 and the human homolog of mouse keratinocyte chemotactic substance (KC).

[0142] d. Treatment As used herein, the term "dysfunctional" includes unresponsiveness or non-responsiveness to antigen recognition, specifically, impairment of the ability to convert antigen recognition into downstream T cell effector functions such as proliferation, cytokine production (e.g., IL-2), and / or target cell killing.

[0143] The term "anergy" refers to an incomplete or insufficient signal delivered via the T cell receptor (e.g., intracellular Ca in the absence of ras activation) 2+refers to a state of unresponsiveness to antigenic stimulation, which is caused by, e.g., the rise of

[0144] The term "exhaustion" refers to T cell exhaustion as a state of T cell dysfunction resulting from persistent TCR signaling that occurs during the development of many chronic infections and cancers. It is distinguished from anergy in that it results from persistent signaling rather than through incomplete or insufficient signaling. It is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector T cells or memory T cells. Exhaustion impedes the optimal control of infections and tumors.

[0145] "Enhancing T cell function" means inducing, causing, or stimulating T cells to have a sustained or amplified biological function, or regenerating or reactivating exhausted or inactive T cells. Examples of enhancing T cell function include an increase in the secretion of interferon-γ from CD8+ T cells, an increase in proliferation, and an increase in antigen responsiveness (e.g., clearance of a virus, pathogen, or tumor), as compared to the level prior to the intervention. In one embodiment, the level of enhancement is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to those of skill in the art.

[0146] "T cell dysfunctional disorder" refers to a disorder or condition of T cells characterized by a reduced responsiveness to antigen stimulation. In certain embodiments, the T cell dysfunctional disorder is a disorder specifically related to an inappropriate elevated level of PD-1. The T cell dysfunctional disorder can also be related to an inappropriate elevated level of PD-L1 within a tumor, which results in suppression of the anti-tumor function(s) of T cells. In another embodiment, the T cell dysfunctional disorder is one in which the T cells are anergic or have a reduced ability to secrete cytokines, proliferate, or execute cytolytic activity. In a specific aspect, the reduced responsiveness results in ineffective control of pathogens or tumors that express immunogens. Examples of T cell dysfunctional disorders characterized by T cell dysfunction include unexplained acute infections, chronic infections, and tumor immunity.

[0147] "Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated and the tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0148] "Sustained response" refers to a lasting effect on reducing tumor growth after treatment discontinuation. For example, the tumor size can remain the same or become smaller compared to its size at the start of the dosing period. In some embodiments, the sustained response has a duration that is at least as long as the treatment period, at least 1.5-fold, 2.0-fold, 2.5-fold, or 3.0-fold the length of the treatment period.

[0149] As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological state of a mammal in which a population of cells is characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinomas, blastomas, sarcomas, and blood cancers such as lymphomas and leukemias.

[0150] As used herein, the terms "tumor" and "neoplasm" refer to any mass of tissue resulting from either excessive cell growth or proliferation, whether benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions. Tumor growth is generally uncontrolled and progressive, and does not induce or inhibit the proliferation of normal cells. Tumors can affect a variety of cells, tissues or organs, including but not limited to those selected from the organs or tissues of the bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tube, gallbladder, heart, intestine, kidney, liver, lung, lymph node, nerve tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, ureter, uterus, vagina or the corresponding cells. Tumors include cancers such as sarcomas, carcinomas, plasmacytomas, or myelomas (malignant plasma cells). The tumors of the present invention include leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia diseases, heavy chain diseases, and solid tumors such as sarcoma carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endotheliosarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendotheliosarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, 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 adenocarcinoma, carcinoma, bronchogenic carcinoma, medullary carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choroid carcinoma, spermatoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder cancer, kidney cancer, multiple myeloma, but are not limited thereto. Preferably, "tumor" includes, but is not limited to, pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, renal cell carcinoma, leukemia, multiple myeloma, ovarian cancer, cervical cancer, and glioma.

[0151] As used herein, the term "metastasis" refers to the process by which cancer spreads or metastasizes from its site of origin to other areas of the body and gives rise to similar cancerous lesions in new locations. "Metastatic" or "metastasized" cells are those that have lost their adhesive contact with adjacent cells and move away from the primary site of the disease via the bloodstream or lymph and invade adjacent body structures.

[0152] The terms "cancer cell" and "tumor cell" refer to the total population of cells derived from a cancer or tumor or pre-cancerous lesion and include both non-tumorigenic cells and tumorigenic stem cells (cancer stem cells) that make up the majority of the cancer cell population. 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 ability to reproduce and differentiate in order to distinguish those tumor cells from cancer stem cells.

[0153] As used herein, the term "effective amount" refers to the amount for providing a therapeutic or prophylactic benefit.

[0154] As used herein, "complete response" or "CR" refers to the disappearance of all target lesions, "partial response" or "PR" refers to at least a 30% decrease in the sum of the SLD of target lesions based on the baseline longest diameter (SLD), and "stable disease" or "SD" refers to no shrinkage of target lesions sufficient to qualify for PR or increase sufficient to qualify for PD based on the minimum SLD since the start of treatment.

[0155] As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment that the disease being treated (e.g., cancer) does not worsen. Progression-free survival may include the amount of time the patient has experienced a complete response or partial response, as well as the amount of time the patient has experienced stability.

[0156] As used herein, "overall response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0157] As used herein, "overall survival rate" refers to the proportion in a group of individuals who are likely to be alive after a specified period.

[0158] As used herein, the term "treatment" refers to an individual who attempts to alter the process or treatment of a clinical disease caused by cell intervention, which may be either a preventive intervention process in clinical pathology. Treatments include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or achieving remission of a disease, or improving the prognosis.

[0159] The term "subject" refers to any animal (e.g., a mammal) including, but not limited to, humans, non-human primates, dogs, cats, rodents, etc., that is the recipient of a particular treatment. Usually, the terms "subject" and "patient" are used interchangeably herein with reference to a human subject.

[0160] As used herein, the terms "agonist" and "agonistic" refer to an agent that can directly or indirectly substantially induce, activate, promote, enhance, or increase the biological activity of a target or target pathway. The term "agonist" is used herein to include any agent that partially or fully induces, activates, promotes, enhances, or increases the activity of a target protein or other target.

[0161] As used herein, the terms "antagonist" and "antagonist of" 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" is used herein to include any agent that partially or completely blocks, inhibits, reduces, or neutralizes the activity of a target protein or other target.

[0162] As used herein, the terms "modulate" and "modulating" refer to a change or alteration in biological activity. Modulation includes, but is not limited to, stimulating activity or inhibiting activity. Modulation may be an increase or decrease in activity, a change in binding properties, or any other change in biological, functional, or immunological properties associated with the activity of a target protein, pathway, system, or other biological target.

[0163] As used herein, the term "immune response" includes responses from both the innate and adaptive immune systems. It includes both cell-mediated and / or humoral immune responses. It includes responses from both T cells and B cells, as well as responses from other cells of the immune system such as natural killer (NK) cells, monocytes, macrophages, and the like.

[0164] The term "pharmaceutically acceptable" refers to substances approved or approvable by a regulatory agency of the Federal or a State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals including humans.

[0165] The term "pharmaceutically acceptable excipient, carrier or adjuvant" or "pharmaceutically acceptable carrier" refers to an excipient, carrier or adjuvant that can be administered to a subject together with at least one active agent of the present disclosure, is non-toxic when administered in a dosage sufficient to deliver a therapeutic effect without destroying its pharmacological activity. Generally, those skilled in the art and the US FDA consider pharmaceutically acceptable excipients, carriers, or adjuvants to be the inactive ingredients of any formulation.

[0166] The term "effective amount" or "therapeutically effective amount" or "therapeutic effect" refers to the amount of the binder-drug conjugate described herein that is effective to "treat" a disease or disorder in a subject such as a mammal. In the case of cancer or tumor, the therapeutically effective amount of the binder-drug conjugate that binds to PD-L1 has a therapeutic effect, so it enhances the immune response, enhances the anti-tumor response, enhances the cytolytic activity of immune cells, increases the killing of tumor cells by immune cells, and reduces the number of tumor cells; reduces tumorigenicity, tumorigenic frequency or tumorigenic ability; reduces the number and frequency of cancer hepatocytes; reduces the size of the tumor; reduces the cancer cell population; for example, inhibits or stops the infiltration of cancer cells into peripheral organs including the spread of cancer to soft tissue and bone; suppresses and stops the metastasis of tumors or cancer cells; inhibits and stops the growth of tumors or cancer cells; alleviates to some extent one or more of the symptoms associated with cancer; reduces morbidity and mortality; improves the quality of life; or improves a combination of such effects.

[0167] The terms "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both (1) therapeutic measures to cure, slow down, reduce and / or stop the progression of symptoms of a diagnosed medical condition or disorder, and (2) prophylactic or preventive measures to prevent or delay the onset of a targeted medical condition or disorder. Thus, those in need of treatment include those already having a disorder; those having a tendency to have a disorder; and those in need of preventing a disorder. In the case of cancer or a tumor, if a patient exhibits one or more of the following: an increase in immune response, an increase in anti-tumor response, an increase in the cytolytic activity of immune cells, an increase in the killing of tumor cells by immune cells, a decrease or complete absence in the number of cancer cells; a decrease in tumor size; inhibition or absence of cancer cell infiltration into peripheral organs, including spread of cancer cells into soft tissue and bone; inhibition or absence of metastasis of a tumor or cancer cells; inhibition or absence of cancer growth; alleviation of one or more symptoms associated with a particular cancer; a decrease in morbidity and mortality; an improvement in quality of life; a decrease in tumorigenicity; a decrease in the number or frequency of cancer stem cells; or one or more combinations of effects, then the subject is being successfully "treated" according to the methods of the present invention.

[0168] e. Others The term "alkyl" refers to radicals of saturated aliphatic groups including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In certain embodiments, straight-chain or branched-chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C 30 , for branched-chain C3-C 30 ), e.g., 20 or fewer carbon atoms. Similarly, certain cycloalkyl has 3 to 10 carbon atoms in its ring structure, e.g., 5, 6 or 7 carbons in the ring structure. "Alkyl" (or "lower alkyl") as used throughout this specification and claims is intended to include both "unsubstituted alkyl" and "substituted alkyl".

[0169] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group (e.g., aromatic or heteroaromatic).

[0170] The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups of similar length that are capable of substitution relative to the alkyl described above and that each contain at least one double bond or triple bond, respectively.

[0171] Unless otherwise specified as to carbon number, as used herein "lower alkyl" means an alkyl group having, as defined above, but having 1 to 10 carbons, e.g., 1 to 4 or 1 to 6 carbon atoms, in its backbone structure. Similarly, "lower alkenyl" and "lower alkynyl" have similar chain lengths. In some embodiments, the alkyl group is lower alkyl. In some embodiments, the substituent designated as alkyl herein is lower alkyl.

[0172] As used herein, the term "aryl" includes, for example, monocyclic aromatic groups of 5-, 6-, and 7-membered rings which may contain 0 to 4 heteroatoms such as benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics". The aromatic ring can be substituted at one or more ring positions with substituents such as those described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic moiety or heteroaromatic moiety, -CF3, -CN, etc. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings (the rings are "fused rings") and at least one of the rings is aromatic, and for example, the other cyclic ring or rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclyl.

[0173] The term "heterocyclyl" or "heterocyclic group" refers to a ring structure having from 3 to 10 ring members, for example, a ring having from 3 to 7 ring members, the ring structure containing from 1 to 4 heteroatoms. The heterocycle can also be polycyclic. Examples of heterocyclyl groups include thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolidine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinone and pyrrolidinone, sultams and sultones and the like. The heterocycle can be substituted at one or more positions with substituents such as those described above, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic moiety or heteroaromatic moiety, -CF3, -CN, etc.

[0174] The term "heteroaryl" refers to a monovalent aromatic monocyclic 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 having 5 ring atoms. Examples of 5-membered heteroaryl groups include pyrrolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, furazanyl, imidazolinyl, and triazolyl.

[0175] The term "heterocycloalkyl" refers to a monocyclic or bicyclic monovalent saturated or non-aromatic unsaturated ring system in which 1 to 4 ring atoms are heteroatoms independently selected from the group consisting of O, N, and S. The term "3- to 10-membered heterocycloalkyl" refers to a heterocycloalkyl having 3 to 10 ring atoms. Examples of 3- to 10-membered heterocycloalkyl include 3- to 6-membered heterocycloalkyl. Bicyclic ring systems include fused ring systems, bridged ring systems, and spirocyclic ring systems. More specific examples of heterocycloalkyl groups include azepanyl, azetidinyl, aziridinyl, imidazolidinyl, morpholinyl, oxazolidinyl, oxazolidinyl, piperazinyl, piperidinyl, pyrazolidinyl, pyrrolidinyl, quinuclidinyl, and thiomorpholinyl.

[0176] The term "policyclic" or "polycyclic group" refers to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclyl) in which two or more carbons are common to two adjacent rings, e.g., the rings are "fused rings". Rings connected through non-adjacent atoms are called "bridged" rings. Each of the polycyclic rings can be substituted with substituents such as those described above, e.g., halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic moiety or heteroaromatic moiety, -CF3, -CN, etc.

[0177] The term "carbocyclic" as used herein refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.

[0178] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Exemplary heteroatoms are nitrogen, oxygen, sulfur, and phosphorus.

[0179] 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-.

[0180] "Halogen" or "halo" refers to fluorine, chlorine, bromine and iodine, or fluoro, chloro, bromo and iodo, by itself or as part of another substituent.

[0181] It will be understood that "substituted" or "substituted with" includes the implicit condition that such substitution follows the valence numbers of the substituting atoms and substituents and that the substitution results in a stable compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, etc.

[0182] 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. Exemplary substituents include, for example, those described above. Permissible substituents can be one or more for a suitable organic compound and can be the same or different. Substituents include, for example, halogen, hydroxyl, carbonyl (e.g., carboxyl, ester, formyl, or ketone), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphonate, phosphinate, amino, amide, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. Those skilled in the art will understand that moieties substituted on a hydrocarbon chain may themselves be substituted where appropriate. For example, substituents of substituted alkyl include amino, azide, imine, amide, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfate, sulfonamide, sulfamoyl, and sulfonate), and silyl groups, as well as substituted and unsubstituted forms of ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylate, and ester), -CF3, -CN, etc. Exemplary substituted alkyls are described below. Cycloalkyl can be further substituted with alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl, -CF3, -CN, etc. For the purposes of the present invention, a heteroatom such as nitrogen may have a hydrogen substituent and / or any permissible substituent of the organic compounds described herein that satisfies the valence of the heteroatom. The present invention is not intended to be limited in any way by the permissible substituents of the organic compounds.

[0183] The terms "amino acid residue" and "peptide residue" mean a molecule of an amino acid or peptide that does not include the -OH of its carboxyl group. Generally, the abbreviations used herein to designate amino acids and protecting groups are based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry, (1972), 11:1726-1732). For example, Met, Ile, Leu, Ala, and Gly represent the "residues" of methionine, isoleucine, leucine, alanine, and glycine, respectively. A residue means a radical 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" is the portion of an amino acid excluding the -CH(NH2)COOH portion, as defined by K.D. Kopple, "Peptides and Amino Acids", W.A. Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33.

[0184] In most cases, the amino acids used in the application of the present invention are naturally occurring amino acids found in proteins, or naturally occurring catabolic or anabolic products of such amino acids containing amino and carboxyl groups. Particularly preferred amino acid side chains include those of 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, and those of amino acids and amino acid analogs identified as components of peptidylglycan bacterial cell walls.

[0185] The term amino acid residue further includes analogs, derivatives and homologs of any specific amino acid referred to herein. For example, the subject compounds can include amino acid analogs such as, for example, cyanoalanine, canavanine, genisteic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diminiopimelic acid, ornithine, or diaminobutyric acid. Other naturally occurring amino acid metabolites or precursors having side chains suitable herein will be recognized by those skilled in the art and are included within the scope of the present invention.

[0186] Also included are the (D) and (L) stereoisomers of such amino acids when the structure of the amino acid admits of stereoisomers. The configuration of amino acids and amino acid residues herein is indicated by the appropriate symbols (D), (L) or (DL), and further, when no configuration is specified, an amino acid or residue can have the configuration (D), (L) or (DL). It will be mentioned that some structures of the compounds of the present invention contain asymmetric carbon atoms. Accordingly, it should be understood that isomers arising from such asymmetry are included within the scope of the present invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereocontrolled synthesis. For the purposes of the present application, unless expressly stated to the contrary, the specified amino acids should be construed to include both (D) and (L) stereoisomers.

[0187] As mentioned above, certain compounds of the present invention may exist in the form of specific geometric or stereoisomers. The present invention contemplates that all such compounds, including cis and trans isomers, R and S enantiomers, diastereomers, (D) isomers, (L) isomers, their racemic mixtures, and other mixtures thereof, fall within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. It is intended that all such isomers, as well as mixtures thereof, be included in the present invention.

[0188] For example, if a particular enantiomer of a compound of the invention is desired, it may be prepared by asymmetric synthesis or by derivation with a chiral auxiliary, in which case the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group such as an amino or an acidic functional group such as a carboxyl, diastereomeric salts are formed with a suitable optically active acid or base, followed by resolution of the thus formed diastereomers by means of fractional crystallization or chromatography well known in the art, and subsequent recovery of the pure enantiomer.

[0189] The term "IC 50 " refers to the concentration of an inhibitor at which the response (or binding) is reduced by half and can be measured in whole cells, animals, or a cell-free (purified enzyme) system in vitro. Inhibition of cell-free enzymes may also be reported as the Ki value in some formal kinetic measurements.

[0190] The term "ICIC 50 " or "IIC 50 " is a criterion for the evaluation of DPP8 and DPP9 inhibition in the context of whole cells such that cell permeability is a factor (DPP8 and DPP9, which are cell permeable, and purified enzymes have no cell permeability requirements for measuring IC 50 ).

[0191] The term "DPP8" refers to the protein dipeptidyl peptidase 8.

[0192] The term "DPP9" refers to the protein dipeptidyl peptidase 9.

[0193] For the purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements on the inside front cover of the Handbook of Chemistry and Physics, 67th Ed., 1986 - 87, CAS version. Also, for the purposes of the present invention, the term "hydrocarbon" is intended to include all acceptable compounds having at least one hydrogen and one carbon atom. In a broad aspect, acceptable hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non - aromatic organic compounds that can be substituted or unsubstituted.

[0194] The terms "P1 position" and "P2 position", in the case of a dipeptide (or dipeptide analog), refer to the carboxy - terminal and amino - terminal residues, respectively. In the case of the subject I - DASH inhibitor, the P1 position is the amino acid (or amino acid analog) where the boronic acid replaces the carboxy - terminal.

[0195] Whenever embodiments are described herein in the language of "comprising", other similar embodiments are also provided that are described in terms of "consisting of" and / or "consisting essentially of" otherwise. Whenever embodiments are described herein in the language of "consisting essentially of", it is also understood that similar embodiments are provided that are described in terms of "consisting of" otherwise.

[0196] As used herein, references to "about" or "approximately" include (and describe) the embodiments directed to that value or parameter. For example, a description referring to "about X" includes the description of "X".

[0197] As used herein in expressions such as "A and / or B", the term "and / or" is intended to include both A and B; A or B; A alone; and B alone. Similarly, when used in expressions such as "A, B, and / or C", the term "and / or" is intended to include 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.

[0198] III. Exemplary Embodiments One aspect of the invention provides a binder-drug conjugate comprising (i) a cell-binding moiety such as an antibody, antibody fragment, non-antibody scaffold or other polypeptide entity that binds to a cell surface feature such as a protein that is upregulated or otherwise selectively displayed on cells within a tumor, and (ii) one or more drug conjugate moieties attached thereto, wherein the drug conjugate moiety is represented by the formula:

Chemical formula

[0199] When the binder-drug conjugate binds to the surface feature of the target cell, it has an internalization half-life of at least 6 hours, more preferably at least 10, 12, 14, 16, 18, 20, 24, 36, 48, 60, 75, or even 100 hours.

[0200] a. Substrate Recognition Sequence In certain embodiments, the substrate recognition sequence is a portion (usually a peptide portion or peptidyl portion) that is cleaved by an enzyme that is expressed in a tissue in which the cells to which the binding moiety is directed are present. By "cleavage site that can be selectively cleaved in the vicinity of the target cell" we include the meaning of a site that can be cleaved only by an agent that is selectively present in the vicinity of the target cell to reduce the release of the free drug moiety away from the diseased tissue. Preferably, the enzyme that cleaves the substrate recognition sequence is present in the vicinity of the target cell at a concentration that is at least 5-fold or 10-fold higher than the concentration of the enzyme outside the vicinity of the target cell, more preferably at least 100-fold or 500-fold or 1000-fold higher. Most preferably, the enzyme that cleaves the substrate recognition sequence is found only in the vicinity of the target cell. For example, if the target cell is a particular tumor cell (e.g., a breast tumor cell), the substrate recognition sequence may be cleaved by an enzyme that is selectively present in a particular tumor (e.g., a breast tumor) but not present outside the vicinity of the particular tumor (e.g., a breast tumor).

[0201] By "in the vicinity of the cell" we include any meaning of on the surface of the cell or the interstitial fluid within its tissue, or both, or the environment directly surrounding the cell, e.g., in blood, lymph, and other body fluids.

[0202] The substrate recognition sequence is selectively cleaved in the vicinity of the target cell so that the free drug moiety is preferentially released from the conjugate in the vicinity of the target cell to exert its pharmacological activity preferentially in the cells / tissues adjacent to the target cell rather than in the (healthy) cells required. Thus, the substrate recognition sequence is preferably selectively cleaved such that the free drug moiety is released as a drug moiety in the vicinity of the target cell at least 5-fold or 10-fold more than the extent to which the free drug moiety is released in the vicinity of healthy cells / tissues, more preferably at least 100-fold or 500-fold or 1000-fold or more.

[0203] For a given target cell, one of ordinary skill in the art will be able to identify a suitable substrate recognition sequence that can be selectively cleaved in the vicinity of the target cell using methods established in the art. For example, which protease cleaves which peptide can be evaluated by examining a peptide library and considering MS analysis of the fragmentation profile after cleavage. Also, published literature on protease cleavage motifs and peptide cleavage data can be searched as further described below.

[0204] Generally, the substrate recognition sequence is a protease cleavage site. Thus, if the target cell is a tumor cell, the substrate recognition sequence may be selectively cleavable by proteases present in the vicinity of the tumor cell. In other words, the substrate recognition sequence may be cleavable by tumor-associated proteases. During tumor development, it is well known that tumors abnormally express proteases that allow them to invade local tissues and ultimately metastasize.

[0205] The protease may be a matrix metalloproteinase (MMP1-28) including both membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13 and MMP18-23 and MMP26-28) forms. The protease may belong to the A disintegrin and metalloproteinase (ADAM) and A disintegrin with thrombospondin motifs family of proteases or ADAMTS. Other examples include CD10 (CALLA) and prostate specific antigen (PSA). In certain preferred embodiments, the protease is fibroblast activation protein (FAPα). It is fully understood that the protease may be membrane-bound or not.

[0206] Protease cleavage sites are well known in the scientific literature and can readily serve as the basis for a given substrate recognition sequence included in the drug conjugate moiety using established synthetic techniques known in the art.

[0207] In the case of an extracellular concentration, a change in expression, cell transport, or an intracellular enzyme that may become extracellular, a protease that is upregulated / elevated in the target tissue by a change in cell lysis caused by a pathological condition, (MEROPS peptidase database numbers provided in parentheses; Rawlings, N.D., Morton, F.R., Kok, C.Y., Kong, J. & Barrett, A.J. (2008), MEROPS: The Peptidase Database: Nucleic Acids Res. 36 Database Issues, D320 - 325): Pepsin A (MER000885), Gastricsin (MER000894), Memapsin - 2 (MER005870), Renin (MER000917), Cathepsin D (MER000911), Cathepsin E (MER000944), Memapsin - 1 (MER005534), Napsin A (MER004981), Marnelym - AA034 peptidase (MER014038), Pepsin A4 (MER037290), Pepsin A5 (Homo sapiens) (MER037291), hCG1733572 (Homo sapiens) type putative peptidase (MER107386), Napsin B pseudogene (MER004982), CYMPg.p. (Homo sapiens) (MER002929), Subfamily A1A unassigned peptidase (MER181559), Mouse mammary tumor virus retropeptidase (MER048030), Rabbit endogenous retrovirus endopeptidase (MER043650), S71 - related human endogenous retrovirus peptidase (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 (MER047253), RTVL - H - type putative peptidase (MER047260), RTVL - H - type putative peptidase (MER047291), RTVL - H - type putative peptidase (MER047418), RTVL - H - type putative peptidase (MER047440),Putative RTVL-H-type peptidase (MER047479), Putative RTVL-H-type peptidase (MER047559), Putative RTVL-H peptidase (MER047583), Putative RTVL-H-type peptidase (MER015446), Human endogenous retrovirus retropepsin homolog 1 (MER015479), Human endogenous retrovirus retropepsin homolog 2 (MER015481), Endogenous retrovirus retropepsin pseudogene 1 (Homo sapiens chromosome 14) (MER029977), Endogenous retrovirus retropepsin pseudogene 2 (Homo sapiens chromosome 8) (MER029665), Endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER002660), Endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER030286), Endogenous retrovirus retropepsin pseudogene 3 (Homo sapiens chromosome 17) (MER047144), Endogenous retrovirus retropepsin pseudogene 5 (Homo sapiens chromosome 12) (MER029664), Endogenous retrovirus retropepsin pseudogene 6 (Homo sapiens chromosome 7) (MER002094), Endogenous retrovirus retropepsi pseudogene 7 (Homo sapiens chromosome 6) (MER029776), Endogenous retrovirus retropepsin pseudogene 8 (Homo sapiens chromosome Y) (MER030291), Endogenous retrovirus retropepsin pseudogene 9 (Homo sapiens chromosome 19) (MER029680), Endogenous retrovirus retropepsin pseudogene 10 (Homo sapiens chromosome 12) (MER002848), Endogenous retrovirus retropepsin pseudogene 11 (Homo sapiens chromosome 17) (MER004378), Endogenous retrovirus retropepsin pseudogene 12 (Homo sapiens chromosome 11) (MER003344), Endogenous retrovirus retropepsin pseudogene 13 (Homo sapiens chromosome 2 and similar) (MER029779), Endogenous retrovirus retropepsin pseudogene 14 (Homo sapiens chromosome 2) (MER029778),Endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047158), endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER047332), endogenous retrovirus retropepsin pseudogene 15 (Homo sapiens chromosome 4) (MER003182), endogenous retrovirus retropepsin pseudogene 16 (MER047165), endogenous retrovirus retropepsin pseudogene 16 (MER047178), endogenous retrovirus retropepsin pseudogene 16 (MER047200), endogenous retrovirus retropepsin pseudogene 16 (MER047315), endogenous retrovirus retropepsin pseudogene 16 (MER047405), endogenous retrovirus retropepsin pseudogene 16 (MER030292), endogenous retrovirus retropepsin pseudogene 17 (Homo sapiens chromosome 8) (MER005305), endogenous retrovirus retropepsin pseudogene 18 (Homo sapiens chromosome 4) (MER030288), endogenous retrovirus retropepsin pseudogene 19 (Homo sapiens chromosome 16) (MER001740), endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047222), endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047454), endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER047477), endogenous retrovirus retropepsin pseudogene 21 (Homo sapiens) (MER004403), endogenous retrovirus retropepsin pseudogene 22 (Homo sapiens chromosome X) (MER030287), subfamily A2A non-peptidase homolog (MER047046), subfamily A2A non-peptidase homolog (MER047052), subfamily 2A 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 (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 (MER047110), Subfamily A2A non-peptidase homolog (MER047111), Subfamily A2A non-peptidase homolog (MER047114), Subfamily A2A non-peptidase homolog (MER047118), Subfamily A2A non-peptidase homolog (MER047121), Subfamily A2A non-peptidase homolog (MER047122), Subfamily A2A non-peptidase homolog (MER047126), Subfamily A2A non-peptidase homolog (MER047129), Subfamily A2A non-peptidase homolog (MER047130), Subfamily A2A non-peptidase homolog (MER047134), Subfamily A2A non-peptidase homolog (MER047135), 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 (MER047205), 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 (, 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 (MER047276), 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 (MER047389), 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 (MER047490), 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 non-peptidase homolog (MER047514), Subfamily A2A non-peptidase homolog (MER047515), Subfamily A2A non-peptidase homolog (MER047516), Subfamily A2A non-peptidase homolog (MER047520), Subfamily A2A non-peptidase homolog (MER047533), Subfamily A2A non-peptidase homolog (MER047537), Subfamily A2A non-peptidase homolog (MER047569), Subfamily 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 A2A non-peptidase homolog (MER048023), Subfamily A2A non-peptidase homolog (MER048037), Subfamily A2A unassigned peptidase (MER047164), Subfamily A2A unassigned peptidase (MER047231), Subfamily A2A unassigned peptidase (MER047386), Skin AspArtIC protease (MER057097), Presenilin 1 (MER005221), Presenilin 2 (MER005223), IMPAS1 peptidase (MER019701), IMPAS1 peptidase (MER184722), IMPAS4 peptidase (MER019715)Imipase 2 peptidase (MER019708), Imipase 5 peptidase (MER019712), Imipase 3 peptidase (MER019711), putative family A22 pseudogene (Homo sapiens chromosome 18) (MER029974), putative family A22 pseudogene (Homo sapiens chromosome 11) (M, ER023159), CA trypsin V (MER004437), CA trypsin X (MER004508), CA trypsin F (MER004980), CA trypsin L (MER000622), CA trypsin S (MER000633), CA trypsin O (MER001690), CA trypsin K (MER000644), CA trypsin W (MER003756), CA trypsin H (MER000629), CA trypsin B (MER000686), dipeptidyl - peptidase I (MER001937), bleomycin hydrolase (animal) (MER002481), tubulointerstitial nephritis antigen (MER016137), tubulointerstitial nephritis antigen - related protein (MER021799), CA trypsin L - like pseudogene 1 (Homo sapiens) (MER002789), CA trypsin B - like pseudogene (chromosome 4, Homo sapiens) (MER029469), CA trypsin B - like pseudogene (chromosome 1, Homo sapiens) (MER029457), CTSLL2g.p. (Homo sapiens) (MER005210), CTSLL3gp (Homo sapiens) (MER005209), calpain - 1 (MER000770), calpain - 2 (MER000964), calpain - 3 (MER001446), calpain - 9 (MER004042), calpain - 8 (MER021474), calpain - 15 (MER004745), calpain - 5 (MER002939), calpain - 11 (MER005844), calpain - 12 (MER029889), calpain - 10 (MER013510), calpain - 13 (MER020139), calpain - 14 (MER029744), marenostrin - AA253 peptidase (MER005537), calmodulin (MER000718), hippotetin ICAl protein flj40251 (MER003201), ubiquitinyl hydrolase - L1 (MER000832), ubiquitinyl hydrolase - L3 (MER000836), ubiquitinyl hydrolase - BAP1 (MER003989), ubiquitinyl 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 30 (MER014649), Marname-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), Marname-AA129 peptidase (MER016485), Ubiquitin-specific peptidase 49 (MER016486), Marname-AA187 peptidase (MER052579), USP17-like peptidase (MER030192), Ubiquitin-specific peptidase 54 (MER028714), Ubiquitin-specific peptidase 53 (MER027329), Ubiquitin-specific endopeptidase 39 [prone to misunderstanding] (MER064621), Marname-AA090 non-peptidase homolog (MER014739), Ubiquitin-specific peptidase [prone to misunderstanding] (MER030140), Ubiquitin-specific peptidase 52 [prone to misunderstanding] (MER030317), NEK2 pseudogene (MER014736), C19 pseudogene (Homo sapiens: chromosome 5) (MER029972), Marname-AA088 peptidase (MER014750), Autophagin-2 (MER013564), Autophagin-1 (MER013561), Autophagin-3 (MER014316), Autophagin-4 (MER064622), Cezanne deubiquitinating peptidase (MER029042), Cezanne-2 peptidase (MER029044), Tumor necrosis factor alpha-induced protein 3 (MER029050), Trabid peptidase (MER029052), VCIP135 deubiquitinating peptidase (MER152304), Otubain-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), glycosyltransferase 28 domain-containing 1, isoform CRA_C (Homo sapiens)-like (MER123606), hin1L g.p. (Homo sapiens) (MER139816), ataxin-3 (MER099998), ATXN3L putative peptidase (MER115261), Josephin domain-containing 1 (Homo sapiens) (MER125334), Josephin domain-containing 2 (Homo sapiens) (MER124068), YOD1 peptidase (MER116559), legumain (plant alpha type) (MER044591), legumain (MER001800), glycosylphosphatidylinositol:protein transamidase (MER002479), legumain pseudogene (Homo sapiens) (MER029741), family C13 unassigned peptidase (MER175813), caspase-1 (MER000850), caspase-3 (MER000853), caspase-7 (MER002705), caspase-6 (MER002708), caspase-2 (MER001644), caspase-4 (MER001938), caspase-5 (MER002240), caspase-8 (MER002849), caspase-9 (MER002707), caspase-10 (MER002579), caspase-14 (MER012083), paracaspase (MER019325), Marnesin-AA143 peptidase (MER021304), Marnesin-AA186 peptidase (MER020516), putative caspase (Homo sapiens) (MER021463), FLIP protein (MER003026), Marnesin-AA142 protein (MER021316), caspase-12 pseudogene (Homo sapiens) (MER019698), Marnesin-AA093 caspase pseudogene (MER014766),Subfamily C14A non-peptidase homolog (MER185329), Subfamily C14A non-peptidase homolog (MER179956), Separase (Homo sapiens type) (MER011775), Separase-like pseudogene (MER014797), SENP1 peptidase (MER011012), SENP3 peptidase (MER011019), SENP6 peptidase (MER011109), SENP2 peptidase (MER012183), SENP5 peptidase (MER014032), SENP7 peptidase (MER014095), SENP8 peptidase (MER016161), SENP4 peptidase (MER005557), Pyroglutamyl peptidase I (Chordate) (MER011032), Marneame-AA073 peptidase (MER029978), Sonic hedgehog protein (MER002539), Indian hedgehog protein (MER002538), Desert hedgehog protein (MER012170), Dipeptidyl peptidase III (MER004252), Marneame-AA164 protein (MER020410), LOC138971 gp (Homo sapiens) (MER020074), Atp23 peptidase (MER060642), Prenyl peptidase 1 (MER004246), Aminopeptidase N (MER000997), Aminopeptidase A (MER001012), Leukotriene A4 hydrolase (MER001013), Pyroglutamyl-peptidase II (MER012221), Cytoplasmic alanyl aminopeptidase (MER002746), Cystinyl aminopeptidase (MER002060), Aminopeptidase B (MER001494), Aminopeptidase PILS (MER005331), Arginyl aminopeptidase-like 1 (MER012271), Leukocyte-derived arginine aminopeptidase (MER002968), Aminopeptidase Q (MER052595), Aminopeptidase 0 (MER019730), TAtA-binding protein-related factor (MER026493), Angiotensin-converting enzyme peptidase unit 1 (MER004967), Angiotensin-converting enzyme peptidase unit 2 (MER001019),Angiotensin-converting enzyme-2 (MER011061), Marnename-AA153 protein (MER020514), Trimetrigopeptidase (MER001737), Neuroripsin (MER010991), Mitochondrial intermediate peptidase (MER003665), Marnename-AA154 protein (MER021317), Rhizopus neuroripsin-2 (MER014492), Rhizopus neuroripsin-, 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 (MER001089), Matrix metallopeptidase-13 (MER001411), Membrane-type matrix metallopeptidase-1 (MER001077), Membrane-type matrix metallopeptidase-2 (MER002383), Membrane-type matrix metallopeptidase-3 (MER002384), Membrane-type matrix metallopeptidase-4 (MER002595), Matrix metallopeptidase-20 (MER003021), Matrix metallopeptidase-19 (MER002076), Matrix metallopeptidase-23B (MER004766), Membrane-type matrix metallopeptidase-5 (MER005638), Membrane-type 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), Marname-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 homolog (MER180000)Meprin alpha subunit (MER001111), Meprin beta subunit (MER005213), Procollagen C - peptidase (MER001113), Mammalian tolloid - like 1 protein (MER005124), Mammalian - type 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), ADAMS peptidase (MER003902), ADAM9 peptidase (MER001140), ADAM10 peptidase (MER002382), ADAM12 peptidase (MER005107), ADAM19 peptidase (MER012241), ADAM15 peptidase (MER002386), ADAM17 peptidase (MER003094), ADAM20 peptidase (MER004725), ADAMDEC1 peptidase (MER000743), ADAMTS3 peptidase (MER005100), ADAMTS4 peptidase (MER005101), ADAMTS1 peptidase (MER005546), ADAM28 peptidase (Homo sapiens - type) (MER005495), ADAMTS5 peptidase (MER005548), ADAMTS8 peptidase (MER005545), ADAMTS6 peptidase (MER005893), ADAMTS7 peptidase (MER005894), ADAM30 peptidase (MER006268), ADAM21 peptidase (Homo sapiens type) (MER004726), ADAMTS10 peptidase (MER014331), ADAMTS12 peptidase (MER014337), ADAMTS13 peptidase (MER015450), ADAM33 peptidase MER029996), ADAMTS20 peptidase (Homo sapiens 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 type) (MER005199), ADAM11 protein (MER001146), ADAM22 protein (MER005102), ADAM23 protein (MER005103), ADAM29 protein (MER006267), ADAM21 peptidase preproprotein (Homo sapiens) (MER026944)-similar protein, marenostrin-AA225 peptidase homolog (Homo sapiens) (MER047474), putative 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), neprilysin (MER001050), endothelin converting enzyme 1 (MER001057), endothelin converting enzyme 2 (MER004776), DINE peptidase (MER005197), neprilysin-2 (MER013406), Kell blood group protein (MER001054), pHEX peptidase (MER002062), i-AAA peptidase (MER001246), i-AAA peptidase (MER005755), paraplegin (MER004454), Afg3-like protein 2 (MER005496),Afg3-like protein 1A (MER014306), papain-1 (MER002217), papain-2 (MER014521), farnesylated protein converting enzyme 1 (MER002646), metalloprotease-related 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 (MER001205), 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 0 (MER016044), cytoplasmic IC carboxypeptidase-like protein 5 (MER033174), cytoplasmic IC carboxypeptidase 3 (MER033176), cytoplasmic IC carboxypeptidase 6 (MER033178), cytoplasmic IC carboxypeptidase 1 (MER033179), cytoplasmic IC 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 IC carboxypeptidase (MER026952), family M14 non-peptidase homolog (MER199530), insulin (MER001214), mitochondrial processing peptidase beta-subunit (MER004497), nalziricin (MER003883)Eupitrilysin (MER004877), mitochondrial processing peptidase non-peptidase alpha subunit (MER001413), ubiquinol-cytochrome c reductase core protein I (MER003543), ubiquinol-cytochrome c reductase core protein II (MER003544), ubiquinol-cytochrome c reductase core protein domain 2 (MER043998), insulysin unit 2 (MER046821), nalzlysin unit 2 (MER046874), insulysin unit 3 (MER078753), mitochondrial processing peptidase subunit alpha unit 2 (MER124489), nalzlysin unit 3 (MER142856), LOC133083g.p. (Homo sapiens) (MER021876), superfamily M16B non-peptidase homolog (MER188757), leucyl aminopeptidase (animal) (MER003100), marnname-AA040 peptidase (MER003919), leucyl aminopeptidase-1 (Caenorhabditis type) (MER013416), methionyl aminopeptidase 1 (MER001342), methionyl aminopeptidase 2 (MER001728), aminopeptidase P2 (MER004498), Xaa-Pro dipeptidase (eukaryote) (MER001248), aminopeptidase P1 (MER004321), mitochondrial intermediate peptidase 55 kDa (MER013463), mitochondrial methionyl aminopeptidase (MER014055), marnname-AA020 peptidase homolog (MER010972), proliferation-associated protein 1 (MER005497), chromatin-specific transcription elongation factor 140 kDa subunit (MER026495), proliferation-associated protein 1-like (Homo sapiens chromosome X) (MER029983), marnname-AA226 peptidase homolog (Homo sapiens) (MER056262), marnname-AA227 peptidase homolog (Homo sapiens) (MER047299), superfamily M24A non-peptidase homolog (MER179893), aspartyl aminopeptidase (MER003373),Gly-Xaa carboxypeptidase (MER033182), carnosine dipeptidase II (MER014551), carnosine dipeptidase I (MER015142), Marname-AA161 protein (MER021873), aminoacylase (MER001271), glutamate carboxypeptidase II (MER002104), NAALADASE L peptidase (MER, 005239), Glutamate carboxypeptidase III (MER005238), Plasma glutamate carboxypeptidase (MER005244), Marname-AA103 peptidase (MER015091), Fxna peptidase (MER029965), Transferrin receptor protein (MER002105), Transferrin receptor 2 protein (MER005152), Glutaminyl cyclase (MER015095), Glutamate carboxypeptidase II (Homo sapiens) type non-peptidase homolog (MER026971), Nicarin (MER044627), Membrane dipeptidase (MER001260), Membrane-bound dipeptidase-2 (MER013499), Membrane-bound dipeptidase-3 (MER013496), Dihydro-orotase (MER005767), Dihydropyrimidinase (MER033266), Dihydropyrimidinase-related protein-1 (MER030143), Dihydropyrimidinase-related protein-2 (MER030155), Dihydropyrimidinase-related protein-3 (MER030151), Dihydropyrimidinase-related protein-4 (MER030149), Dihydropyrimidinase-related protein-5 (MER030136), Hypothetical protein-like 5730457F11RIK (MER033184), 1300019j08rik protein (MER033186)), Guanine aminohydrolase (MER037714), Kae1 putative peptidase (MER001577), OSGEPL1-like protein (MER013498), S2P peptidase (MER004458), Superfamily M23B non-peptidase homolog (MER199845), Superfamily M23B non-peptidase homolog (MER199846), Superfamily M23B non-peptidase homolog (MER199847), Superfamily M23B non-peptidase homolog (MER137320), Superfamily M23B non-peptidase homolog (MER201557), Superfamily M23B non-peptidase homolog (MER199417), Superfamily M23B non-peptidase homolog (MER199418), Superfamily M23B non-peptidase homolog (MER199419),Superfamily M23B non-peptidase homolog (MER199420), Superfamily M23B non-peptidase homolog (MER175932), Superfamily M23B non-peptidase homolog (MER199665), Poh1 peptidase (MER020382), Jab1 / MPN domain metalloenzyme (MER022057), Marne-AA165 peptidase (MER021865), Brcc36 isopeptidase (MER021890), Histone H2A deubiquitinase MYSM1 (MER021887), AMSH deubiquitinating peptidase (MER030146), Putative peptidase (Homo sapiens chromosome 2) (MER029970), Marne-AA168 protein (MER021886), COP9 signalosome subunit 6 (MER030137), 26S proteasome non-ATPase regulatory subunit 7 (MER030134), Eukaryotic translation initiation factor 3 subunit 5 (MER030133), 1FP38 peptidase homolog (MER030132), Superfamily M67A non-peptidase homolog (MER191181), Superfamily M67A unassigned peptidase (MER191144), Granzyme B (Homo sapiens type) (MER000168), Testisin (MER005212), Tryptase beta (MER000136), Kallikrein-related peptidase 5 (MER005544), Chymase (MER005881), Kallikrein-related peptidase 12 (MER006038), DESC1 peptidase (MER006298), Tryptase gamma1 (MER011036), Kallikrein-related peptidase 14 (MER011038), Hyaluronan-binding peptidase (MER003612), Transmembrane peptidase, serine 4 (MER011104), Intestinal serine peptidase (rodent) (MER016130), Adrenal-secreted serine peptidase (MER003734), Tryptase delta1 (Homo sapiens) (MER005948), Matriptase-3 (MER029902), Matrilysin (MER006119), Tryptase-6 (MER006118), Ovokininase-1 domain 1 (MER099182), Transmembrane peptidase,Serine 3 (MER005926), Kallikrein-related peptidase 15 (MER000064), Marnam-AA031 peptidase (MER014054), TMPRSS13 peptidase (MER014226), Marnam-AA038 peptidase (MER062848), Marnam-AA204 peptidase (MER029980), Cationic trypsin (Homo sapiens type) (MER000020), Elastase-2 (MER000118), Mannan-binding lectin-associated serine peptidase-3 (MER031968), Cathepsin G (MER000082), Myeloblastin (MER000170), Granzyme A (MER001379), Granzyme M (MER001541), Chymase (Homo sapiens type) (MER000123), Tryptase alpha (MER000135), Granzyme K (MER001936), Granzyme H (MER000166), Chymotrypsin B (MER000001), Elastase-1 (MER003733), Pancreatic endopeptidase E (MER000149), Pancreatic elastase II (MER000146), Enteropeptidase (MER002068), Chymotrypsin C (MER000761), Prostasin (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 activation C1r (MER000238), Complement component activation C1s (MER000239), 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)Thrombin (MER000188), Protein C (activated) (MER000222), Acrosin (MER000078), Hepsin (MER000156), Hepatocyte growth factor activator (MER000186), Mannan-binding lectin-associated serine peptidase 2 (MER002758), u-Plasminogen activator (MER000195), t-Plasminogen activator (MER000192), Plasmin (MER000175), Kallikrein-related peptidase 6 (MER002580), Neurotrypsin (MER004171), Kallikrein-related peptidase 8 (MER005400), Kallikrein-related peptidase 10 (MER003645), Epicellarin (MER003736), Kallikrein-related peptidase 4 (MER005266), Proseminin (MER004214), Chymopasin (MER001503), Kallikrein-related peptidase 11 (MER004861), Kallikrein-related peptidase 11 (MER216142), Trypsin-2 type 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-related peptidase 7 (MER002001), Matriptase (MER003735), Kallikrein-related peptidase 13 (MER005269), Kallikrein-related peptidase 9 (MER005270), Matriptase-2 (MER005278), Umbilical vein peptidase (MER005421), LCLP peptidase (MER001900), Spinensin (MER014385), Marapsin-2 (MER021929), Complement factor D-like putative peptidase (MER056164), Ovokimase-2 (MER022410), HAT-like 4 peptidase (MER044589), Ovokimase 1 domain 1 (MER022412),Epidermis-specific SP-like putative peptidase (MER029900), testis serine peptidase 5 (MER029901), Marneame-AA258 peptidase (MER000285), polycerase-IA unit 1 (MER030879), polycerase-IA unit 2 (MER030880), testis serine peptidase 2 (human type) (MER033187), putative acrosin-like peptidase (Homo sapiens) (MER033253), HAT-like 5 peptidase (MER028215), polycerase-3 unit 1 (MER061763), polycerase-3 unit 2 (MER061748), tryptophan-similar peptidase / serine protease (MER056263), polycerase-2 unit 1 (MER061777), Marneame-AA123 peptidase (MER021930), HAT-like 2 peptidase (MER099184), hCG2041452-like protein (MER099172), hCG22067 (Homo sapiens) (MER099169), brain rescue factor-1 (Homo sapiens) (MER098873), hCG2041108 (Homo sapiens) (MER099173), polycerase-2 unit 2 (MER061760), polycerase-2 unit 3 (MER065694), Marneame-AA201 (peptidase homolog) MER099175, secreted trypsin-like serine peptidase homolog (MER030000), polycerase-1A unit 3 (MER029880), azurocidin (MER000119), heptoglobin-1 (MER000233), heptoglobin-related 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 (MER015077), Trypsin pseudogene II (MER015078), Trypsin pseudogene III (MER015079), Superfamily S1A unassigned peptidase (MER216982), Superfamily S1A unassigned peptidase (MER216148), Amidophosphoribosyltransferase precursor (MER003, 314), Glutamine-fructose-6-phosphate transaminase 1 (MER003322), Glutamine: fructose-6-phosphate amidotransferase (MER012158), Marname-AA144 protein (MER021319), Asparagine synthetase (MER033254), Family C44 non-peptidase homolog (MER159286), Family C44 unassigned peptidase (MER185625), Family C44 unassigned peptidase (MER185626), Secretinin 1 (MER045376), Secretinin 2 (MER064573), Secretinin 3 (MER064582), Acid ceramidase precursor (MER100794), N-acylethanolamine acid 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 (MER001515), Proteasome catalytic subunit 3i (MER000555), Proteasome catalytic subunit 5t (MER026203), Protein serine kinase c17 (MER026497), Proteasome subunit alpha 6 (MER000557), Proteasome subunit alpha 2 (MER000550), Proteasome subunit alpha 4 (MER000554), Proteasome subunit alpha 7 (MER033250), Proteasome subunit alpha 5 (MER000558), Proteasome subunit alpha 1 (MER000549), Proteasome subunit alpha 3 (MER000553), Proteasome subunit XAPC7 (MER004372), Proteasome subunit beta 3 (MER001710), Proteasome subunit beta 2 (MER002676), Proteasome subunit beta 1 (MER000551), Proteasome subunit beta 4 (MER001711), Marname-AA230 peptidase homolog (Homo sapiens) (MER047329),Marnename-AA231 pseudogene (Homo sapiens) (MER047172), Marnename-AA232 pseudogene (Homo sapiens) (MER047316), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), taspease-1 (MER016969), gamma-glutamyltransferase 5 (mammalian type) (MER001977), gamma-glutamyltransferase 1 (mammalian type) (MER001629), gamma-glutamyltransferase 2 (Homo sapiens) (MER001976), gamma-glutamyltransferase-like protein 4 (MER002721), gamma-glutamyltransferase-like protein 3 (MER016970), similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026204), similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026205), Marnename-AA211 putative peptidase (MER026207), gamma-glutamyltransferase 6 (MER159283), gamma-glutamyl transpeptidase homolog (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824), KIAA1879 protein (MER159329), autosomal dominant polycystic kidney disease 1-like 3 (MER172554), gamma-glutamyl hydrolase (MER002963), guanosine 5'-monophosphate synthase (MER043387), carbamoyl-phosphate synthetase (Homo sapiens type) (MER078640), dihydro-orotase (N-terminal unit) (Homo sapiens type) (MER060647) DJ-1 putative peptidase (MER003390), Marnename-AA100 putative peptidase (MER014802), Marnename-AA101 non-peptidase homolog (MER014803), KIAA0361 protein (Homo sapiens type) (MER042827), F1134283 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), EGF-like module-containing mucin-like hormone receptor-like 2 (MER037230), CD97 antigen (human type) (MER037286), EGF-like module-containing mucin-like hormone receptor-like 3 (MER037288), EGF-like module-containing mucin-like hormone receptor-like 1 (MER037278), EGF-like module-containing mucin-like hormone receptor-like 4 (MER037294), cadherin EGF LAG seven-pass transmembrane G-type receptor 2 precursor (Homo sapiens) (MER045397), Gpr64 (Mus musculus) type protein (MER123205), GPR56 (Homo sapiens) type protein (MER122057), latrophilin 2 (MER122199), latrophilin-1 (MER126380), latrophilin 3 (MER124612), protocadherin Flamingo2 (MER124239), ETL protein (MER126267), G protein-coupled receptor 112 (MER126114), seven transmembrane helix receptor (MER125448), Gpr114 protein (MER159320), GPR126 vascular-inducible G protein-coupled receptor (MER140015), GPR125 (Homo sapiens) type protein (MER159279), 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), unassigned homolog (MER166269), GPR113 protein (MER159352),Brain-specific angiogenesis inhibitor 2 (MER159746), PIDD self-processing protein unit 1 (MER020001), PIDD self-processing protein unit 2 (MER063690), MUC1 self-cleaving mucin (MER074260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site-1 protease (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), superfamily S8A non-peptidase homolog (MER201339), superfamily S8A non-peptidase homolog (MER191613), superfamily S8A unassigned peptidase (MER191611), superfamily S8A unassigned peptidase (MER191612), superfamily S8A unassigned peptidase (MER191614), tripeptidyl-peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl-peptidase IV (eukaryote) (MER000401), acylaminoacyl-peptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPL A protein (MER004227), dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Marnem-AA194 putative peptidase (MER017353), Marnem-AA195 putative peptidase (MER017367), Marnem-AA196 putative peptidase (MER017368), Marnem-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical 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 similar to Mus musculus chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198), carboxylesterase D1 (MER033213), liver carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile salt-dependent lipase (MER033227), carboxylesterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4, X-linked (MER033235), neuroligin 4, Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 non-peptidase homolog (MER212939), family S9 non-peptidase homolog (MER211490), superfamily S9C unassigned peptidase (MER192341), family S9 unassigned peptidase (MER209181), family S9 unassigned peptidase (MER200434), family S9 unassigned peptidase (MER209507), family S9 unassigned peptidase (MER209142), serine carboxypeptidase A (MER000430), vitellogenic carboxypeptidase-like protein (MER005492),RISC peptidase (MER010960), unassigned peptidase of family S15 (MER199442), unassigned peptidase of family S15 (MER200437), unassigned peptidase of family S15 (MER212825), lysosomal Pro-Xaa carboxypeptidase (MER000446), dipeptidyl-peptidase II (MER004952), thymus-specific serine peptidase (MER005538), epoxide hydrolase-like putative peptidase (MER031, 614), proteins similar to Loc328574-like protein (MER033246), abhydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm-specific transcript protein (MER199890), mesoderm-specific transcript protein (MER017123), cytoplasmic epoxide hydrolase (MER029997), cytoplasmic epoxide hydrolase (MER213866), CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical 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), glycosylasparaginase precursor (MER003299), isoaspartyl dipeptidase (threonine type) (MER031622), taspease-1 (MER016969), gamma-glutamyltransferase 5 (mammalian type) (MER001977), gamma-glutamyltransferase 1 (mammalian type) (MER001629), gamma-glutamyltransferase 2 (Homo sapiens) (MER001976), gamma-glutamyltransferase-like protein 4 (MER002721), gamma-glutamyltransferase-like protein 3 (MER016970), similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026204), similar to gamma-glutamyltransferase 1 precursor (Homo sapiens) (MER026205), Marnename-AA211 putative peptidase (MER026207), gamma-glutamyltransferase 6 (MER159283), gamma-glutamyltranspeptidase homolog, (chromosome 2, Homo sapiens) (MER037241), polycystin-1 (MER126824),KIAA1879 protein (MER159329). Polycystic kidney 1-like 3 (MER172554). Gamma-glutamyl hydrolase (MER002963). Guanylate 5'-monophosphate synthase (MER043387). Carbamoyl-phosphate synthetase (Homo sapiens type) (MER078640). Dihydro-orotase (N-terminal unit) (Homo sapiens type) (MER060647). DJ-1 putative peptidase (MER003390). Marne-AA100 putative peptidase (MER014802). Marne-AA101 non-peptidase homolog (MER014803). KIAA0361 protein (Homo sapiens type) (MER042827). FLJ34283 protein (Homo sapiens) (MER044553). Non-peptidase homolog on 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). EGF-like module containing mucin-like hormone receptor-like 2 (MER037230). CD97 antigen (human type) (MER037286). EGF-like module containing mucin-like hormone receptor-like 3 (MER037288). EGF-like module containing mucin-like hormone receptor-like 1 (MER037278). EGF-like module containing mucin-like hormone receptor-like 4 (MER037294). Cadherin EGFLAG 7-pass G-type receptor 2 precursor (Homo sapiens) (MER045397),Gpr64 (Mus musculus) protein (MER123205). GPR56 (Homo sapiens) protein (MER122057). Latrophilin 2 (MER122199). Latrophilin-1 (MER126380). Latrophilin 3 (MER124612). Protocadherin Flamingo 2 (MER124239). ETL protein (MER126267). G protein-coupled receptor 112 (MER126114). Seven-transmembrane helix receptor (MER125448). Gpr114 protein (MER159320). GPR126 vascular-inducible G protein-coupled receptor (MER140015). GPR125 (Homo sapiens) protein (MER159279). 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), unassigned homolog (MER166269), GPR113 protein (MER159352), brain-specific angiogenesis inhibitor 2 (MER159746), PIDD self-processing protein unit 1 (MER020001), PIDD self-processing protein unit 2 (MER063690), MUC1 autocleaving mucin (MER074260), dystroglycan (MER054741), proprotein convertase 9 (MER022416), site-1 protease (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 non-peptidase homolog (MER201339), subfamily S8A non-peptidase homolog (MER191613), subfamily S8A unassigned peptidase (MER191611), subfamily S8A unassigned peptidase (MER191612), subfamily S8A unassigned peptidase (MER191614), tripeptidyl-peptidase I (MER003575), prolyl oligopeptidase (MER000393), dipeptidyl-peptidase IV (eukaryote) (MER000401), acylaminoacyl-peptidase (MER000408), fibroblast activation protein alpha subunit (MER000399), PREPLA protein (MER004227), dipeptidyl-peptidase 8 (MER013484), dipeptidyl-peptidase 9 (MER004923), FLJ1 putative peptidase (MER017240), Marnelym-AA194 putative peptidase (MER017353), Marnelym-AA195 putative peptidase (MER017367), Marnelym-AA196 putative peptidase (MER017368), Marnelym-AA197 putative peptidase (MER017371), C14orf29 protein (MER033244), hypothetical protein (MER033245), hypothetical esterase / lipase / thioesterase (MER047309), protein BAt5 (MER037840), hypothetical protein flj40219 (MER033212), hypothetical protein flj37464 (MER033240), hypothetical protein flj33678 (MER033241), dipeptidyl peptidase homolog DPP6 (MER000403), dipeptidyl peptidase homolog DPP10 (MER005988), protein similar to Musmusculus chromosome 20 open reading frame 135 (MER037845), kynurenine formamidase (MER046020), thyroglobulin precursor (MER011604), acetylcholinesterase (MER033188), cholinesterase (MER033198), carboxylesterase D1 (MER033213)Liver carboxylesterase (MER033220), carboxylesterase 3 (MER033224), carboxylesterase 2 (MER033226), bile salt-dependent lipase (MER033227), carboxylesterase-related protein (MER033231), neuroligin 3 (MER033232), neuroligin 4, X-linked (MER033235), neuroligin 4, Y-linked (MER033236), esterase D (MER043126), arylacetamide deacetylase (MER033237), KIAA1363-like protein (MER033242), hormone-sensitive lipase (MER033274), neuroligin 1 (MER033280), neuroligin 2 (MER033283), family S9 non-peptidase homolog (MER212939), family S9 non-peptidase homolog (MER211490), subfamily S9C unassigned peptidase (MER192341), family S9 unassigned peptidase (MER209181), family S9 unassigned peptidase (MER200434), family S9 unassigned peptidase (MER209507), family S9 unassigned peptidase (MER209142), serine carboxypeptidase A (MER000430), vitellogenic carboxypeptidase-like protein (MER005492), RISC peptidase (MER010960), family S15 unassigned peptidase (MER199442), family S15 unassigned peptidase (MER200437), family S15 unassigned 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), AB hydrolase domain-containing protein 4 (MER031616), epoxide hydrolase (MER000432), mesoderm-specific transcript protein (MER199890), mesoderm-specific transcript protein (MER017123)A substrate recognition sequence designed to be selectively cleavable by one or a selected subgroup of human proteases selected from the group consisting of cytoplasmic IC epoxide hydrolase (MER029997), cytoplasmic IC epoxide hydrolase (MER213866), putative protein FLJ22408 (MER031608)-like, CGI-58 putative peptidase (MER030163), Williams-Beuren syndrome critical region protein 21 epoxide hydrolase (MER031610), epoxide hydrolase (MER031612), putative protein flj22408 (epoxide hydrolase) (MER031617), monoglyceride lipase (MER033247), putative protein (MER033249), valganciclovir hydrolase (MER033259), CCg1-interacting factor b (MER210738) may be utilized.

[0208] In some embodiments, the substrate recognition sequence is a peptide portion up to 15 amino acids in length. The substrate recognition sequence is cleaved by a protease. In some embodiments, the protease is co-localized with a target at a cell-binding moiety within a tissue, and when the binder-drug conjugate is exposed to the protease, the protease cleaves the substrate recognition sequence in the drug conjugate portion. In some embodiments, the protease is inactive or significantly less active in tissues that do not significantly express cell surface features. In some embodiments, the protease is inactive or significantly less active in healthy, e.g., non-diseased tissues.

[0209] In certain embodiments, the substrate recognition sequence is cleaved by a protease selected from: - ADAMS or ADAMTS, e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4 or ADAMTS5. - Aspartic proteases, e.g., BACE or renin. - Aspartic cathepsin (upregulated or released to the extent by cell lysis in the extracellular space), for example, cathepsin D or cathepsin E. - Caspase (upregulated or released to the extent by cell lysis in the extracellular space), for example, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10 or caspase 14. - Cysteine cathepsin, for example, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P. - Cysteine protease, for example, cruzain, legumain or oubain-2. - KLK, for example, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14. - Metalloprotease, for example, meprin, neprilysin, PSMA or BMP-1, - MMP, for example, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27. - Serine protease, for example, activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor protease (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidino benzoatase, HtrA1, human neutrophil elastase, lactoferrin, malapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase or uPA. - Type II transmembrane serine protease (TTSP), for example, DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3, TMPRSS4.

[0210] For example, suitable substrate recognition sequences that may be included in the binder-drug conjugate, i.e., SRSs, are peptide moieties selected from the group consisting of TGRGPSWV, SARGPSRW, TARGPSFK, LSGRSDNH, GGWHTGRN, HTGRSGAL, PLTGRSGG, AARGPAIH, RGPAFNPM, SSRGPAYL, RGPATPIM, RGPA, GGQPSGMWGW, FPRPLGITGL, VHMPLGFLGP, SPLTGRSG, SAGFSLPA, LAPLGLQRR, SGGPLGVR, PLGL, GPRSFGL, and GPRSFG.

[0211] In some embodiments, the substrate recognition sequence is a substrate for MMP, such as a sequence selected from the group consisting of ISSGLLSS, QNQALRMA, AQNLLGMV, STFPFGMF, PVGYTSSL, DWLYWPGI, MIAPVAYR, RPSPMWAY, WATPRPMR, FRLLDWQW, LKAAPRWA, GPSHLVLT, LPGGLSPW, MGLFSEAG, SPLPLRVP, RMHLRSLG, LAAPLGLL, AVGLLAPP, LLAPSHRA, PAGLWLDP, and ISSGLSS.

[0212] In some embodiments, the substrate recognition sequence is a substrate for MMP, such as a sequence selected from the group consisting of ISSGLSS, QNQALRMA, AQNLLGMV, STFPFGMF, PVGYTSSL, DWLYWPGI, ISSGLLSS, LKAAPRWA, GPSHLVLT, LPGGLSPW, MGLFSEAG, SPLPLRVP, RMHLRSLG, LAAPLGLL, AVGLLAPP, LLAPSHRA, and PAGLWLDP.

[0213] In some embodiments, the substrate recognition sequence is a substrate for thrombin, such as GPRSFGL or GPRSFG.

[0214] In certain embodiments of the subject binder-drug conjugate, the substrate recognition sequence is cleaved by fibroblast activation protein alpha (FAPα) and is represented by the formula [Chemical formula] In the formula, R 2 represents H or (C1-C6) alkyl, preferably H; R 3 represents H or (C1-C6) alkyl, preferably methyl, ethyl, propyl, or isopropyl, more preferably methyl; R 4 is absent or represents (C1-C6) alkyl, -OH, -NH2, or halogen; X represents O or S; and -NH- is part of L 2 when L is a self-cleavable linker, or represents an amine that is part of DM when L 2 is a bond. 2 In certain embodiments, R

[0215] 2 is H, R 3 3 is methyl, R 4 is absent, and X is O.

[0216] b. Self-cleavable The binder-drug conjugate of the present invention can employ a heterocyclic self-destructing moiety covalently attached to the drug moiety and a cleavable substrate recognition sequence moiety. The self-destructing moiety can covalently bond two spaced chemical moieties together to form a generally stable molecule, and by enzymatic cleavage, one of the spaced chemical moieties is liberated from the molecule; following the enzymatic cleavage, it may be defined as a bifunctional chemical group that spontaneously cleaves from the remainder of the bifunctional chemical group to liberate the other of the spaced chemical moieties. According to the present invention, the self-destructing moiety is covalently bonded to the ligand by an amide bond, directly or indirectly via a spacer unit, at one of its ends, and covalently bonded to a chemical reaction site (functional group) hanging from the drug at the other of its ends. Derivatization of the drug moiety by the self-destructing moiety may render the drug pharmacologically less active (e.g., less toxic) until the drug is cleaved, or may render it completely inactive.

[0217] Binder-drug conjugates are generally stable in circulation, or at least should be so in the absence of an enzyme capable of cleaving the amide bond between the substrate recognition sequence and the self-destructing moiety. However, upon exposure of the binder-drug conjugate to a suitable enzyme, the amide bond is cleaved, initiating a spontaneous self-destruction reaction, as a result of which the bond covalently attaching the self-destructing moiety to the drug undergoes cleavage, thereby achieving the liberation of the free drug moiety in its non-derivatized or pharmacologically active form.

[0218] The self-destructing moiety in the conjugate of the present invention incorporates one or more heteroatoms, thereby providing improved solubility, improving the cleavage rate, and reducing the tendency of the conjugate to aggregate. These improvements of the heterocyclic self-destructing linker construct of the present invention over the non-heterocyclic PAB-type linker may result in surprising and unexpected biological properties such as increased potency, reduced toxicity, and more desirable pharmacokinetics.

[0219] In certain embodiments, L 2 is a benzyloxycarbonyl group.

[0220] In certain embodiments, L 2 is of the formula

Chemical formula

[0221] In certain embodiments, L 2 is selected from the following.

Chemical formula

[0222] In certain embodiments, the self - destructing moiety L2 is selected from the following,

Chemical formula

[0223] When T is NH, it is derived from a primary amine (-NH2) hanging from the drug moiety (before attachment to the self-destructing moiety), and when T is N, it is derived from a secondary amine from the (-MH-) drug moiety (before attachment to the self-destructing moiety). Similarly, when T is O or S, it is derived from a hydroxyl group (-OH) or a sulfhydryl (-SH) group hanging from the drug moiety, respectively, before attachment to the self-destructing moiety.

[0224] In certain embodiments, the self-destructing linker L 2 is -NH-(CH2)4-C(=O)- or -NH-(CH2)3-C(=O)-.

[0225] In certain embodiments, the self-destructing linker L 2 is p-aminobenzyloxycarbonyl (PABC).

[0226] In certain embodiments, the self-destructing linker L 2 is 2,4-bis(hydroxymethyl)aniline.

[0227] Other exemplary self-immolative linkers that are readily adaptable for use in the present invention are taught, for example, in U.S. Patent US7754681, WO2012074693A1, US9089614, EP1732607A2, WO2015038426A1 (all of which are incorporated by reference), Walther, et al. “Prodrugs in medicinal chemistry and enzyme prodrug therapies”, Adv. Drug Deliv. Rev. 2017, Sep. 1; 118:65 - 77, and Tranoy-Opalinski, et al. “Design of self-immolative linkers for tumour-activated prodrug therapy”, Anticancer Agents Med Chem. 2008, Aug; 8(6):618 - 37; the teachings of each are incorporated herein by reference.

[0228] c. Drug moiety A wide range of drug entities can be used as the drug moiety, DM, of the subject binder-drug conjugate.

[0229] In certain embodiments, the free drug moiety is an immunomodulatory agent, which includes drug moieties that act as immune activators and / or inducers of innate immune pathway responses. In certain embodiments, the free drug moiety induces the production of IFN-α. In certain embodiments, the free drug moiety induces the production of pro-inflammatory cytokines. In certain embodiments, the free drug moiety induces the production of IL-1β. In certain embodiments, the free drug moiety induces the production of IL-18.

[0230] In certain embodiments, the free drug moiety promotes the proliferation and survival of effector cells including NK, γδT, and CD8+ T cells.

[0231] In certain embodiments, the free drug moiety induces macrophage pyroptosis.

[0232] (i) Exemplary ImmunoDASH inhibitors In certain embodiments, the ImmunoDASH inhibitors for use in the methods of the invention are represented by the general formula

Chemical formula

Chemical formula

[0233] In a preferred embodiment, ring A is a 5-, 6-, or 7-membered ring, for example, represented by the formula, and more preferably, a 5- or 6-membered ring (i.e., n may be 3 or 4, but n is 1 or 2).

Chemical formula

[0234] Optionally, the ring can be further substituted.

[0235] In a preferred embodiment, W is

Chemical formula

[0236] In a preferred embodiment, R’1 is as follows,

Chemical formula

[0237] In a preferred embodiment, R’2 is absent, or represents a small hydrophobic group such as lower alkyl or halogen.

[0238] In a preferred embodiment, R'3 is hydrogen, or a small hydrophobic group such as lower alkyl or halogen.

[0239] In a preferred embodiment, R'5 is hydrogen, or halogenated lower alkyl.

[0240] In a preferred embodiment, X1 is fluorine, and when it is a halogen, X2 and X3 are fluorine.

[0241] Also considered to be equivalents are any compounds that can be converted by hydrolysis into any of the aforementioned compounds including boronic acid esters and halides, as well as carbonyl equivalents including acetals, hemiacetals, ketals, and hemiketals, and cyclic dipeptide analogs.

[0242] In certain preferred embodiments, the subject method utilizes a boronic acid analog of an amino acid as an immunodash inhibitor. For example, the present invention contemplates the use of boroprolyl derivatives in the subject method. Exemplary boronic acid-derived inhibitors of the present invention are represented by the general formula,

Chemical formula

[0243] In certain embodiments, the immunodash inhibitor comprises a prolyl group or an analog thereof at the P1 specificity position and a nonpolar (and preferably hydrophobic) amino acid, such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan or methionine, or an analog thereof at the P2 specificity position, and is a peptide or peptidomimetic. In other embodiments, P2 is disposed with an amino acid having a charged side chain, such as arginine, lysine, aspartic acid or glutamic acid. For example, the immunodash inhibitor may comprise an Ala-Pro or Val-Pro dipeptide sequence or an equivalent thereof, and may be represented by the general formula: [Chemical formula]

[0244] In a preferred embodiment, ring A is a 5-, 6- or 7-membered ring and is represented, for example, by the formula. [Chemical formula]

[0245] In certain preferred embodiments, R32 is a small hydrophobic group, such as lower alkyl or halogen.

[0246] In certain preferred embodiments, R32 is -(CH2) m -NH-C(=N)(NH2), -(CH2) m -NH2 or -(CH2) m -lower alkyl-guanidine, -lower alkyl-amine, lower alkyl-C(O)OH such as -COOH, where m is 1 to 6, preferably 1 to 3.

[0247] In a preferred embodiment, R'2 is absent or represents a small hydrophobic group such as lower alkyl or halogen.

[0248] In a preferred embodiment, R’3 is hydrogen or a small hydrophobic group such as lower alkyl or halogen.

[0249] Another aspect of the invention relates to an immunodash inhibitor represented by Formula III, or a pharmaceutically acceptable salt thereof:

Chemical formula

Chemical formula

[0250] Another aspect of the present invention relates to an immunodash inhibitor represented by formula IV, or a pharmaceutically acceptable salt thereof:

Chemical formula

Chemical formula

[0251] In certain preferred embodiments, the ImmunoDASH inhibitor is a boronic acid inhibitor of the DASH enzymes DPP8 and DPP9 (and optionally also DPP-4 and / or FAP).

[0252] In certain preferred embodiments, the ImmunoDASH inhibitor is a dipeptide boronic acid inhibitor of the DASH enzymes DPP8 and DPP9 (and optionally also DPP-4 and / or FAP). In certain preferred embodiments, the ImmunoDASH inhibitor dipeptide boronic acid has a proline or proline analog at the P1 position. The subject ImmunoDASH inhibitor can mediate tumor regression by an immune-mediated mechanism. The subject ImmunoDASH inhibitor induces macrophage pyroptosis, has activities such as immunogenicity regulation directly or indirectly, sensitizes tumor cells to antigen-specific CTL killing, changes the subset and function of immune cells, accelerates T cell priming through regulation of dendritic cell trafficking, and induces general T cell-mediated anti-tumor activity.

[0253] In certain embodiments, the subject combination of an ImmunoDASH inhibitor and a PD-1 inhibitor can be administered as part of a treatment comprising one or more other chemotherapeutic agents, immuno-oncology agents, or radiation. It can also be used as part of treatment methods including tumor vaccines, adoptive cell therapy, gene therapy, oncolytic virus therapy, etc.

[0254] In certain embodiments, the ImmunoDASH inhibitor of the method is represented by Formula I or a pharmaceutically acceptable salt thereof,

Chemical formula

[0255] In certain embodiments, the immuno-DASH inhibitor of formula I is represented by formula Ia, or a pharmaceutically acceptable salt thereof, [Chemical formula] wherein X, W, Z, R 1 , R 2 , R 9 and R 10 are as defined above for formula I, and p is 1, 2, or 3.

[0256] In certain preferred embodiments of Ia, R 1 is lower alkyl; R 9 is absent or, independently for each occurrence, is 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 is hydrogen, R 10 is absent or represents a single substitution of -OH, -NH2, -CN or -N3; W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0257] In certain embodiments, the immuno-DASH inhibitor of formula I is represented by formula Ib, or a pharmaceutically acceptable salt thereof, [Chemical formula] wherein X, W, R 1 , R 2 , R 9 and R 10 are as defined above for formula I, and p is 1, 2, or 3.

[0258] In certain preferred embodiments of Ib, R1 is a lower alkyl. R 9 is absent or, independently for each occurrence, is 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が is hydrogen, and R 10 is absent or represents a single substitution with -OH, -NH2, -CN or -N3; W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0259] In certain embodiments, the immunomodulatory DASH inhibitor is represented by Formula Ic or a pharmaceutically acceptable salt thereof,

Chemical formula

[0260] In certain preferred embodiments of Ic, R 1 is lower alkyl; R 9 is absent or, independently for each occurrence, is 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 is hydrogen, and R 10 is absent or represents a single substitution with -OH, -NH 2、 -CN or -N3; and W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0261] In some embodiments, the immunomodulatory DASH inhibitor is represented by:

Chemical formula

[0262] Another aspect of the present invention relates to an immunodash inhibitor represented by Formula II, or a pharmaceutically acceptable salt thereof,

Chemical formula

Chemical formula

[0263] In certain embodiments, the immunomodulatory inhibitor of Formula II is represented by Formula IIa or a pharmaceutically acceptable salt thereof,

Chemical formula

[0264] In certain preferred embodiments of IIa: R 9is, independently for each occurrence, 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 is hydrogen, and R 10 is absent or represents a single substitution with -OH, -NH2, -CN or -N3; W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0265] In certain embodiments, the immunomodulatory DASH inhibitor of formula II is represented by formula IIb or a pharmaceutically acceptable salt thereof,

Chemical formula

[0266] In certain preferred embodiments of IIb: R 9 is, independently for each occurrence, 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 is hydrogen, and R 10 is absent or represents a single substitution with -OH, -NH2, -CN or -N3; W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0267] In certain embodiments, the immunomodulatory DASH inhibitor of formula II is represented by formula IIc or a pharmaceutically acceptable salt thereof,

Chemical formula

[0268] In certain preferred embodiments of IIc: R9 is, for each occurrence independently, 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 is hydrogen, R 10 is absent, or represents a single substitution of -OH, -NH2, -CN or -N3; W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0269] In certain embodiments, the immunomodulatory DASH inhibitor of formula II is represented by formula IId, or a pharmaceutically acceptable salt thereof,

Chemical formula

[0270] In certain preferred embodiments of IId: R 9 is, for each occurrence independently, 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 is hydrogen, R 10 is absent, or represents a single substitution of -OH, -NH2, -CN or -N3; W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0271] In certain embodiments, the immunomodulatory DASH inhibitor of formula II is represented by formula IIe, or a pharmaceutically acceptable salt thereof,

Chemical formula

[0272] In a particular preferred embodiment of IIe: R 9 is, independently for each occurrence, 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 is hydrogen, R 10 is absent, or represents a single substitution of -OH, -NH2, -CN or -N3; Z is a pyrrolidine ring or a piperidine ring (more preferably a pyrrolidine ring); W is -B(OH)2 or -CN (more preferably -B(OH)2).

[0273] In some embodiments, the immunoDASH inhibitor is one of the following:

Chemical formula

[0274] (ii) Exemplary STING agonists Non-limiting examples of STING agonists include agonists represented by one of the general formulas,

Chemical formula

[0275] In certain embodiments, the STING agonist is represented by one of the formulas:

Chemical formula

[0276] In the above STING agonist structure, X 3 or one of X4 is L 2が When L is a self-cleaving linker, L2 contains a functional group sharing the bond, or when L2 is the (said) bond, DM contains a functional group sharing the bond, X3 and X4 may each independently be, for example, 9-purine, 9-adenine, 9-guanine, 9-hypoxanthine, 9-xanthine, 9-uric acid, or 9-isoguanine.

[0277] X3 and X4 may be the same or different.

[0278] In some embodiments, the STING agonist may be provided mainly in the form of Rp, Rp or Rp, Sp stereoisomers. In some embodiments, the STING agonist may be provided mainly in the form of Rp, Rp stereoisomers.

[0279] Exemplary STING agonists include the following:

Chemical formula

Chemical formula

Chemical formula

[0280] In certain embodiments, the STING agonist is represented by one of the following structures. [Chemical formula]

[0281] Yet another STING agonist that can be used as a drug moiety in current conjugate conjugates is as follows. [Chemical formula]

[0282] Other exemplary STING agonists that can be readily adapted for use as drug moieties in the conjugates of the present invention are taught, by way of example only, in PCT Publications WO2017123669A1, WO2015077354A1, and U.S. Patent Publication US20150056224A1 (each incorporated herein by reference).

[0283] It will also be well understood by those skilled in the art that, particularly by using a self-immolative linker, the STING agonist can be attached to the linker via a functional group other than an amine as shown above, for example, via a free hydroxyl group.

[0284] (iii) Exemplary TLR agonists Examples of "Toll-like receptor (TLR) agonists" include, but are not limited to, TLR1 / 2 agonists, TLR2 agonists, TLR3 agonists (e.g., poly:C), TLR4 agonists (e.g., S-type lipopolysaccharide, paclitaxel, lipid A, monophosphoryl lipid A), TLR5 agonists (e.g., flagellin), TLR6 / 2 agonists (e.g., MALP-2), TLR7 agonists, TLR7 / 8 agonists (e.g., gardiquimod, imiquimod, loxoribine, and resiquimod (R848)), TLR7 / 9 agonists (e.g., hydroxychloroquine sulfate), TLR8 agonists (e.g., motolimod (VTX-2337)), TLR9 agonists (e.g., CPG-ODN), and TLR11 agonists (e.g., profilin).

[0285] Exemplary TRL agonists that can be used as the drug moiety in the binder conjugates of the present invention include 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, or, optionally, their analogs having appropriate functional groups for the indicated binding and release from the substrate recognition sequence, or having appropriate functional groups by attachment to a self-cleaving linker.

[0286] Exemplary agonists of TRL, particularly TRL7 agonists, TRL8 agonists and TRL7 / 8 agonists, include the following:

Chemical formula

Chemical formula

[0287] In certain embodiments, the drug moiety is a TRL7 / 8 agonist represented by the general formula [Chemical formula] In the formula, X is CH2, O, S or N, preferably CH2, O or N, more preferably CH2 or O; n is 0 (direct bond from N to O), or an integer from 1 to 5, preferably 1 or 2; z is an integer from 1 to 5; m is an integer from 1 to 20, preferably from 1 to 16; p is 0 (direct bond from the ring to X), or an integer from 1 to 5, preferably 1 or 2; and q is an integer from 1 to 5, preferably 1 or 2.

[0288] For example, the TRL agonist is a TRL7 / 8 agonist such as one of the following. [Chemical formula]

[0289] Publication numbers WO2008135791 and WO2016141092 also describe a class of imidazoquinoline compounds having immunomodulatory properties acting via TLR7.

[0290] Other exemplary TRL agonists that are readily adaptable for use as the drug moiety of the binder conjugate of the present invention are disclosed, for example, in 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.

[0291] It will also be fully understood by those skilled in the art that, particularly by using a self-destructing linker, the TRL agonist can be bound to the linker via a functional group other than an amine as shown above, for example, via a free hydroxyl group.

[0292] (iv) Exemplary RIG-1 agonists The conjugate according to any one of the preceding embodiments, wherein the immunostimulatory agonist is a RIG-1 agonist, and the RIG-1 agonist is KIN700, KIN1148, KIN600, KIN500, KIN100, KIN101, KIN400, KIN2000, or SB-9200.

[0293] (v) Exemplary anthracyclines In certain embodiments, the drug moiety is an anthracycline or a derivative thereof, preferably doxorubicin or other analogs capable of inducing immunogenic cell death of tumor cells.

[0294] Anthracyclines and their analogs include, but are not limited to, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, valrubicin, aclarubicin, mitoxantrone, actinomycin, bleomycin, peplomycin, and mitomycin. For example, the anthracycline moiety can be represented by the formula,

Chemical formula

[0295] (vi) Exemplary proteasome inhibitors In certain embodiments, the drug moiety is a proteasome inhibitor. Exemplary proteasome inhibitors include the following.

Chemical formula

Chemical formula

[0296] d. Cell-binding moiety In certain embodiments, the diseased tissue is a tumor. In certain embodiments, the cell-binding portion of the binder-drug conjugate is selected to bind to a cell surface protein on the tumor cells. In other embodiments, the cell-binding portion of the binder-drug conjugate is selected to bind to cell surface proteins on macrophages, monocyte-derived suppressor cells (MDSC), dendritic cells, fibroblasts, T cells, NK cells, mast cells, granulocytes, eosinophils, and B cells.

[0297] In certain 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 the target cell, it has an internalization half-life of at least 6 hours, more preferably at least 10, 12, 14, 16, 18, 20, 24, 36, 48, 60, 75, or even 100 hours.

[0298] In certain embodiments, the cell-binding portion of the binder-drug conjugate binds to a cell surface protein that is selectively expressed or upregulated by the target cells in the diseased tissue as compared to normal cells from a healthy state of the tissue. For example, the protein is detectable on the surface of the target cells at a level that is at least 2-fold higher, and even more preferably at least 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, or even 1000-fold higher than that of normal cells from the tissue.

[0299] In certain embodiments, the cell-binding portion of the binder-drug conjugate is selected to bind to a cell surface protein that is selectively expressed or upregulated by the target cells in the diseased tissue as compared to cells from other tissues, particularly cells from vital organs. For example, the protein is detectable on the surface of the target cells at a level that is at least 2-fold higher, and even more preferably at least 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, or even 1000-fold higher than that of cells from other tissues.

[0300] In certain embodiments, the cell-binding portion of the binder-drug conjugate is selected to bind to a checkpoint protein, and preferably, the cell-binding portion is an antagonist of that checkpoint. Examples of checkpoint proteins include 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, CSF-1R, and more preferably those selected from the group consisting of CTLA-4, PD-1, LAG-3, TIM-3, BTLA, VISTA, HVEM, TIGIT, PVR, PD-L1 and CD160.

[0301] In certain 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 agonist of the receptor. Examples include 4-1BB, 4-1BB-L, OX40, OX40-L, GITR, CD28, CD40, CD40-L, ICOS, ICOS-L, LIGHT, and CD27, and more preferably surface features that are co-stimulatory receptors or ligands selected from the group consisting of 4-1BB, OX40, GITR, CD40 and ICOS.

[0302] In certain embodiments, the cell-binding moiety is an antibody such as, for example, a humanized antibody, a human antibody, or a chimeric antibody, or an antigen-binding portion thereof that binds to a cell surface feature such as, for example, Fab, F(ab)2, F(ab’), F(ab’)2, F(ab’)3, Fd, Fv, disulfide-bonded Fv, dAb or sdAb (or nanobody), CDR, scFv, (scFv)2, diabody-scFv, bis-scFv, tascFv (tandem scFv), AVIBODY (e.g., diabody, tribody, tetrabody), T cell engager (BiTE), scFv-Fc, Fcab, mAb2, small modular immunopharmaceutical (SMIP), Genmab / unibody or duo-body, V-NAR domain, IgNAR, minibody, IgGACH2, DVD-Ig, probody, intrabody, or a multispecific antibody.

[0303] In other embodiments, the cell-binding agent moiety is a non-antibody scaffold selected from the group consisting of, for example, affibody, affimer, affilin, anticalin, atrimer, avimer, DARPins, FN3 scaffolds (e.g.,adnectin and centyrin), finomer, knotted domain, nanofitin, pronectin, OBodys, tribody, avimer, bicyclic peptide, and Cys-knots.

[0304] (i) PD-L1-binding affimer In certain embodiments, the cell-binding moiety is an affimer that binds to PD-L1. An affimer is a scaffold based on stefin A and means having a sequence derived from stefin A, preferably mammalian stefin A, more preferably human stefin A. One aspect of the embodiments of the present application provides an affimer that binds to PD-L1 (also referred to as an “anti-PD-L1 affimer”) comprising an affimer having an amino acid sequence derived from a wild-type stefin A protein that preferably selectively binds PD-L1 with a Kd of 10 -6 M or less.

[0305] In certain embodiments, the anti-PD-L1 affimer has a framework sequence and one or both of loop 2 [(Xaa) n named] and loop 4 [(Xaa) m named] are replaced with alternative loop sequences (Xaa) n and (Xaa) m and is derived from the wild-type human stefin A protein having the general formula (i), FR1-(Xaa) n -FR2-(Xaa) m -FR3(I) wherein, 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 thereto; FR2 is a polypeptide sequence represented by GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2) or a polypeptide sequence having at least 70% homology thereto; FR3 is a polypeptide sequence represented by EDLVLTGYQV DKNKDDELTG F (SEQ ID NO: 3) or a polypeptide sequence having at least 70% homology thereto; Xaa is an amino acid residue individually for each occurrence, and n and m are each independently an integer of 3 to 20.

[0306] In certain embodiments, FR1 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% homology with SEQ ID NO: 1. In certain embodiments, FR1 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 1; In certain embodiments, FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% homology with SEQ ID NO: 2. In certain embodiments, FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 2; In certain embodiments, FR3 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% homology with SEQ ID NO: 3. In certain embodiments, FR3 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 3.

[0307] For those embodiments in which at least one drug conjugate moiety is attached to the affimer sequence via the thiol side chain of a cysteine introduced into the affimer sequence, the cysteine is preferably provided in a part of the affimer sequence region corresponding to FR1, FR2 and / or FR3, and more preferably, its side chain is solvent accessible and substitution of the amino acid residue in the affimer that is not involved in hydrogen bonding with other parts of the affimer will be provided. Generally, cysteine will not be introduced into loop (Xaa) n or (Xaa) m will not be introduced.

[0308] In certain embodiments, the anti-PD-L1 affimer has an amino acid sequence represented by the general formula (SEQ ID NO: 4): MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(Xaa) n -Xaa2-TNYYIKVRAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa) m -Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF wherein, Xaa is an amino acid residue individually for each occurrence; n and m are each independently an integer of 3 to 20; Xaa1 is Gly, Ala, Val, Arg, Lys, Asp, or Glu, more preferably Gly, Ala, Arg or Lys, 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, even more preferably Lys or Asn; Xaa4 is Gly, 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, even more preferably Leu or Pro; Xaa6 is Gly, Ala, Val, Asp, or Glu, more preferably Ala, Val, Asp, or Glu, even more preferably Ala or Glu; Xaa7 is Ala, Val, Ile, Leu, Arg or Lys, more preferably Ile, Leu or Arg, even more preferably Leu or Arg.

[0309] For those embodiments in which at least one drug conjugate moiety is attached to the affimer sequence via the thiol side chain of cysteine introduced into the affimer sequence, the cysteine is preferably in the loop sequence (Xaa) n or (Xaa) m and would be provided as part of the affimer sequence other than as provided. Thus, SEQ ID NO: 4 may contain 1 to 5 cysteines in place of amino acid residues at various positions in the sequence.

[0310] For example, the anti-PD-L1 affimer can have an amino acid sequence represented by the general formula (SEQ ID NO: 5): MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(Xaa) n-STNYYIKVRAGDNKYMHLKVFNGP-(Xaa) m -ADRVLTGYQVDKNKDDELTGF In the formula, Xaa is an amino acid residue that is individually distinct for each occurrence; n and m are each independently an integer from 3 to 20.

[0311] In certain embodiments, 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.

[0312] In certain embodiments, 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.

[0313] In certain embodiments, Xaa is an amino acid that can be added to the polypeptide by recombinant expression in prokaryotic or eukaryotic cells independently for each occurrence, and more preferably is one of the 20 naturally occurring amino acids.

[0314] For those embodiments in which at least one drug conjugate moiety is added to the affimer sequence via the thiol side chain of cysteine introduced into the affimer sequence, the cysteine is preferably in the loop sequence (Xaa) n or (Xaa) m and will be provided as part of the affimer sequence other than where provided. Thus, SEQ ID NO: 5 may contain 1 to 5 cysteines in place of amino acid residues at various positions in the sequence.

[0315] In certain embodiments of the above sequences and formulas, (Xaa) n is an amino acid sequence represented by the general formula (II), -aa1-aa2-aa3-Gly-Pro-aa4-aa5-Trp-aa6- (II) In the formula, aa1 represents an amino acid residue having a basic side chain, more preferably Lys, Arg or His, and even more preferably Lys or Arg; aa2 is an amino acid residue, preferably 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, even more preferably, Ala, Gln, Asp or Glu; aa3 is an amino acid residue with an aromatic side chain or a basic side chain, preferably Phe, Tyr, Trp, Lys, Arg or His, more preferably Phe, Tyr, Trp, even more preferably His or Tyr, Trp or His; aa4 is a neutral polar or nonpolar side chain or a charged (acidic or basic) side chain, preferably an amino acid residue with 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, even more preferably, Gln, Lys, Arg, His, Asp or Glu; aa5 is a neutral polar or charged (acidic or basic) or small aliphatic side chain or an aromatic side chain, preferably an amino acid residue with a neutral polar side chain or a charged side chain, more preferably, Ser, Thr, Asn, Gln, Asp, Glu, Arg or His, even more preferably, Ser, Asn, Gln, Asp, Glu or Arg; and aa6 is an amino acid residue with an aromatic side chain or an acidic side chain, preferably Phe, Tyr, Trp, Asp or Glu, more preferably, Trp or Asp; and even more preferably Trp.

[0316] In certain embodiments of the above sequences and formulas, (Xaa) n is an amino acid sequence represented by the general formula (III), -aa1-aa2-aa3-Phe-Pro-aa4-aa5-Phe-Trp- (III) wherein, 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, even more preferably Lys or Asn; aa2 is 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 having a small aliphatic side chain, a neutral polar side chain or an acidic or basic side chain, even more preferably representing Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg or His, even more preferably representing Ala, Gln, Asp or Glu; aa3 represents an amino acid residue having an aromatic side chain or a basic side chain, preferably Phe, Tyr, Trp, Lys, Arg or His, more preferably Phe, Tyr, Trp or His, even more preferably Tyr, Trp or His; aa4 is a neutral polar side chain or a nonpolar side chain or a charged (acidic or basic) side chain, preferably an amino acid residue having a neutral polar side chain or a charged (acidic or basic) side chain, more preferably representing Ala, Pro, Ile, Gln, Thr, Asp, Glu, Lys, Arg or His, even more preferably representing Gln, Lys, Arg, His, Asp or Glu; and aa5 is a neutral polar side chain or a charged (acidic or basic) side chain or a small aliphatic or aromatic side chain; preferably an amino acid residue having a neutral polar side chain or a charged side chain, more preferably representing Ser, Thr, Asn, Gln, Asp, Glu, Arg or His, even more preferably representing Ser, Asn, Gln, Asp, Glu or Arg.

[0317] In certain embodiments of the above sequences and formulas, (Xaa) n is an amino acid sequence selected from SEQ ID NOs: 6-40, or an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% homology to a sequence selected from SEQ ID NOs: 6-40. In certain embodiments, (Xaa) nis an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% identity with a sequence selected from SEQ ID NOs: 6 to 40. TIFF2025100616000052.tif247101

[0318] In certain embodiments of the above sequences and formulas, (Xaa) m is an amino acid sequence represented by general formula (IV), -aa7-aa8-aa9-aa10-aa11-aa12-aa13-aa14-aa15- (IV) wherein, aa7 is an amino acid residue having a neutral polar side chain or nonpolar side chain or acidic side chain, preferably representing Gly, Ala, Val, Pro, Trp, Gln, Ser, Asp or Glu, even more preferably representing Gly, Ala, Trp, Gln, Ser, Asp or Glu; aa8 is an amino acid residue, preferably having a neutral polar or nonpolar side chain or charged (acidic or basic) side chain or aromatic side chain, more preferably an amino acid residue having a charged (acidic or basic) side chain, even more preferably representing Asp, Glu, Lys, Arg, His, Gln, Ser, Thr, Asn, Ala, Val, Pro, Gly, Tyr or Phe, even more preferably representing Asp, Glu, Lys, Arg, His or Gln; aa9 is an amino acid residue, preferably having a neutral polar or nonpolar side chain or charged (acidic or basic) side chain or aromatic side chain, more preferably an amino acid residue having a neutral polar side chain or acidic side chain, even more preferably representing Gln, Ser, Thr, Asn, Asp, Glu, Arg, Lys, Gly, Leu, Pro or Tyr, even more preferably representing Gln, Thr or Asp; aa10 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 or an aromatic side chain, more preferably an amino acid residue having a neutral polar side chain or a basic or acidic side chain, preferably Asp, Glu, Arg, His, Lys, Ser, Gln, Asn, Ala, Leu, Tyr, Trp, Pro or Gly, even more preferably Asp, Glu, His, Gln, Asn, Leu, Trp or Gly; aa11 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 amino acid residue having a neutral polar side chain or a basic or acidic side chain, even more preferably Asp, Glu, Ser, Thr, Gln, Arg, Lys, His, Val, Ile, Tyr or Gly, even more preferably Asp, Glu, Ser, Thr, Gln, Lys or His; 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 amino acid residue having an acidic side chain, even more preferably Asp, Glu, Ser, Thr, Gln, Asn, Lys, Arg, Val, Leu, Ile, Trp, Tyr, Phe or Gly, even more preferably Asp, Glu, Ser, Tyr, Trp, Arg or Lys; aa13 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 amino acid residue having an acidic side chain, even more preferably Ser, Thr, Gln, Asn, Val, Ile, Leu, Gly, Pro, Asp, Glu, His, Arg, Trp, Tyr or Phe, even more preferably Ser, Thr, Gln, Asn, Val, Ile, Leu, Gly, Asp or Glu; 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 aa15 represents an amino acid residue, preferably an amino acid residue having a neutral polar side chain or a neutral non-polar 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.

[0319] In certain embodiments of the above sequences and formulas, (Xaa) m is an amino acid sequence selected from SEQ ID NOs: 41-75, or an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% homology with a sequence selected from SEQ ID NOs: 41-75. In certain embodiments, (Xaa) m is an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% identity with a sequence selected from SEQ ID NOs: 41-75. TIFF2025100616000053.tif247101

[0320] In certain embodiments, the anti-PD-L1 affimer has an amino acid sequence selected from SEQ ID NOs: 76-84, 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 NOs: 76-84. In certain embodiments, the anti-PD-L1 affimer 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 NOs: 76-84.

[0321] For those embodiments in which at least one drug conjugate moiety is added to the Affimer sequence via the thiol side chain of cysteine introduced into the Affimer sequence, the cysteine is preferably in the loop sequence (Xaa) n or (Xaa) m and will be provided as part of the Affimer sequence other than where n or m is provided. Thus, the anti-PD-L1 Affimer will have a sequence different from SEQ ID NOs: 76-84 by including at least 1-5 cysteines in place of amino acid residues at various positions in its sequence, preferably but not in the sequence of loop 2 or loop 4. TIFF2025100616000054.tif255170

[0322] In certain embodiments, the anti-PD-L1 Affimer 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: 85-92, or an amino acid sequence that can be encoded by a nucleic acid having a coding sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or even 98% identical to nucleotides 1-336 of one of SEQ ID NOs: 85-92, or an amino acid sequence that can be encoded by a nucleic acid having a coding sequence that hybridizes to nucleotides 1-336 of one of SEQ ID NOs: 85-92 under stringent conditions (e.g., in the presence of 6× sodium chloride / sodium citrate (SSC) at 45°C followed by washing in 0.2× SSC at 65°C).

[0323] For those embodiments in which at least one drug conjugate moiety is added to the Affimer sequence via the thiol side chain of cysteine introduced into the Affimer sequence, the cysteine is preferably in the loop sequence (Xaa) n or (Xaa) mIt is provided in a part of the affimer array other than being provided otherwise. Thus, the anti-PD-L1 affimer will have a sequence different from the amino acid sequence encoded by SEQ ID NOs: 85-92 by including at least 1 to 5 cysteines in place of amino acid residues at various positions in the sequence, although preferably not in the sequence of loop 2 or loop 4. TIFF2025100616000055.tif233170TIFF2025100616000056.tif227170

[0324] Furthermore, minor modifications may also include small deletions or additions to the sequence of stefin A or an affimer polypeptide derived from stefin A as disclosed herein, such as addition or deletion of up to 10 amino acids, beyond the regions of loop 2 and loop 4 insertions described above.

[0325] In certain embodiments, the PD-L1 binding affimer polypeptide binds PD-L1 as a monomer having a dissociation constant (K D ) 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.

[0326] In certain embodiments, the PD-L1 binding affimer polypeptide moiety has an off-rate constant (K -3 S -1 (i.e., 1 unit / second) or less, about 10 -4 s -1 or less, or even about 10 -5 s -1 or less, as measured by Biacore, and binds human PD-L1 as a monomer. off

[0327] In certain embodiments, the PD-L1 binding affimer polypeptide moiety has an on-rate constant of at least about 10 3 M -1 s -1 or more, at least about 104 M -1 seconds -1 or more, at least about 10 5 M -1 seconds -1 or more, or even at least about 10 6 M -1 seconds -1 or more of the association constant (K on ) binds human PD-L1 as a monomer.

[0328] In certain embodiments, the PD-L1 binding affimer polypeptide moiety binds human PD-L1 as a monomer with 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 in a competitive binding assay with human PD-1.

[0329] Fusion proteins: General In some embodiments, the affimer polypeptide may further include additional insertions, substitutions or deletions that modulate the biological activity of the affimer polypeptide. For example, the addition, substitution, or deletion may modulate one or more properties or activities of the modified affimer. For example, the addition, substitution or deletion may modulate, for example, the affinity for the affimer polypeptide for binding to and inhibiting PD-1, may modulate the circulation half-life, may modulate the therapeutic half-life, may modulate the stability of the affimer polypeptide, may modulate cleavage by proteases, may modulate the dosage, may modulate the release or bioavailability, may facilitate purification, may reduce deamidation, may improve the shelf life, or may improve or alter a particular route of administration. Similarly, the affimer polypeptide 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), or a linking molecule (including but not limited to biotin) that improves the detection, purification or other properties of the polypeptide.

[0330] In some cases, these additional arrays are added to one or both ends of the affimer polypeptide in the form of a fusion protein. Thus, in certain embodiments of the present invention, the binder-drug conjugate is a fusion protein having at least one affimer polypeptide sequence and one or more heterologous polypeptide sequences (referred to herein as "fusion domains"). The fusion domains may be selected, by way of example only, to confer desired properties such as secretion from cells or retention on the cell surface (i.e., for the encoded affimer), function as a substrate sequence for post-translational modification or other recognition sequences, create multimeric structures that aggregate via protein-protein interactions, alter (often extend) the serum half-life, or alter tissue localization or tissue clearance and other ADME properties.

[0331] For example, some fusion domains are particularly useful for the isolation and / or purification of the fusion protein, such as by affinity chromatography. Well-known examples of such fusion domains that facilitate expression or purification include, by way of illustration only, for example, polyhistidine (i.e., His6 tag), Strep II tag, streptavidin binding peptide (SBP) tag, calmodulin binding peptide (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), S tag, HA tag, C-Myc tag, thioredoxin, protein A, and protein G.

[0332] For an Affimer to be secreted when made recombinantly, it will generally contain a signal sequence that directs transport of the protein into the lumen of the endoplasmic reticulum and is ultimately secreted (or retained at the cell surface in the case of a transmembrane domain or other cell surface retention signal). Signal sequences (also called signal peptides or leader sequences) are located at the N-terminus of the nascent polypeptide. They target the polypeptide to the endoplasmic reticulum, where the protein is sorted to its destination, such as the internal space of an organelle, the inner membrane, the outer cell membrane, or outside the cell via secretion. Most signal sequences are cleaved from the protein by signal peptidase after the protein has been transported to the endoplasmic reticulum. Cleavage of the signal sequence from the polypeptide usually occurs at specific sites in the amino acid sequence and depends on the amino acid residues within the signal sequence.

[0333] In some embodiments, the signal peptide is an amino acid of about 5 to about 40 in length (e.g., amino acids of 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 in length).

[0334] In some embodiments, the signal peptide is a native signal peptide from a human protein. In other embodiments, the signal peptide is a non-native signal peptide. For example, in some embodiments, the non-native signal peptide is a variant native signal peptide from the corresponding native secreted human protein and can contain one or more (2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitutions, insertions, or deletions.

[0335] In some embodiments, the signal peptide is a signal peptide or variant thereof from a non-IgSF protein family such as an immunoglobulin (e.g., IgG heavy chain or IgG kappa light chain), a cytokine (e.g., interleukin-2 (IL-2) or CD33), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, luciferase, trypsinogen (e.g., chymotrypsinogen or trypsinogen), or other signal peptide capable of efficiently secreting proteins from cells. Exemplary signal peptides include, but are not limited to: TIFF2025100616000057.tif206158

[0336] The fusion protein of the subject may also include one or more linkers that separate the sequences or domains of the heterologous protein - that is, separate the cell-binding moieties where more than one is included in the conjugate drug conjugate. As used herein, the term "linker" refers to a linker amino acid sequence inserted between a first polypeptide (e.g., an affimer) and a second polypeptide (e.g., a second affimer, Fc region, receptor trap, albumin, etc.). Empirical linkers designed by researchers are generally classified into three categories according to their structure: flexible linkers, rigid linkers, and cleavable linkers in vivo. In addition to the basic role of linking functional domains together (such as flexible and rigid linkers) or releasing free functional domains in vivo (such as cleavable linkers in vivo), linkers may provide many other advantages for the production of fusion proteins, such as improved biological activity, increased expression yield, and achievement of a desirable pharmacokinetic profile. The linker must not adversely affect the expression, secretion, or biological activity of the fusion protein. The linker must not be antigenic and must not induce an immune response.

[0337] Suitable linkers are known to those skilled in the art and often contain a mixture of glycine and serine residues and often contain amino acids with no steric hindrance. Other amino acids that can be incorporated into useful linkers include threonine and alanine residues. The linker can be, for example, 1 to 50 amino acids in length, 1 to 22 amino acids in length, 1 to 10 amino acids in length, 1 to 5 amino acids in length, or 1 to 3 amino acids in length and can have a range of lengths. In some embodiments, the linker may include a cleavage site. In some embodiments, the linker may include an enzymatic cleavage site so that the second polypeptide may be separated from the first polypeptide.

[0338] In certain preferred embodiments, the linker can be characterized as being flexible. Flexible linkers are typically applied when the attached domains require some degree of movement or interaction. They are generally composed of small non-polar (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 allows movement of the functional domains being connected. Incorporation of Ser or Thr can reduce unfavorable interactions between the linker and the protein moiety, as it can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules. The most commonly used flexible linkers have sequences consisting mainly of stretches of Gly and Ser residues (the “GS” linker). An example of the most widely used flexible linker has the sequence (Gly-Gly-Gly-Gly-Ser)n. By adjusting the copy number “n”, the length of this GS linker can be optimized to achieve appropriate separation of the functional domains or to maintain the necessary domain-domain interactions. In addition to the GS linker, many other flexible linkers have been designed for recombinant fusion proteins. These flexible linkers are rich in small amino acids such as Gly and Ser or polar amino acids, but can also contain additional amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility.

[0339] In certain preferred embodiments, the linker can be characterized as rigid. While flexible linkers have the advantage of passively connecting functional domains and allowing some degree of movement, the lack of rigidity of these linkers can be a limitation in certain fusion protein embodiments such as expression yield or bioactivity. The ineffectiveness of flexible linkers in these cases was due to inefficient separation of protein domains or insufficient reduction of their mutual interference. Under these circumstances, rigid linkers have been successfully applied to maintain a fixed distance between domains and maintain their independent functions.

[0340] Many natural linkers exhibited an α-helical structure. The α-helical structure was rigid and stable, with a tightly packed backbone and hydrogen bonds within the segment. Thus, a rigid α-helical linker can act as a rigid spacer 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 adopt an α-helical structure or exhibit a relatively rigid structure by containing multiple Pro residues. In many situations, they separate functional domains more efficiently than flexible linkers. The length of the linker can be easily adjusted by varying the copy number to achieve the optimal distance between domains. As a result, when spatial separation of domains is important to maintain the stability or bioactivity of the fusion protein, a rigid linker is selected. In this regard, α-helix-forming linkers with the sequence (EAAAK)n have been applied in the construction of 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.

[0341] For illustrative purposes only, exemplary linkers include the following: TIFF2025100616000058.tif104141

[0342] Other linkers that may be used in the subject fusion proteins 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 regions of the Fc fusions described below may also be considered linkers.

[0343] Still other modifications that can be made to the affimer polypeptide sequence itself or to adjacent polypeptide portions provided as part of a fusion protein are one or more sequences that are sites for post-translational modification by enzymes. These include, but are not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitic acid addition, phosphorylation, glycolipid attachment modification, and the like.

[0344] Manipulating PK and ADME properties In certain embodiments, the binder-drug conjugate may not have a half-life and / or PK properties that are optimal for an administration route 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. The biological half-life can be affected by elimination, excretion, degradation (e.g., enzymatic), or absorption and concentration in specific organs or tissues of the body. In some embodiments, the biological half-life can be evaluated by determining the time it takes for the plasma concentration of the substance to reach half of its steady-state level ("plasma half-life"). To address this drawback, there are various general strategies for half-life extension that have been used in the case of other protein therapeutics, including the incorporation of a half-life extension moiety as part of the binder-drug conjugate.

[0345] The term "half-life extension moiety" refers to a pharmaceutically acceptable moiety, domain or molecule that prevents or reduces modifications that decrease proteolysis or other activities in vivo of an affimer polypeptide, increases the half-life, and / or increases absorption rate, decreases toxicity, improves solubility, decreases protein aggregation, increases bioactivity and / or target selectivity of the modified affimer polypeptide, improves manufacturability, and / or decreases immunogenicity of the modified affimer polypeptide, compared to a reference such as an unconjugated modified affimer polypeptide that is covalently conjugated ("conjugated" or "fused") directly or via a linker to an affimer polypeptide through an optionally non-naturally encoded amino acid. The term "half-life extension moiety" includes, for example, non-proteinaceous half-life extension moieties such as water-soluble polymers such as polyethylene glycol (PEG) or individual PEGs, 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; proteinaceous half-life extension moieties such as serum albumin, transferrin, adnectin (e.g., albumin-binding or pharmacokinetic extension (PKE) adnectin), Fc domain, and unstructured polypeptides such as, for example, XTEN and PAS polypeptides (e.g., a stereochemically disordered polypeptide sequence composed of the amino acids Pro, Ala, and / or Ser); and fragments of any of the foregoing. Examination of the crystal structure of an affimer and its interaction with its target, such as the anti-PD-L1 affimer complex with PD-1 shown in the figure, can show which specific amino acid residues have side chains that are fully or partially solvent-accessible.

[0346] In certain embodiments, the half-life extension moiety extends the half-life of the binder-drug conjugate that results from circulating in mammalian serum compared to the half-life of a protein that is not so conjugated to that moiety (e.g., compared to the affimer polypeptide alone). In some embodiments, the half-life is extended by 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or more than 6.0-fold. 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 compared to a protein without a half-life extension moiety after in vivo administration.

[0347] As a means for further illustration, half-life extension moieties that can be used in the production of the binder-drug conjugates of the present invention include the following: Genetic fusions to natural proteins or protein domains with long half-lives of pharmacologically active Affimer arrays (e.g., Fc fusions, transferrin [Tf] fusions, or albumin fusions). 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-Fc fusion therapeutics, traps, and Mimetibody technology”, Curr. Opin. Biotechnol. 2009;20:692-9; Keefe et al. (2013), “Transferrin fusion protein therapies: acetylcholine receptor-transferrin fusion protein as a model. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; p. 345-56; Weimer et al. (2013), “Recombinant albumin fusion proteins. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013. p. 297-323; Walker et al. (2013), “Albumin-binding fusion proteins in the development of novel long-acting therapeutics. In: Schmidt S, editor.See Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013. p. 325-43. Genetic fusions to pharmacologically inert polypeptides, such as XTEN (also known as recombinant PEG or “rPEG”), homo-amino acid polymers (HAP; HAPylation), proline-alanine-serine polymers (PAS; PASylation), or elastin-like polypeptides (ELP; ELPylation). 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 recombinant antibody 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 half-life of pharmaceutically active proteins. Protein Eng. Des. Sel. 26:489-501. Floss et al. (2012), “Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application. Trends Biotechnol. 28:37-45. Floss et al.”ELP-fusion technology for biopharmaceuticals. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: application and challenges. Hoboken: Wiley; 2013. p. 372-98. Increasing the hydrodynamic radius by chemically conjugating a pharmacologically active peptide or protein to a repetitive chemical moiety, such as PEG (PEGylation) or hyaluronic acid. See, for example, Caliceti et al. (2003), “Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates”, Adv. Drug Delivery Rev. 55:1261-77; Jevsevar et al. (2010), PEGylation of therapeutic proteins. Biotechnol. J. 5:113-28; Kontermann, (2009), “Strategies to extend plasma half-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. Fusing a pharmacologically active peptide or protein by polysialylation or, alternatively, significantly increasing the negative charge of fusing a negatively charged, highly sialylated peptide known to extend the half-life of a native protein, such as the human CGb subunit (e.g., the carboxy-terminal peptide [CTP; chorionic gonadotropin (CG) b chain]), to a biological drug candidate. 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 serum hormones 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 the chorionic 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." Binding non-covalently to a protein with a normal long half-life, such as HSA, human IgG, transferrin or fibronectin, through the binding of a peptide or protein binding domain to a bioactive protein. 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 a short peptide that binds immunoglobulin G to a recombinant protein substantially increases its plasma half-life in mice”, PLoS.One. 2014;9:e102566.

[0348] Classical gene fusions to long-lived serum proteins offer an alternative method of half-life extension that is distinct from chemical conjugates to PEG or lipids. Conventionally, two major proteins, the antibody Fc domain and human serum albumin (HSA), have been used as fusion partners. Fc fusions include the fusion of peptides, proteins, or receptor ectodomains to the Fc portion of an antibody. Both Fc and albumin fusions not only achieve half-life extension by increasing the size of the peptide drug, but also utilize the neonatal Fc receptor, FcRn, which is a natural recycling mechanism of the body. The pH-dependent binding of these proteins to FcRn prevents the degradation of the fusion protein within the endosome. Fusions based on these proteins can have half-lives in the range of 3 to 16 days, much longer than typical pegylated or lipidated peptides. Fusion to the antibody Fc domain can improve the solubility and stability of peptide drugs 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 utilized by fatty acylated peptides, is another well-regarded fusion partner. Albiglutide is a GLP-1 receptor agonist based on this platform. The main difference between Fc and albumin is the dimeric nature of Fc versus the monomeric structure of HSA, which results in the presentation of the fusion peptide as a dimer or monomer depending on the choice of fusion partner. The dimeric nature of an Affimer-Fc fusion can produce a binding activity effect if the targets of the Affimer, such as PD-L1 on tumor cells, are arranged in close proximity or are themselves dimeric. This may or may not be desirable depending on the target.

[0349] Fc fusion In some embodiments, the affimer polypeptide may be part of a fusion protein with an immunoglobulin Fc domain (“Fc domain”), or a fragment or variant thereof, such as a functional Fc region. In this context, an Fc fusion (an “Fc-fusion”), such as a binder-drug conjugate created as an affimer-Fc fusion protein, is a polypeptide that contains one or more affimer sequences covalently bound to the Fc region of an immunoglobulin via a peptide backbone (either directly or indirectly). The Fc fusion may, for example, include the Fc region of an antibody (which promotes effector function and pharmacokinetics) and an affimer sequence as part of the same polypeptide. The immunoglobulin Fc region may also be indirectly linked to one or more affimers. A variety of linkers are known in the art and optionally can be used to link Fc to a polypeptide containing an affimer sequence to generate an Fc fusion. In certain embodiments, the Fc fusion can be dimerized to form an Fc fusion homodimer, or non-identical Fc domains can be used to form an Fc fusion heterodimer.

[0350] There are several reasons for selecting the Fc region of a human antibody for use in generating the binder-drug conjugate of the subject as an affimer fusion protein. The main rationale is to generate a stable protein large enough to exhibit a similar pharmacokinetic profile compared to that of an antibody and to utilize the properties conferred by the Fc region; this includes the salvage neonatal FcRn receptor pathway involving FcRn-mediated recycling of the fusion protein to the cell surface after endocytosis, avoiding lysosomal degradation, and resulting in re-release back into the bloodstream, thus contributing to an extended serum half-life. Another obvious advantage is the binding of the Fc domain to protein A, which can simplify downstream processing during the generation of the binder-drug conjugate and enable the production of a high-purity preparation of the binder-drug conjugate.

[0351] Generally, the Fc domain will include the constant regions of the antibody, excluding the first constant region immunoglobulin domain. Thus, the Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. In the case of IgA and IgM, the Fc may include the J chain. For IgG, the Fc includes the immunoglobulin domains Cγ2 and Cγ3, and the hinge between Cγ1 and Cγ2. The boundaries of the Fc domain may vary, but the human IgG heavy chain Fc region is typically defined to include residues from C226 or P230 to its carboxyl terminus, numbered according to the EU index described in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)). Fc may refer to this region separately, or in the context of the whole antibody, an antibody fragment, or an Fc fusion protein. Polymorphisms have been observed at many different Fc positions and are also included as Fc domains when used herein.

[0352] In certain embodiments, as used herein, Fc, "functional Fc region" refers to an Fc domain or fragment thereof that retains the ability to bind to FcRn. The functional Fc region binds to FcRn but has no effector function. The ability of an Fc region or fragment thereof to bind to FcRn can be measured by standard binding assays known in the art. Exemplary "effector 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. Such effector functions can be evaluated using various assays known in the art for evaluating such antibody effector functions.

[0353] In an exemplary embodiment, the Fc domain is derived from the IgG1 subclass, although other subclasses (e.g., IgG2, IgG3, and IgG4) may also be used. Exemplary sequences of the human IgG1 immunoglobulin Fc domain that can be used are as follows. DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 93)

[0354] In some embodiments, the Fc region used in the fusion protein may include the hinge region of the Fc molecule. An exemplary hinge region includes the core hinge residues (i.e., DKTHTCPPCPAPELLG) spanning positions 1 to 16 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In certain embodiments, the affimer-containing fusion protein may adopt a multimeric structure (e.g., dimer) due in part to the 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 further include residues derived from the CH1 and CH2 regions adjacent to the core hinge sequence of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In still other embodiments, the hinge sequence may include or consist of GSTHTCPPCPAPELLG or EPKSCDKTHTCPPCPAPELLG.

[0355] In some embodiments, the hinge sequence may include one or more substitutions that confer desirable pharmacokinetic, biophysical, and / or biological properties. Some exemplary hinge sequences include the following: EPKSCDKTHTCPPCPAPELLGGPS EPKSSDKTHTCPPCPAPELLGGPS; EPKSSDKTHTCPPCPAPELLGGSS; EPKSSGSTHTCPPCPAPELLGGSS; DKTHTCPPCPAPELLGGPS and DKTHTCPPCPAPELLGGSS.

[0356] In one embodiment, the residue P at position 18 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above may be replaced with S to remove the effector function of Fc; this substitution is exemplified by the hinge having the sequences EPKSSDKTHTCPPCPAPELLGGSS, EPKSSGSTHTCPPCPAPELLGGSS, and DKTHTCPPCPAPELLGGSS. In another embodiment, the residues 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; this substitution is exemplified by the sequence EPKSSGSTHTCPPCPAPELLGGSS. In another embodiment, the C at position 103 of the heavy chain constant region of human IgG1 (i.e., domains CH1-CH3) may be replaced with S to prevent inappropriate disulfide bond formation in the absence of the light chain; this substitution is exemplified by EPKSSDKTHTCPPCPAPELLGGPS, EPKSSDKTHTCPPCPAPELLGGSS, and EPKSSGSTHTCPPCPAPELLGGSS.

[0357] In some embodiments, Fc is a mammalian Fc such as human Fc that includes an Fc domain 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 native Fc region and / or the Fc region of the parent polypeptide. In some embodiments, the Fc region may have at least about 90% sequence identity with the native Fc region and / or the Fc region of the parent polypeptide.

[0358] In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NO: 93, or an Fc sequence from an example provided by SEQ ID NOs: 94-106. It should be understood that the C-terminal lysine of the Fc domain is any component of the fusion protein comprising the Fc domain. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 93-106, except that its 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, except that its C-terminal lysine is omitted. TIFF2025100616000059.tif204170TIFF2025100616000060.tif199170TIFF2025100616000061.tif123170

[0359] Exemplary Fc fusions of a PD-L1 binding affimer and Fc are provided in the Examples and Figures, demonstrating that the affimer sequence can be located at either the N-terminus or C-terminus of the Fc domain, and may be directly linked, or the fusion protein may have other polypeptide sequences intervening between the Fc domain and the affimer polypeptide sequence. In the illustrated example, an unstructured (flexible) linker (Gly4Ser) n is used with the PD-L1 binding affimer "251" (SEQ ID NO: 84) and the Fc domain of human IgG1 (SEQ ID NO: 93) whose hinge region is EPKSCDKTHTCPPCPAPELLG. Both constructs included the CD33 secretion signal sequence MPLLLLLPLLWAGALA, which is cleaved from the mature form of the protein. TIFF2025100616000062.tif133170

[0360] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to an Fc receptor (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to target cells bearing an antigen and subsequently kill the target cells with cytotoxins.

[0361] In certain embodiments, the fusion protein comprises an Fc domain sequence such that the resulting binder-drug conjugate does not have ADCC and / or does not have complement activation or effector function (or has reduced ADCC and / or complement activation or effector function). For example, the Fc domain may comprise the naturally inactivated constant region of the IgG2 or IgG4 isotype, or a mutated 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 numbering).

[0362] In other embodiments, the fusion protein comprises an Fc domain sequence such that the resulting binder-drug conjugate will retain some or all of the Fc functionality, e.g., an Fc domain sequence that will enable one or both of the activities of ADCC and CDC if the fusion protein comprises an Fc domain derived from human IgG1 or IgG3. The level of effector function can be altered according to known techniques, e.g., by mutation of the CH2 domain, e.g., wherein the CH2 domain of IgG1 has one or more mutations at positions selected from 239 and 332 and 330, e.g., the mutations are selected from S239D and I332E and A330L such that the antibody enhances effector function, and / or, e.g., by altering the glycosylation profile of the antigen-binding protein of the invention such that fucosylation of the Fc region is decreased.

[0363] Albumin fusion In other embodiments, the binder-drug conjugate is a fusion protein comprising an albumin sequence or albumin fragment in addition to at least one affimer sequence. In other embodiments, the binder-drug conjugate is conjugated to an albumin sequence or albumin fragment via a chemical bond other than incorporation into a polypeptide sequence comprising an affimer. In some embodiments, the albumin, albumin variant, or albumin fragment is human serum albumin (HSA), a human serum albumin variant, or a human serum albumin fragment. Albumin serum proteins comparable to HSA are found, for example, in cynomolgus monkeys, cows, dogs, rabbits, and rats. Among non-human species, bovine serum albumin (BSA) is most structurally similar to HSA. See, for example, Kosa et al., (2007), J. Pharm. Sci. 96(11):3117-24. The present disclosure contemplates the use of albumin from non-human species, including but not limited to, albumin sequences derived from cynomolgus monkey serum albumin or bovine serum albumin.

[0364] Mature HSA, a 585 amino acid polypeptide (˜67 kDa) with a serum half-life of approximately 20 days, is primarily involved in maintaining colloid osmotic blood pressure, blood pH, and the transport and distribution of numerous endogenous and exogenous ligands. This protein has three structurally homologous domains (domains I, II, III), is almost entirely alpha-helical in structure, and is highly stabilized by 17 disulfide bridges. In certain preferred embodiments, the binder-drug conjugate can be an albumin fusion protein comprising one or more affimer 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 in vivo distribution characteristics of mature albumin to the desired extent in the fusion protein. DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL(SEQ ID NO: 111)

[0365] The albumin sequence can be separated from the affimer polypeptide sequence or other adjacent sequences in the binder-drug conjugate by using a linker sequence as described above.

[0366] Unless otherwise indicated, references to "albumin" or "mature albumin" in this specification are meant to refer to HSA. However, full-length HSA is noted to have an 18-amino acid signal peptide (MKWVTFISLLFLFSSAYS) followed by a 6-amino acid prodomain (RGVFRR); this 24-amino acid residue peptide may be referred to as the preprodomain. Affimer-HSA fusion proteins can be expressed and secreted using the HSA preprodomain of the recombinant protein coding sequence. Alternatively, Affimer-HSA fusions can be expressed and secreted via the inclusion of other secretion signal sequences as described above.

[0367] In alternative embodiments, the serum albumin polypeptide is not provided as part of a fusion protein with an Affimer polypeptide, but can be covalently attached to the Affimer-containing polypeptide via a bond other than the backbone amide bond, such as a chemical conjugate between the respective amino acid side chains of the albumin polypeptide and the Affimer-containing polypeptide.

[0368] Albumin binding domain In certain embodiments, the binder-drug conjugate can include a serum binding moiety that is chemically conjugated via a site that is not part of a fusion protein (when the polypeptide is also included) with the Affimer polypeptide sequence or part of an adjacent polypeptide chain.

[0369] In certain embodiments, the serum binding polypeptide is an albumin binding moiety. Albumin contains multiple hydrophobic binding pockets and, as such, serves as a transporter for various drugs, similar to various ligands such as fatty acids and steroids. Additionally, the surface of albumin is negatively charged, making it highly water soluble.

[0370] As used herein, the term "albumin binding moiety" refers to any chemical group that can bind to albumin, i.e., has 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. Since the binding of these drugs to albumin is reversible, the albumin-drug complex serves as a drug reservoir that can enhance the biodistribution and bioavailability of the drug. Incorporation of components that mimic endogenous albumin-binding ligands such as fatty acids has been used to enhance albumin association and improve drug efficacy.

[0371] In certain embodiments, a chemical modification method that can be applied to the generation of the subject binder-drug conjugate to increase protein half-life is lipidation, which involves covalent attachment of a fatty acid to a peptide side chain. Lipidation, originally devised and developed as a method to extend the half-life of insulin, shares the same basic mechanism of half-life extension as PEGylation, i.e., increasing the hydrodynamic radius and reducing renal filtration. However, the lipid moiety itself is relatively small, and its effect is mediated indirectly through non-covalent attachment of the lipid moiety to circulating albumin. One consequence of lipidation is that it decreases the water solubility of the peptide, but this can be modulated by manipulation of the linker between the peptide and the fatty acid, for example, by using glutamate or mini-PEG in the linker. Manipulation of the linker and changes in the lipid moiety can affect self-aggregation, which can contribute to half-life extension by slowing down biodistribution, independent of albumin. See, e.g., Jonassen et al. (2012), Pharm. Res. 29(8):2104-14.

[0372] Other examples of albumin-binding moieties for use in the generation of specific binder-drug conjugates include albumin-binding (PKE2) adnectin (WO2011140086 "Serum Albumin-Binding Molecule", WO2015143199 "Serum Albumin-Binding Fibronectin Type III Domain", and WO2017053617 "Fast Off-Rate Serum Albumin-Binding Fibronectin Type III Domain"), the albumin-binding domain 3 (ABD3) of protein G from Streptococcus strain G148, and the albumin-binding domain antibody GSK2374697 of ATN-103 ("AlbudAb") or an albumin-binding nanobody moiety (ozoralizumab).

[0373] PEGylation, XTEN, PAS and other polymers A variety of polymeric polymers and other molecules can be linked to an affimer containing the polypeptide of the present disclosure to modulate the biological properties of the resulting binder-drug conjugate and / or provide new biological properties to the binder-drug conjugate. These polymeric polymers can be linked to the affimer-containing polypeptide via naturally encoded amino acids, via 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 polymer can be in a wide range including but not limited to between about 100 Da and about 100,000 Da or more. 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, 30,000 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 about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da.

[0374] For this purpose, various methods have been developed that include pegylation, polysialylation, HESylation, glycosylation, or recombinant PEG mimetics fused to a flexible hydrophilic amino acid chain (500-600 amino acids) (see Chapman, (2002), Adv. Drug. Deliv. Rev. 54, 531-545; Schlapschy et al., (2007), Prot. Eng. Des. Sel. 20, 273-283; Contermann, (2011), Curr. Op. Biotechnol. 22, 868-876; Jevsevar et al., (2012), Methods Mol. Biol. 901, 233-246).

[0375] Examples of polymers include polyalkyl ethers and their alkoxy-capped analogs (e.g., polyoxyethylene glycol, polyoxyethylene / propylene glycol, and their methoxy- or ethoxy-capped analogs, especially polyoxyethylene glycol, the latter also known as polyethylene glycol or PEG); individual PEGs (dPEG); polyvinyl pyrrolidone; polyvinyl alkyl ethers; polyoxazolines, polyalkyl oxazolines, and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropyl methacrylamide and their derivatives); polyhydroxyalkyl acrylates; polysialic acids and their analogs; hydrophilic peptide sequences; polysaccharides and their derivatives including dextran and dextran derivatives, e.g., carboxymethyl dextran, dextran sulfate, aminodextran; cellulose and its derivatives, e.g., carboxymethyl cellulose, hydroxyalkyl cellulose; chitin and its derivatives, e.g., chitosan, succinyl chitosan, carboxymethyl chitin, carboxymethyl chitosan; hyaluronic acid and its derivatives; starch; alginates; chondroitin sulfate; albumin; pullulan and carboxymethyl pullulan; polyamino acids and their derivatives, e.g., polyglutamic acid, polylysine, polyaspartic acid, polyaspartamide; maleic anhydride copolymers such as: styrene maleic anhydride copolymer, divinyl ethyl ether maleic anhydride copolymer; polyvinyl alcohol; their copolymers; their terpolymers; their mixtures; and derivatives of the foregoing, including but not limited to these.

[0376] The selected polymer may be water-soluble so that the binder-drug conjugate to which it is linked does not precipitate in an aqueous environment such as a physiological environment. The water-soluble polymer may be in any structural form including, but not limited to, linear, branched, or dendritic. Usually, the water-soluble polymer is a poly(alkylene glycol) such as poly(ethylene glycol) (PEG), although other water-soluble polymers can also be employed. By way of example, PEG is used to illustrate certain embodiments of the present disclosure. For the therapeutic use of the binder-drug conjugate, the polymer may be pharmaceutically acceptable.

[0377] The term "PEG" is used broadly to encompass any polyethylene glycol molecule, regardless of size or modification at the termini of the PEG, and can be represented as being linked to an affimer-containing polypeptide by the formula, XO-(CH2CH2O) n -CH2CH2- or XO-(CH2CH2O) n - wherein n is from 2 to 10,000 and X is H, or a C1-4 alkyl, a protecting group, or a terminal modification including, but not limited to, a terminal functional group. In some cases, the PEG used in the polypeptides of the present disclosure terminates with hydroxy or methoxy at one terminus, i.e., X is H or CH3 ("methoxy PEG").

[0378] It is mentioned that the other end of the PEG represented by the terminal "-" in the above formula may be linked to the affimer-containing polypeptide via a naturally occurring or non-naturally encoded amino acid. For example, the linkage may be via an amide bond, a carbamate bond or a urea bond to an amine group of the polypeptide (including but not limited to the epsilon amine of lysine or the N-terminus). Alternatively, the polymer is linked to a thiol group (including but not limited to the thiol group of cysteine) by a maleimide bond, which requires changing the residues of the affimer sequence to cysteine when linking to the affimer polypeptide sequence itself.

[0379] The number of water-soluble polymers linked to the affimer-containing polypeptide (i.e., the degree of pegylation or glycosylation) can be adjusted to provide, for example, changes (including but not limited to increases or decreases) in pharmacological, pharmacokinetic or pharmacodynamic properties such as the 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 percent, 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 over the unmodified polypeptide.

[0380] Another variation of the polymeric system useful for modifying the PK or other biological properties of the resulting binder-drug conjugate is the use of an unstructured hydrophilic amino acid polymer that is a functional analog of PEG, particularly as part of a fusion protein with an Affimer polypeptide sequence. The inherent biodegradability of the polypeptide platform makes it attractive as a potentially safer alternative to PEG. Another advantage is the exact molecular structure of the recombinant molecule, in contrast to the polydispersity of PEG. Unlike fusions of HSA and Fc peptides that need to maintain the three-dimensional folding of the fusion partner, recombinant fusions to unstructured partners can often be exposed to harsh conditions such as high temperature or HPLC purification.

[0381] One of the more advanced members of this class of polypeptides is called XTEN (Amunix), which is 864 amino acids in length and is composed of 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. Enabled by the biodegradable nature of the polymer, this is much larger than the commonly used 40 kDa PEG and confers a much greater half-life extension at the same time. Fusing XTEN to an Affimer-containing polypeptide should result in a final binder-drug conjugate with a half-life 60 - 130 times longer than the unmodified polypeptide.

[0382] A 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. A random coil polymer composed of an even more restricted set of just three small uncharged amino acids: proline, alanine, and serine. Similar to Fc, HAS, and XTEN, PAS modification can be genetically encoded with an affimer polypeptide sequence and, when expressed, can result in an in-line fusion protein.

[0383] Multispecific fusion protein In certain embodiments, the binder-drug conjugate is a multispecific polypeptide comprising, for example, a first anti-PD-L1 affimer polypeptide and at least one additional binding domain. The additional binding domain may be a polypeptide sequence selected from, by way of example, a second affimer polypeptide sequence (which may be the same as or different from the first affimer polypeptide sequence), an antibody or fragment thereof, or other antigen-binding polypeptide, the ligand-binding portion of a receptor (e.g., a receptor trap polypeptide), a receptor-binding ligand (e.g., a cytokine, growth factor, etc.), an engineered T cell receptor, an enzyme or catalytic fragment thereof, or other polypeptide sequences conferring some other function.

[0384] In certain embodiments, the binder-drug conjugate comprises one or more additional affimer polypeptide sequences that are also directed to PD-L1. The additional anti-PD-L1 affimers may be the same as or different from the first anti-PD-L1 affimer polypeptide (or mixtures thereof) to create a multispecific affimer fusion protein. The binder-drug conjugate can bind to the same or overlapping sites of PD-L1, or can bind to two different sites such that the binder-drug conjugate can simultaneously bind two sites (two paratopes) or more than two sites (multiple paratopes) of the same PD-L1 protein.

[0385] In certain embodiments, the binder-drug conjugate comprises one or more antigen-binding sites derived from an antibody. The resulting binder-drug conjugate can be a single chain that includes both an anti-PD-L1 affimer and an antigen-binding site (e.g., in the case of a scFv), or can be a multimeric protein complex such as in an antibody constructed from a heavy chain and / or a light chain to which the sequence of the anti-PD-L1 antibody is also fused. An exemplary affimer / antibody fusion of this format is the ipilimumab-AVA04-141 bispecific antibody shown in FIG. 11A, which is bivalent for each of CTLA-4 and PD-L1. Another is the bevacizumab-AVA04-251 bispecific antibody shown in FIG. 13A, which is bivalent for each of VEGF-A and PD-L1.

[0386] In the case of the illustrated ipilimumab-AVA04-141 bispecific antibody, the anti-PD-L1 affimer polypeptide is provided as an in-line fusion at the C-terminus of the heavy chain of the anti-CTLA-4 antibody, and the heavy chain (including a secretion signal sequence MPLLLLLPLLWAGALA that can be removed, and a Gly4-Ser repeat linker) has an affimer fusion sequence: MPLLLLLPLLWAGALAQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLAAAHFPEHFWSTNYYIKVRAGDNKYMHLKVFNGPQPADMSAEFADRVLTGYQVDKNKDDELTGF (SEQ ID NO: 112)

[0387] And the light chain (including the secretory signal sequence MPLLLLLPLLWAGALA that can be removed) has the sequence of the native ipilimumab antibody: MPLLLLLPLLWAGALAEIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 113)

[0388] Similarly, in the case of the illustrated bevacizumab-AVA04-251 bispecific antibody, the anti-PD-L1 affimer polypeptide is provided as an in-line fusion at the C-terminus of the heavy chain of the anti-VEGF-A antibody, and the heavy chain (including the secretory signal sequence MPLLLLLPLLWAGALA which can be removed and a flexible Gly4-Ser repeat linker) has an affimer fusion sequence: MPLLLLLPLLWAGALAEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAREGRQDWVLSTNYYIKVRAGDNKYMHLKVFNGPWVPFPHQQLADRVLTGYQVDKNKDDELTGF (SEQ ID NO: 114)

[0389] And the light chain (including the secretory signal sequence MPLLLLLPLLWAGALA which can be removed) has the sequence of the native bevacizumab antibody: MPLLLLLPLLWAGALADIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 115)

[0390] To further illustrate the flexibility in formatting the affimer of the present invention, a format is also generated in which the light chain is the same as above, but the heavy chain contains a rigid linker between the antibody heavy chain and the anti-PD-L1 affimer, in the form of the bevacizumab-AVA04-251 bispecific antibody. At that time, the heavy chain (including the secretion signal sequence MPLLLLLPLLWAGALA and the rigid A(EAAAK)3 linker that can be removed) has an affimer fusion sequence: MPLLLLLPLLWAGALAEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAEAAAKEAAAKEAAAKIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAREGRQDWVLSTNYYIKVRAGDNKYMHLKVFNGPWVPFPHQQLADRVLTGYQVDKNKDDELTGF(SEQ ID NO: 116)

[0391] As will be apparent to those skilled in the art, as shown in Figure 17, the anti-PD-L1 affimer polypeptide sequence can be added to either the N-terminus or C-terminus of the heavy or light chain of the antibody, or to any combination / permutation thereof. Further, as shown in Figure 9 in the context of multimeric affimers, more than one affimer sequence can be included in any given antibody chain.

[0392] In some embodiments of the multispecific binding agent-drug conjugate comprising a full-length immunoglobulin, the fusion of the affimer polypeptide sequence to the antibody will preserve the Fc function of the Fc region of the immunoglobulin. For example, in certain embodiments, the binding agent-drug conjugate will be able to bind to the Fc receptor of Fc receptor-positive cells via its Fc portion. In some further embodiments, the binding agent-drug conjugate may activate Fc receptor-positive cells by binding to them, thereby initiating or increasing the expression of cytokines and / or costimulatory antigens. Further, the binding agent-drug conjugate may transmit at least a second activation signal required for the physiological activation of T cells to the T cells via costimulatory antigens and / or cytokines.

[0393] In some embodiments, as a result of the binding of its Fc portion to other cells expressing Fc receptors present on the surface of effector cells derived from the immune system, such as immune cells, hepatocytes, and endothelial cells, the binding agent-drug conjugate may have an antibody-dependent cell-mediated cytotoxicity (ADCC) function, which is a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells, and since the membrane surface antigen of the target cell is bound by the antibody, it causes tumor cell death via ADCC. In some further embodiments, the binding agent-drug conjugate can demonstrate the ADCC function.

[0394] As described above, apart from Fc-mediated cytotoxicity, the Fc portion may contribute to the maintenance of the serum level of the binding agent-drug conjugate, which is important for ...

Claims

Claim 1 A conjugate of a binder and a drug, comprising: (i) a cell-binding moiety that binds to a cell surface feature on a target cell in a diseased tissue, wherein the cell surface feature undergoes slow internalization upon binding of the conjugate of the binder and the drug; (ii) a drug moiety that has a pharmacological effect on bystander cells in proximity to the target cell, having an EC50 for a pharmacological effect that is at least 10-fold attenuated when the drug moiety is part of the conjugate of the binder and the drug compared to the free drug moiety released from the conjugate of the binder and the drug; and (iii) a linker moiety that covalently attaches the polypeptide binder moiety to the drug moiety, comprising a substrate recognition sequence cleavable by an enzyme present extracellularly in the diseased tissue, wherein the linker moiety is cleaved in the presence of the enzyme to release the free drug moiety; A conjugate of a binder and a drug.

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