JP2005518332A - Conjugates activated by cell surface proteases and their therapeutic use - Google Patents

Abstract

Treatment, prevention, or amelioration of one or more symptoms of cell surface protease-related diseases, including MTSP-related, urokinase-type plasminogen activator (uPA) or endotheliase-related diseases Conjugates, compositions and methods for providing are provided. The conjugates used in the compositions and methods are peptidic conjugates containing therapeutic agents, including cytotoxic agents.

Description

Related Applications US Provisional Application No. 60 / 293,267, filed May 23, 2001, Edwin L. Madison, Joseph Edward Semple and George P. Vlasuk, entitled “Conjugates Activated by Cell Surface Proteases” Claims the privilege of "and its therapeutic use". If the claim is granted, the contents of this application are hereby incorporated by reference in their entirety.

TECHNICAL FIELD Provided are methods for local delivery of conjugates, compositions and therapeutic agents for treating various disorders such as proliferative diseases, autoimmune diseases, infectious diseases and inflammatory diseases. Conjugates that act as prodrugs contain peptide substrates that are cleaved by the therapeutic agent and cell surface proteases to release the therapeutic agent in the vicinity of the targeted cell.

Background Art Effective treatment of cancer and other proliferative diseases involves administration of chemotherapeutic agents, typically systemic administration. Typically, chemotherapeutic agents are cytotoxic agents that act by inhibiting proliferation or other processes, so that actively proliferating and growing cells will be targeted by the drug . However, such targeting is not highly specific and side effects are often devastating.

  Thus, the goal in pharmacology is the design of specific drugs that act with high specific activity on the targeted cells or tissues. The purpose of this is, for example, proliferative diseases, including neoplastic diseases, and diseases of viral origin, where the ratio of toxic dose to therapeutic dose is generally close to 1 and the dose must be limited Of particular importance in the design of drugs for the treatment of disease. Numerous approaches have been developed to achieve this goal. Among these are the use of conjugates containing targeting agents such as antibodies and / or growth factors and therapeutic agents that act on specific cells; of antisense technology targeted to specific genes and / or proteins Use; for example, the use of a deleted gene or gene therapy that provides a correct copy of a pharmaceutically active compound, as well as the use of a toxic agent that is relatively non-toxic if not delivered intracellularly. So far success has been limited. There are a very limited number and type of potential targeting agents, and the specificity of such agents is optimal.

  Thus, there is a need to develop means for delivering therapeutic agents to targeted cells and tissues. Therefore, it is an object herein, inter alia, to provide methods and compounds for targeted delivery of therapeutic agents.

SUMMARY OF THE INVENTION Compounds and methods for targeted delivery of therapeutic agents are provided herein. The compound is a peptide substrate for a cell surface protease or soluble, shed or released form thereof, and a form that can be activated by a targeted cell or tissue upon cleavage by the protease or targeted by a cell or tissue, or A conjugate containing a drug in its local ter. Agents include cytotoxic agents, drugs, therapeutic agents such as therapeutic nucleic acid molecules, and diagnostic agents such as labeling components and contrast agents. Cell surface proteases are proteases that are localized on the cell surface and include, for example, membrane-bound proteases such as membrane-bound serine protease (SP), including proteases called MTSP and endotheliase. It is not limited. Also contemplated are proteases that are localized on the cell surface thanks to specific binding interactions with their receptors. Among such proteases are urokinase plasminogen activator (u-PA; eg Hung (1984) Adv. Exp. Med. Biol. 172: 281- conjugated to the urokinase plasminogen activator receptor (u-PAR). 293; Cheng et al. (1989) Gene 69: 357-363). The conjugate is either directly or via a linker, one or more to one or more cell surface proteases conjugated to a targeted agent, including a therapeutic agent such as a cytotoxic agent. Contains substrate.

The conjugates provided herein contain the following components: (peptidic substrate) _s , (linker) _q and (targeted agent) _t , where: at least one peptidic substrate The moiety is attached to at least one therapeutic agent with or without a linker (L), s is 1 or more, and each substrate is the same or different, typically 1-6 , Generally 1, 2 or 3; q is 0 or the cell surface protease (s) cleave the peptidic substrate (s) to release the active therapeutic agent, or the cells, As long as the drug is released in a form that is converted to the active form by the tissue or the surrounding environment, q is 1 or more, q is 0 to t, generally 1 to 4; t is 1 or more, generally 1 Or 2 and each mark The targeted drug is the same or different; the linker refers to any linker; and the targeted drug is any drug, typically a therapeutic agent such as a cytotoxic agent, nucleic acid, imaging Diagnostic agents such as agents or labeling components or include but are not limited to anti-tumor, anti-cancer, anti-angiogenesis, pro-apoptosis, and anti-cell division agents or treatments.

  The therapeutic agent includes any biologically active molecule. These agents include nucleic acid molecules, dsRNA and DNA molecules, such as toxins, cytokines and lymphokines, growth factors, antisense nucleic acids. Therapeutic agents include those that are active intracellularly, such as a cytotoxin, or active extracellularly, such as a modulator of the activity of an extracellular receptor. When in a conjugate, the therapeutic agent is substantially inactive and, when cleaved, releases in active form or in a form that can be activated by the targeted cell or tissue or its environment. Is done.

In exemplary embodiments, conjugates for use in the methods and compositions provided herein have the formula:
(Peptide ⁱ ) _S- (linker) _q- (therapeutic agent) _t ,
Or a derivative thereof, wherein peptide ⁱ is a peptidic substrate for cell surface protease; s is equal to or greater than 0, or 1-6, or 1 or 2, or 1; the linker is any linker Yes; q is greater than or equal to 0, or 0-4, or 0 or 1; therapeutic agents include, for example, antitumor, antiangiogenic, anticancer, pro-apoptotic, and anti-apoptotic A cytotoxic agent, including but not limited to a cell division agent; and t is 1 or more, or 1 or 2.]. In such conjugates, the therapeutic agent is covalently attached to the C-terminus or N-terminus of the peptidic substrate, optionally via a linker L.

In certain embodiments, peptide ⁱ is a substrate for a cell surface protease, whereby a substantially inactive conjugate is cleaved at a point on the peptide substrate chain by the action of the protease, in vitro and / or Formula showing the therapeutic activity in vivo:
(Peptide ^a ) _S- (linker) _q- (therapeutic agent) _t
Or a derivative thereof. In these conjugates, the therapeutic agent is, for example, a cytotoxic agent, and peptide ^a is a truncated form of peptide ⁱ obtained by cleavage at the P1-P1 ′ bond.

  Conjugates can be used to target to specific cells and deliver targeted drugs and thus express cell surface proteases, including proteases associated with and localized to cells Can be used in the treatment of any disease associated with cells or tissues. Cells in which such protease is expressed on or near it are not necessarily involved in the disease or disease process, but may be present and serve to present a protease that cleaves the targeted conjugate. .

  Methods are provided for the treatment of diseases associated with cells or tissues expressing cell surface proteases, including proteases associated with and localized to cells. Diseases include, but are not limited to, proliferative diseases, autoimmune diseases, infectious diseases and inflammatory diseases. For example, diseases include rheumatoid arthritis, lupus, multiple sclerosis, psoriasis, diabetic retinopathy, recurrence of pterygium, excimer laser surgery for scars and glaucoma filtration surgery, and other eyes including various diseases of the anterior eye Diseases, cardiovascular diseases, restenosis, chronic inflammatory diseases, wounds, cardiovascular diseases, CREST syndrome, bacterial infections, viral diseases including AIDS, dermatological diseases, and solid neoplasia and lung cancer, colon cancer Vascular tumors, including but not limited to, esophageal cancer, breast cancer, ovarian cancer and prostate cancer.

Also provided are methods for identifying proteases that target conjugates for treatment of disease. The method involves identifying a cell surface protease-related disease by identifying a cell involved in or near a cell involved in the disease process; and identifying a cell surface protease on the cell . Conjugates can then be made that target proteases as provided herein.
BRIEF DESCRIPTION OF THE FIGURES FIGS. 1-5 provide in vitro CT ₅₀ (time to 50% cleavage) for the exemplary conjugates provided herein, where: A = 0.1-25 minutes B = 25-100 minutes; C = 100-250 minutes; D ≧ 250 minutes.

FIG. 1A shows the in vitro CT ₅₀ (min) for cleavage of the conjugate by the exemplary doxorubicin conjugate and MTSP1 provided herein. FIG. 1B shows the in vitro CT ₅₀ (min) for cleavage of the conjugate by the exemplary doxorubicin conjugate and MTSP1 provided herein. FIG. 1C shows the in vitro CT ₅₀ (min) for cleavage of the conjugate by the exemplary doxorubicin conjugate and MTSP1 provided herein. FIG. 1D shows the in vitro CT ₅₀ (min) for cleavage of the conjugate by the exemplary doxorubicin conjugate provided herein and MTSP1. FIG. 1E shows the in vitro CT ₅₀ (min) for cleavage of the conjugate by the exemplary doxorubicin conjugate provided herein and MTSP1. FIG. 2A shows the in vitro CT ₅₀ (min) for cleavage of the conjugate by the exemplary doxorubicin conjugate and u-PA provided herein. FIG. 2B shows the in vitro CT ₅₀ (min) for cleavage of the exemplary doxorubicin conjugate provided herein and the conjugate by u-PA. FIG. 2C shows the in vitro CT ₅₀ (min) for cleavage of the exemplary doxorubicin conjugate provided herein and the conjugate by u-PA. FIG. 2D shows the in vitro CT ₅₀ (min) for cleavage of the exemplary doxorubicin conjugate provided herein and the conjugate by u-PA. FIG. 2E shows the in vitro CT ₅₀ (min) for cleavage of the conjugate by the exemplary doxorubicin conjugate and u-PA provided herein. FIG. 2F shows the in vitro CT ₅₀ (min) for cleavage of the exemplary doxorubicin conjugate provided herein and the conjugate by u-PA. FIG. 2G shows in vitro CT ₅₀ (min) for cleavage of the conjugate by the exemplary doxorubicin conjugate provided herein and u-PA. FIG. 2H shows the in vitro CT ₅₀ (min) for cleavage of the conjugate by the exemplary doxorubicin conjugate and u-PA provided herein. FIG. 3 shows in vitro CT ₅₀ (min) for cleavage of the conjugate by the exemplary taxol conjugate and MTSP1 provided herein. FIG. 4 shows the in vitro CT ₅₀ (min) for cleavage of the exemplary taxol conjugate provided herein and the conjugate by u-PA. FIG. 5 shows the in vitro CT ₅₀ (min) for cleavage of the exemplary doxorubicin and taxol conjugates provided herein and the conjugate by ET1 (endotheliase 1).

(Detailed description of embodiments)
A. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, published applications and publications, Genbank sequences, websites and other materials referenced throughout the entire disclosure herein are hereby incorporated by reference in their entirety, unless otherwise indicated. Part of the description. If there are multiple definitions for a term in this specification, the definition in this section prevails. If a reference is made to a URL or other such identifier or address, such an identifier may change and certain information on the Internet may or may not be posted, but search the Internet It is understood that equivalent information can be found by doing so. Reference to it establishes the availability and public dissemination of such information.

  As used herein, a targeted agent is any agent that is intended for targeted delivery, and therapeutic and diagnostic agents and any other agent that are intended for targeted delivery. including.

As used herein, targeted delivery means delivery to a selected cell or tissue that expresses a protease that releases the targeted agent. Such delivery need not be dedicated to such selected cells or tissues, but must be included and generally higher doses of such selected cells or Delivered to the tissue. Delivery includes introduction into the cell or tissue, or binding to the cell or tissue or release in the vicinity of the cell or tissue. For example, in some cases, the tumor induces the production of a substrate associated with proteases, receptors, cofactors or stroma; thus, delivery may involve proteases, receptors in infiltrating cells or cells in the targeted tumor. Targeting the activity of such induced stroma, such as body and / or enzyme cofactors.
As used herein, a therapeutic index _is the ratio, LD 50 / _{ED 50.}

  As used herein, a therapeutic agent is a targeted cell or tissue, such as a proliferating cell, including tumor cells and cells that are involved in a proliferative, typically unwanted response. Any drug or other agent intended for delivery to. Therapeutic agents include anticancer agents, anti-angiogenic agents, pro-apoptotic agents, anti-mitotic growth factors, cytokines such as tumor necrosis factor and interleukins, as well as cytotoxic agents and those described herein. Such agents are well known to those skilled in the art, but are not limited thereto. Therapeutic agents include those that are active at internalization, and also those that act extracellularly, such modulators of the activity of certain cell surface receptors such as G proteins that transduce extracellular signals.

  As used herein, an inactive therapeutic agent binds to a peptide and thereby has no activity, either due to conformational changes or size or any other factor such as steric hindrance. A therapeutic agent that does not show or exhibits substantially reduced activity compared to the released active therapeutic agent. For example, bound doxorubicin is not toxic to the cell until it is released from the conjugate in a form that allows it to enter the cell. Upon cleavage of the drug from the conjugate, it is in the active form, or in a form that is further processed into the active form by one or more steps, including enzymatic or chemical, in or on the cell.

  As used herein, an active therapeutic agent is a therapeutic agent that has been released from the conjugate by cleavage of the peptidic substrate portion of the conjugate. An active therapeutic agent can demonstrate its intended activity, typically by entering the cell, thanks to cleavage. When bound, the therapeutic agent has reduced or no activity as a therapeutic agent and is released in the vicinity of the cell upon cleavage.

  As used herein, an anti-cancer agent (used interchangeably with “anti-tumor or anti-neoplastic agent”) refers to any agent used in the treatment of cancer. Do these reduce, reduce, ameliorate, prevent, or put remission of clinical symptoms or diagnostic markers associated with neoplasia, tumors or cancer when used alone or in combination with other compounds? Alternatively, any agent that can be or can be maintained and used in the methods, combinations, and compositions provided herein. Non-limiting examples of anti-neoplastic agents include anti-angiogenic agents, alkylating agents, antimetabolites, certain natural products, platinum coordination complexes, anthracenediones, urea substitutes, methyl hydrazine derivatives, adrenal cortex Inhibitors, specific hormones, antagonists and anti-cancer polysaccharides.

  As used herein, substantially inactive with reference to a bound therapeutic agent is recognized in the standard or art of assessing the therapeutic activity of a drug, such as an in vitro or in vivo assay. Means at least 1%, generally 10, 20, 30, 50, 60, 70, 80 or 90 or 100% inactive compared to an unbound therapeutic agent.

  As used herein, a targeted cell or tissue refers to a cell or tissue that contains a cell surface protease that cleaves the conjugate. The cell or tissue can be involved in the disease, or can be present at one or more loci of the disease, thanks to involvement in the disease process, or simply by chance.

  As used herein, angiogenesis broadly encompasses the entire process directly or indirectly involved in the establishment and maintenance of new vasculature (neovascularization), and neovascularization associated with tumors. Including, but not limited to.

As used herein, an anti-angiogenic treatment or agent is one or more associated with undesirable and / or uncontrolled angiogenesis when used alone or in combination with other compounds. Refers to any therapeutic regimen and compound that can reduce, reduce, ameliorate, prevent, or place or maintain a remission state of these clinical symptoms or diagnostic markers. Thus, for purposes herein, an anti-angiogenic agent refers to an agent that inhibits the establishment or maintenance of the vasculature. Such agents include, but are not limited to, anti-tumor agents and agents for the treatment of diabetic retinopathy, hyperproliferative disorders and other disorders associated with undesirable angiogenesis such as others. Not.

As used herein, a non-anti-angiogenic anti-tumor agent refers to an anti-tumor agent that does not act primarily by inhibiting angiogenesis. Whether an anti-tumor agent acts primarily by inhibiting angiogenesis can be determined by using the assays provided herein or by using other assays well known to those skilled in the art. .

  As used herein, undesired and / or uncontrolled angiogenesis refers to pathological angiogenesis in which the effect of an angiogenic agent is superior to that of an angiogenesis inhibitor. As used herein, deficient angiogenesis refers to a pathological angiogenesis associated with a disorder that has abnormal angiogenesis or a defect in normal angiogenesis that results in the absence or substantial reduction of angiogenesis. Point to.

  As used herein, a cell surface protease is either on the cell surface thanks to a specific binding interaction with any protease located on or in the cell surface and / or a receptor thereto. A protease that is localized or localized at or near the cell surface or associated with the cell surface. An exemplary protease localized on the cell surface due to its specific binding interaction with its receptor is urokinase plasminogen activator (u-PA) bound to urokinase plasminogen activator receptor (u-PAR). Accordingly, cell surface proteases contemplated herein include cell surface related proteases. It also includes all of its types that can circulate or be within cells. To be classified as a cell surface protease, it is localized on the surface of the cell at some point in the cycle (i.e. it binds to the surface as a transmembrane protease or to a receptor for it). There must be at least one type. Cell surface proteases include serine proteases such as, but not limited to, transmembrane serine protease (MTSP) and endotheliase and urokinase.

  As used herein, serine protease (SP) refers to a diverse family of proteases where serine residues are involved in the hydrolysis of proteins or peptides. The serine residue is part of a catalytic triad mechanism that includes serine, histidine and aspartic acid in catalysis, or a hydroxyl / ε-amine or hydroxyl / α-amine catalytic bipartite mechanism that includes serine and lysine in catalysis. Can be part. Of particular interest are SPs from mammals including humans. Those skilled in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide generally does not substantially alter biological activity (see, eg, Watson et al. (1987) “Molecular biology of genes, 4th edition ", The Bejacmin / Cummings Pub. Co., P.224).

  As used herein, soluble and released types of cell surface proteases are contemplated. Such forms include, for example, forms found in serum by proteolysis or other removal of extracellular components of membrane-bound proteases, and splice variants that do not contain a transmembrane domain.

As demonstrated herein, the protease activity of cell surface proteases and cell-associated proteases is such as cytotoxic agents by providing conjugates that are activated upon cleavage by such enzymes. It can be utilized to provide a means of concentrating therapeutic agents near such cells. Such conjugates release the therapeutic agents, such as cytotoxic agents, or derivatives thereof that can be converted locally to therapeutic agents at the site of action under the action of cell surface proteases or cell-associated proteases. As described above, the substrate is expressed on the surface of a cell, such as a tumor cell or endothelial cell, that is involved in the disease or is present at the site (s) of the disease or at one or more loci. Designed to be a substrate for a targeted protease that is either active or active. Thanks to the specific expression, localization or activation of such proteases, or the presence of co-factors for their receptors, substrates or enzymes, administration of the conjugates provided herein to such cells Allows targeting of therapeutic agents. Upon contact with the protease, the active therapeutic agent is released in close proximity to the targeted cell. For example, the specific profiles of some MTSPs are as follows:
0

  As used herein, “transmembrane serine protease (MTSP)” refers to a family of transmembrane serine proteases that share common structural features as described herein (Hooper et al. (2001) J. Biol. Chem. 276: 857-860). Thus, for example, a reference to “MTSP” includes all that is encoded by the MTSP gene, including but not limited to MTSP1, MTSP3, MTSP4, MTSP6, MTSP7, MTSP9, MTSP10, MTSP12, MTSP20, MTSP22 and MTSP25 Or equivalent molecules obtained from any other source or made synthetically or exhibiting the same activity. Other MTSPs include, but are not limited to, choline, enteropeptidase, human airway trypsin-like protease (HAT), TMPRSS2 and TMPRSS4. Other MTSPs can be identified using the MTSPs described herein. Full-length molecular and splice variants, and other animals of interest to primates, rodents, dogs, cats, and domestic animals, agricultural and zoo animals, including cattle, sheep, goats, pigs, horses, chimpanzees and gorillas Methods for isolating nucleic acids encoding other MTSPs, including nucleic acid molecules encoding MTSPs from animal species such as species, are well known to those of skill in the art and are outlined herein. A human supply to screen a suitable library with nucleic acid molecules or primers or probes selected based thereon using the nucleic acids described herein, including those shown in SEQ ID NOs: Nucleic acid molecules encoding full length MTSP polypeptides from sources or from other animals can be obtained.

Sequences encoding nucleic acid molecules and encoded amino acid sequences of exemplary MTSPs and / or domains thereof are shown in SEQ ID NOs: 1-45, 269-270 and 272-276. The term also encompasses MTSP with amino acid substitutions that do not substantially alter the activity of the respective member, and also includes polypeptides encoded by splice variants thereof. Thus, the resulting polypeptide is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the proteolytic activity of the unchanged polypeptide Are included, and are also encoded by allelic variants such as MTSP and single nucleotide polymorphisms (SNPs) encoded by the splice variant thereof. Includes MTSP. Suitable substitutions, including but not necessarily, amino acid conservative substitutions are known to those skilled in the art and can be made without removing biological activity such as catalytic activity of the resulting molecule. MTSP includes those of animals of mammalian origin including humans.
1

  As used herein, “MTSP protease domain” refers to an MTSP extracellular protease domain that exhibits proteolytic activity and shares homology and structural characteristics with the protease domain of the chymotrypsin / trypsin family. Point to. Thus, it is at least a minimal portion of a domain that exhibits proteolytic activity as assessed by standard in vitro assays. As used herein, such protease domains and catalytically active portions thereof are contemplated.

  Exemplary MTSP polypeptides that exhibit protease domains are set forth in SEQ ID NOs: 1-45, 269-270, and 272-276, and include smaller portions thereof that retain or exhibit protease activity. Protease domains contain insertions and deletions in the surface loop and vary in size and configuration. They retain a conserved structure that includes at least one of the active site triad, the major specificity pocket, the oxyanion hole and / or other features of the serine protease domain of the protease. Thus, for purposes herein, a protease domain is part of an MTSP as defined herein and is homologous to other MTSP domains. As MTSP, MTSP1, MTSP3, MTSP4, MTSP6, MTSP7, MTSP9, MTSP10, MTSP12, MTSP20, MTSP22 and MTSP25 (see SEQ ID NOS: 1-19, 42-45, 269-270 and 272-276; also international PCT See application WO 02/00860 (see SEQ ID NOs: 38 and 97 therein which provide variants of MTSP12); choline (SEQ ID NOs: 28 and 29), enteropeptidase (SEQ ID NOs: 30 and 31), These include human airway trypsin-like protease (HAT) (SEQ ID NOs: 32 and 33), hepsin (SEQ ID NOs: 34 and 35), TMPRSS2 (SEQ ID NOs: 36 and 37) and TMPRSS4 (SEQ ID NOs: 38 and 39). With folding Like the larger class of enzymes (see, eg, the internet accessible MEROPS database), the protease domains of MTSP share a high degree of amino acid sequence identity: His, Asp and The Ser residue is present in a conserved motif, among those activated by cleavage, the activation site that results in the N-terminus of the second strand in the double-stranded form has a conserved motif. (E.g., amino acids 801-806 of SEQ ID NO: 29, amino acids 406-410 of SEQ ID NO: 31; amino acids 186-190 of SEQ ID NO: 33; amino acids 161-166 of SEQ ID NO: 35; SEQ ID NO: 37 amino acids 255-259; amino acids 190-194 of SEQ ID NO: 39 and those of skill in the art and in the art See others as described in known and / or herein Te).

For example, MTSP10 there is a disulfide bond as follows with respect to (SEQ ID NO: 44 and 45, a _{reference): C 488 -C 504, C} 587 -C 653; C 619 -C 632; C 643 -C 673 ( SEQ ID NO: 44 and 45) (42-58 by chymotrypsin numbers; 136-201; 168-182 and 191-220). A disulfide bond is formed between the Cys residues of C ₅₇₃ -C ₂₉₆ linking the protease domain to another domain, and thus activated cleavage (between the R ₄₆₂ and I ₄₆₃ residues of SEQ ID NO: 45). The polypeptide obtained in is a double-stranded molecule. C ₅₇₃ (SEQ ID NO: 45) is the free Cys in the single chain protease domain. As described, the protease can also be provided as a double-stranded molecule. Single and double stranded forms are proteolytically active. Double stranded forms are typically generated by binding between Cys outside the protease domain, such as C ₅₇₃ and Cys ₂₉₆ . Upon activation cleavage, the disulfide bond remains, resulting in a double-stranded polypeptide. The size of the “A” chain is a function of the starting length of the polypeptide prior to activating cleavage between _R462 and _I463 . Any length of polypeptide comprising a protease domain (residues 463-692 of SEQ ID NO: 45) or a catalytically active fragment thereof is contemplated herein. The double stranded form includes at least the protease domain of a polypeptide from and including _C296 to _C573 .

As used herein, a double-stranded protease domain refers to, for example, Cys ₅₇₃ (SEQ ID NO: 45 for MTSP), which links the protease domain to the remaining polypeptide “A” chain. Such as, for example, a double-stranded form formed from a single-chain type protease in which Cys forms a pair with Cys outside the protease domain. A double-stranded protease domain refers to any type in which the “remaining polypeptide”, ie, the “A” chain, is shortened and contains Cys from outside the protease domain.

  As used herein, the catalytically active domain of MTSP refers to the protease domain. A reference to the protease domain of MTSP generally refers to a single chain protein. If a double-stranded or both types are intended, they are identified as such. The zymogen form of each protein is a single chain, which is converted to an active double-stranded or multi-stranded form by activating cleavage. An active form means a form active in vivo or in vitro.

As used herein, activated cleavage refers to cleavage of the protease (generally between R and I or V in the full-length protein) at the N-terminus of the protease domain. Thanks to Cys-Cys pairing between Cys outside the protease domain and Cys inside the protease domain (see, eg, Cys _{573 of} SEQ ID NO: 45), the polypeptide resulting from cleavage has a double chain ( "A" chain and MTSP protease domain, "B" chain). Cleavage can be done with another protease or autocatalytically. The conjugates provided herein conveniently contain a site recognized by an active cell surface protease (or cell-associated protease) and are thereby cleaved to the active form or inactivity of the therapeutic agent. Release a prodrug form.

As used herein, MTSP1 is always referred to herein,
A polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1 or 40;
A polypeptide encoded by a sequence of nucleotides that hybridizes under low, moderate, or high stringency conditions to the sequence of nucleotides set forth in SEQ ID NO: 1 or 40;
A polypeptide comprising the sequence of amino acids set forth in SEQ ID NO: 2 or 41;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 with the amino acid sequence shown in SEQ ID NO: 2 or 41 A polypeptide comprising a sequence of amino acids having%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; / Or a polypeptide encoded by a splice variant of MTSP1 as shown in SEQ ID NO: 1 or 40:
Including at least one or all or any of the combinations.

  MTSP1 can be from any animal, particularly mammals, and includes but is not limited to humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated. MTSP1 is also referred to as TADG-15 and matriptase. As explained below, the protein originally called matriptase appears to be a splice variant or processed product of MTSP1.

As used herein, MTSP3 is always referred to herein.
A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 3;
A polypeptide encoded by a sequence of nucleotides that hybridizes under low, moderate or high stringency conditions to the sequence of nucleotides set forth in SEQ ID NO: 3;
A polypeptide comprising the sequence of amino acids set forth as amino acids 205-437 of SEQ ID NO: 4;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, and the sequence of amino acids set forth in SEQ ID NO: 4 A polypeptide comprising a sequence of amino acids having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and / or A polypeptide encoded by a splice variant of MTSP3 as shown in SEQ ID NOs: 3 and 4:
Including at least one or all or any of the combinations.

  MTSP3 can be from any animal, particularly mammals, and includes but is not limited to humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated.

As used herein, MTSP4 is always referenced when referred to herein.
A polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 5, 7 or 9;
A polypeptide encoded by a sequence of nucleotides that hybridizes to the nucleotide sequence set forth in SEQ ID NO: 5, 7, or 9 under conditions of low, moderate, or high stringency;
A polypeptide comprising a sequence of amino acids set forth in any of SEQ ID NOs: 6, 8, or 10;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% with the amino acid sequence shown in SEQ ID NO: 6, 8 or 10 , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequences comprising a sequence identity And / or a polypeptide encoded by a splice variant of MTSP4 shown in SEQ ID NOs: 7-10:
Including at least one or all or any of the combinations.

  MTSP4 can be from any animal, particularly mammals, and includes but is not limited to humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated.

As used herein, MTSP6 is always referred to herein,
A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 11;
A polypeptide encoded by a sequence of nucleotides that hybridizes under low, moderate, or high stringency conditions to the sequence of nucleotides set forth in SEQ ID NO: 11;
A polypeptide comprising the sequence of the amino acid set forth in any of SEQ ID NO: 12;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, and the sequence of the amino acid set forth in SEQ ID NO: 12 A polypeptide comprising a sequence of amino acids having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and / or :
The polypeptide encoded by the splice variant of MTSP6 shown in SEQ ID NO: 12:
Including at least one or all or any of the combinations.

  MTSP6 can be from any animal, particularly mammals, and includes but is not limited to humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated. Of particular interest herein is MTSP6 of SEQ ID NO: 12.

As used herein, MTSP7 is whenever referred to herein,
The polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 13;
A polypeptide encoded by a sequence of nucleotides that hybridizes under low, moderate, or high stringency conditions to the sequence of nucleotides set forth in SEQ ID NO: 13;
A polypeptide comprising the sequence of the amino acid set forth in SEQ ID NO: 13;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the amino acid sequence shown in SEQ ID NO: 14, A polypeptide comprising a sequence of amino acids having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and / or A polypeptide encoded by a splice variant of MTSP7 shown in SEQ ID NO: 13:
Including at least one or all or any of the combinations.

    MTSP7 can be from any animal, particularly mammals, and includes but is not limited to humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated.

As used herein, MTSP9 is always referred to herein.
A polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 17 or SEQ ID NO: 42;
A polypeptide encoded by a sequence of nucleotides that hybridizes to the sequence of nucleotides set forth in SEQ ID NO: 17 or 42 under conditions of low, moderate, or high stringency;
A polypeptide comprising the sequence of amino acids set forth in SEQ ID NO: 18 or 43;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 with the amino acid sequence shown in SEQ ID NO: 18 or 270 A polypeptide comprising a sequence of amino acids having%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; / Or a polypeptide encoded by a splice variant of MTSP9 as shown in SEQ ID NO: 17:
Including at least one or all or any of the combinations.

  MTSP9 can be from any animal, particularly mammals, and includes, but is not limited to, humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated.

As used herein, MTSP 10 is whenever referred to herein,
The polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 44;
A polypeptide encoded by a sequence of nucleotides that hybridizes under low, moderate or high stringency conditions to the sequence of nucleotides set forth in SEQ ID NO: 44;
A polypeptide comprising the sequence of the amino acid set forth in SEQ ID NO: 45;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, and the sequence of the amino acid set forth in SEQ ID NO: 45 A polypeptide comprising a sequence of amino acids having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and / or The polypeptide encoded by the splice variant of MTSP10 shown in SEQ ID NO: 44:
Including at least one or all or any of the combinations.

  MTSP10 can be from any animal, particularly mammals, and includes but is not limited to humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated.

  Provided herein are MTSP10 polypeptides, and nucleic acids encoding MTSP, and domains, derivatives and analogs thereof, including but not limited to splice variants thereof. Also provided is a single-chain protease domain having an N-terminus functionally equivalent to that produced by activation of zymogen-type MTSP10. The cleavage site for the protease domain of MTSP10 is between amino acid R and amino acid I (R ↓ IIGGT) (residues 462-467 of SEQ ID NO: 45).

As used herein, MTSP12 is the sequence 19 and 20 whenever referred to herein.
A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 19 or by a sequence of nucleotides comprising a nucleotide that encodes the sequence of amino acids set forth in SEQ ID NO: 20;
A polypeptide encoded by a sequence of nucleotides that hybridizes under low, moderate, or high stringency conditions to the sequence of nucleotides shown as SEQ ID NO: 19;
A polypeptide comprising the sequence of amino acids set forth in SEQ ID NO: 20, or a catalytically active portion thereof;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the amino acid sequence shown in SEQ ID NO: 20, A polypeptide comprising a sequence of amino acids having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and / or A polypeptide encoded by a splice variant of MTSP12 comprising the amino acid sequence set forth in SEQ ID NO: 20:
Including at least one or all or any of the combinations.

In particular, MTSP12 polypeptides are provided having a protease domain as shown in SEQ ID NOs: 19 and 20. The polypeptide is a single chain or a multi-chain polypeptide. The protease domain of MTSP12, whenever referred to herein, includes the sequence of amino acids set forth in SEQ ID NO: 20, or a catalytically active portion thereof, but does not include the sequence of amino acids set forth in SEQ ID NO: 271. A polypeptide;
A polypeptide comprising the sequence of amino acids set forth in SEQ ID NO: 272 or a catalytically active fragment thereof;
Including a polypeptide containing amino acids 237-456 of SEQ ID NO: 6, a polypeptide containing amino acids 538-765 of SEQ ID NO: 20, and a polypeptide containing amino acids 861-1087 of SEQ ID NO: 20, but SEQ ID NO: 271 A polypeptide not comprising the amino acid sequence shown in;
A polypeptide encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to a sequence of nucleotides encoding any of the polypeptides of a) -c) ;
Encoded by a nucleotide sequence that hybridizes to the nucleotide sequence set forth in SEQ ID NO: 20 under low, moderate, or high stringency conditions but does not encode the amino acid sequence set forth in SEQ ID NO: 271 A polypeptide;
At least about 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with the sequence of the amino acid set forth in SEQ ID NO: 20, A polypeptide comprising a sequence of amino acids having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity;
a) -e) polypeptide amino acid sequence and at least about 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% A polynucleotide comprising a sequence of amino acids having a sequence identity of 89, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%;
A polypeptide encoded by a splice variant of the sequence of nucleotides encoding any of the above MTSP12:
At least one or all of the combinations or any or catalytically active moiety.

  Its smaller portion that retains protease activity is also provided. MTSP12 can be from any animal, particularly mammals, and includes, but is not limited to, humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated. MTSP12 also includes variants described in International PCT Application No. WO 02/00860 (see SEQ ID NOs: 38 and 97 therein).

As used herein, MTSP 20 is always referred to herein,
The polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 273;
A polypeptide encoded by a sequence of nucleotides that hybridizes to the sequence of nucleotides set forth in SEQ ID NO: 273 under low, moderate, or high stringency conditions;
A polypeptide comprising the sequence of the amino acid set forth in SEQ ID NO: 273;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, and the sequence of amino acids set forth in SEQ ID NO: 274 A polypeptide comprising a sequence of amino acids having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and / or A polypeptide encoded by a splice variant of MTSP20 encoded by a sequence of nucleotides comprising the sequence set forth in SEQ ID NO: 273:
Including at least one or all or any of the combinations.

  MTSP20 can be from any animal, especially mammals, and includes, but is not limited to, humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated.

As used herein, MTSP 22 is whenever referred to herein,
The polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 275;
A polypeptide encoded by a sequence of nucleotides that hybridizes under low, moderate, or high stringency conditions to the sequence of nucleotides set forth in SEQ ID NO: 275;
A polypeptide comprising the sequence of amino acids set forth in SEQ ID NO: 276;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, and the sequence of amino acids set forth in SEQ ID NO: 276 A polypeptide comprising a sequence of amino acids having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and / or The polypeptide encoded by the splice variant of MTSP22 shown in SEQ ID NO: 275:
Including at least one or all or any of the combinations.

  MTSP22 can be from any animal, particularly mammals, and includes but is not limited to humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated.

As used herein, MTSP25 is whenever referred to herein,
The polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 269;
A polypeptide encoded by a sequence of nucleotides that hybridizes to the sequence of nucleotides set forth in SEQ ID NO: 269 under low, moderate, or high stringency conditions;
A polypeptide comprising the sequence of amino acids set forth in SEQ ID NO: 270;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, and the sequence of the amino acid set forth in SEQ ID NO: 270 A polypeptide comprising a sequence of amino acids having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and / or The polypeptide encoded by the splice variant of MTSP25 shown in SEQ ID NO: 269:
Including at least one or all or any of the combinations.

MTSP25 can be from any animal, particularly mammals, and includes, but is not limited to, humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated.
As used herein, a human protein is one that is encoded by a nucleic acid present in the human genome and allows all allelic and conservative mutations to be transferred to other mammals. Including unless found mutant.

As used herein, a cell surface membrane targeted therein is not substantially cleaved by plasmin or prostate specific antigen (PSA) (or other non-cell surface related protease). In a comparable in vitro assay in which the activity against cleavage of a conjugate of a protease, or a catalytically active portion of a protease domain of prostate specific antigen (PSA) or plasmin (or its catalytically active form) is compared. Means (under optimal conditions for each enzyme) a relative activity of at least 2: 1, 3: 1, 4: 1, 5: 1, 10: 1 or 100: 1.
As used herein, activity refers to the K _cat / K _m ratio, where K _cat is the rate of catalytic rotation for a particular enzyme, and K _m is Michaelis for substrate binding. It is a constant.

  As used herein, a “nucleic acid encoding a protease domain or catalytically active portion of MTSP” encodes only the single-chain protease domain described above or an active portion thereof and other MTSP domains. It shall refer to a nucleic acid that does not encode adjacent portions as a continuous sequence.

  As used herein, CUB domains are motifs that mediate protein-protein interactions in complement components C1r / C1s and have been identified in various proteins involved in developmental processes.

  As used herein, zymogens are enzymatically inactive proteins (ie, not necessarily so) that are converted to proteolytic enzymes by the action of activators, including those due to self-activation. But typically less than 1% of the active form). Inactive means less activity than one of ordinary skill in the art would consider the active form of the enzyme. The ratio of zymogen activity to activated form varies from enzyme to enzyme.

  As used herein, “disease or disorder” refers to a condition in an organism that results, for example, from an infection or genetic defect and is characterized by identifiable symptoms. Diseases contemplated for treatment herein include targeted cells or cell surface proteases, including proteases that are localized to or associated with cells, or that are involved in the disease or process of disease. Any disease associated with the tissue. Such an association may be because the protease is involved in the disease or because it is accidentally associated with a cell involved in the disease. In the present specification, such diseases are referred to as cell surface protease-related diseases. Thus, to treat a disease, for example, it is expressed on a cell associated with the disease, such as an immune cell for the treatment of an inflammatory disease and a virus-infected cell for the treatment of a viral disease. However, cell surface proteases are identified. As described herein, the conjugate is designed to be cleaved by the selected protease.

As used herein, neoplasia (tumor formation) refers to abnormal new growth and thus has the same meaning as a tumor that can be benign or malignant. Unlike hyperplasia, neoplastic growth persists even in the absence of the original stimulus.
As used herein, neoplastic disease refers to any disorder associated with cancer, including tumorigenesis, growth, metastasis and progression.

As used herein, cancer refers to the general term for diseases caused by any type of malignancy.
As used herein, malignancy applied to a tumor refers to a primary tumor with metastatic potential that has lost growth and position control.

  As used herein, an endotheliase is a transmembrane domain that is expressed or active on the surface of an endothelial cell and contains a protease domain, particularly an extracellular protease domain. , And refers to mammalian proteins, including human proteins, which are generally serine proteases (see also US Patent Application Serial No. 09 / 717,473 and International PCT Application No. WO 01/36604). Thus, for example, a reference to endotheliase may be derived from all molecules encoded by the endotheliase gene family, or any other source, or made synthetically, or has the same activity. Includes the equivalent molecule shown. The endotheliase gene family is a transmembrane protease that is expressed or active in endothelial cells. These proteases include serine proteases. These include proteins containing protease domains that have these characteristics and also show sequence homology to endotheliases 1 and 2. Endothelins 1 and 2 exhibit, for example, about 40% or 45% identity. Sequence homology is at least about 25%, 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83 of residues when aligned to maximize equivalence. %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or More numerical values mean sequence identity along its length.

  Sequence homology may also be at least moderate to the nucleic acid molecule in which the coding sequence of the nucleic acid encodes a nucleic acid molecule provided herein or to the same protein but differs in sequence due to the degeneracy of the genetic code, or More closely related proteins are evaluated by measuring whether they hybridize with a high degree of stringency. In addition, “endotheliase” has amino acid substitutions including those shown in Table 1, and the resulting polypeptide is at least 1%, 5%, 10%, 20% of the proteolytic activity of the unconverted polypeptide. , 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc. Appropriate substitutions of amino acids are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. As described, one of ordinary skill in the art recognizes that substitution of a single amino acid in a non-essential region of a polypeptide generally does not substantially change biological activity (eg, Watson et al. Biology, 4th edition ", 1987, The Bejacmin / Cummings Pub. Co., p.224). Also, a catalytically active fragment or a truncated form of endotheliase is included within the definition of “endotheliase”.

As used herein, endotheliase 1 is whenever referred to herein.
The polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 21;
A polypeptide encoded by a sequence of nucleotides that hybridizes to the sequence of nucleotides set forth in SEQ ID NO: 21 under conditions of low, moderate, or high stringency;
A polypeptide comprising the sequence of the amino acid set forth in SEQ ID NO: 22;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the amino acid sequence shown in SEQ ID NO: 22, A polypeptide comprising a sequence of amino acids having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and / or A polypeptide encoded by a splice variant of a nucleic acid molecule that encodes a protein containing the polypeptide set forth in SEQ ID NO: 22:
Including at least one or all of or any combination thereof.

  Endoceliase 1 can be from any animal, particularly a mammal, and includes but is not limited to humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated.

As used herein, endotheliase 2 is whenever referred to herein,
A polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23 or 25;
A polypeptide encoded by a sequence of nucleotides that hybridizes under low, moderate or high stringency conditions to the sequence of nucleotides set forth in SEQ ID NO: 23 or 25;
A polypeptide comprising the sequence of amino acids set forth in SEQ ID NO: 24 or 26;
At least about 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 with the amino acid sequence shown in SEQ ID NO: 24 or 26 A polypeptide comprising a sequence of amino acids having%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; / Or a polypeptide encoded by a splice variant of the nucleic acid shown in SEQ ID NO: 23 or 25:
Including at least one or all of or any combination thereof.

  Endoceliase 2 can be from any animal, particularly a mammal, and includes but is not limited to humans, rodents, birds, ruminants and other animals. A full-length zymogen or double-stranded activated form, or any domain thereof, including a protease domain, which can be double-stranded activated form, or single-chain form is contemplated.

As used herein, the protease domain of endotheliase refers to the polypeptide portion of endotheliase, the extracellular portion that exhibits protease activity. A protease domain is a polypeptide that contains at least the minimum number, generally more than 50 or 100, amino acids required for protease activity. Protease activity can be assessed empirically, such as by testing a polypeptide for its activity acting as a protease. Assays as described in the examples can be used to assess protease activity, except that known endotheliases are used in place of test compounds. Furthermore, since proteases, particularly serine proteases, have a characteristic structure and sequence or motif, protease domains are easily identified by such structure and sequence or motif.

As used herein, a portion of an endotheliase protease domain is an endotheliase extracellular domain that is localized within the endotheliase and exhibits serine protease activity. Refers to the portion of the protease domain of endotheliase. Thus, it is at least the minimal portion of the extracellular domain that exhibits proteolytic enzyme activity as assessed by standard assays. An exemplary protease domain of endotheliase is shown in SEQ ID NO: 22 and as amino acids 321 to 688 and 321 to 562 of SEQ ID NOs: 24 and 26, respectively. Smaller portions thereof that retain protease activity are contemplated. Protease domains differ in size and configuration, including insertions and deletions in the surface loop. Such domains are conserved, including at least one structural feature, such as an active site triad, major specificity pocket, oxyanion hole and / or other structural features of the serine protease domain of the protease. The structure is shown. Thus, for purposes herein, a protease domain is part of an endotheliase, as defined herein, but in retaining structural features and sequences of similarity or homology. Homologous to the protease domain of chymotrypsin or trypsin.

As used herein, homology means about 25%, 40%, 60%, 80%, 90%, 95%, 98% or greater sequence identity. By sequence identity is meant the number of conserved amino acids measured by standard alignment algorithm programs using the default gap penalty established by each supplier. Homology can also be assessed by conserved nucleic acid sequences, including any that hybridize at least under low stringency conditions and encode domains. Similarly, nucleic acid sequence alignment programs are commercially available (DNAStar “MegAlign” program (Madison, Wis.) And University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison, Wis.)). Substantially homologous nucleic acid molecules will typically hybridize all along the entire length of the nucleic acid of interest with moderate or high stringency. Also contemplated are nucleic acid molecules that contain degenerate codons instead of codons in the hybridizing nucleic acid molecule.

As used herein, a statement that a polypeptide consists essentially of a protease domain means that only the endotheliase portion of the polypeptide is the protease domain or a catalytically active portion thereof. . The polypeptide can optionally further comprise an amino acid sequence derived from another non-endotheliase.
As used herein, a domain refers to a portion of a molecule, such as a protein or nucleic acid, that is structurally and / or functionally distinct from other parts of the molecule.

As used herein, an active form of a protease refers to an enzyme that catalyzes the hydrolysis of a protein or peptide. Reference to protease includes active forms and zymogens or other less active forms.
As used herein, nucleic acids include DNA, RNA, and analogs thereof, including peptide nucleic acids (PNA) and mixtures thereof. The nucleic acid can be single-stranded or double-stranded. When referring to a probe or primer, a single-stranded molecule optionally labeled with a detectable label such as a fluorescent or radioactive label is contemplated. Such molecules typically have their targets statistically unique for probing or priming the library, or low copy numbers (typically less than 5, typically 3 Have a length that has less than In general, a probe or primer contains at least 14, 16, or 30 consecutive sequences that are complementary or identical to the gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids in length.

As used herein, a nucleic acid that encodes a fragment or portion of an endotheliase refers to another sequence of endotheliase that encodes only the described fragment or portion of the endotheliase protein. Means a nucleic acid that does not encode the sequence as a continuous sequence.

As used herein, a heterologous nucleic acid encodes RNA if it is DNA, or if it is RNA, is it generally not normally produced in vivo by the cell in which it is expressed? Mediate changes in the expression of endogenous nucleic acids such as DNA by affecting transcription, translation or other controllable biochemical processes, or encode such mediators or are normal It is a nucleic acid that encodes a protein that is located at a locus different from the locus of. A heterologous nucleic acid is generally not endogenous to the cell into which it is introduced, but has been obtained from another cell or made synthetically. Generally, although not necessarily, such a nucleic acid encodes an RNA or protein that is not normally produced by the cell in which it is currently expressed.

  Heterologous nucleic acids such as DNA are also referred to as exogenous nucleic acids such as DNA. Any nucleic acid, such as DNA, recognized or considered by a person skilled in the art as being heterologous or exogenous to the expressing cell is encompassed by the heterologous nucleic acid herein; Also include exogenously added nucleic acids that are also expressed. Examples of heterologous nucleic acids include nucleic acids encoding traceable marker proteins such as proteins that confer drug resistance, nucleic acids encoding therapeutically effective substances such as anticancer agents, enzymes and hormones, As well as other types of proteins such as antibodies, RNA interference (RNAi) or other double stranded RNA, and RNA such as antisense RNA. An antibody encoded by a heterologous nucleic acid can be secreted or expressed on the surface of a cell into which the heterologous nucleic acid has been introduced.

  For example, the nucleic acid can be a targeted agent, such as a therapeutic or diagnostic agent in a conjugate. Nucleic acids are RNA interference (RNAi) used to inhibit the expression of targeted genes by creating loss of function (eg, Chuang et al. (2000) Proc. Natl. Acad. Sci. USA 97: 4985, The use of dsRNA for Methods relating to the use of RNAi to silence genes in mammals, C. elegans, Drosophila and plants, and organisms including humans are known (eg Fire et al. (1998) Nature 391: 806-811 Fire ( 1999) Trends Genet. 15: 358-363; Sharp (2001) Genes Dev. 15: 485-490; Hammond, et al. (2001) Nature Rev. Genet. 2: 110-1119; Tuschl (2001) Chem. Biochem 2: 239-245; Hamilton et al. (1999) Science 286: 950-952; Hammond et al. (2000) Nature 404: 293-296; Zamore et al. (2000) Cell 101: 25-33; Bernstein et al. (2001) Nature 409: 363-366; Elbashir et al. (2001) Genes Dev. 15: 188-200; Elbashir et al. (2001) Nature 411: 494-498; International PCT Application No. WO 01 / 29058; see International PCT Application No. WO 99/32619). By choosing the appropriate sequence, the expression of dsRNA can interfere with the accumulation of endogenous mRNA that encodes the targeted gene product. A region that contains at least about 21 nucleotides and is selective for a nucleic acid that encodes the targeted gene product (ie, the target is unique) is used to create RNAi. The

  As used herein, gene therapy is a heterologous nucleic acid such as DNA into a specific cell, target cell of a mammal, particularly a human having a disorder or abnormality for which such treatment is sought. With the introduction of. The nucleic acid molecule is contained in a conjugate that is bound via a cell surface protein cleavage site. Nucleic acids such as DNA are introduced into selected target cells in such a manner that a heterologous nucleic acid such as DNA is expressed, thereby producing the therapeutic product encoded. Alternatively, the heterologous nucleic acid, such as DNA, can mediate the expression of the DNA encoding the therapeutic product in some manner, or it can directly express the therapeutic product in some manner or Products such as indirectly mediated peptides or RNA can be encoded.

  Gene therapy can also be used to deliver a nucleic acid encoding a gene product that replaces the defective gene or supplements the gene product produced by the mammal or cell into which it is introduced. The introduced nucleic acid is not normally produced in a mammalian host or is a therapeutic compound, such as its growth factor inhibitor, which is not produced in a therapeutically effective amount or therapeutically useful time, or tumor necrosis factor or The inhibitor, such as a receptor for can be encoded. A heterologous nucleic acid, such as DNA, encoding a therapeutic product is modified prior to introduction into the cells of the affected host in order to promote or otherwise alter the expression of the product or its expression. Can. Gene therapy also involves the delivery of inhibitors or repressors or other modulators of gene expression such as dsRNA or antisense or other nucleic acid molecules. The conjugates herein can be used to deliver products such as nucleic acids for gene therapy.

  As used herein, a therapeutically effective product for gene therapy improves or eliminates the symptoms, manifestations of hereditary or acquired disease upon introduction of a nucleic acid into a host, or A product encoded by a heterologous nucleic acid, typically DNA, in which the product that cures the disease is expressed. Also included are biologically active nucleic acid molecules such as RNAi and antisense.

  As used herein, a sequence complementary to at least a portion of RNA with respect to an antisense oligonucleotide generally hybridizes to RNA under conditions of moderate or high stringency and is stable 2 Means a sequence with sufficient complementarity to be able to form a double helix; in the case of double stranded SP antisense nucleic acids, thus testing a single strand of double helix DNA (ie dsRNA) Or triple helix formation can be assayed. The ability to hybridize depends on the degree and length of complementarity of the antisense nucleic acid. In general, the longer the nucleic acid that hybridizes, the more bases mismatch with the RNA encoding SP, which contains and still forms a stable double helix (or even a triple helix). be able to. One skilled in the art can ascertain the tolerability of the mismatch by using standard procedures to determine the melting point of the hybrid complex.

  Amino acid substitutions can be made or caused in any SP or its protease. Amino acid substitutions include conservative substitutions such as those shown in Table 1 that do not remove proteolytic activity. As described herein, substitutions that alter the properties of the protein, such as removal of cleavage sites and other such sites are also contemplated; such substitutions are generally non-conservative Can be easily achieved by those skilled in the art.

Appropriate conservative substitutions of amino acids are known to those skilled in the art and can be made without generally altering the biological activity of the resulting molecule, eg, enzymatic activity. Also included within the definition are catalytically active fragments of SP, particularly single chain protease moieties.
Conservative amino acid substitutions are made, for example, according to those shown in Table 1 below.

  Other substitutions are also allowed and can be determined empirically or according to known conservative substitutions. For example, one or more amino acid residues in a sequence can be replaced with another amino acid of similar polarity that serves as a functional equivalent, resulting in a silent change. Substitutions for amino acids in the sequence can be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

  As used herein, amino acids present in the various amino acid sequences appearing herein are identified according to well-known three letter or single letter ellipsis. Nucleotides present in various DNA fragments are referred to using standard single letter names routinely used in the art. Other abbreviations include: hR or hArg for homoarginine; hY or hTyr for homotyrosine; Cha for cyclohexylalanine; Amf for 4-aminomethylphenylalanine; 2- (4, DPL for 6-dimethylpyrimidinyl) lysine; (imidazolyl) K for N ′-(2-imidazolyl) lysine; Me2OP3-Y for O-dimethylphosphotyrosine; O-Me-Y for O-methyltyrosine; MeL for isoquinoline carboxylic acid; DAP for 1,3-diaminopropane; TFA for trifluoroacetic acid; AA for acetic acid.

As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.
As used herein, a probe or primer based on a nucleotide sequence disclosed herein comprises at least 10, 14, generally at least 16 or 30, or 100 contiguous nucleotide sequences. .

  As used herein, an antisense polynucleotide refers to a synthetic sequence of nucleotide bases complementary to the sense strand of mRNA or double-stranded DNA. Mixing sense and antisense polynucleotides under appropriate conditions leads to the binding of two molecules, ie hybridization. When these polynucleotides bind (hybridize) to mRNA, inhibition of protein synthesis (translation) occurs. When these polynucleotides bind to double-stranded DNA, inhibition of RNA synthesis (transcription) occurs. The resulting inhibition of translation and / or transcription leads to an inhibition of the synthesis of the protein encoded by the sense strand. An antisense nucleic acid molecule typically has a sufficient number of nucleotides to bind specifically to the target nucleic acid, generally at least 5 consecutive nucleotides, often at least 14 or 16 or 30 consecutive nucleotides. Or a modified nucleotide complementary to the coding region of a nucleic acid molecule encoding a gene of interest, eg, a nucleic acid encoding a short protease domain of SP.

  As used herein, an array refers to a collection of elements, such as antibodies, that contain three or more members. An addressable array is one in which members of the array are typically identifiable by their location on a solid support. Thus, in general, the members of the array are immobilized at separate identifiable loci on the solid surface.

  As used herein, an antibody refers to an immunoglobulin, whether natural or partially or fully synthetic, and retains the specific binding ability of the antibody. Any derivative thereof is also included. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. Antibodies include any immunoglobulin class member, including IgG, IgM, IgA, IgD and IgE.

As used herein, an antibody fragment refers to any derivative of an antibody that retains at least a portion of the full-length antibody's specific binding ability and is shorter than the full length. Examples of antibody fragments include, but are not limited to, Fab, Fab ′, F (ab) ₂ , single chain Fv (scFV), FV, dsFV diabody and Fd fragment. Fragments can contain multiple chains linked together, such as disulfide bridges. Antibody fragments generally contain at least about 50 amino acids and typically at least 200 amino acids.
As used herein, an Fv antibody fragment consists of one variable heavy chain domain (V _H ) and one variable light chain domain joined in a non-covalent interaction.
As used herein, dsFV refers to an Fv with an engineered intermolecular disulfide bond that stabilizes the V _H -V _L pair.

As used herein, an F (ab) ₂ fragment is an antibody fragment that results from digesting an immunoglobulin with pepsin at pH 4.0-4.5; Equivalent fragments can be produced.
As used herein, Fab fragments are antibody fragments that result from digestion of immunoglobulins with papain; they can be expressed recombinantly to produce equivalent fragments.

As used herein, scFV refers to an antibody fragment that contains a variable light chain (V _L ) and a variable heavy chain (V _H ) joined in any order by a polypeptide linker. The linker has a length such that the two variable domains are cross-linked without substantial interference. Exemplary linkers include, but are not limited to, for example, (Gly-Ser) _n residues that can include several Glu or Lys residues dispersed throughout to increase solubility. Not.

As used herein, a humanized antibody refers to an antibody that is modified to include a human sequence of amino acids such that administration to a human does not elicit an immune response. Methods for producing such antibodies are known. For example, to produce such antibodies, hybridomas expressing monoclonal antibodies or E. coli. Nucleic acids encoding in other prokaryotic or eukaryotic cells, such as E. coli or CHO cells, are altered by recombinant DNA techniques to express antibodies in which the amino acid composition of the non-variable regions is based on human antibodies. Computer programs have been designed to identify such non-variable regions.
As used herein, diabodies are dimeric scFVs; diabodies typically have a peptide linker that is shorter than the scFv and they generally dimerize.

As used herein, production by recombinant means using recombinant DNA methods means the use of well-known molecular biology methods to express the protein encoded by the cloned DNA. .
As used herein, the evaluation of a term is in the sense of obtaining the absolute value of the activity of an SP or domain thereof present in a sample, or an indicator, ratio, percentage, visual or In the sense of obtaining other values, we shall include quantitative and qualitative measurements. The assessment can be direct or indirect, and the chemical species actually detected need not of course be the proteolytic product itself, but can be, for example, a derivative thereof or some further substance.

As used herein, biological activity refers to the in vivo activity of a compound, ie, the physiological response obtained as a result of in vivo administration of a compound, composition or other mixture. Thus, biological activity encompasses the therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Biological activity can be observed in in vitro systems designed to test or use such activity.
As used herein, a combination refers to any collection between two or more items.

As used herein, fluid refers to any composition that can flow. Thus, fluids include compositions in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.
As used herein, an effective amount of a compound for treating a particular disease is an amount sufficient to ameliorate or reduce in some manner the symptoms associated with the disease. Such an amount, if effective, can be administered as a single dose or according to a regimen. The amount can cure the disease but is typically administered to ameliorate the symptoms of the disease. Repeated administration may be required to achieve the desired improvement in disease.

  As used herein, equivalent when referring to two sequences of nucleic acids means that the two sequences in question encode the same amino acid sequence or equivalent protein. When equivalent is used in referring to two proteins or peptides, it means that the two proteins or peptides do not substantially alter the activity or function of the protein or peptide (ie retain at least 1% of the activity). It means having substantially the same sequence with substitutions (see, eg, Table 1 above). When referring to equivalents of properties, the properties need not be present to the same extent (e.g., two peptides can exhibit different rates of the same type of enzyme activity), but the activities are generally substantially the same. . Complementarity, when referring to two nucleotide sequences, typically results in a mismatch between the two nucleotide sequences that is less than 25%, often less than 15%, or even lower, 5% or not at all. It means that it has the ability to hybridize. In general, two molecules hybridize under conditions of high stringency.

  As used herein, a method for treating or preventing a disease or disorder associated with unfavorable and / or uncontrolled angiogenesis is associated with unfavorable and / or uncontrolled angiogenesis. It means that the disease or disorder is alleviated, reduced, improved, prevented or placed in or maintained in remission. It also means that the features of pathological angiogenesis are eliminated, reduced and prevented by treatment. Non-limiting examples of pathological angiogenesis features include uncontrolled degradation of endothelial cell basement membrane and proximal extracellular matrix, migration of endothelial cells to the generation of new functional capillaries, division and organization, As well as the persistence of such functional capillaries.

  As used herein, operably linked or functionally related includes nucleotide regulation and effector sequences such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. And the functional relationship of DNA. For example, functional binding of DNA to a promoter refers to DNA and the promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds and transcribes the DNA. Refers to the physical as well as functional relationship between. To optimize expression and / or in vitro transcription, the 5 ′ untranslated portion of the clone can be removed, added or altered to prevent or reduce expression at either the transcriptional or translational level, It may be necessary to remove extra potentially inappropriate further translation initiation (ie start) codons or other sequences. Alternatively, a consensus ribosome binding site (see, eg, Kozak J. Biol. Chem. 266: 19867-19870 (1991)) may be inserted immediately 5 ′ of the start codon to promote expression. it can. The desirability (or necessity) of such modifications can be determined empirically.

  As used herein, a promoter region or promoter element refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operably linked. The promoter region contains specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. This part of the promoter region is called the promoter. In addition, the promoter region contains sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences can respond to factors acting on cis or acting on trans. A promoter can be constitutive or regulatable, depending on the nature of the regulation. Exemplary promoters intended for use in prokaryotes include the bacteriophage T7 and T3 promoters.

  As used herein, a sample refers to anything that may contain an analyte for which an analyte assay is desired. The sample can be a biological sample such as a biological fluid or biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, and the like. Biological tissues are usually those intercellular substances that form one of the structural substances of human, animal, plant, bacterial, fungal, or viral structure, including connective, epithelial, muscle and nerve tissues Is an aggregate of certain types of cells together. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cells.

As used herein, hybridizing under conditions of particular stringency is used to describe the stability of a hybrid formed between two single stranded DNA fragments and washed Refers to ionic strength and temperature conditions at which such hybrids are washed following annealing under conditions of stringency below or equal to the stage. Typically, high, medium and low stringency includes the following conditions or equivalent conditions.
1) Altitude strictness: 0.1 × SSPE or SSC, 0.1% SDS, 65 ° C.
2) Medium stringency: 0.2 x SSPE or SSC, 0.1% SDS, 50 ° C
3) Low severity: 1.0 x SSPE or SSC, 0.1% SDS, 50 ° C

  Equivalent conditions refer to conditions that select substantially the same percentage of mismatches in the resulting hybrid. The addition of components such as formamide, Ficoll and Denhardt solutions affects parameters such as the temperature at which hybridization should be performed and the reaction rate. Thus, hybridization at 42 ° C. in 5 × SSC in 20% formamide is substantially the same as the hybridization conditions under the low stringency conditions described above. See, for example, Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Chapter 8, for recipes for SSPE, SSC and Denhardt solutions and deionized formamide; Sambrook et al. ., vol. 3, p. B.13, as well as numerous catalogs describing commonly used laboratory solutions). It will be appreciated that equivalent stringency can be achieved by using additional buffers, salts and temperatures.

The term substantially the same or similar depends on the context as understood by one skilled in the art and generally means at least 40, 60, 80, 90, 95 or 98%.
As used herein, substantially identical to a product is sufficient for the property of interest so that a substantially identical product can be used in place of the product. Means similar enough to be unchanged.
As used herein, a target cell refers to a cell that expresses a cell surface protease.

  As used herein, a test substance, including a compound provided herein, is a cell surface protein or cell surface related protein, or a method herein that affects or interacts with its domain. Chemically defined compounds (such as organic molecules, inorganic molecules, organic / inorganic molecules, proteins, peptides, nucleic acids, oligonucleotides, lipids, polysaccharides, saccharides, or glycoproteins) Refers to a hybrid between molecules, etc.) or a mixture of compounds (eg, a library of test compounds, natural extracts, or culture supernatants, etc.).

  As used herein, therapeutic agents, treatment regimens, radioprotectants, and chemotherapeutic agents mean conventional drugs and drug therapies, including vaccines, that are well known to those skilled in the art. Radiotherapeutic agents are well known in the art.

  As used herein, a vector (or plasmid) refers to an individual element used to introduce heterologous DNA into a cell for its expression or replication. Vectors typically remain episomes, but can be designed to incorporate a gene or a portion thereof into the genomic chromosome. Also contemplated are vectors that are artificial chromosomes such as yeast artificial chromosomes and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art. Expression vectors include vectors capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of effecting expression of such DNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA construct such as a plasmid, phage, recombinant virus or other vector that results in expression of the cloned DNA upon introduction into a suitable host cell. Suitable expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic and / or prokaryotic cells and those that remain episomes or that integrate into the genome of the host cell.

  As used herein, chemically stable means that the compound is sufficiently stable to be formulated for pharmaceutical use. Such chemical stability is well known to those skilled in the art and can be measured by well known routine methods. Whether a given compound is sufficiently stable to be formulated for pharmaceutical use includes the type of formulation and the desired route of administration, the disease to be treated, and the method of preparation of the pharmaceutical formulation, Depends on a number of factors, including but not limited to:

[式中、Ｘが０もしくは１である]のように配座的に制約されたアルギニン側鎖(例えば、Webb et al. (1991) J. Org. Chem. 56:3009、を参照)、または

[式中、ｄが０〜５、もしくは１〜３の整数であり；そしてＷがＮもしくはＣＨであるかまたは上記グループの１もしくは２置換Ｎ−アルキル誘導体[そこではアルキルが、特定の実施態様では、メチルのような低級アルキルである]である]ように配座的に制約されたアルギニン側鎖が挙げられるがそれらに限定されない。 As used herein, a “functional equivalent” of an amino acid side chain functions in substantially the same manner as a naturally occurring side chain to achieve substantially the same result. A group or moiety (eg, a substrate for a cell surface protease). For example, functional equivalents of the side chain of arginine include homoarginine, guanidinoaminopropyl, guanidinoaminoethyl, (Me) ₂ -arginine side chain, (Et) ₂ -arginine side chain, (4-aminomethyl) phenyl Methyl, 4-amidinophenylmethyl, 4-guanidinophenylmethyl, or

An arginine side chain that is conformationally constrained such that X is 0 or 1 (see, eg, Webb et al. (1991) J. Org. Chem. 56: 3009), or

Wherein d is an integer from 0 to 5, or from 1 to 3; and W is N or CH or a 1 or 2 substituted N-alkyl derivative of the above group wherein alkyl is a specific embodiment Is a lower alkyl such as methyl]], but is not limited to arginine side chains that are conformationally constrained.

  As used herein, a pharmaceutically acceptable derivative of a compound includes a salt, ester, enol ether, enol ester, acid, base, solvate, hydrate or prodrug thereof. . Such derivatives can be readily made by those skilled in the art using known methods for such derivatization. The prepared compounds can be administered to animals or humans without substantial toxic effects and are either pharmaceutically active or prodrugs. Pharmaceutically acceptable salts include N, N′-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethylethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine. Amine salts, such as, but not limited to, 1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine and tris (hydroxymethyl) aminomethane; Alkali metal salts such as but not limited to lithium, potassium and sodium; alkaline earth metal salts such as but not limited to barium, calcium and magnesium; Transition metal salts; and other metal salts such as but not limited to sodium hydrogen phosphate and disodium phosphate, including but not limited to; and hydrochloride and sulfuric acid Salts of mineral acids, such as but not limited to: salts of acetate, lactate, malate, tartrate, citrate, ascorbate, oxalate, butyrate, valerate and Examples include, but are not limited to, salts of organic acids, such as but not limited to fumarate. Pharmaceutically acceptable esters include, but are not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids, and boronic acids, alkyl groups, alkenyls, alkynyls, aryls, heteroaryls of acidic groups , Aralkyl, heteroaralkyl, cycloalkyl, and heterocyclic esters. Pharmaceutically acceptable enol esters include those of the formula C═C (OR), where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclic. The derivative | guide_body of this is mentioned, However, It is not limited to them. Pharmaceutically acceptable enol esters include those of the formula C = C (OC (O) R), wherein R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or complex. Cyclic)] derivatives, but is not limited thereto. Pharmaceutically acceptable solvates and hydrates include one or more solvents or water molecules, generally 1 to about 100, typically 1 to about 2, 3 or 4. Or a complex of a compound with 1 to about 10 solvents or water molecules.

  As used herein, treatment means any manner in which one or more symptoms of an abnormality, disorder or disease are alleviated or otherwise altered to be beneficial. . Treatment also encompasses any pharmaceutical use of the compositions herein, such as use for the treatment of cancer.

  As used herein, can the reduction of symptoms of a particular disorder by administration of a particular pharmaceutical composition be attributed to administration of the composition, whether permanent or temporary, persistent or transient? Or any mitigation that can be related.

  As used herein, a prodrug is metabolized or otherwise converted to a biologically, pharmaceutically, or therapeutically active form of a compound upon in vivo administration. A compound. To produce a prodrug, the pharmaceutically active compound is modified such that the active compound is regenerated by metabolic processes. Prodrugs are designed to alter the metabolic stability or transport properties of the drug, mask side effects or toxicity, improve the flavor of the drug, or alter other characteristics or properties of the drug Can be done. Thanks to knowledge of in vivo pharmacokinetic processes and drug metabolism, one of ordinary skill in the art can design prodrugs of compounds once pharmaceutically active compounds are known (eg, Nogrady (1985), (See Medicinal Chemistry Biochemical Approach, Oxford University Press, New York, pages 388-392).

  It should be understood that the conjugates provided herein can contain chiral centers. Such chiral centers can have either the (R) or (S) configuration, or can be a mixture thereof. Thus, the compounds provided herein can be enantiomerically pure or stereoisomeric or diastereomeric mixtures. In the case of amino acid residues, such residues can have either the L or D form. The configuration of naturally occurring amino acid residues is generally L. When not specified, the residue is L-shaped. It should be understood that the chiral centers of the compounds provided herein can undergo epimerization in vivo. As such, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent to administration of the compound in its (S) form for a compound that undergoes epimerization in vivo. .

  The conjugates provided herein are prodrugs because they contain an inactive therapeutic agent that is ultimately converted to the active form in the targeted cell or tissue or in its environment. is there. Upon exposure to the targeted protease, it can be further metabolized into a biologically, pharmaceutically or therapeutically active form of the compound, or a biologically, pharmaceutically or therapeutically active form of the compound. Either of the derivatives is released.

As used herein, substantially pure is thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC) and mass used by those skilled in the art to assess such purity. Is homogeneous enough to appear free of detectable impurities as measured by standard analytical methods such as analytical methods, or further purification is likely to be the enzymatic and biological activity of the substance Means that it is sufficiently pure that it does not appear to change its physical and chemical properties for its intended purpose. Methods of purifying compounds to produce substantially chemically pure compounds are well known to those skilled in the art. However, a substantially chemically pure compound can be a mixture of stereoisomers. In such cases, further purification will increase the specific activity of the compound.
As used herein, peptidic substrates include peptides, including molecules such as peptides and peptidomimetics and peptide binding surrogates.

  As used herein, reference to a specific subsite of a protease substrate: Pn ... P3-P2-P1 ↓ P1'-P2'-P3 '... Pn' is conventional terminology. (Schecter et al. (1967) Biochem. Biophys. Res. Commun. 27: 157-162) is used. Substrate cleaved bonds (ie, cleavage sites) are indicated by arrows. The site at the N-terminal side of the bond is referred to as the position without the prime symbol. Subsites are then assigned numerical values based on their distance from the cleavable bond. The amino acid (or amino acid surrogate) that forms the cleavable bond is assigned number 1, the adjacent residue is number 2, and so on, counted from the cleavable bond. Therefore, each particular subsite of the substrate is uniquely identified by a number and a symbol with or without a prime.

のようなシクロプロピル同配体が挙げられるが、それらに限定されない。 As used herein, a peptide bond surrogate is a divalent group possessing steric and / or electronic properties similar to -C (O) NH-. Peptide bond surrogates include alkene isosteres (—CR═CR—), particularly (E) -alkene isosteres of the formula —CH═CH—, hydroxyethylene isosteres (—CH (OH) CH ₂ -), enamine isostere (-C (= CRR) NH-) , amino alcohols isostere _{(-CH (OH) CH 2 NH-} ), difluoroketone isostere _{(-C (O) CF 2 -} ) A reverse compound (—NHC (O) —), a divalent heterocyclic or heteroaryl group, and

Cyclopropyl isosteres such as, but not limited to.

  As used herein, alkyl, alkenyl, and alkynyl carbon chains, unless specified, contain 1-20, generally 1-16 carbons, and are straight or branched. . An alkenyl carbon chain of 2 to 20 carbons typically contains 1 to 8 double bonds, and an alkenyl carbon chain of 2 to 16 carbons typically has 1 to 5 carbon atoms. Contains double bonds. An alkynyl carbon chain of 2-20 carbons typically contains 1-8 triple bonds, and an alkynyl carbon chain of 2-16 carbons typically contains 1-5 triple bonds. contains. Exemplary alkyl, alkenyl, and alkynyl groups herein include methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl, and isohexyl. However, it is not limited to them. The alkyl, alkenyl and alkynyl groups can be optionally substituted with one or more groups, generally the same or different alkyl group substituents, unless otherwise specified. As used herein, lower alkyl, lower alkenyl, and lower alkynyl refer to carbon chains having less than about 6 carbons. As used herein, “al (ke) (quin) nyl” refers to an alkyl group containing at least one double bond and at least one triple bond.

  As used herein, “cycloalkyl” is typically a saturated monocyclic or polycyclic of 3 to 10 carbon atoms, such as, for example, 3 to 6 carbon atoms. Cycloalkenyl and cycloalkynyl refer to monocyclic or polycyclic ring systems containing at least one double bond and at least one triple bond, respectively. Cycloalkenyl and cycloalkynyl groups contain, for example, 3 to 10 carbon atoms, cycloalkenyl groups typically contain 4 to 7 carbon atoms, and cycloalkynyl groups contain, for example, 8 to 10 carbon atoms. Containing carbon atoms. The ring system of cycloalkyl, cycloalkenyl and cycloalkynyl groups can consist of one ring or two or more rings which can be joined in a fused, bridged or spiro-linked manner, and optionally one or It can be substituted with further alkyl group substituents. “Cycloal (ke) (x) nyl” refers to a cycloalkyl group containing at least one double bond and at least one triple bond.

  As used herein, “substituted alkyl”, “substituted alkenyl”, “substituted alkynyl”, “substituted cycloalkyl”, “substituted cycloalkenyl”, and “substituted cycloalkynyl” are alkyl, halo, Haloalkyl such as halo lower alkyl, pseudohalo, aryl, amino, dialkylamino, nitro, cyano, azide, alkylsulfinyl, alkylsulfonyl, alkylcarbonylamino, alkoxycarbonylamino, aminoimino, hydroxy, alkoxy, aryloxy, alkyloxy, One or more of each independently selected from alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkylcarbonyl, alkoxycarbonyl, oxo and cycloalkyl, It refers groups, alkyl substituted with 1-3 substituents in certain embodiments, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl groups.

  As used herein, “aryl” refers to a cyclic group containing from 6 to 19 carbon atoms. Aryl groups include, but are not limited to groups such as fluorenyl, substituted fluorenyl, phenyl, substituted phenyl, naphthyl and substituted naphthyl. As used herein, “aryl” also refers to aryl-containing groups, including but not limited to aryloxy, arylthio, arylcarbonyl and arylamino groups.

As used herein, “heteroaryl” refers to one or more heteroatoms, ie, elements other than carbon, such as 1 to 3 atoms in the ring system, For example, it refers to a generally about 5 to about 15 membered monocyclic or polycyclic aromatic ring system which is a nitrogen, oxygen and sulfur atom. A heteroaryl group can optionally be fused to a benzene ring. Exemplary heteroaryl groups include, for example, furyl, imidazolyl, pyrrolidinyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl and ioquinolinyl, examples of which include pyridinyl, thienyl and quinolinyl.
As used herein, “heteroaryl” also refers to heteroaryl-containing groups including, but not limited to, heteroaryloxy, heteroarylthio, heteroarylcarbonyl, and heteroarylamino.

  As used herein, “heterocyclic” means one or more heteroatoms, ie elements other than carbon atoms, such as 1 to 3 atoms in a cyclic system. Monocyclic or polycyclic non-aromatic ring systems, such as 3 to 10 membered, eg 4 to 7 or 5 to 6 membered systems, for example nitrogen, oxygen and / or sulfur atoms. Point to.

  As used herein, “substituted aryl”, “substituted heteroaryl” and “substituted heterocyclic” are halo, haloalkyl and alkyl, aralkyl, heteroaralkyl, 1 to 2 doubles, respectively. Alkenyl with bond, alkynyl with 1-2 triple bonds, alk (ke) (quin) nyl group, halo, pseudohalo, cyano, hydroxyl, halo lower alkyl, especially haloalkyl and polyhaloalkyl such as trifluoromethyl , Formyl, alkylcarbonyl, halo, haloalkyl and alkyl, heteroarylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminoimino, alkoxycoubonylamino, aryloxycarbonylamino, aminocarbonyl, alkylaminocarbonyl, dial Killaminocarbonyl, arylaminocarbonyl, diarylaminocarbonyl, aralkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, amino , Alkylamino, dialkylamino, arylamino, alkylarylamino, alkylcarbonylamino, arylcarbonylamino, azide, nitro, mercapto, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, aryl Sulfonyl, aminosulfonyl, alkylaminosulfur 1-3 selected from arylcarbonyl optionally substituted with one or more substituents, such as 1-3 selected from nyl, dialkylaminosulfonyl and arylaminosulfonyl One or more substituents independently selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, optionally substituted with one or more substituents, in certain embodiments 1 Refers to aryl, heteroaryl and heterocyclic groups substituted with ~ 3 substituents.

As used herein, “aralkyl” refers to an alkyl group in which one of the alkyl hydrogen atoms has been replaced with an aryl group.
As used herein, “heteroaralkyl” refers to an alkyl group in which one of the alkyl hydrogen atoms is replaced with a heteroaryl group.

  As used herein, nomenclature such as alkyl, alkoxy, carbonyl, etc. is used as commonly understood by one of ordinary skill in the art. For example, as used herein, alkyl refers to a saturated carbon chain containing one or more carbons; the chain can be straight or branched or contain a cyclic moiety or It can be annular.

Where the number of any given substituent is not specified (eg, haloalkyl), one or more substituents may be present. For example, “haloalkyl” can include one or more of the same or different halogens. As another example, “C _1-3 alkoxyphenyl” may include the same or different alkoxy groups containing 1, 2 or 3 carbons.
As used herein, “halo”, “halogen” or “halide” refers to F, Cl, Br or I.

As used herein, a pseudohalide is a compound that behaves substantially similar to a halide. Such compounds can be used and treated in the same manner as halides (X ⁻ , where X is a halogen such as Cl or Br). Pseudohalides include, but are not limited to, cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethoxy, difluoromethoxy, dichloromethoxy and azide.

As used herein, “haloalkyl” refers to a lower alkyl group in which one or more of the hydrogen atoms has been replaced with a halogen. Such groups include, but are not limited to, chloromethyl, trifluoromethyl, 1-chloro-2-fluoroethyl, and the like.
As used herein, “haloalkoxy” refers to RO— in which R is a haloalkyl group.

As used herein, “sulfinyl” or “thionyl” refers to —S (O) —. As used herein, “sulfo” refers to —S (O) ₂ O—. As used herein, “sulfonyl” or “sulfuryl” refers to —S (O) ₂ —.
As used herein, “carboxy” refers to the divalent group C (O) O—.

As used herein, “aminocarbonyl” refers to —C (O) NH ₂ .
As used herein, “alkylaminocarbonyl” refers to —C (O) NHR in which R is hydrogen or an alkyl such as, for example, lower alkyl.

As used herein, “dialkylaminocarbonyl” refers to —C (O) NR in which R ′ and R are independently selected from hydrogen or alkyl such as, for example, lower alkyl. 'R refers to; "carboxamide" refers to a group of formula -NR'COR.
As used herein, “diarylaminocarbonyl” refers to —C (O) NRR in which R and R ′ are independently selected from aryl such as lower aryl, eg, phenyl. Point to '.
As used herein, “aralkylaminocarbonyl” is one in which one of R and R ′ is an aryl such as lower aryl, eg, phenyl, and other R and R ′ One refers to -C (O) NRR ', for example alkyl such as lower alkyl.

As used herein, “arylaminocarbonyl” refers to —C (O) NHR in which R is aryl such as lower aryl, eg, phenyl.
As used herein, “hydroxycarbonyl” refers to —COOH.
As used herein, “alkoxycarbonyl” refers to —C (O) OR in which R is alkyl, such as lower alkyl.

As used herein, “aryloxycarbonyl” refers to —C (O) OR in which R is aryl such as lower aryl, eg, phenyl.
As used herein, “alkoxy” and “alkylthio” refer to RO— and RS—, in which R is alkyl, eg, lower alkyl.
As used herein, “aryloxy” and “arylthio” refer to RO— and RS— in which R is lower aryl, eg, aryl such as phenyl.

As used herein, “alkylene” refers to a linear, branched or cyclic, such as, for example, 1 to about 20 carbon atoms, such as 1 to 12 carbons. Refers to a divalent aliphatic hydrocarbon group such as linear or branched and is, for example, lower alkylene. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms can be optionally inserted along the alkylene group, where the nitrogen substituent is alkyl as described above. Exemplary alkylene groups include methylene (—CH ₂ —), ethylene (—CH ₂ CH ₂ —), propylene (— (CH ₂ ) ₃ —), cyclohexylene (C ₆ H ₁₀ —), methylenedioxy ( _-O-CH 2 -O-) and ethylenedioxy _{(-O- (CH 2) 2 -O-} ) can be mentioned. The term “lower alkylene” refers to alkylene groups having 1 to 6 carbons. Exemplary alkylene groups are lower alkylene, such as alkylene of 1 to 3 carbon atoms.

As used herein, “alkenylene” refers to a straight chain having, for example, 2 to about 20 carbon atoms and at least one double bond, typically 1 to 12 carbons. A linear, branched or cyclic, typically linear or branched, divalent aliphatic hydrocarbon group and, for example, a lower alkenylene. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms can be optionally inserted along the alkenylene group, where the nitrogen substituent is alkyl as described above. Exemplary alkenylene groups include —CH═CH—CH═CH— and —CH═CH—CH ₂ —. The term “lower alkenylene” refers to alkenylene groups having 2 to 6 carbons. Exemplary alkenylene groups are lower alkenylene such as, for example, alkenylene of 3 to 4 carbon atoms.

As used herein, “alkynylene” refers to 2 to about 20 carbon atoms and at least one triple bond, generally 1 to 12 carbons such as, for example, lower alkynylene. It is a linear, branched or cyclic, typically linear or branched, divalent aliphatic hydrocarbon group such as those having. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms can be optionally inserted along the alkynylene group, where the nitrogen substituent is alkyl as described above. Exemplary alkynylene groups include —C≡C—C≡C—, —C≡C—, and —C≡C—CH ₂ —. The term “lower alkynylene” refers to alkynylene groups having 2 to 6 carbons. Exemplary alkynylene groups are, for example, lower alkynylenes such as alkynylenes of 3 to 4 carbon atoms.

As used herein, “al (ke) (ki) nylene” means, for example, having 2 to about 20 carbon atoms and at least one triple bond, and at least one double Bond; typically linear, branched or cyclic, generally linear, having 1 to 12 carbons such as, for example, lower al (ke) (ki) nylene It refers to a cyclic divalent aliphatic hydrocarbon group. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms can be optionally inserted along the alkynylene group, where the nitrogen substituent is alkyl as described above. Exemplary al (ke) (ki) nylene includes —C═C— (CH ₂ ) _n —C≡C—, where n is 1 or 2. The term “lower al (ke) (ki) nylene” refers to an al (ke) (x) nylene group having up to 6 carbons. Exemplary al (ke) (x) nylene groups are, for example, lower alk (ke) (x) nylene such as al (ke) (x) nylene of 4 carbon atoms.

  As used herein, “cycloalkylene” refers to a divalent saturated monocyclic or polycyclic system of generally 3 to 10 carbon atoms, such as 3 to 6 carbon atoms. Cycloalkenylene and cycloalkynylene refer to monocyclic or polycyclic systems containing at least one double bond and at least one triple bond, respectively. Cycloalkenylene and cycloalkynylene groups can contain 3 to 10 carbon atoms, for example, cycloalkenylene groups contain 4 to 7 carbon atoms, and cycloalkynylene groups contain 8 to 10 carbon atoms. Containing carbon atoms. Cycloalkylene, cycloalkenylene and cycloalkynylene ring systems can consist of one ring or two or more rings that can be joined together in a fused, bridged or spiro-linked manner. “Cycloal (ke) (x) nylene” refers to a cycloalkylene group containing at least one double bond and at least one triple bond.

  As used herein, “substituted alkylene”, “substituted alkenylene”, “substituted alkynylene”, “substituted cycloalkylene”, “substituted cycloalkenylene” and “substituted cycloalkynylene” are halo, For example, one or more independently selected from haloalkyl such as halo lower alkyl, aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxyalkoxycarbonyl, oxo and cycloalkyl Refers to alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, and cycloalkynylene groups substituted with the above substituents, and in certain embodiments 1 to 3 substituents.

  As used herein, “arylene” refers to a monocyclic having at least one aromatic ring such as, for example, 5 to about 20 carbon atoms and 5 to 12 carbons. A monocyclic or polycyclic divalent aromatic group such as, for example, lower arylene. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms can optionally be inserted around the arylene group, where the nitrogen substituent is alkyl as described above. Exemplary arylene groups include 1,2-, 1,3- and 1,4-phenylene. The term “lower arylene” refers to an arylene group having 5 or 6 carbons. An exemplary arylene group is a lower arylene.

  As used herein, “heteroarylene” refers to one or more, typically, for example, 1 to 3 atoms of a cyclic system having a heteroatom, ie an element other than carbon. Refers to a divalent monocyclic or polycyclic aromatic ring system, such as from about 2 to about 15 members, which is, for example, a nitrogen, oxygen and / or sulfur atom (s).

  As used herein, “heterocyclylene” refers to one or more, for example, 1 to 3 atoms in a cyclic system having a heteroatom, ie, an element other than carbon, such as nitrogen. An oxygen and / or sulfur atom (s), for example 4 to 7 or 5 to 6 members, typically 3 to 10 members, divalent monocyclic or polycyclic Refers to the aromatic ring system of formula

  As used herein, “substituted arylene”, “substituted heteroarylene” and “substituted heterocyclylene” are halo, haloalkyl and alkyl, aralkyl, heteroaralkyl, 1 to 2 doubles, respectively. Alkenyl having a bond, alkynyl having 1 to 2 triple bonds, alk (ke) (quin) nyl group, halo, pseudohalo, cyano, hydroxy, halo lower alkyl, eg haloalkyl such as trifluoromethyl and poly Haloalkyl, formyl, alkylcarbonyl, eg, halo, haloalkyl and alkyl, heteroarylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl , Diarylaminocarbonyl, aralkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, amino, alkylamino, dialkylamino, Arylamino, alkylarylamino, alkylcarbonylamino, arylcarbonylamino, azide, nitro, mercapto, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylamino Sulfonyl, dialkylaminosulfonyl and aryl 1 or more substituents such as 1 to 3 selected from arylcarbonyl optionally substituted with one or more substituents such as 1 to 3 selected from aminosulfonyl Substituted with one or more substituents independently selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, optionally substituted with 1 to 3 substituents in certain embodiments. The term arylene, heteroarylene and heterocyclylene groups are pointed out.

As used herein, “alkylidene” refers to a divalent group such as ═CR′R ”that is attached to one atom of another group to form a double bond. Exemplary alkylidene groups are methylidene (═CH ₂ ) and ethylidene (═CHCH ₃ ) As used herein, “aralkylidene” refers to either R ′ or R ″. Refers to an alkylidene group in which is an aryl group, a “cycloalkylidene” group is the combination of R ′ and R ″ to form a carbocyclic ring. At least one of 'and R''contains a heteroatom in the chain, and R' and R '' are joined to form a heterocycle.

  As used herein, “amide” refers to the divalent group —C (O) NH—. “Thioamido” refers to the divalent group —C (S) NH—. “Oxyamido” refers to the divalent group —OC (O) NH—. “Thiaamido” refers to the divalent group —SC (O) NH—. “Dithiaamido” refers to the divalent group —SC (S) NH—. “Ureido” refers to the divalent group —HNC (O) NH—. “Thioureido” refers to the divalent group —HNC (S) NH—.

As used herein, “semicarbazide” refers to —NHC (O) NHNH—. “Carbazate” refers to the divalent group —OC (O) NHNH—. “Isothiocarbazate” refers to the divalent group —SC (O) NHNH—. “Thiocarbazate” refers to the divalent group —OC (S) NHNH—. “Sulfonylhydrazide” refers to the group —SO ₂ NHNH—. “Hydrazide” refers to the divalent group —C (O) NHNH—. “Azo” refers to the divalent group —N═N—. “Hydrazinyl” refers to the divalent group —NH—NH—.

  As used herein, “amino acid” refers to an α-amino acid in either racemic or D- or L-configuration. The name “d” (eg, dAla, dSer, dVal, etc.) preceding the amino acid name refers to the D-isomer of the amino acid. The name “dl” (eg, dlPip) preceding the amino acid name refers to a mixture of L- and D-isomers of amino acids.

  As used herein, when any particular group is specified, such as phenyl or pyridyl, this means that the group is unsubstituted or substituted. Exemplary substituents not specified are halo, halo lower alkyl, and lower alkyl.

  As used herein, the abbreviations for any protecting groups, amino acids and other compounds relate to their common usage, accepted abbreviations, or biochemical nomenclature unless otherwise indicated. Conforms to the IUPAC-IUB Committee (see (1972) Biochem. 11: 942-944).

  As used herein, HHT and CHT refer to hexahydrotyrosyl (also known as cyclohexatyrosyl or p-hydroxycyclohexylalanyl) and CHA is cyclohexylalanyl. , Pyr and pyroGlu refer to pyroglutamic acid, Pip is pipecolic acid, Sar is sarcosine, nLeu and Nle are norleucine, nVal is norvaline, Aib is 2-aminoisobutyric acid, Quat is (R ) -Glu (α- (3-amidinobenzyl)) and Abu and But are 2-aminobutyric acid.

を表し、そして用語ＰＥＧ(６)は：

を表す。 As used herein, PEG represents a polyethylene glycol containing substituents having a specified number of ethyleneoxy subunits. Thus, the term PEG (2) is:

And the term PEG (6) is:

Represents.

が挙げられるが、それらに限定されない。 When R ¹ and R ² are combined to form — (CH ₂ ) _n —, the cyclic moiety so defined and the cyclic moiety containing a heteroatom include:

But are not limited to these.

  As used herein, the term “hydroxylated” refers to a substitution on a substitutable carbon of a cyclic system so described by a hydroxy moiety. As used herein, the term “polyhydroxylated” refers to two or more substitutable carbons of a cyclic system so described by 2, 3 or 4 hydroxy moieties. Represents the above substitution.

As used herein, the term “(d) (2,3-dihydroxypropionyl)” represents the following structure:

As used herein, the term “(2R, 3S) -2,3,4-trihydroxybutanoyl” represents the following structure:

もしくはそのジアステレオマーを表す。 As used herein, the term “quinyl” has the structure:

Or the diastereomer is represented.

もしくはそのジアステレオマーを表す。 As used herein, the term “Glonyl” has the structure:

Or the diastereomer is represented.

もしくはそのジアステレオマーを表す。 As used herein, the term “cotininyl” has the structure:

Or the diastereomer is represented.

As used herein, the term “galyl” represents the following structure:

As used herein, the term “4-ethoxyscuaryl” represents the following structure:

As used herein, 1-methylHis or (1Me) H refers to the following structure:

As used herein, 3-methylHis or (3Me) H refers to the following structure:

を指す。 As used herein, Quat ² is

Point to.

を指す。 Quat ³

Point to.

を指す。 Quat ⁴

Point to.

を指す。

Quat ⁵

Point to.

B. Protease Targets The conjugates herein are designed to target proteases that are localized on the surface of cells, particularly tumor cells and cells involved in tumorigenic processes and angiogenesis and other growth processes. . The conjugates described in detail below are conjugated either directly or via a linker to a therapeutic agent, particularly a cytotoxic agent, which is substantially inert when in the conjugate. Contains peptidic substrates for selected and targeted cell surface proteases. The therapeutic agent is released in a form that is active or can be activated in the vicinity of the targeted cell or tissue to which it is delivered.

  Targeted proteases are localized on cells or tissues that are involved in (or related to) the disease process or happen to be present in the field of cells or tissues involved in the disease or disease process, and generally all Not at all or not present on many other cells or tissues, or active at low levels, generally substantially at low levels, or altered activity or specificity Is selected by identifying the protease that exhibits The type and number of non-targeted cells or tissues that express an active protease will vary for the particular protease and disease intended for treatment. One skilled in the art will select a target based on the tolerable or acceptable level of disease, targeted drug and side effects. The goal is to achieve an increased therapeutic index compared to administration of the targeted drug itself.

  The targeted protease may or may not be involved in the disease process and its expression may be accidental; for the purposes herein, its specific role or lack thereof is not important; The fact that it is active at the site of the designated tissue or cell is important. For example, many of the cell surface proteases of interest herein are expressed or active on tumor cells or cells involved in oncogenic processes. Any method known to those of skill in the art for measuring or detecting the expression profile of a tissue or cell can be used. For example, RNA blots consisting of RNA from various tissues (e.g., multiple tissue expression (MTE) arrays available from CLONTECH, Palo Alto, CA) can be screened with probes based on the nucleic acid sequence of the protease of interest. Cells that express the protease can be identified. Also, Northern analysis of blots can be used to test expression.

  Included in the targeted proteases are those referred to as type II membrane-bound serine proteases (MTSP; eg, US Application Serial No. 09 / 776,191, filed February 2, 2001, and International PCT See International PCT Application No. PCT / US01 / 03471, published as application WO 01/57194; see International PCT Application No. PCT / US02 / 07903; and also US Provisional Application Serial No. 60/275, 592, No. 60 / 278,166, No. 60 / 279,228, No. 60 / 291,001, No. 60 / 291,501, No. 60 / 316,818, No. 60 / 302,939 60 / 316,818, 60 / 328,529, 60 / 328,530, 60 / 332,015, 60 / 328,939, and Provisional application filed on May 20, 2002 with the registration number 24745-P1624; US application serial number 10/099, filed on May 14, 2002 with the registration number 24745-1616 , 700, 10 / 104,271, 10 / 112,221), and those found on endothelial cells and referred to as endotheliases (US application filed November 20, 2000) Serial number 09 / 717,473 and international PCT application No. PCT / US00 / 31803 published as international PCT application WO 01/36604); SEQ ID NOs: 3-26, 269-270 and See also 272-276.

  Also contemplated are proteases that localize on the cell surface thanks to specific interactions with cell surface proteins. Urokinase plasminogen activator (u-PA) bound to urokinase plasminogen activator receptor (u-PAR) is an example of such a protease. Exemplary MTSP and endotheliase nucleic acid sequence information and expression profiles are as follows (see also Example 6).

1. MTSP
Cell surface proteolysis is a mechanism for the production of bioactive proteins that mediate various cellular functions. Proteins immobilized on these membranes include disintegrin-like and metalloproteinases (ADAM) and membrane matrix metalloproteinases (MT-MMP). In addition to MMPs, serine proteases have been implicated in the progression of neoplastic diseases. Most serine enzymes, which are secreted enzymes or sequestered in cytoplasmic storage organelles, have a role in blood clotting, wound healing, digestion, immune response and tumor invasion and metastasis.

  Transmembrane serine protease (MTSP) appears to be involved in tumor pathogenesis and pathogenesis. These enzymes are expressed in certain cancerous and tumor cells, and in other cells involved in other disease states such as other proliferative disorders and inflammatory cells, and tissues Or it may be organ specific. In mammals, more than 21 members of the family are known (see Hooper et al. (2001) J. Biol. Chem. 276: 857-860) and a series of US applications filed February 2, 2001. No. 09 / 776,191 and International PCT Application No. PCT / US01 / 03471; see also US Provisional Application Serial Nos. 60 / 275,592 and 60 / 278,166; and sequences Nos. 1-37).

  These include choline (accession numbers AF133845 and AB013844; Yan et al. (1999) J. Biol. Chem. 274: 14926-14938; Tomia et al. (1998) J. Biochem. 124: 784- 789; Uan et al. (2000) Proc. Natl. Acad. Sci. USA 97: 8525-8529); enteropeptidase (also called enterokinase; accession number U09860 for human protein; Kitamoto et al. (1995) Biochem. 27: 4562-4568; Yahagi et al. (1996) Biochem. Biophys. Res. Commun. 219: 806-812; Kitamoto et al. (1994) Proc. Natl. Acad. Sci. USA 91: 7588-7592; see Matsushima et al. (1994) J. Biol. Chem. 269: 19976-19982); human airway trypsin-like protease (HAT; accession number AB002134; Yamaoka et al. J. Biol. Chem). 273: 11894-11901); MTSP1 (also referred to as TADG-15 and matriptase, see SEQ ID NOs: 1 and 2; accession no. A 1333086 / AF118224, AF04280022; Takeuchi et al. (1999) Proc. Natl. Acad. Sci. USA 96: 11054-1161; Lin et al. (1999) J. Biol. Chem. 274: 18231-18236; Takeuchi et al. (2000) J. Biol. Chem. 275: 26333-26342; and Kim et al. (1999) Immunogenetics 49: 420-429); hepsin (accession numbers M18930, AF030065, X70900). Leytus et al. (1988) Biochem. 27: 11895-11901; Vu et al. (1997) J. Biol. Chem. 272: 31315-31320; and Farley et al. (1993) Biochem. Biophys. Acta 1173: 350-352; and U.S. Pat. No. 5,972,616); TMPRS2 (accession numbers U75329 and AF113596; Paoloni-Giacobino et al. (1997) Genomics 44: 309-320; And Jacquinet et al. (2000) FEBS Lett. 468: 93-100); and TMPRSS4 (accession number NM 016425; Wallrapp et al. (2000) Cancer 60: 2602-2606).

  Also, MTSP3, MTSP4, MTSP6, MTSP7, MTSP9, MTSP10, MTSP12, MTSP20, MTSP22 and MTSP25 (see SEQ ID NOs: 3-26, 269-270 and 272-276; also US application filed February 2, 2001 See Serial Number 09 / 776,191 and International PCT Application No. PCT / US01 / 03471, published as International PCT Application No. WO 01/57194; see International PCT Application No. PCT / US02 / 07903. And US provisional application serial numbers 60 / 275,592, 60 / 278,166, 60 / 279,228, 60 / 291,001, 60 / 291,501, 60 / 316,818, 60 / 302,939, 60 / 316,818, 60 / Provisional application filed on May 20, 2002 under 328,529, 60 / 328,530, 60 / 332,015, 60 / 328,939 and agent registration number 24745-P1624; (See U.S. Application Serial Nos. 10 / 099,700, 10 / 104,271, and 10 / 112,221, filed May 14, 200, with agent registration number 24745-1616) ,It has been known.

  Serine proteases, including transmembrane serine proteases, have been thought to be involved in processes involved in neoplastic development and progression. Although the exact role of these proteases has not been clarified, serine proteases and their inhibitors are many intracellular and extracellular, including tumor angiogenesis, which are involved in cancer cell invasion, metastatic spread, and tumor progression Involved in the physiological process. It is believed that proteases are involved in extracellular matrix (ECM) degradation, contribute to tissue remodeling and are required for cancer invasion and metastasis. The activity and / or expression of certain proteases has been shown to correlate with tumor progression and development, and has also been shown to be active on specific cell types.

  For example, it is expressed in epithelial cancer and normal tissues (Takeucuhi et al. (1999) Proc. Natl. Acad. Sci. USA 96: 11054-61). See SEQ ID NOS: 1 and 2 from 5,972,616; and GenBank accession number AF118224; (1999) J. Biol. Chem. 274: 18231-18236; US Pat. No. 5,792,616. Also see Takeuchi (1999) Proc. Natl. Acad. Sci. USA 96: 11054-1161). It has been proposed to play a role in breast cancer metastasis. Its main cleavage specificity is the Arg-Lys residue. Matriptase is also expressed at high levels of activity and / or expression in various epithelial tissues in the human gastrointestinal tract and prostate.

  Hepsin, a cell surface serine protease identified in hepatocellular carcinoma, is overexpressed in ovarian cancer (Tanimoto et al. (1997) Cancer Res., 57: 2884-7). Hepsin transcripts appear to be present in large amounts in carcinoma tissues and are almost never expressed in normal adult tissues, including normal ovaries. It has been suggested that hepsin is often overexpressed in ovarian tumors and therefore may be a candidate protease in the invasion process and proliferative capacity of ovarian tumor cells.

  A serine protease-like gene (Liu et al. (1996) Cancer Res. 56: 3371-9) called normal epithelial cell specific 1 (NES1) has been identified. Although NES1 mRNA expression is observed in all normal and immortalized non-neoplastic epithelial cell lines, most human breast cancer cell lines show a dramatic decrease or complete lack of their expression. The structural similarity of NES1 to polypeptides known to modulate growth factor activity and the negative correlation of NES1 expression with breast cancer development may be direct or indirect of this protease-like gene product in suppressing tumor development. Suggest a role.

Exemplary MTSP
Each MTSP has a characteristic tissue expression profile; in particular, MTSP is not expressed or activated exclusively in tumors, but exhibits an expression or activation profile in characteristic tumor tissues . In some cases, MTSP may have a different activity in tumor cells than non-tumor tissue due to changes in the substrate or cofactors for it or other factors that would change the functional activity of MTSP. it can. Thus, thanks to the level or function of activity and / or expression in a subject (eg, a mammal, particularly a human) with a neoplastic disease, as compared to the subject or subjects who do not have a neoplastic disease. Each can serve as a diagnostic marker for a particular tumor. In addition, detection of activity (and / or expression) in specific tissues can be indicative of a neoplastic disease. Also, thanks to the respective activity and / or expression profiles, they may serve as therapeutic targets by administration of a modulator of that activity or by administration of a prodrug that is specifically activated by one of the MTSPs. it can. Each or any MTSP can exhibit an activity or expression level or substrate specificity that differs from that in normal cells in tumor cells. Such tumor cells include, but are not limited to, colon, lung, prostate, breast, esophagus, pancreas, uterus, endometrium and other solid tumors and blood and lymph tumors. Thus, the conjugates provided herein can be designed by selection of substrate specificity for any treatment of such tumors and neoplastic abnormalities.

Tissue expression profile The following is an exemplary tissue and gene (see also Example 8) profile of an exemplary MTSP. These profiles are not intended to define the full range of expression or activation of these MTSPs, but MTSP is expressed in tumors and therefore their expression or activation or substrate on the surface of tumor cells Specificity can be utilized in the methods herein and is designed according to the methods herein and as exemplified herein, and is cleaved by one or more of these MTSPs. Have been prepared and can be used to treat neoplastic or other diseases or abnormalities or to target cells expressing these proteins on their surface .

MTSP1 (matriptase)
MTSP1 (also called matriptase) is a trypsin-like serine protease with a broad spectrum cleavage activity and two potential regulatory modules. It was named “matriptase” based on its activity of degrading the extracellular matrix and its trypsin-like activity. When isolated from breast cancer cells (or conditioned medium of T-47 cells), MTSP1 has been reported to be predominantly uncomplexed. MTSP1 has been isolated from human milk; when isolated from human milk, it has been reported to be one of two complex types, 95 kDa (preferred type) and 110 kDa. Uncomplexed MTSP1 was not detected (Liu, et al. (1999) J. Biol. Chem. 274: 18237-18242). MTSP1 has been proposed to exist as an uncomplexed protease when in the active state. In breast milk, it is reported to exist in a complex with a fragment of stem cell growth factor inhibitor-1 (HAI-1), a Kunitz-type serine protease inhibitor that has activity against trypsin-like serine proteases.

  A nucleic acid encoding a protein called matriptase has been cloned from conditioned medium of human breast cancer cells T-47D (Lin et al. (1999) J. Biol. Chem. 274: 18231-18236). Upon analysis of the cDNA, the full-length protease has 683 amino acids and has three major structural regions: a serine protease domain near the carboxyl terminal region, four tandem low-density lipoprotein receptor domains, and two Of complement complement components C1r and C1s (see SEQ ID NO: 1). Studies to identify additional serine proteases made by cancer cells were performed using PC-3 cells. A serine protease, reported as a transmembrane protease and named “MT-SP1” by the authors, has been cloned (Takeuchi et al. (1999) Proc. Natl. Acad. Sci. USA 96: 11054-11061 ). It was subsequently found that the originally identified sequence of matriptase was included in the translated sequence of the cDNA encoding MTSP1. The nucleic acid that originally encodes a protein called matriptase is a partial MTSP1 clone that lacks 516 of the encoding nucleotide (Takeuchi, et al., J. Biol. Chem 275: 26333-26342 ( 2000).). It was proposed that the reported cDNA sequence encoding matriptase encoded a possible initiating methionine so that alternative splicing could yield a protein lacking the N-terminal region of MTSP1. Accordingly, matriptase herein is a mutant form of MTSP1.

  MTSP1 is a type II transmembrane protein that exhibits trypsin-like protease activity and has an extracellular protease domain. A study of the substrate specificity of MTSP1 shows that protease-activated receptor 2 (PAR2), pro-hepatocyte growth factor (pro-HGF) and single-chain urokinase-type plasminogen activator (sc-uPA) Clarify that it is a polymer substrate. PAR2 functions in inflammation, cytoprotection and / or cell adhesion, while sc-uPA functions in tumor cell invasion and metastasis. HGF acts as a growth and pro-angiogenic factor.

Exemplary nucleotide sequences encoding human MTSP1 are shown in SEQ ID NOs: 1 and 2. As noted above, SEQ ID NO: 1 shows the nucleic acid sequence encoding MTSP1. This sequence is a longer variant and contains a protease domain common to both variants.
MTSP1 is expressed in breast, prostate and colorectal tumors. Thus, conjugates with substrates thereto can be used for the treatment of such tumors.

MTSP3
MTSP3 transcript was detected in lung carcinoma (LX-1), colon adenocarcinoma (CX-1), colon adenocarcinoma (GI-112) and ovarian carcinoma (GI-102). No obvious signal was detected in another form of lung carcinoma (GI-117), breast carcinoma (GI-101), pancreatic adenocarcinoma (GI-103) and prostate adenocarcinoma (PC3).

MTSP4
From 76 different human tissues (Catalog No. 77775-1; Human Multiple Tissue Expression (MTE) Array; CLONTECH) using MTSP4 transcripts, DNA fragments encoding part of the LDL receptor domain and the protease domain The resulting RNA blot was probed. A very strong signal was observed in the liver, as in Northern analysis of gel blots. Signals were observed in other tissues (in order of decreasing signal level): fetal liver> heart = kidney = adrenal = testis = fetal heart and kidney = skeletal muscle = bladder = placenta> brain = spinal cord = colon = stomach = Spleen = lymph node = bone marrow = trachea = uterus = pancreas = salivary gland = mammary gland = lung. MTSP4 also has several tumor cells including HeLa S3 = leukemia K-562 = Burkitt lymphoma (Raji and Daudi) = colorectal adenocarcinoma (SW480)> lung carcinoma (A549) = leukemia MOLT4 = leukemia HL-60 Less expressed. PCR of MTSP4 transcripts from cDNA libraries made from several human primary tumors xenografted into nude mice (human tumor multi-tissue cDNA panel, catalog number K1522-1, CLONTECH), with MTSP4-specific primers Implemented. MTSP4 transcript was detected in breast carcinoma (GI-101), lung carcinoma (LX-1), colon adenocarcinoma (GI-112) and pancreatic adenocarcinoma (GI-103). No obvious signal is detected in another type of lung carcinoma (GI-117), colon adenocarcinoma (CX-1), ovarian carcinoma (GI-102) and prostate adenocarcinoma (PC3) It was. MTSP4 transcripts were also detected in the LNCaP and PC-3 prostate adenocarcinoma cell lines and the human fibrosarcoma cell line HT-1080.

MTSP6
MTSP6 is expressed at high levels in the colon. It is also expressed in the stomach, trachea, mammary gland, thyroid, salivary gland, pituitary gland and pancreas. It is expressed at lower levels in other tissues (see Example 6).

  MSTP6 is also expressed in several tumor cell lines including HeLa S3> colorectal adenocarcinoma (SW480)> leukemia MOLT-4> leukemia K-562. In a mouse xenograft model, MTSP6 transcript is strongly detected in lung carcinoma (LX-1), moderately detected in pancreatic adenocarcinoma (GI-103), and ovarian carcinoma (GI-102). Weakly detected in; and weakly detected in colon adenocarcinoma (GI-112 and CX-1), breast carcinoma (GI-101), lung carcinoma (GI-117) and prostate adenocarcinoma (PC3) . MTSP6 was also detected in breast cancer cell line MDA-MB-231, prostate adenocarcinoma cell line PC-3, but not in human fibrosarcoma cell line HT-1080. MTSP6 is also expressed in mammary carcinoma cDNA (Clontech). MTSP6 is also overexpressed in ovarian tumor cells.

MTSP7
MTSP7 transcripts were detected in lung carcinoma (A549 cell line), leukemia (K-562 cell line) and cervical carcinoma (HeLaS3 cell line). MTSP7 is believed to be expressed in lung, colon, prostate, mammary gland, cervix and other tumors.

MTSP9
MTSP9 is expressed, for example, in esophageal tumor tissue, in lung carcinoma, in colorectal carcinoma, lymphoma, cervical carcinoma (HeLaS3) and leukemia cell lines, and in certain normal cells and tissues . MTSP9 can also be a marker for breast cancer, prostate cancer, cervical cancer and colon cancer.
MTSP9 is highly expressed in the esophagus and is expressed at low levels in many other tissues. MTSP9 transcripts are found in kidney (adult and fetus), spleen (adult and fetus), placenta, liver (adult and fetus), thymus, peripheral blood leukocytes, lung (adult and fetus) pancreas, lymph node, bone marrow, trachea, uterus Found in the prostate, testis, ovary and glandular organs (mammary gland, adrenal gland, thyroid, pituitary and salivary glands). MTSP9 is also expressed in esophageal tumor tissue, lung carcinoma, and at low levels in colorectal carcinoma, lymphoma, cervical carcinoma (HeLaS3) and leukemia cell lines.

MTSP10
MTSP10 is expressed, for example, in esophageal tumor tissue, in lung carcinoma, prostate cancer, pancreas and breast cancer, and in cell lines and in certain normal cells and tissues (eg, for tissue-specific expression profiles). For example). Activated MTSP10 levels can be for the diagnosis of prostate, uterus, lung, esophagus, or colon cancer or leukemia or other cancers. Expression and / or activation of MTSP10 on or near cells in a subject or in body fluids can be a marker for breast cancer, prostate cancer, lung cancer, colon cancer, esophageal cancer and other cancers.

  MTSP10 transcript was detected in pancreas, lung and kidney. The MTSP10 transcript was also detected in Marathon-Ready cDNA (Clontech) in the small intestine. MTSP10 transcripts were detected in breast carcinoma (GI-101), lung carcinoma (LX-1 and GI-117), ovarian carcinoma (GI-102), and pancreatic adenocarcinoma (GI-103). MTSP10 transcript was weakly detected in prostate adenocarcinoma (PC3). The MTSP10 transcript was also detected in prostate tumor CWR22R grown in nude mice. No obvious signal was detected in the two types of colon adenocarcinoma (GI-112 and CX-1).

MTSP12
MTSP12 transcript was detected in pancreas, lung and kidney. The MTSP12 transcript was also detected in Marathon-Ready cDNA (Clontech) in the small intestine. MTSP12 transcripts are breast carcinoma (GI-101), lung carcinoma (LX-1 and GI-117), ovarian carcinoma (GI-102), lung carcinoma (LX-1 and GI-117), ovarian cancer Detected in tumors (GI-102) and pancreatic adenocarcinoma (GI-103). MTSP12 transcripts were also detected in CWR22R prostate tumors grown on nude mice. No obvious signal was detected in the two types of colon adenocarcinoma (GI-112 and CX-1).

MTSP20
MTSP20 is expressed in lung, colon, cervical tumors and in leukemia cells. It can also be expressed in breast, ovary, pancreas, prostate and other tumors. MTSP20 transcripts were detected in the liver, lymph nodes, cerebellum, pancreas, prostate, uterus, testis, glands (adrenal, thyroid and salivary glands), thymus, kidney and spleen. Even lower transcription levels were found in the lung, placenta, bladder, ovary, digestive system, circulatory system, and other parts of the brain. MTSP20 is also a lung carcinoma (A519), colorectal carcinoma (SW480), lymphoma (Raji and Daudi), cervical carcinoma (HeLaS3) and leukemia (HL-60, K-562 and MOLT-4) cell lines. Is expressed in certain tumor cells.

MTSP22
MTSP22 is expressed in uterine tissue, thymus, adipose tissue and lymph nodes. It can also be expressed in lung, stomach, uterus, breast, ovary, prostate or other tumors. MTSP22 transcripts were detected in certain uterine tissue samples but not in those matched tumor samples. In one of the 42 uterine samples, MTSP22 is expressed in the tumor and its metastatic tissue, but not in the corresponding normal tissue. MTSP22 is also expressed in certain gastric and lung tumors, but not in their corresponding normal tissues. MTSP22 is also expressed in normal tissues of pancreatic matched cDNA pairs. CDNA encoding MTSP22 was detected in thymus, adipose tissue, and lymph nodes.

MTSP25
MTSP25 is expressed in breast, colon, uterus, ovary, kidney, prostate, testicular cancer tissues. It can also be expressed in lung, stomach, prostate and other tumors. MTSP25 transcript is weakly expressed in lymph nodes. In cancer profiling array analysis, MTSP25 is highly expressed in prostate samples (normal and cancer samples). MTSP25 was highly expressed in kidney tumor samples but not in its corresponding normal tissue. MTSP25 was also expressed in breast cancer samples but not in its corresponding normal tissue. MTSP25 was expressed in normal uterine samples but not in their corresponding tumors. MTSP25 expression was also detected in ovarian cancer samples. Of these three samples, MTSP25 expression was also detected in one of the corresponding matched normal tissues. MTSP25 expression was also detected in tumor samples in colon cDNA pairs.
PCR analysis showed that MTSP25 cDNA was strongly detected in testicular and mammary gland carcinomas, weakly detected in brain, placenta, lung, spleen, prostate, small intestine, colon, and white blood cells, and heart, liver and pancreas It was detected very weakly.

2. Endoceliases Endoceliases are a class of cell surface proteases that are expressed on cells, especially endothelial cells, especially those proliferating endothelial cells, which are tumor growth and metastasis, as well as restenosis, scarring Has been implicated in a variety of proliferative processes, including undesirable angiogenesis associated with other hyperproliferative disorders such as diabetic retinopathy, anterior segment diseases and disorders (filed November 20, 2000). (See US Application Serial No. 09 / 717,473 and International PCT Application No. PCT / US00 / 31803).

Proliferative diseases Endoceliases are particularly useful targets for the delivery of therapeutic agents for the treatment of any disorder involving abnormal angiogenesis. Endothelial cells play an important role in angiogenesis, which is the generation of new blood vessels from parental microvessels. Angiogenesis plays a major role in cancer metastasis and various other lesion lesions.

  Controlled and uncontrolled angiogenesis proceeds in a similar manner. Endothelial cells and pericytes surrounded by a matrix membrane form capillaries. Angiogenesis begins with the erosion of the matrix membrane by enzymes released by endothelial cells and leukocytes. Endothelial cells lining the lumen of the blood vessel then protrude from the matrix membrane. Angiogenic stimulants induce endothelial cells to migrate through the eroded matrix membrane. Migrating cells form “budding” from the parent's blood vessels, where the endothelial cells undergo cell division and proliferation. Endothelial budding merges with each other to form a capillary loop and create new blood vessels.

Angiogenesis, modulators and related diseases Angiogenesis is highly regulated by a system of angiogenic stimulants and inhibitors. Known examples of angiogenic stimulants include certain growth factors, cytokines, proteins, peptides, carbohydrates and lipids (Norrby (1997) APMIS 105: 417-437); Polverini (1995) Crit. Rev. Oral Biol. Med. 6: 230-247). Various endogenous and exogenous angiogenesis inhibitors are known in the art (Jackson et al. (1997) FASEB 11: 457-465; Norrby (1997) APMIS 105: 417-437); and O'Reilly ( 1997) Investigational New Drugs, 15: 5-13).

  Angiogenesis is essential for normal placenta, embryo, fetus and postnatal development and growth, but rarely occurs physiologically in adults except in very specific limited situations. For example, angiogenesis is commonly observed in wound healing, fetal and embryonic development and the formation of the corpus luteum, endometrium and placenta. Angiogenesis in adults is often associated with a disease state.

  Persistent unregulated angiogenesis occurs in numerous disease states, tumor metastasis, and abnormal growth by endothelial cells, supporting the pathological damage seen in these abnormalities. Various pathological disease states where unregulated angiogenesis exists are grouped together as angiogenesis-dependent or angiogenesis-related diseases.

  The control of angiogenesis changes in certain disease states, and in many cases, pathological damage associated with the disease is associated with uncontrolled angiogenesis (generally Norrby (1997) APMIS 105: 417-437); and O'Reilly (1997) Investigational New Drugs 15: 5-13). Thus, angiogenesis can occur in various diseases such as rheumatoid arthritis, psoriasis, various inflammatory diseases such as diabetic retinitis, pterygium recurrence, scars from excimer laser surgery and glaucoma filtration surgery, anterior segment Specific eye diseases, cardiovascular disorders, chronic inflammatory diseases, wound repair, circulatory system disorders, CREST syndrome, skin diseases (eg, US Pat. No. 5,593,990, 5, 629,327 and 5,712,291) and in particular involved in the development or progression of cancer, including solid neoplasia and vascular tumors. Angiogenesis is essential for the growth and survival of solid tumors and their metastases. Suppression, elimination or modulation of this activity affects the etiology of these diseases and should serve as a point of therapeutic intervention. In disease states, blocking angiogenesis could avoid damage caused by infiltration of new microvasculature. Treatment aimed at controlling the angiogenic process could lead to the prevention or alleviation of these diseases. Therefore, there is a need to develop therapeutic agents that target angiogenesis and modulate, in particular inhibit, abnormal or uncontrolled angiogenesis.

  Therefore, conjugates containing endotheliase substrates can be used to treat rheumatoid arthritis, psoriasis, diabetic retinopathy, pterygium recurrence, scars from excimer laser surgery and glaucoma filtration surgery, and various anterior segment disorders. Other eye diseases, including cardiovascular diseases, autoimmune diseases, chronic inflammatory diseases, wounds, circulatory system disorders, CREST syndrome, psoriasis and other skin diseases (eg, US Pat. Nos. 5,593,990, 5, 629,327 and 5,712,291), and delivering therapeutic agents for the treatment of diseases including, but not limited to, cancers including solid neoplasia and vascular tumors, among others. Can do.

Endoceliase 1 and 2
Illustrative of endotheliases are two different endotheliases, referred to as endotheliase 1 and endotheliase 2, and variants thereof (see SEQ ID NOs: 21-27). Other members of the family have sequence identity, in particular at least 40%, 60%, 80%, 90%, 95%, 98% or greater sequence identity with the endotheliase protease domain identified herein. Genes for genes that have sex or hybridize under high stringency conditions under high stringency conditions and are expressed on endothelial cells of the nucleic acid encoding the protease domain of endotheliase provided herein Can be identified by scrutinizing or by searching a library.

  As an alternative and as a way to identify endotheliases that may have even lower sequence identity, endotheliases can identify ESTs or other nucleic acid fragments that have sequences similar to proteases, and then their To identify and select cDNA clones encoding full-length proteases or their protease domains, identify those with transmembrane protein characteristics, and then measure gene expression profiles And can be identified by such methods as identifying those expressed on endothelial cells. An encoded protein that contains a transmembrane domain and an extracellular domain and has protease activity expressed in endothelial cells is an endotheliase. Any method of identifying a gene (or protein) that encodes a protein that encodes a transmembrane protease expressed on an endothelial cell is contemplated herein.

Endoceliase 1
Examples of endotheliase are endotheliase 1 and endotheliase 2. They are expressed on endothelial cells. An example of full-length endotheliase 1 is that comprising the sequence of the amino acid set forth in SEQ ID NO: 42 (International PCT Application No. WO 00 describing a gene designated DESC1, expressed in squamous cell carcinoma and prostate tumor). / 5006). As described, endotheliase is expressed on endothelial cells. Its protease domain is shown in SEQ ID NO: 22.

Expression profile of endotheliase 1 To obtain information on the tissue distribution of endotheliase 1, using a DNA insert of clone H117, an RNA blot consisting of 76 different human tissues (Cat. No. 77775-1; human multiple tissues) Expression (MTE) arrays; CLONTECH, Palo Alto, CA) were probed. Significant expression was observed in the esophagus, with mild expression levels in the stomach, salivary glands, pancreas, prostate, bladder, trachea, and uterus. Northern analysis using RNA blots (catalog numbers 7765-1 and 7782-1; human muscle and digestive multiple tissue northern (MTN) blots; CLONTECH) confirmed that expression was restricted to the esophagus. Two transcripts (approximately 1.7 and 2 kb) were detected in the esophagus. Endoceliase 1 is also expressed in umbilical vein endothelial cells, PC3 and LnCAP cells.

Nucleic acids encoding endotheliase 2 and endotheliase 2 Two splice variants of endotheliase 2, termed endotheliase 2-S and endotheliase 2-L, are exemplified herein. (See SEQ ID NOs: 23-26). The open reading frame encoding SEQ ID NO: 23 (SEQ ID NO: 23) consists of 1,689 bp and translates into a 562 amino acid protein (SEQ ID NO: 24), while the ORF of endotheliase 2-L Consists of 2,067 bp (SEQ ID NO: 25) and translates into a 688 amino chain protein (SEQ ID NO: 26).

  The nucleic acid encoding the protease domain of endotheliase 2-S consists of 729 bp that translates into a 242 amino acid protein (amino acids 321 to 562 of SEQ ID NOs: 23 and 24), while endotheliase 2-L. It consists of 107 bp that translates into a 368 amino acid protein (amino acids 321 to 688 of SEQ ID NOs: 25 and 26).

Endoceliase 2 protein The above endotheliase and / or its amino acid sequence comprising the amino acid sequence of SEQ ID NO: 22, 24, 26 and 27 or encoded by a nucleic acid which hybridizes thereto under the conditions as described above Any as well as all of the protease domains are contemplated for use in the methods herein. Also contemplated herein are proteins that contain amino acid sequence changes such as those shown in Table 1 above and retain protease activity.

Gene expression profile and transcript size of endotheliase 2 in normal and tumor tissues In addition to expression in endothelial cells, endotheliase 2 is expressed in placenta, pancreas, thyroid, liver and lung tissues. It is also expressed at lower levels in the mammary gland, salivary gland, kidney, trachea, esophagus, appendix, heart and fetal lung. Endoceliase 2 also includes in several tumor cell lines, and thus includes lung and colon, breast carcinoma, lung carcinoma, colon adenocarcinoma, pancreatic adenocarcinoma (GI-103), and ovarian cancer. It is expressed in certain tumors, including tumors. It is also detected in prostate and fibrosarcoma cell lines.

C. Conjugates Conjugates are provided that are substrates for proteases on the surface of cells, particularly serine proteases including type II membrane-bound serine proteases and endotheliases. Any cell surface protease is contemplated herein, including proteases associated with or localized to cells. In general, proteases that are expressed at high levels in an active form in essential tissues are not ideal target candidates. Proteases are expressed on a relatively limited number of cells, or expressed at high levels on cells such as tumor cells and endothelial cells and immune cells, are involved in disease states, or Examples of the disease state include those present in the disease state at the site of cells involved in the disease state. For example, endothelial cells, due to their role in angiogenesis, are involved in many proliferative disorders; immune cells are involved in many disease processes, including cancer and disease as well as inflammatory disorders. Other cell surface proteases are expressed at higher levels in certain tumors than in normal cells. Regardless of whether such proteases have a role in the disorder, their higher expression in cells involved in the disease state is for targeting therapeutic agents in the conjugates provided herein. It is enough for use.

  Conjugates containing therapeutic agents such as cytotoxic agents are activated upon cleavage by cell surface proteases, including cell-associated or cell-localized proteases. Examples of such proteases are MTSPs, urokinase and endotheliaase, such as but not limited to MTSP1, MTSP3, MTsP4, MTSP6, MTSP7, MTSP9, MTSP10, MTSP12, MTSP20, MTSP22, MTSP25. . Thus, conjugates targeted to such proteases are prodrugs in that the therapeutic agent is inactive upon administration and is ultimately activated in the vicinity of the targeted cell or tissue. Cell surface proteases such as transmembrane proteases are intended targets, but proteases and any other secreted, shed or soluble forms can also be targeted.

  Thus, conjugates containing therapeutic agents, such as cytotoxic agents, act by cell surface proteases, i.e., peptide moieties that are substrates for targeted cell surface proteases (i.e., peptidic substrates), and optionally linkers. Formerly substantially inert. The therapeutic agent in the conjugate is activated by cleavage of the conjugate peptide substrate by the cell surface protease. A therapeutic agent, such as a cytotoxic agent, is released as a free drug, or alternatively a peptidic substrate (P1-P2-P3) to which the drug is optionally linked via a linker in the conjugate. -Etc. (ie N-terminal) or P1'-P2'-etc. These forms of cytotoxic agents are released in the vicinity of cells expressing the protease. In certain embodiments, as a result of the action of cell surface proteases, therapeutic agents such as cytotoxic agents can cross the cell membrane or otherwise interact with cells or tissues and exhibit therapeutic activity. So activation is achieved. In other embodiments, any remaining peptidic moiety or amino acid can be cleaved from the therapeutic agent to make it active. Since the therapeutic agent is substantially inactive when bound, the conjugate acts as a prodrug. Upon cleavage by the targeted protease, the therapeutic agent is released either in an active form or in a form activated by the targeted cell, tissue or surrounding environment.

In one exemplary embodiment, the targeted agent is a cytotoxic agent and the conjugate for use in the methods and compositions provided herein has the formula:
(Peptide ⁱ ) _S- (linker) _q- (cytotoxic agent) _t
Or a derivative thereof, wherein peptide ⁱ is a cell surface protease, or a membrane protease that is released, shed, or otherwise detached from binding, such as MTSP; Greater than or equal to, or 1 to 6, or 1 or 2, or 1; the linker is any linker; q is greater than or equal to 0, or 0 to 4, or 0 or 1 Yes; the cytotoxic agent is an anti-cell mitotic agent including an anti-tumor, anti-cancer or anti-anti-angiogenic agent; t is 1 or more, or 1 or 2. In these conjugates, the cytotoxic agent is covalently attached to either the C-terminus or N-terminus of the peptidic substrate, optionally via a linker. In embodiments where a therapeutic agent such as a cytotoxic agent is attached to the C-terminus of the peptidic substrate, the N-terminus is optionally capped. N-terminal caps for use herein include, but are not limited to, acyl, sulfonyl and carbamoyl groups. In embodiments where the therapeutic agent is attached to the N-terminus of the peptidic substrate, the C-terminus is a carboxamide derivative.

In a particular embodiment, peptide ⁱ is a peptidic substrate for cell surface proteases or soluble MTSP, whereby a substantially inactive conjugate is cleaved at the P1-P1 ′ bond by the action of the protease. A formula that expresses a therapeutic activity such as a cytotoxic activity in vitro and in vivo:
(Peptide ^a ) _S- (linker) _q- (cytotoxic agent) _t
Or a derivative thereof. In these compounds, peptide ^a is a truncated form of peptide ⁱ resulting from cleavage at the P1-P1 ′ bond.

In another embodiment, the conjugates for use in the methods and compositions provided herein are optionally linkers at the C-terminus and N-terminus of the peptide substrate for cell surface protease or soluble MTSP, respectively. We own two therapeutic agents, such as cytotoxic agents, which are the same or different, linked via certain linker ¹ and linker ² . In this embodiment, the conjugate is of the formula:
(Therapeutic Agent ¹ ) _x- (Linker ¹ ) _w- (Peptide ⁱ ) _s- (Linker ² ) _q- (Therapeutic Agent ² ) _t
Or a derivative thereof, wherein peptide ⁱ is a peptidic substrate for cell surface protease or soluble MTSP; s is greater than or equal to 1, or 1-6, or 1 or 2, or 1 Linker ¹ and Linker ² are any independent linkers, and are the same or different; q and w are each independently greater than or equal to 0, or 0 to 4, or 0 or 1; The same or different therapeutic agent is an anti-tumor, anti-cancer or anti-cell mitotic agent; t and x are each independently 1 or more, or 1 or 2.

In these embodiments, peptide ⁱ is a peptidic substrate for cell surface proteases or soluble MTSP, whereby conjugates that are substantially inactive under the action of the protease are cleaved at points on the peptide chain. And the formula:
(Therapeutic agent ¹ ) _x- (linker ¹ ) _w- (peptide ^a1 ) _s ;
(Peptide ^a2 ) _s- (linker ² ) _q- (therapeutic agent ² ) _t
The two compounds or their derivatives. The released therapeutic agent is active or further activated by the cell, tissue or surrounding environment. In these compounds, peptide ^a1 and peptide ^a2 are the N-terminal and C-terminal cleavage portions, respectively, of peptide ⁱ resulting from cleavage at the P1-P ′ bond.

In one embodiment, the conjugate for use in the compositions and methods provided herein is of the formula 1:
_{^{X n - (P6) m -}} (P5) p - (P4) i - (P3) j - (P2) l -P1- (P1 ') u - (P2') k - (P3 ') r - (L ) _n -Z
Or a derivative thereof, wherein Z is a therapeutic agent; L is a linker; l, j, i, p and m are selected as follows:
l is 0 or 1; when l is 0, j, i, p and m are 0; when l is 1, j is 0 or 1; when j is 0 , I, p and m are 0; when j is 1, i is 0 or 1; when i is 0, p and m are 0; when i is 1 , P is 0 or 1; when p is 0, m is 0; when p is 1, m is 0 or 1;
u, k and r are selected as follows:
u is 0 or 1; when u is 0, k and r are 0; when u is 1, k is 0 or 1; when k is 0, r is 0; when k is 1, r is 0 or 1;
n is 0 or 1; X ⁿ is hydrogen or acyl, sulfonyl or carbamoyl cap; and P6-P3 ′ are amino acid residues as defined below. In this embodiment, the P6-P3 ′ residues are linked by a peptide bond or peptide bond surrogate. Thus, the P6-P3 ′ portion of the conjugate is a peptidic substrate, as defined herein.

In another embodiment, the conjugate for use in the compositions and methods provided herein is of formula II:
_{Z- (L) n - (P6} ) m - (P5) p - (P4) i - (P3) j - (P2) l -P1- (P1 ') u - (P2') k - (P3 ') _r -X ^c
Or having a derivative thereof, wherein Z is a therapeutic agent; L is a linker; l, j, i, p and m are selected as follows:
l is 0 or 1; when l is 0, j, i, p and m are 0; when l is 1, j is 0 or 1; when j is 0 I, p and m are 0; when j is 1, i is 0 or 1; when i is 0, p and m are 0; when i is 1, p is 0 or 1; when p is 0, m is 0; when p is 1, m is 0 or 1;
u, k and r are selected as follows:
u is 0 or 1; when u is 0, k and r are 0; when u is 1, k is 0 or 1; when k is 0, r is 0; when k is 1, r is 0 or 1;
n is 0 or 1; X ^c together with the carbonyl group of the amino acid residue to which it is attached forms a carboxylic acid or carboxamide group; and P6-P3 ′ are as defined below It is an amino acid residue. In this embodiment, the P6-P3 ′ residues are linked by a peptide bond or peptide bond surrogate. Thus, the P6-P3 ′ portion of the conjugate is a peptidic substrate, as defined herein.

In a further embodiment, the conjugate for use in the compositions and methods provided herein is of formula III:
_{^{Z 1 - (L 1) n}} - (P6) m - (P5) p - (P4) i - (P3) j - (P2) l -P1- (P1 ') u - (P2') k - (P3 ') _r- (L ² ) _v -Z ²
Or a derivative thereof, wherein Z ¹ and Z ² are each a therapeutic agent and are the same or different; L ¹ and L ² are each a linker and are the same or different; , J, i, p and m are selected as follows:
l is 0 or 1; when l is 0, j, i, p and m are 0; when l is 1, j is 0 or 1; when j is 0 I, p and m are 0; when j is 1, i is 0 or 1; when i is 0, p and m are 0; when i is 1 P is 0 or 1; when p is 0, m is 0; when p is 1, m is 0 or 1;
u, k and r are selected as follows:
u is 0 or 1; when u is 0, k and r are 0; when u is 1, k is 0 or 1; when k is 0, r is 0; when k is 1, r is 0 or 1;
n and v are each independently 0 or 1; and P6-P3 ′ are amino acid residues as defined below. In this embodiment, the P6-P3 ′ residues are linked by a peptide bond or peptide bond surrogate. Thus, the P6-P3 ′ portion of the conjugate is a peptidic substrate, as defined herein.

In another embodiment, the conjugate for use in the compositions and methods provided herein is a compound of formula IV:
_{^{X n - (P6) m -}} (P5) p - (P4) i - (P3) j - (P2) l -P1- (P1 ') u - (P2') k - (P3 ') r - (P4 ') _s- (L) _n -Z
Or having a derivative thereof, wherein Z is a therapeutic agent; L is a linker; l, j, i, p and m are selected as follows:
l is 0 or 1; when l is 0, j, i, p and m are 0; when l is 1, j is 0 or 1; when j is 0 I, p and m are 0; when j is 1, i is 0 or 1; when i is 0, p and m are 0; when i is 1, p is 0 or 1; when p is 0, m is 0; when p is 1, m is 0 or 1;
u, k, r and s are selected as follows:
u is 0 or 1; when u is 0, k, r and s are 0; when u is 1, k is 0 or 1, and when k is 0, r and s are 0; when k is 1, r is 0 or 1; when r is 0, s is 0; when r is 1, s is 0 or 1 Is;
n is 0 or 1; X ⁿ is hydrogen or an acyl, sulfonyl or carbamoyl cap; and P6-P4 ′ are amino acid residues as defined below. In this embodiment, the P6-P4 ′ residues are linked by a peptide bond or peptide bond surrogate. Thus, the P6-P4 ′ portion of the conjugate is a peptidic substrate as defined herein.

In another embodiment, the conjugate for use in the compositions and methods provided herein is of formula V:
_{Z- (L) n - (P6} ) m - (P5) p - (P4) i - (P3) j - (P2) l -P1- (P1 ') u - (P2') k - (P3 ') _r − (P4 ′) _s −X ^c
Or having a derivative thereof, wherein Z is a therapeutic agent; L is a linker; l, j, i, p and m are selected as follows:
l is 0 or 1; when l is 0, j, i, p and m are 0; when l is 1, j is 0 or 1; when j is 0 I, p and m are 0; when j is 1, i is 0 or 1; when i is 0, p and m are 0; when i is 1 P is 0 or 1; when p is 0, m is 0; when p is 1, m is 0 or 1;
u, k, r and s are selected as follows:
u is 0 or 1; when u is 0, k, r and s are 0; when u is 1, k is 0 or 1, and when k is 0, r and s are 0; when k is 1, r is 0 or 1; when r is 0, s is 0; when r is 1, s is 0 Or 1;
n is 0 or 1; X ^C together with the carbonyl group of the amino acid residue to which it is attached forms a carboxylic acid or carboxamide group; and P6-P4 ′ are defined below Such amino acid residues. In this embodiment, the P6-P4 ′ residues are linked by a peptide bond or peptide bond surrogate. Thus, the P6-P4 ′ portion of the conjugate is a peptidic substrate as defined herein.

In a further embodiment, the conjugate for use in the compositions and methods provided herein is of formula VI:
_{^{Z 1 - (L 1) n}} - (P6) m - (P5) p - (P4) i - (P3) j - (P2) l -P1- (P1 ') u - (P2') k - (P3 ') _r- (P4') _s- (L ² ) _v -Z ²
Or a derivative thereof, wherein Z ¹ and Z ² are each independently a therapeutic agent and are the same or different; L ¹ and L ² are each a linker and are the same or different L, j, i, p and m are selected as follows:
l is 0 or 1; when l is 0, j, i, p and m are 0; when l is 1, j is 0 or 1; when j is 0 I, p and m are 0; when j is 1, i is 0 or 1; when i is 0, p and m are 0; when i is 1 P is 0 or 1; when p is 0, m is 0; when p is 1, m is 0 or 1;
u, k, r and s are selected as follows:
u is 0 or 1; when u is 0, k, r and s are 0; when u is 1, k is 0 or 1, and when k is 0, r and s are 0; when k is 1, r is 0 or 1; when r is 0, s is 0; when r is 1, s is 0 Or 1;
n and v are each independently 0 or 1; and P6 to P4 ′ are amino acid residues as defined below. In this embodiment, the P6-P4 ′ residues are linked by a peptide bond or peptide bond surrogate. Thus, the P6-P4 ′ portion of the conjugate is a peptidic substrate as defined herein.

  Exemplary peptide substrates, therapeutic agents, linkers, and exemplary conjugates of Formulas I-VI are described in further detail below. As used herein, all combinations and / or permutations of the groups described below for the variables of Formulas I-VI are intended to be encompassed within this disclosure.

1. Peptidic substrate A peptidic substrate intended for use in a conjugate is a substrate for the targeted cell surface protease or its soluble, detached or released form, and any treatment in the conjugate Contains a sufficient number of amino acid residues to make the drug a substantially inactive conjugate. In an exemplary embodiment where the therapeutic agent is, for example, doxorubicin, the conjugate is substantially inactive because the bound therapeutic agent cannot pass through the cell membrane. In certain embodiments, the peptidic substrate contains at least 1, 2, 3, 4 or 5 amino acid residues and can contain up to 9 or 10 residues. Longer peptidic substrates can be used in the conjugate as long as the resulting therapeutic agent or therapeutic agent-amino acid or -peptide moiety conjugate exhibits the desired therapeutic effect in vivo and in vitro upon cleavage. .

  Accordingly, exemplary peptidic substrates for use in the conjugates provided herein are at least one amino acid (P1), two amino acids (P1-P1 ′), three amino acids (P2- P1-P1 ') and typically 4, 5 or 6 amino acid residues (P3-P2-P1-P1', P4-P3-P2-P1-P1 ', P4-P3-P2- P1-P1′-P2 ′), where P1-P1 ′ binding includes, for example, serine proteases, including but not limited to uPA, MTSPs and endotheliaase bound to its receptor. A cell surface protease or a soluble, shed, or released form of a cleavage site, including but not limited to any cell surface protease. The peptidic substrate optionally further possesses P5, P6 or P3 'amino acid residues, and in certain embodiments possesses P7, P8, P9, P10, P4', P5 ', P6' residues. Thus, peptidic substrates for use in the conjugates provided herein are penta-, hexa-, hepta-, octa-, and non-peptidic substrates, with cleavage of the conjugate by proteases. The resulting therapeutic agent or conjugate of the therapeutic agent-amino acid or -peptide moiety contains 10, 11, 12, 13, 14, 15 or more residues so long as it exhibits the desired therapeutic effect in vivo and in vitro. can do.

  Peptidic substrates can be attached to therapeutic agents via C-terminal residues (ie P1 ′, P2 ′ or P3 ′) or N-terminal residues (ie P6, P5 or P4), or optionally internal residues. (Or to the linker to which the therapeutic agent is attached). Peptidic substrates can be, for example, linear, but can be cyclic or contain a cyclic moiety.

  In embodiments where attachment is through the C-terminus of the peptidic substrate, the peptidic substrate optionally possesses a cap, such as an acyl or carbamoyl cap, at the N-terminus. In embodiments where attachment is through the N-terminus of the peptidic substrate, the peptidic substrate further possesses a terminal group at the C-terminus, such as a carboxamide group.

  The conjugate can contain multiple peptidic substrates and multiple therapeutic agents. For example, in conjugates containing two identical or different therapeutic agents, binding to the therapeutic agent (s) or the linker attached thereto can be via the C-terminal and N-terminal residues of the peptidic substrate. .

  Appropriate substrates can be designed using the methods described above for substrate selection. In addition, substrates can be designed based on the known specificity of other proteases. For example, the specificity of trypsin-like and trypsin family members can help in the design of possible substrates. The following summarizes substrate preferences for specific serine proteases (see, eg, Harris et al. (2000) PNAS 97 (14): 7754-7759).

  Typical protocols for the preparation of conjugates are: 1) identification of targeted proteases; 2) expression and assay development; 3) selected, for example, by testing chromogenic or fluorogenic substrates By identifying what is cleaved by the targeted protease, using a substrate phage display, identifying the peptidic substrate cleaved by the targeted protease, the natural protein or peptide substrate or the native protein of the protease Selection of substrates such as by using inhibitors and using combinatorial libraries to identify substrates that are cleaved by the targeted substrate; 4) Synthesis of conjugates containing the identified substrate And 5) in vitro assays, cell culture assays, biological assays, and in vivo motility Including model, but not limited to, the biological evaluation may include the step (e.g., see Example 10).

  The conjugate can be designed by any method known to those skilled in the art. The following provides an exemplary protocol. First, a series of commercially available chromogenic and fluorogenic peptide substrates can be tested for cleavage by the protease of interest (for exemplary chromogenic and fluorogenic substrates and tables, see See examples below). The peptidic portion of these substrates occupies the unprimed binding site of the protease, while the reporter group is located on the primed side of the cleavable bond. Effective conjugates can then be designed based on the structure of the substrate that is efficiently cleaved by the protease.

  The peptidic portion of these efficiently cleaved substrates can be used as an unprimed region of the conjugate, and Ser-therapeutics such as cytotoxic agents (eg doxorubicin), Ser-Leu-therapeutics Alternatively, a Ser-Ser-Leu-therapeutic can be used as the primed region of the conjugate. Cleavage of these conjugate prodrugs releases either the Ser-therapeutic, Ser-Leu-therapeutic or Ser-Ser-Leu-therapeutic compound. In another embodiment, the Ser in the free Ser-therapeutic may be substituted with other amino acid residues, including but not limited to Ala, hSer, Abu, Thr, Met, nLeu and Val. Good. In another embodiment, such as when the therapeutic agent is doxorubicin, the amino acid residue attached to the therapeutic agent possesses a hydrophobic side chain. Such amino acid residues include, but are not limited to, Leu, Abu, nLeu, nVal, CHA, hCHA, (hexyl) Gly, (allyl) Gly, (propargyl) Gly, and (cyclopropyl) Ala. . In another embodiment, such as when the therapeutic agent is taxol, the amino acid residue attached to the therapeutic agent possesses a side chain that is not sterically bulky. Such amino acid residues include, but are not limited to, Gly and Ala. The resulting P1'-therapeutic, P1'-P2'-therapeutic, or P1'-P2'-P3'-therapeutic compound can be further processed in vivo into an active therapeutic agent.

  Another approach to designing conjugate prodrugs for protease substrates is to elucidate optimal subsite occupancy for proteases using substrate phage display. The information obtained can then be used to design the peptidic, non-primed portion of the conjugate. As explained above, the primed region of the conjugate can be immobilized as a Ser-therapeutic, Ser-Leu-therapeutic, or Ser-Ser-Leu-therapeutic.

  A third approach to designing effective prodrug conjugates involves the use of a combinatorial fluorogenic substrate library to determine optimal residues for the unprimed region of the protease substrate. These selected sequences can then be used as the unprimed portion of the conjugate prodrug and Ser-therapeutic (eg, doxorubicin), Ser-Leu-therapeutic, or Ser-Ser -Leu therapeutics can be used as primed regions of the conjugate.

  These methods have been used to design the peptidic substrate portion of the conjugates provided herein. For example, sequences including GSGR (and related sequences such as TGR, SGR, extended variants and others herein) are based on or derived from substrate phage display experiments using u-PA as the target protease. It was done. Many matriptase conjugates, such as (R / K) -XSR and X- (R / K) -SR, as well as related sequences provided herein are derived from combinatorial libraries. Based on the data. In another embodiment, a natural substrate or natural inhibitor of a protease target such as uPA, comprising VSAR, PGR (from P3-P1 of plasminogen) and related sequences, is targeted binding to u-PA. Used for body design. In other embodiments, sequences from chromogenic substrates such as D-HHT-Gly-Arg and related sequences were used in the design of conjugates targeted to ET-1.

ａ カップルアッセイ、Spec ＰＬ存在下でのプラスミノーゲンの活性化 Chromogenic / Fluorogenic Substrate

a Coupled assay, plasminogen activation in the presence of Spec PL

Briefly, for couple assays, the activity of a protease that activates an enzyme, such as plasminogen or trypsinogen, is tested. In order to perform these assays, the protease can be expressed as plasminogen or trypsinogen in the presence of a known substrate labeled for zymogen, such as lys-plasminogen or Spec PL (for plasminogen). Incubate with zymogen. If the protease activates the zymogen, activated enzymes such as plasmin and trypsin will degrade the substrate, thereby changing the spectral properties of the substrate.

Exemplary Peptidic Substrate The following description provides a general discussion of exemplary peptidic substrates for cleavage by proteases, such as MTSP1 (ie, matriptase), endoseriase 1 and urokinase, and the nature of the residues To do. Similarly, peptidic substrates for cleavage by other cell surface proteases, or soluble, shed or released forms thereof, identify peptidic substrates for the selected protease and then such peptidic properties. It can be similarly designed by making conjugates containing the substrate.

[式中、ｚが０もしくは１](例えば、Webb et al. (1991) J. Org. Chem. 56: 3009、を参照)のような配座的に制約されたアルギニン類似体である；または側鎖が：
式：

[式中、ｄは、０〜５、もしくは１〜３の整数であり；そしてＷは、ＮもしくはＣＨである]；または上の基のモノ−もしくはジ−置換Ｎ−アルキル誘導体[そこでは、アルキル基は、特定の実施態様では、例えばメチルのような低級アルキルである]のような、配座的に制約されたアルギニン側鎖類似体である。
本明細書における特定の実施態様では、Ｐ１残基は、Ａｒｇである。 a. P1 Residues Amino acid residues used at the P1 position of peptidic substrates for use in the conjugates provided herein include Arg, Arg surrogate and Lys. As an Arg surrogate, it functions in a manner substantially similar to the naturally occurring side chain of arginine and achieves substantially similar results (eg, a P1 residue in a substrate for MTSP1, urokinase or endotheliaase). And non-natural amino acids that possess groups or moieties. Arg surrogates include: homoarginine side chain; guanidinoaminopropyl; guanidinoaminoethyl; (Me) ₂ arginine side chain; (Et) ₂ arginine side chain; (4-aminomethyl) phenylmethyl; 4-amidino Phenylmethyl; 4-guanidinophenylmethyl, which includes, but is not limited to, an α-amino acid carrying any of the side chains; or Arg surrogate:
formula:

A conformationally constrained arginine analog such as [wherein z is 0 or 1] (see, eg, Webb et al. (1991) J. Org. Chem. 56: 3009); or The side chain is:
formula:

Wherein d is an integer from 0 to 5, or from 1 to 3; and W is N or CH; or a mono- or di-substituted N-alkyl derivative of the above group [wherein The alkyl group is a conformationally constrained arginine side chain analog, such as in certain embodiments lower alkyl such as methyl.
In certain embodiments herein, the P1 residue is Arg.

b. P2 residue Among the conjugates provided herein, the P2 residue is Phe, Ser, Gly, Ala, Ser (OMe), hSer, 1-methylHis, 3-methylHis, His, nVal, selected from nLeu, Abu, (hS) Gly, Thr, Aib, CHA and Tyr. In another embodiment, the P2 residue is selected from Phe, Ser, Gly and Ala. In certain embodiments herein, the P2 residue is Ser or Ala. In another embodiment, the P2 residue is Gly or Ala.

c. P3 residues The amino acid residues for use at the P3 position of the conjugates provided herein include Arg, Lys, Gln, Quat, Arg surrogate, Ser, Thr, hSer, dSer, Pro, (hS ) Gly, Tyr, 4,4-dimethylThr, Asn, Met (O ₂ ), Quat ² , Quat ³ , Quat ⁴ and Quat ⁵ . In another embodiment, the P3 residue is selected from Arg, Lys, Gln, Quat and Arg surrogates. Arg surrogates include those described above for the P1 residue.
In certain embodiments, the P3 residue is Gln or Ser.

d. P4 residue In the conjugates provided for use in the compositions and methods provided herein, the P4 residue is a Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu , Phe, Val, N, N-dimethyl Gly, β-Ala, Cys (Me), Gln, t-butyl Gly and nVal. In another embodiment, the P4 residue is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val. In a further embodiment, the P4 residue is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Phe or Val. In certain embodiments herein, the P4 residue is Arg or Gly.

e. P5 and P6 residues In certain embodiments herein, the peptidic substrate used in the conjugate comprises P5 and optionally P6 residues. P5 residues include Ile, Arg and Arg surrogates. In another embodiment, P5 residues include Arg and Arg surrogates. Arg surrogates include those described above for the P1 residue. Examples of the P6 residue include Leu, Val and Arg. In another embodiment, the P6 residue includes, for example, Leu.

f. P1 ′ residue The P1 ′ residue of the conjugates provided herein is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexa Noil, Thr or hSer. In another embodiment, the P1 ′ residue of the conjugates provided herein is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6 -Aminohexanoyl. In another embodiment, the P1 ′ residue is Ser, Ala, hSer, Abu, Thr, Met, nLeu or Val. In another embodiment, the P1 ′ residue is Gly or Ala. In another embodiment, the P1 ′ residue is Ser, Ala or Gly. In another embodiment, the P1 ′ residue is Leu, Abu, nLeu, nVal, CHA, hCHA, (hexyl) Gly, (allyl) Gly, (propargyl) Gly or (cyclopropyl) Ala. In certain embodiments herein, the P1 ′ residue is Ala, Ser, Gly, Ile or d-Ile.

g. P2 ′ Residues In certain embodiments herein, the conjugates provided herein carry a P2 ′ residue. P2 ′ residues for use herein include Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, hCHA, CHA , Hexyl Gly, allyl Gly, and Phe, but are not limited thereto. In another embodiment, P2 ′ residues for use herein include Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6- Aminohexanoyl can be mentioned, but is not limited thereto. In another embodiment, the P2 ′ residue is Ser, hSer, Abu, nLeu, nVal, CHA, hCHA, allyl Gly or hexyl Gly. In another embodiment, the P2 ′ residue is Gly or Ala. In another embodiment, the P2 ′ residue is Leu, Abu, nLeu, nVal, CHA, hCHA, (hexyl) Gly, (allyl) Gly, (propargyl) Gly or (cyclopropyl) Ala. In a further embodiment, the P2 ′ residue is Ala, Gly, Ile or d-Ile.

h. P3 ′ residue In other embodiments herein, the peptidic substrate used in the conjugates provided herein comprises a P3 ′ residue. P3 ′ residues for use herein include Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA and allyl Gly. However, it is not limited to them. In another embodiment, the P2 ′ residue is Ser, hSer, Abu, nLeu, nVal, CHA, hCHA, (allyl) Gly or (hexyl) Gly. In another embodiment, P3 ′ residues for use herein include Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. But not limited to. In another embodiment, the P3 ′ residue is Gly or Ala. In another embodiment, the P3 ′ residue is Leu, Abu, nLeu, nVal, CHA, hCHA, (hexyl) Gly, (allyl) Gly, (propargyl) Gly or (cyclopropyl) Ala.

i. P4 ′ Residue In other embodiments herein, the peptidic substrate used in the conjugates provided herein comprises a P4 ′ residue. P4 ′ residues for use herein include Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA, and allyl Gly. However, it is not limited to them. In another embodiment, P4 ′ residues for use herein include Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. But not limited to. In another embodiment, the P4 ′ residue is Gly or Ala. In another embodiment, the P4 ′ residue is Leu, Abu, nLeu, nVal, CHA, hCHA, (hexyl) Gly, (allyl) Gly, (propargyl) Gly or (cyclopropyl) Ala. In another embodiment, the P4 ′ residue is Leu.

j. Cap 1) ^Xn (N-terminal cap)
In embodiments herein where the therapeutic agent is attached to the C-terminus of the peptidic substrate (ie, the conjugate has Formula I), the N-terminus of the peptidic substrate is optionally acyl, sulfonyl or Capped with a carbamoyl derivative. In certain embodiments, a cap is selected to increase the hydrophilicity of the conjugate. In embodiments where the peptidic substrate-therapeutic agent conjugate is sufficiently hydrophilic and does not require further hydrophilicity, a non-hydrophilic N-terminal cap such as an acetyl group may be used. In embodiments where increased hydrophilicity is desired, the N-terminal amino acid is modified with a hydrophilic protecting group. Such protecting groups are selected based on the presence of hydrophilic functional groups. Such protection of the terminal amino group can also reduce or eliminate enzymatic degradation of such peptidic therapeutics by the action of exogenous aminopeptidases present in the plasma of warm-blooded animals.

  N-terminal protecting groups that increase the hydrophilicity of the conjugate and therefore increase the water solubility of the conjugate include alkanoyl hydroxide, polyalkanoyl hydroxide, polyethylene glycol, glycosylates, sugars and crown ethers. However, it is not limited to them.

式中、Ｒ^１およびＲ^２は、以下のような(i)もしくは(ii)から選択される：
(i)Ｒ^１およびＲ^２は、それぞれ独立に：
ａ)水素；
ｂ)未置換もしくは置換アリール、未置換もしくは置換ヘテロサイクリル、Ｃ_３〜Ｃ_１０シクロアルキル、Ｃ_２〜Ｃ_６アルケニル、Ｃ_２〜Ｃ_６アルキニル、ハロゲン、Ｃ_１〜Ｃ_６パーフルオロアルキル、Ｒ^４Ｏ−、Ｒ^３Ｃ(Ｏ)ＮＲ^３−、(Ｒ^３)_２ＮＣ(Ｏ)−、(Ｒ^３)_２Ｎ−Ｃ(ＮＲ^３)−、Ｒ^４Ｓ(Ｏ)_ｅＮＨ−、−ＣＮ、−ＮＯ_２、Ｒ^３Ｃ(Ｏ)−、−Ｎ_３、−Ｎ(Ｒ^３)_２もしくはＲ^４ＯＣ(Ｏ)ＮＲ^３−；
ｃ)未置換Ｃ_１〜Ｃ_６アルキル；
ｄ)置換Ｃ_１〜Ｃ_６アルキル、[そこでは置換Ｃ_１〜Ｃ_６アルキル上の置換基が未置換もしくは置換アリール、未置換もしくは置換へテロサイクリル、Ｃ_３〜Ｃ_１０シクロアルキル、Ｃ_２〜Ｃ_６アルケニル、Ｃ_２〜Ｃ_６アルキニル、Ｒ^３Ｏ−、Ｒ^４Ｓ(Ｏ)_ｅＮＨ−、Ｒ^３Ｃ(Ｏ)ＮＲ^３−、(Ｒ^３)_２ＮＣ(Ｏ)−、(Ｒ^３)_２Ｎ−Ｃ (ＮＲ^３)−、−ＣＮ、Ｒ^３Ｃ(Ｏ)−、−Ｎ_３、−Ｎ(Ｒ^３)_２およびＲ^４ＯＣ(Ｏ)−ＮＲ^３−から選択される]であるか；または
(ii)Ｒ^１およびＲ^２は、結合して−(ＣＨ_２)_ｆ[そこでは一つの炭素原子が：−Ｏ−、−Ｓ(Ｏ)_ｅ−、−ＮＣ(Ｏ)−、−ＮＨ−および−Ｎ(ＣＯＲ^４)−から選択される部分によって任意に置換され；
Ｒ^３は：水素、未置換もしくは置換アリール、未置換もしくは置換ヘテロサイクリル、Ｃ_１〜Ｃ_６アルキルおよびＣ_３〜Ｃ_１０シクロアルキル、から選択される；
Ｒ^４は：未置換もしくは置換アリール、未置換もしくは置換ヘテロサイクリル、Ｃ_１〜Ｃ_６アルキルおよびＣ_３〜Ｃ_１０シクロアルキル、から選択され；
ｅは、０、１もしくは２であり；
ａは、１、２、３もしくは４であり；
ｂは、ゼロもしくは１〜１００の整数であり；および
ｃは、もしｂがゼロであるならば、ｃは１〜１０であるという条件で、０〜１０であり；ならびに
ｆは、３、４もしくは５である]を形成する。 In certain embodiments herein, the N-terminal protecting group is one of the following:

Wherein R ¹ and R ² are selected from (i) or (ii) as follows:
(i) R ¹ and R ² are each independently:
a) hydrogen;
b) unsubstituted or substituted aryl, unsubstituted or substituted _{heterocyclyl,} C 3 _{-C 10 cycloalkyl,} C 2 -C _{6 alkenyl,} C 2 -C ₆ alkynyl, _halogen, C 1 -C ₆ perfluoroalkyl, R ⁴ O-, R ³ C (O) NR ^3- , (R ³ ) ₂ NC (O)-, (R ³ ) ₂ N-C (NR ³ )-, R ⁴ S (O) _e NH-,- CN, —NO ₂ , R ³ C (O) —, —N ₃ , —N (R ³ ) ₂ or R ⁴ OC (O) NR ³ —;
c) unsubstituted _C 1 -C ₆ alkyl;
d) substituted _C 1 -C ₆ alkyl, [where the substituents _C 1 -C ₆ alkyl substituent on the unsubstituted or substituted aryl, unsubstituted or Terosaikuriru to _{substitution,} C 3 _{-C 10 cycloalkyl,} C 2 -C _{6 alkenyl,} C 2 -C ₆ ^{alkynyl, R 3 O-, R 4 S} (O) e NH-, R 3 C (O) NR 3 -, (R 3) 2 NC (O) -, (R 3) ₂ N—C (NR ³ ) —, —CN, R ³ C (O) —, —N ₃ , —N (R ³ ) ₂ and R ⁴ OC (O) —NR ³ —. Or;
(ii) R ¹ and R ² are bonded to — (CH ₂ ) _f [wherein one carbon atom is: —O—, —S (O) _e —, —NC (O) —, —NH— And optionally substituted by a moiety selected from: —N (COR ⁴ ) —;
R ³ is selected from: hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclyl, C ₁ -C ₆ alkyl and C ₃ -C ₁₀ cycloalkyl;
R ⁴ is selected from: unsubstituted or substituted aryl, unsubstituted or substituted heterocyclyl, C ₁ -C ₆ alkyl and C ₃ -C ₁₀ cycloalkyl;
e is 0, 1 or 2;
a is 1, 2, 3 or 4;
b is zero or an integer from 1 to 100; and c is 0 to 10, provided that if b is zero, c is 1 to 10; and f is 3, 4 Or 5].

In certain embodiments, R ¹ and R ² are each independently hydrogen, OH, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ aralkyl or aryl. In these embodiments, a is 1, 2, 3 or 4; b is 0 or an integer from 1 to 100; c is c is 1 to 10 if b is zero. Under certain conditions, it is 0-10.

または(ii)

または(iii)

または(iv)

式中、Ｒ^１およびＲ^２は、それぞれ独立に、水素、Ｃ_１〜Ｃ_６アルキルおよびアリールであり；ａは、１、２、３もしくは４であり；ａ´は、０、１、２もしくは３であり；ｂは、０もしくは１〜１４の整数であり；ｃは、もしｂが０であるならば、ｃは１であるという条件で、０もしくは１である。 In another embodiment, the N-terminal cap (X ⁿ ) is hydrogen or (i), (ii), (iii) or (iv) as follows:
(i)

Or (ii)

Or (iii)

Or (iv)

Wherein R ¹ and R ² are each independently hydrogen, C ₁ -C ₆ alkyl and aryl; a is 1, 2, 3 or 4; a ′ is 0, 1, 2 or B is 0 or an integer from 1 to 14; c is 0 or 1 provided that if b is 0, c is 1.

In another embodiment, X ⁿ is R ³⁰ O—C (O) —, R ³¹ R ³² N—C (O) —, R ³³ (CH ₂ ) _k C (O) — or HC ( Wherein k is an integer from 1 to 4, or 1 or 2; R ³⁰ is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; R ³¹ and R ³² are Each independently hydrogen, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and R ³³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, Aralkyl, aralkoxy, heteroaralkyl or heteroaralkoxy].

In certain embodiments herein, X ⁿ is hydrogen, acetyl, hydroxyacetyl, 2,3-dihydroxypropionyl, 2,3,4-trihydroxybutanoyl, PEG (1), PEG (2), PEG (4), PEG (6), PEG (14), PEG (15), PEG (16), PEG (17), PEG (18) or PEG (19). In other embodiments herein, ^Xn is hydrogen, acetyl, hydroxyacetyl, succinyl, quinyl, galyl, 4-imidazolylacetyl, cotininyl, 3-phosphonylpropionyl, glonyl, 4-phosphonylbutyryl, glutaryl. Ethoxy squalyl or PEG (2). In a further embodiment, X ⁿ is hydrogen, acetyl, —C (O) NH ₂ , HOCH ₂ CH ₂ C (O) —, diaminopropanoyl or NH ₂ — (CH ₂ ) ₅ —C (O) —. is there. In another embodiment, ^Xn is hydrogen, acetyl, succinyl, glutaryl, PEG (2) or malonyl. In another embodiment, X ⁿ is hydrogen, acetyl, succinyl, glutaryl, PEG (2), malonyl, methoxycarbonyl, phenylsulfonyl, 3-methoxypropanoyl, ethoxycarbonyl, isobutoxycarbonyl, benzyloxycarbonyl, tert -Butoxycarbonyl, 4-oxopentanoyl, 2- (2-methoxyethoxy) ethoxy) acetyl, 3,4-methylenedioxyphenylacetyl, 2-pyridylacetyl, phenoxyacetyl, phenylacetyl, methoxyacetyl, 2-methoxyethoxy With carbonyl, 2-methoxyethoxyacetyl, 3-phenyl-2-hydroxypropanoyl, pent-4-inoyl, 1-naphthylacetyl, hydroxyacetyl, 3-methoxycarbonylpropanoyl or formyl is there.

In certain embodiments herein, the N-terminal cap (X ⁿ ) is acetyl, glutaryl, or related acyl, sulfonyl, or carbamoyl derivatives. Capping groups include, but are not limited to, simple N-acetyl residues through larger fragments that affect the overall physicochemical properties of the conjugate. Appropriate selection of capping groups allows either relatively hydrophilic or hydrophobic molecules to be delivered to the target site. In one embodiment, ^Xn is acetyl.

2) ^Xc (C-terminal cap)
In embodiments herein where the therapeutic agent is attached to the N-terminus of the peptidic substrate (ie, the conjugate has formula II), the C-terminus of the peptidic substrate is a carboxylic acid or a carboxamide derivative. . Appropriate selection of capping groups allows either relatively hydrophilic or hydrophobic molecules to be delivered to the target site.

であるか；または、
(ii)Ｒ^ｄおよびＲ^ｅは、一緒に−ＣＨ_２ＣＨ_２ＯＣＨ_２ＣＨ_２−ジラジカル；
[ｂは０もしくは１〜１００の整数であり；
ｃは、もしｂが０であるならば、ｃは１であるという条件で、０もしくは１である]を形成する。
一つの実施態様では、Ｒ^ｄは、水素であり、Ｒ^ｅは、２−ヒドロキシエチルである。 In one embodiment, X ^c , together with the carbonyl group to which it is attached, has the formula —C (O) NR ^d R ^e , wherein R ^d and R ^e are as follows (i ) Or (ii) is selected:
(i) R ^d and R ^e are each independently hydrogen, C ₁ -C ₆ alkyl, —C ₁ -C ₆ alkyl-OH, —C ₁ -C ₆ alkyl-di-OH, —C ₁ -C ₆ alkyl-tri-OH and at least one of R ^d and R ^e is not hydrogen or C ₁ -C ₆ alkyl,

Or
(ii) R ^d and R ^e are taken together —CH ₂ CH ₂ OCH ₂ CH ₂ -diradical;
[b is 0 or an integer of 1 to 100;
c is 0 or 1 if b is 0, provided that c is 1.]
In one embodiment, R ^d is hydrogen and R ^e is 2-hydroxyethyl.

2. The linker conjugate optionally contains a linker (ie, L, L ¹ or L ^{2 of} formulas I, II and III) that conjugates the peptidic substrate to the therapeutic agent. A linker is a substrate in which a peptidic moiety is a substrate for a cell surface protease and the therapeutic agent is substantially inactive when in a conjugate, in an active form or of a targeted tissue. Any that results in a conjugate that is released in a form that is subsequently activated by a cell, tissue or environment.

  For example, the linker can include carbohydrates, peptides, diamines, arylamines and / or hydrocarbon core structures. The linker is desirably synthetically available, provides a stable product and does not possess any inherent biological activity that interferes with the conjugate activity. They can add desirable properties to increase solubility or to aid in the transport of cleaved therapeutic agents in the cell. In certain embodiments, certain linkers will be enzymatically cleaved in vitro and in vivo and fragmented to release an active or activatable therapeutic agent. In embodiments where the therapeutic agent is doxorubicin, the linker is a sugar and / or peptide, such as, for example, the amino sugar daunosamine.

  In one embodiment, a linker for use herein includes an amine moiety on a therapeutic agent linked to a linker unit to form an amide bond, and the amino terminus of the peptidic substrate is the other of the linker unit. Examples include, but are not limited to, biscarbonylalkyl diradicals that are linked to the termini to form amide bonds. Conversely, the carbonyl moiety on the cytotoxic agent is covalently attached to one of the amines of the linker moiety, while the other amine of the linker unit is covalently attached to the C-terminus of the peptidic substrate. Diaminoalkyl diradical linker units may also be useful. Other such linkers that are stable to physiological environments in the absence of cell surface proteases but that are cleavable by cleavage of cell surface protease proteolytic cleavage sites are described herein. Intended for use. Furthermore, in the cleavage of the cell surface protease proteolytic cleavage site, it remains attached to the therapeutic agent, but when compared to the unmodified therapeutic agent, the therapeutic activity of the therapeutic agent derivative after such cleavage is Linker units that are not significantly reduced can be utilized.

  In other embodiments, the linker is a diamine that includes a cyclic alkyl moiety, and in certain embodiments, the diamine includes a bicycloalkylene moiety. Examples of such diamine linkers include 1,4-bis (aminomethyl) cyclohexane, 1,4-bis (aminomethyl) cycloheptane, 1,3-bis (aminomethyl) cyclopentane, 1-amino-4 Examples include, but are not limited to,-(aminomethyl) cyclohexane, 1,4-diaminocyclohexane and 1,4-bis (aminomethyl) bicyclo [2.2.2] octane.

  Other linkers include, but are not limited to, 1,3-diaminopropane, 1, ω-diaminoalkanes, and oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid and pivalic acid, Non-limiting examples include 1, ω-dicarbonylalkane.

[式中、ＡはＮＨもしくはＯであり；ＤはＮ(Ｈもしくはアルキル)またはＯであり；Ｒ^２５はＨ、アルキル、シクロアルキル、シクロアルキルアルキル、アリール、例えば、１〜３個のような１個またはそれ以上のハロ、ハロアルキルおよびアルキルから選択される置換基で任意に置換されたヘテロアリール、アラルキル、ヘテロアラルキル、１〜２個の二重結合を含むアルケニル、１〜２個の三重結合を含むアルキニル、アルケ(エン)(イン)イル基、ハロ、擬似ハロ、シアノ、ヒドロキシ、例えば、ハロ低級アルキル、特にトリフルオロメチル、のようなハロアルキルおよびポリハロアルキル、ホルミル、アルキルカルボニル、例えば１〜３個のような１個もしくはそれ以上の、例えば、ハロ、ハロアルキルおよびアルキルから選択される置換基で任意に置換されたところのアリールカルボニル、ヘテロアリールカルボニル、カルボキシ、アルコキシカルボニル、アリールオキシカルボニル、アミノイミノ、アルコキシカルボニルアミノ、アリールオキシカルボニルアミノ、アミノカルボニル、アルキルアミノカルボニル、ジアルキルアミノカルボニル、アリールアミノカルボニル、ジアリールアミノカルボニル、アラルキルアミノカルボニル、アルコキシ、アリールオキシ、パーフルオロアルコキシ、アルケニルオキシ、アルキニルオキシ、アリールアルコキシ、アミノアルキル、アルキルアミノアルキル、ジアルキルアミノアルキル、アリールアミノアルキル、アミノ、アルキルアミノ、ジアルキルアミノ、アリールアミノ、アルキルアリールアミノ、アルキルカルボニルアミノ、アリールカルボニルアミノ、アジド、ニトロ、メルカプト、アルキルチオ、アリールチオ、パーフルオロアルキルチオ、チオシアノ、イソチオシアノ、アルキルスルフィニル、アルキルスルホニル、アリールスルフィニル、アリールスルホニル、アミノスルホニル、アルキルアミノスルホニル、ジアルキルアミノフルホニルおよびアリールアミノスルホニル、であり；ならびにｙは、１〜３の整数である]のもののような自己除去するリンカーが挙げられる。 Additional linkers for use in the conjugates provided herein include the following formulas:

[Wherein A is NH or O; D is N (H or alkyl) or O; R ²⁵ is H, alkyl, cycloalkyl, cycloalkylalkyl, aryl, such as 1-3 Heteroaryl optionally substituted with one or more substituents selected from halo, haloalkyl and alkyl, aralkyl, heteroaralkyl, alkenyl containing 1-2 double bonds, 1-2 triple bonds Including alkynyl, alk (en) (yn) yl groups, halo, pseudohalo, cyano, hydroxy, haloalkyl and polyhaloalkyl such as halo lower alkyl, especially trifluoromethyl, formyl, alkylcarbonyl, such as 1 to One or more such as three, eg, selected from halo, haloalkyl and alkyl Arylcarbonyl, heteroarylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminoimino, alkoxycarbonylamino, aryloxycarbonylamino, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl optionally substituted with groups , Diarylaminocarbonyl, aralkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, amino, alkylamino, dialkylamino, Arylamino, alkylarylamino, alkylcarbonylamino , Arylcarbonylamino, azide, nitro, mercapto, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl and arylamino Sulfonyl, and as well as y is an integer from 1 to 3].

3. Therapeutic agent conjugates are intended to modify various biological responses. Thus, a therapeutic agent is any agent, including proteins and polypeptides, small molecules and other molecules that possess or enhance the desired biological activity. Such molecules include, but are not limited to, cytotoxic agents and toxic moieties thereof such as abrin, ricin A, Pseudomonas aeruginosa exotoxin, Shiga toxin, diphtheria toxin and other such toxins. And / or subunits or chains; tumor necrosis factor, α-interferon, γ-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, such as but not limited to a protein; or, for example, , Lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor (G -CSF), erythropoietin (EPO), tissue factor and tissue factor variants Biological response modifiers such as clotting factors, pro-apoptotic agents such as FAS-ligands, fibroblast growth factor (FGF), nerve growth factors and other growth factors. Each must be in a form that can enter the cell or otherwise exhibit a therapeutic effect when in its vicinity.

  Thus, therapeutic agents include, but are not limited to, anti-tumor agents, anti-angiogenic agents, pro-apoptotic agents, anti-cancer agents and anti-cell division agents. These are optionally linked via a linker to a substrate such as a peptidic substrate that is a substrate for the protease.

  Among the therapeutic agents are generally cytotoxic agents, including but not limited to alkylating agents, toxins, antiproliferative agents and tubulin binding agents. Classes of cytotoxic agents for use herein include, for example, anthracycline family drugs, vinca drugs, mitomycin, bleomycin, cytotoxic nucleosides, pteridine family drugs, diynenes, maytansinoids, epothilones, taxanes and Podophyllotoxins are mentioned.

  Exemplary members of these classes include, for example, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, metopterin, dichloromethotrexate, mitomycin C, porphyromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside. , Podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leulocidin, vindesine, leucine, maytansinol, epothilone A or B, taxotere, taxol, etc. It is done. Other such therapeutic agents include estramustine, cisplatin, combretastatin and analogs, and cyclophosphamide. One skilled in the art can make chemical modifications to the desired therapeutic agent to make the reaction of the compound more convenient for the purpose of creating conjugates.

  Specific therapeutic agents include the following drugs. Those skilled in the art will appreciate that these structural formulas are merely exemplary and that such compounds or their derivatives or analogs have different common or common names in the art.

式中、
Ｒ^１２は、アミノもしくはヒドロキシであり；
Ｒ^７は、水素もしくはメチルであり；
Ｒ^８は、水素、フルオロ、クロロ、ブロモもしくはヨードであり；
Ｒ^９は、ヒドロキシもしくはカルボン酸の塩を完成するところの部分である。 a. Methotrexate group of formula (1):

Where
R ¹² is amino or hydroxy;
R ⁷ is hydrogen or methyl;
R ⁸ is hydrogen, fluoro, chloro, bromo or iodo;
R ⁹ is the part where the hydroxy or carboxylic acid salt is completed.

式中、Ｒ^１０は水素もしくはメチルである。 b. Mitomycin group of formula (2):

In the formula, R ¹⁰ is hydrogen or methyl.

式中、Ｒ^１１は、ヒドロキシ、アミノ、Ｃ_１〜Ｃ_３アルキルアミノ、ジ(Ｃ_１〜Ｃ_３アルキル)アミノ、Ｃ_４〜Ｃ_６ポリメチレンアミノ、−ＮＨＣＨ_２ＣＨ_２ＣＨ_２ＣＨ_２ＮＨ−Ｃ(ＮＨ)ＮＨ_２もしくは−ＮＨＣＨ_２ＣＨ_２ＣＨ_２Ｓ^＋(ＣＨ_３)_２である。 c. Bleomycin group of formula (3):

In the formula, R ¹¹ represents hydroxy, amino, C ₁ -C ₃ alkylamino, di (C ₁ -C ₃ alkyl) amino, C ₄ -C ₆ polymethyleneamino, —NHCH ₂ CH ₂ CH ₂ CH ₂ NH—. C (NH) NH ₂ or —NHCH ₂ CH ₂ CH ₂ S ⁺ (CH ₃ ) ₂ .

d. Melphalan of formula (4):

e. Mercaptopurine of formula (5):

f. Cytosine arabinoside of formula (6):

式中、
Ｒ^１３は水素もしくはメチルであり；および
Ｒ^１４はメチルもしくはチエニルまたはそのリン酸塩である。 g. Podophyllotoxin of formula (7):

Where
R ¹³ is hydrogen or methyl; and R ¹⁴ is methyl or thienyl or a phosphate thereof.

式中、
Ｒ^１７およびＲ^１８が単一に用いられるときには、Ｒ^１５はＨ、ＣＨ_３もしくはＣＨＯであり：そしてＲ^１８はＨであり、そしてＲ^１６およびＲ^１７の一つはエチルであり、他はＨもしくはＯＨであり；
Ｒ^１７およびＲ^１８が、それらが付着されるところの炭素と一緒に用いられるときには、それらは、Ｒ^１６がエチルである場合にはオキシラン環を形成し；ならびに、
Ｒ^１９は、水素、(Ｃ_１〜Ｃ_３アルキル)−ＣＯもしくはクロロ置換された(Ｃ_１〜Ｃ_３アルキル)−ＣＯである。 h. Drug group of vinca alkaloids of formula (8):

Where
When R ¹⁷ and R ¹⁸ are used singly, R ¹⁵ is H, CH ₃ or CHO: and R ¹⁸ is H and one of R ¹⁶ and R ¹⁷ is ethyl and the other is H Or OH;
When R ¹⁷ and R ¹⁸ are used together with the carbon to which they are attached, they form an oxirane ring when R ¹⁶ is ethyl; and
R ¹⁹ is hydrogen, (C ₁ -C ₃ alkyl) -CO or chloro-substituted (C ₁ -C ₃ alkyl) -CO.

[式中、ペプチド性基質は、式ＩおよびＩＩに対する上記の通りであり；Ｌは、−ＮＨ−(ＣＨ_２)_ｕ−Ｔ−(ＣＨ_２)_ｕ−ＮＨ−のようなリンカーであり；Ｘ^ｎは、
ａ)水素、
ｂ)−(Ｃ＝Ｏ)Ｒ^１ａ、
ｃ)

ｄ)

ｅ)

ｆ)エトキシスクアリン酸エステル；ならびに
ｇ)コチニニル、であり；
Ｒ^１およびＲ^２は、独立に、水素、ＯＨ、Ｃ_１〜Ｃ_６アルキル、Ｃ_１〜Ｃ_６アルコキシ、Ｃ_１〜Ｃ_６アラルキルおよびアリールであり；
Ｒ^１ａは、Ｃ_１〜Ｃ_６アルキル、水酸化Ｃ_３〜Ｃ_８シクロアルキル、多水酸化Ｃ_３〜Ｃ_８シクロアルキル、水酸化アリール、多水酸化アリールもしくはアリールであり、
Ｒ^１９は、水素、(Ｃ_１〜Ｃ_３アルキル)−ＣＯもしくはクロロ置換された(Ｃ_１〜Ｃ_３アルキル)−ＣＯであり；
Ｔは、シクロペンチル、シクロヘキシル、シクロヘプチルもしくはビシクロ[２.２.２]オクタニルから選択され；
ａは、１、２、３もしくは４であり；
ｂは、ゼロもしくは１〜１００の整数であり；
ｃは、もしｂが０であるならばｃは１であることを条件として、０もしくは１であり；
ｇは、１、２もしくは３であり；
ｕは、０、１、２もしくは３である；]のもの
または、それらの薬学的に許容される誘導体、が挙げられる。 The conjugates provided herein wherein the therapeutic agent is the vinca alkaloid vinblastine include the formula:

[Wherein the peptidic substrate is as described above for Formulas I and II; L is a linker such as —NH— (CH ₂ ) _u —T— (CH ₂ ) _u —NH—; ⁿ is
a) hydrogen,
b)-(C = O) R ^1a ,
c)

d)

e)

f) ethoxy squarate ester; and g) cotininyl;
R ¹ and R ² are independently hydrogen, OH, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ aralkyl and aryl;
R ^1a is C ₁ -C ₆ alkyl, hydroxylated C ₃ -C ₈ cycloalkyl, polyhydroxylated C ₃ -C ₈ cycloalkyl, aryl hydroxide, polyhydroxylated aryl or aryl,
R ¹⁹ is hydrogen, (C ₁ -C ₃ alkyl) -CO or chloro-substituted (C ₁ -C ₃ alkyl) -CO;
T is selected from cyclopentyl, cyclohexyl, cycloheptyl or bicyclo [2.2.2] octanyl;
a is 1, 2, 3 or 4;
b is zero or an integer from 1 to 100;
c is 0 or 1 provided that c is 1 if b is 0;
g is 1, 2 or 3;
u is 0, 1, 2 or 3;] or a pharmaceutically acceptable derivative thereof.

式中、Ｒ^２１は式：

[式中、Ｒ^２２は、水素、メチル、ブロモ、フルオロ、クロロもしくはヨードであり；
Ｒ^２３は、ＯＨもしくは−ＮＨ_２であり；
Ｒ^２４は、水素、ブロモ、クロロもしくはヨードである]の一つの塩基である。 i. Difluoronucleoside of formula (9):

Wherein R ²¹ is the formula:

[Wherein R ²² is hydrogen, methyl, bromo, fluoro, chloro or iodo;
R ²³ is OH or —NH ₂ ;
R ²⁴ is hydrogen, bromo, chloro or iodo].

j. Estramustine (10):

k. Cyclophosphamide (11):

式中、
Ｒ^ａは、−ＣＨ_３、−ＣＨ_２ＯＨ、−ＣＨ_２ＯＣＯ(ＣＨ_２)_３ＣＨ_３もしくは−ＣＨ_２ＯＣＯＣＨ(ＯＣ_２Ｈ_５)_２であり；
Ｒ^ｂは、−ＯＣＨ_３、−ＯＨもしくは−Ｈであり；
Ｒ^ｃは、−ＮＨ_２、−ＮＨＣＯＣＦ_３、４−モルホリニル、３−シアノ−４−モルホリニル、１−ピペリジニル、４−メトキシ−１−ピペリジニル、ベンジルアミン、ジベンジルアミン、シアノメチルアミンもしくは１−シアノ−２−メトキシエチルアミンであり；
Ｒ^５は、−ＯＨ、−ＯＴＨＰもしくは−Ｈであり；および
Ｒ^６は、Ｒ^５が−ＯＨもしくは−ＯＴＨＰであるときには、Ｒ^６は−ＯＨではないことを条件として、−ＯＨもしくは−Ｈである。 l. Anthracycline antibiotics of formula (12):

Where
R ^a is —CH ₃ , —CH ₂ OH, —CH ₂ OCO (CH ₂ ) ₃ CH ₃ or —CH ₂ OCOCH (OC ₂ H ₅ ) ₂ ;
R ^b is —OCH ₃ , —OH or —H;
R ^c is —NH ₂ , —NHCOCF ₃ , 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine, cyanomethylamine or 1-cyano. -2-methoxyethylamine;
R ⁵ is —OH, —OTHP, or —H; and R ⁶ is —OH or —H, provided that when R ⁵ is —OH or —OTHP, R ⁶ is not —OH. is there.

Table 2 below provides a number of anthracycline drugs and their common or common names:

^ａダウノルビシンは、ダウノマイシンに対する別名である。
^ｂドキソビシンは、アドリアマイシンに対する別名である。

^a Daunorubicin is another name for daunomycin.
^b doxobicin is another name for adriamycin.

  In one embodiment, when the therapeutic agent is doxorubicin, it is attached to the peptidic substrate via the amino group of the aminoglycoside portion of doxorubicin.

m. Maytansinol:

n. Epothilone A or B:

式中、ＲはＰｈＣ(Ｏ)もしくはｔ−ＢｕＯＣ(Ｏ)である。 o. Taxol:

In the formula, R is PhC (O) or t-BuOC (O).

  In one embodiment, when the therapeutic agent is taxol (R = C (O) Ph), the peptidic substrate is attached to the secondary hydroxyl group of the cyclohexane moiety of taxol.

p. Ribosome-inactivating proteins Ribosome-inactivating proteins (RIPs), including lysine, abrin and saporin, are plant proteins that catalytically inactivate eukaryotic ribosomes. RIP inactivates ribosomes by interfering with the protein elongation step of protein synthesis. For example, RIP saporin (hereinafter also referred to as SAP) has been shown to enzymatically inactivate the 60S ribosome by cleavage of the n-glycoside bond of adenine at position 4324 in rat 28S ribosomal RNA (rRNA). . Certain RIPs, such as the toxins abrin and ricin, have two constituent chains: a cell-binding chain that mediates binding to cell surface receptors and internalization of the molecule; and an enzymatic responsible for protein synthesis inhibitory activity. Containing an active chain. Such RIP is a type II RIP. Other RIPs such as saporin are single chain and are referred to as type I RIPs. Since such RIPs lack a cell binding chain, they are less toxic to the whole cell than RIPs with double chains. As long as the single strand is not further bound to an agent such as a growth factor that mediates binding and internalization, double stranded RIP is generally used herein for binding.

  Several structurally related RIPs have been isolated from seeds and leaves of the plant Saponaria officinalis. Of these, SAP-6 is the most active and abundant, representing 7% of the total seed protein. Saporin is very stable, has a high isoelectric point, does not contain carbohydrates, and is resistant to denaturing agents such as dodecyl sulfate (SDS) and various proteases. The amino acid sequences of several saporin-6 isoforms from seed are known and there appears to be a family of saporin RIPs that differ in a few amino acid residues. Since saporin is a type I RIP, it does not carry a cell binding chain. As a result, its toxicity to whole cells is significantly lower than other toxins such as ricin and abrin. However, when internalized by eukaryotic cells, its cytotoxicity is 100-1000 times more potent than ricin A chain.

4). Exemplary Conjugates The conjugates provided herein identify suitable peptidic substrates or their soluble, shed or released forms for targeted cell surface proteases and peptidic substrates ( It is created by forming a conjugate with the therapeutic agent (s). For example, exemplary conjugates containing peptidic substrates designed for cleavage by MTSP1, endotheliase and urokinase are described. A cell-relevance in or related to a cell involved in a disease or other abnormality of interest, or a cell that is involved or related to a disease or other abnormality of interest Once a cell surface protease, including a cell localized protease or soluble, shed or released form thereof, is identified, a suitable peptidic substrate for it can be empirically designed and then exemplified herein. It is understood that it can be conjugated to a therapeutic agent.

In certain embodiments, conjugates for use in the compositions and methods provided herein include the following:
Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 46);
Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 47);
Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 48);
Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 49);
Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic agent) (SEQ ID NO: 50);
Ac-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (Therapeutic) (SEQ ID NO: 51);
Ac-Pro-Arg-Phe-Lys-Ile-Ile- (Therapeutic) (SEQ ID NO: 52);
Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 53);
Ac-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 54);
Ac-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 55);
Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 56);
Ac-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 57);
Ac-Pro-Arg-Phe-Arg-Ile-Ile- (Therapeutic) (SEQ ID NO: 58);
Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 59);
Ac-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 60); and
Ac-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 61).

In a further embodiment herein, the conjugate is: Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 62); Ac-Leu- Arg-Ala-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 63); Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 63) : 64); and Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 65).
In other embodiments, the conjugate is:
Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 66);
Ac-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (Therapeutic) (SEQ ID NO: 67);
Ac-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 68);
Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 69);
Ac-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 70);
Ac-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 71);
Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 72);
Ac-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 73);
Ac-Pro-Arg-Phe-Arg-Ile-Ile- (Therapeutic) (SEQ ID NO: 74);
Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 75);
Ac-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 76); and
Ac-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 77).

In other embodiments, a binding rod for use herein includes:
Pyro Glu-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 78);
CH ₃ SO ₂ -D-HHT-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 79);
Np-tosyl-Gly-Pro-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 80);
Benzoyl-Val-Gly-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 81);
CH ₃ SO ₂ -D-HHT-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 82);
N-α-ZD-Arg-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 83) (Z = benzyloxycarbonyl);
Pyro Glu-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 84);
HD-Ile-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 85);
Cbo-L- (γ) Glu (α-t-BuO) -Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 86) (Cbo = carbobenzoxy);
HD-Pro-Phe-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 87);
HD-Val-Leu-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 88);
Bz-Ile-Glu (γ-OH) -Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 89) (Bz = benzoyl);
Bz-Ile-Glu (γ-OMe) -Gly-Arg-Ala-Ala- (Therapeutic) (SEQ ID NO: 90);
Benzoyl-Pro-Phe-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 91);
HD-Phe-Pip-Arg-Ala-Ala- (Therapeutic) (SEQ ID NO: 92);
HD-Val-Leu-Lys-Ala-Ala- (therapeutic) (SEQ ID NO: 93);
HD-Nle-HHT-Lys-Ala-Ala- (therapeutic) (SEQ ID NO: 94);
Pyr-Arg-Thr-Lys-Arg-Ala-Ala- (Therapeutic) (SEQ ID NO: 95);
H-Arg-Gln-Arg-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 96);
Boc-Gln-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 97);
Z-Arg-Arg-Ala-Ala- (Therapeutic) (SEQ ID NO: 98);
HD-HHT-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 99);
HD-CHT-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 100);
MeSO ₂ -dPhe-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 101);
δ-ZD-Lys-Pro-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 102); and
CH ₃ SO ₂ -D-CHA-But-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 103).

In other embodiments, conjugates for use in the compositions and methods provided herein include:
Ac-Arg-Gln-Ser-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 104);
Ac-Arg-Arg-Gln-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 105);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 106);
Ac-Arg-Gln-Ser-Arg-Ala- (therapeutic) (SEQ ID NO: 107);
Ac-Arg-Arg-Gln-Ser-Arg-Ala- (therapeutic agent) (SEQ ID NO: 108);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Gly-Gly- (therapeutic) (SEQ ID NO: 109);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ala- (therapeutic) (SEQ ID NO: 110);
Ac-Arg-Arg-Gln-Ser-Arg-Ile- (therapeutic) (SEQ ID NO: 111); and
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ala-Ile- (therapeutic agent) (SEQ ID NO: 112).

In certain embodiments, conjugates for use in the methods and compositions provided herein include:
Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 113);
Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 114);
Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 115);
Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 116);
Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 117);
Ac-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 118);
Ac-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 119);
Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 120);
Ac-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 121);
Ac-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 122);
Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 123);
Ac-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 124);
Ac-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 125);
Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 126);
Ac-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 127); and
Ac-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 128).

In a further embodiment herein, the conjugate is: Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Leu- (therapeutic agent) (SEQ ID NO: 129); Ac-Leu- Arg-Ala-Quat-Ala-Arg-Ser-Leu- (Therapeutic) (SEQ ID NO: 130); Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Leu- (Therapeutic) (SEQ ID NO: : 131); and Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 132).
In another embodiment herein, the conjugate is: Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic agent) (SEQ ID NO: 133);
Ac-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 134);
Ac-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 135);
Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 136);
Ac-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 137);
Ac-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 138);
Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 139);
Ac-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 140);
Ac-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 141);
Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 142);
Ac-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 143); and
Ac-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 144).

In other embodiments, conjugates for use herein include the following:
Pyro Glu-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 145);
CH ₃ SO ₂ -D-HHT-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 146);
Np-tosyl-Gly-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 147);
Benzoyl-Val-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 148);
CH ₃ SO ₂ -D-HHT-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 149);
N-α-ZD-Arg-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 150) (Z = benzyloxycarbonyl);
Pyro Glu-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 151);
HD-Ile-Pro-Arg-Ser-Leu- (Therapeutic) (SEQ ID NO: 152);
Cbo-L- (γ) Glu (α-t-BuO) -Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 153) (Cbo = carbobenzoxy);
HD-Pro-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 154);
HD-Val-Leu-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 155);
Bz-Ile-Glu (γ-OH) -Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 156) (Bz = benzoyl);
Bz-Ile-Glu (γ-OMe) -Gly-Arg-Ser-Leu- (Therapeutic) (SEQ ID NO: 157);
Benzoyl-Pro-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 158);
HD-Phe-Pip-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 159);
HD-Val-Leu-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 160);
HD-Nle-HHT-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 161);
Pyr-Arg-Thr-Lys-Arg-Ser-Leu- (Therapeutic) (SEQ ID NO: 162);
H-Arg-Gln-Arg-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 163);
Boc-Gln-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 164);
Z-Arg-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 165);
HD-HHT-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 166);
HD-CHT-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 167);
MeSO ₂ -dPhe-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 168);
δ-ZD-Lys-Pro-Arg-Ser-Leu- (therapeutic agent) (SEQ ID NO: 169); and
CH ₃ SO ₂ -D-CHA-But-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 170).

In other embodiments, conjugates for use in the compositions and methods provided herein include:
Ac-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 171);
Ac-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 172);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 173);
Ac-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 174);
Ac-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 175);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 176);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 177);
Ac-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 178); and
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 179).
In other embodiments, the conjugates provided herein include:
Ac-Arg-Gln-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 180);
Ac-Arg-Gln-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 181);
Ac-Arg-Gln-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 182);
Ac-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 183);
Ac-Arg-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 184);
Ac-Arg-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 185);
Ac-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 186);
Ac-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 187);
Ac-Gln-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 188);
Ac-Gln-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 189); and
Ac-Gln-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 190).

In further embodiments, conjugates for use in the compositions and methods provided herein include:
Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 191);
Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 192);
Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 193);
Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 194);
Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 195);
Ac-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 196);
Ac-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 197);
Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 198);
Ac-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 199);
Ac-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 200);
Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 201);
Ac-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 202);
Ac-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 203);
Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic agent) (SEQ ID NO: 204);
Ac-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 205); and
Ac-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 206).

  In a further embodiment herein, the conjugate is: Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic agent) (SEQ ID NO: 207); Ac- Leu-Arg-Ala-Quat-Ala-Arg-Ser-Ser-Leu- (Therapeutic) (SEQ ID NO: 208); Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Ser-Leu- ( (Therapeutic) (SEQ ID NO: 209); and Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Ser-Leu- (Therapeutic) (SEQ ID NO: 210).

In other embodiments, the conjugate is:
Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 211);
Ac-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 212);
Ac-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 213);
Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 214);
Ac-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 215);
Ac-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 216);
Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 217);
Ac-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 218);
Ac-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 219);
Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 220);
Ac-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 221); and
Ac-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 222).

In other embodiments, conjugates for use herein include:
Pyro Glu-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 223);
CH ₃ SO ₂ -D-HHT-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 224);
Np-tosyl-Gly-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 225);
Benzoyl-Val-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 226);
CH ₃ SO ₂ -D-HHT-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 227);
N-α-ZD-Arg-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 228) (Z = benzyloxycarbonyl);
Pyro Glu-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 229);
HD-Ile-Pro-Arg-Ser-Ser-Leu- (Therapeutic) (SEQ ID NO: 230);
Cbo-L- (γ) Glu (α-t-BuO) -Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 231) (Cbo = carbobenzoxy);
HD-Pro-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 232);
HD-Val-Leu-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 233);
Bz-Ile-Glu (γ-OH) -Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 234) (Bz = benzoyl);
Bz-Ile-Glu (γ-OMe) -Gly-Arg-Ser-Ser-Leu- (Therapeutic) (SEQ ID NO: 235);
Benzoyl-Pro-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 236);
HD-Phe-Pip-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 237);
HD-Val-Leu-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 238);
HD-Nle-HHT-Lys-Ser-Ser-Leu- (Therapeutic) (SEQ ID NO: 239);
Pyr-Arg-Thr-Lys-Arg-Ser-Ser-Leu- (Therapeutic) (SEQ ID NO: 240);
H-Arg-Gln-Arg-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 241);
Boc-Gln-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 242);
Z-Arg-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 243);
HD-HHT-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 244);
HD-CHT-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 245);
MeSO ₂ -dPhe-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 246);
δ-ZD-Lys-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 247); and
CH ₃ SO ₂ -D-CHA-But-Arg-Ser-Ser-Leu- (Therapeutic) (SEQ ID NO: 248).

In other embodiments, conjugates for use in the compositions and methods provided herein include:
Ac-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 249);
Ac-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 250);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 251);
Ac-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 252);
Ac-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic agent) (SEQ ID NO: 253);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 254);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 255);
Ac-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 256); and
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 257).

In other embodiments, the conjugates provided herein include:
Ac-Arg-Gln-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 258);
Ac-Arg-Gln-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 259);
Ac-Arg-Gln-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 260);
Ac-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 261);
Ac-Arg-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 262);
Ac-Arg-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 263);
Ac-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 264);
Ac-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 265);
Ac-Gln-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 266);
Ac-Gln-Ala-Arg-Ser-Ser-Leu- (therapeutic agent) (SEQ ID NO: 267); and
Ac-Gln-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 268).

In other embodiments, the conjugates provided herein include:
Ac-Gly-dSer-Ala-Arg-Ser-Ala- (therapeutic) (SEQ ID NO: 569);
Ac-Arg-Gly-dSer-Ala-Arg-Ser-Ala- (therapeutic) (SEQ ID NO: 570);
Ac-Gly-Ser-Gly-Arg-Ser-Ala- (therapeutic) (SEQ ID NO: 571);
Ac-Arg-Gly-Ser-Gly-Arg-Ser-Ala- (therapeutic agent) (SEQ ID NO: 572);
Ac-Leu-Arg-Gly-Ser-Gly-Arg-Ser-Ala- (therapeutic) (SEQ ID NO: 573);
Ac-Leu-Arg-Gly-dSer-Ala-Arg-Ser-Ala- (therapeutic) (SEQ ID NO: 574);
Ac-Cys (Me) -Pro-Gly-Arg-Val-Val- (therapeutic) (SEQ ID NO: 575);
Ac-Arg-Cys (Me) -Pro-Gly-Arg-Val-Val- (therapeutic) (SEQ ID NO: 577);
Ac-Arg-Arg-Cys (Me) -Pro-Gly-Arg-Val-Val- (therapeutic agent) (SEQ ID NO: 578);
Ac-Val-Ser-Ala-Arg-Met-Ala- (therapeutic) (SEQ ID NO: 579);
Ac-Ile-Val-Ser-Ala-Arg-Met-Ala- (therapeutic) (SEQ ID NO: 580);
Ac-Val-Ile-Val-Ser-Ala-Arg-Met-Ala- (therapeutic) (SEQ ID NO: 581);
Ac-Val-Ile-Val-Ser-Ala-Arg-nLeu-Ala- (therapeutic) (SEQ ID NO: 582);
Ac-Val-Ser-Ala-Arg-nLeu-Ala- (therapeutic) (SEQ ID NO: 583);
Ac-Ile-Val-Ser-Ala-Arg-nLeu-Ala- (therapeutic) (SEQ ID NO: 584);
Ac-Gly-Ser-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 585);
Ac-Gly-Ser-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 586);
Ac-Gly-Ser-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 587);
Ac-Ser-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 588);
Ac-Ser-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 589);
Ac-Ser-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 590);
Ac-Arg-Gly-Ser-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 591);
Ac-Arg-Gly-Ser-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 592);
Ac-Arg-Gly-Ser-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 593);
Ac-Leu-Arg-Gly-Ser-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 594);
Ac-Leu-Arg-Gly-Ser-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 595); and
Ac-Leu-Arg-Gly-Ser-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 596).

In other embodiments, the conjugates provided herein include:
Ac-RQGRSL- (therapeutic agent) (SEQ ID NO: 491);
Ac-RQGRSSL- (therapeutic agent) (SEQ ID NO: 492);
Ac-RQGRS-nL- (therapeutic) (SEQ ID NO: 493);
Ac-RQGRS-nV- (therapeutic) (SEQ ID NO: 494);
Ac-RQGRSF- (therapeutic agent) (SEQ ID NO: 495);
Ac-RQGRAL- (therapeutic) (SEQ ID NO: 496);
Ac-RQGRAL- (therapeutic agent) (SEQ ID NO: 497);
Ac-RQGRA-nL- (therapeutic agent) (SEQ ID NO: 498);
Ac-RQGRA-nL- (therapeutic agent) (SEQ ID NO: 499);
Ac-RQGRA-nV- (therapeutic agent) (SEQ ID NO: 500);
Ac-RQGRA-Cha- (therapeutic) (SEQ ID NO: 501);
Ac-RQGRAF- (therapeutic agent) (SEQ ID NO: 502);
Ac-RNGRSL- (therapeutic agent) (SEQ ID NO: 503);
Ac-RNGRA-nL- (therapeutic agent) (SEQ ID NO: 504);
Ac-RQARSL- (therapeutic agent) (SEQ ID NO: 505);
Ac-RQARS-nL- (therapeutic) (SEQ ID NO: 506);
Ac-RQARS-nV- (therapeutic) (SEQ ID NO: 507);
Ac-RQAAS-Cha- (therapeutic) (SEQ ID NO: 508);
Ac-RQARSS-Cha- (therapeutic) (SEQ ID NO: 509);
Ac-RQART-nL- (therapeutic agent) (SEQ ID NO: 510);
Ac-RQARAL- (therapeutic agent) (SEQ ID NO: 511);
Ac-RQARA-nL- (therapeutic) (SEQ ID NO: 512);
Ac-RQARA-nV- (therapeutic) (SEQ ID NO: 513);
Ac-RQARA-Cha- (therapeutic) (SEQ ID NO: 514);
Ac-RQSRAA- (therapeutic) (SEQ ID NO: 515);
Ac-RQSRA- (therapeutic agent) (SEQ ID NO: 516);
Ac-RQSRA-nL- (therapeutic) (SEQ ID NO: 517);
Ac-RQSRAL- (therapeutic agent) (SEQ ID NO: 518);
Ac-RQSRA-nV- (therapeutic) (SEQ ID NO: 519);
Ac-RQSRA-Cha- (therapeutic) (SEQ ID NO: 520);
Ac-RQSRSSL- (therapeutic) (SEQ ID NO: 521);
Ac-RQSRSL- (therapeutic agent) (SEQ ID NO: 522);
Ac-RQSRS-nL- (therapeutic) (SEQ ID NO: 523);
Ac-RQSRS-nL- (therapeutic agent) (SEQ ID NO: 524);
Ac-RQSRS-nV- (therapeutic agent) (SEQ ID NO: 525);
Ac-RQSRS-allyl G- (therapeutic) (SEQ ID NO: 526);
Ac-RQSRS-Cha- (therapeutic) (SEQ ID NO: 527);
Ac-RQSRT-nL- (therapeutic) (SEQ ID NO: 528);
Ac-RQTRSSL- (therapeutic) (SEQ ID NO: 529);
Ac-RQTRSL- (therapeutic agent) (SEQ ID NO: 530);
Ac-RNSRS-nL- (therapeutic agent) (SEQ ID NO: 531);
Ac-RQFRSL- (therapeutic agent) (SEQ ID NO: 532);
Ac-RQFRS-nL- (therapeutic) (SEQ ID NO: 534);
Ac-RQFRS-nV- (therapeutic) (SEQ ID NO: 535);
Ac-RQFRS-nL- (therapeutic) (SEQ ID NO: 536);
Ac-RQFRS-Cha- (therapeutic) (SEQ ID NO: 537);
Ac-RQFRAL- (therapeutic agent) (SEQ ID NO: 538);
Ac-RQFRA-nL- (therapeutic agent) (SEQ ID NO: 539);
Ac-RQFRA-nV- (therapeutic) (SEQ ID NO: 540);
Ac-RQFRA-Cha- (therapeutic) (SEQ ID NO: 541);
Ac-QSRSS-nL- (therapeutic agent) (SEQ ID NO: 542);
MeOCO-Quat2-GRSL- (therapeutic) (SEQ ID NO: 483);
MeOCO-Quat3-GRSL- (therapeutic) (SEQ ID NO: 484);
MeOCO-Quat-GRSL- (therapeutic) (SEQ ID NO: 485);
MeOCO-Quat4-GRSL- (therapeutic agent) (SEQ ID NO: 486);
MeOCO-Quat5-GRSL- (therapeutic agent) (SEQ ID NO: 487);
MeOCO-Quat2-GRSSL- (therapeutic) (SEQ ID NO: 488);
MeOCO-Quat4-GRSL- (therapeutic agent) (SEQ ID NO: 489);
MeOCO-Quat2-GRSL- (therapeutic agent) (SEQ ID NO: 490);
Ac-QGRSL- (therapeutic agent) (SEQ ID NO: 445);
Ac-QGRSSL- (therapeutic agent) (SEQ ID NO: 446);
Ac-QGRASL- (therapeutic agent) (SEQ ID NO: 447);
Ac-NGRSSL- (therapeutic agent) (SEQ ID NO: 448);
Ac-QGRSS-nL- (therapeutic) (SEQ ID NO: 449);
Ac-QGRSS-nV- (therapeutic) (SEQ ID NO: 450);
Ac-QGRSS-Cha- (therapeutic) (SEQ ID NO: 451);
Ac-QGRSS-allyl G- (therapeutic) (SEQ ID NO: 452);
Ac-QGRSS-allyl G- (therapeutic) (SEQ ID NO: 453);
Ac-QARSL- (therapeutic agent) (SEQ ID NO: 454);
Ac-QARSSL- (therapeutic agent) (SEQ ID NO: 455);
Ac-QSRSL- (therapeutic agent) (SEQ ID NO: 456);
Ac-QSRSS-nV- (therapeutic) (SEQ ID NO: 457);
Ac-QSRSS-Cha- (therapeutic) (SEQ ID NO: 458);
Ac-QSRSSL- (therapeutic agent) (SEQ ID NO: 459);
Ac-QTRSSL- (therapeutic agent) (SEQ ID NO: 460);
Ac-Q-Aib-RSS-Cha- (Therapeutic) (SEQ ID NO: 461);
Ac-Q-Aib-RSSL- (therapeutic) (SEQ ID NO: 462);
Ac-Q-Abu-RSS-Cha- (therapeutic agent) (SEQ ID NO: 463);
Ac-Q-Abu-RSSL- (therapeutic) (SEQ ID NO: 464);
Ac-Q-Cha-RSS-Cha- (therapeutic agent) (SEQ ID NO: 465);
Ac-QFRSL- (therapeutic agent) (SEQ ID NO: 466);
Ac-QFRSSL- (therapeutic agent) (SEQ ID NO: 467);
Ac-QYRSSL- (therapeutic) (SEQ ID NO: 468);
Ac-RGRSL- (therapeutic agent) (SEQ ID NO: 469);
Ac-RGRSSL- (therapeutic agent) (SEQ ID NO: 470);
Ac-RGRSS-Cha- (therapeutic) (SEQ ID NO: 471);
Ac-RGRS-Cha- (therapeutic) (SEQ ID NO: 472);
Ac-RARSL- (therapeutic agent) (SEQ ID NO: 473);
Ac-RARSSL- (therapeutic agent) (SEQ ID NO: 474);
Ac-RSRSL- (therapeutic agent) (SEQ ID NO: 475);
Ac-RSRSSL- (therapeutic agent) (SEQ ID NO: 476);
Ac-RSRS-Cha- (therapeutic agent) (SEQ ID NO: 477);
Ac-RSRSS-Cha- (therapeutic) (SEQ ID NO: 478);
Ac-RFRSL- (therapeutic agent) (SEQ ID NO: 479);
Ac-RFRS-Cha- (therapeutic) (SEQ ID NO: 480);
Ac-YGRSSL- (therapeutic agent) (SEQ ID NO: 481);
Ac-M (O2) -SRSL- (therapeutic) (SEQ ID NO: 482);
Ac-RRQSRAA- (therapeutic agent) (SEQ ID NO: 105);
Ac-RRQSRI- (therapeutic agent) (SEQ ID NO: 610);
Ac-RRQSRSSL- (therapeutic) (SEQ ID NO: 543);
Ac-RRQSRSL- (therapeutic agent) (SEQ ID NO: 544);
Ac-RGSGRSL- (therapeutic agent) (SEQ ID NO: 545);

Ac-RGSGR--S-nL- (therapeutic agent) (SEQ ID NO: 546);
Ac-RGSGRA-nL- (therapeutic) (SEQ ID NO: 547);
Ac-RGSGRSSL- (therapeutic agent) (SEQ ID NO: 548);
Ac-IVSGRASL- (therapeutic agent) (SEQ ID NO: 549);
Ac-RRQSRA- (therapeutic agent) (SEQ ID NO: 108);
Ac-RRQSRI- (therapeutic) (SEQ ID NO: 111);
Ac-LRRQSRAA- (therapeutic agent) (SEQ ID NO: 106);
Ac-LRRQSRGG- (therapeutic agent) (SEQ ID NO: 109);
Ac-LRRQSRA- (therapeutic agent) (SEQ ID NO: 110);
Ac-LRRQSRAI- (therapeutic agent) (SEQ ID NO: 112);
Ac-LRRQSRAI- (therapeutic) (SEQ ID NO: 611);
Ac-LRRQSRSSL- (therapeutic agent) (SEQ ID NO: 550); and
Ac-LRRQSRSL- (therapeutic agent) (SEQ ID NO: 551);
In other embodiments, the conjugates provided herein are selected from:
Ac-SGRSL- (therapeutic agent) (SEQ ID NO: 362);
Ac-SGRSSL- (therapeutic agent) (SEQ ID NO: 363);
Ac-SGRSSSL- (therapeutic) (SEQ ID NO: 364);
Ac-SGRS-nL- (therapeutic agent) (SEQ ID NO: 365);
Ac-SGRS-nV- (therapeutic) (SEQ ID NO: 366); isomer 1
Ac-SGRS-nV- (therapeutic) (SEQ ID NO: 367); isomer 2
Ac-SGRSG (hex)-(therapeutic) (SEQ ID NO: 368);
Ac-SGRS-Cha- (therapeutic) (SEQ ID NO: 369);
Ac-SGRS-hCha- (therapeutic) (SEQ ID NO: 370);
Ac-SARSL- (therapeutic agent) (SEQ ID NO: 371);
Ac-SARSSL- (therapeutic) (SEQ ID NO: 372);
Ac-SSRS-nL- (therapeutic) (SEQ ID NO: 373);
Ac-TGRS-Abu- (therapeutic agent) (SEQ ID NO: 374);
Ac-TGRSL- (therapeutic agent) (SEQ ID NO: 375);
Ac-TGRS-nV- (therapeutic agent) (SEQ ID NO: 376);
Ac-TGRS-nL- (therapeutic agent) (SEQ ID NO: 377);
Ac-TGRSG (hex)-(therapeutic agent) (SEQ ID NO: 378);
Ac-TGRS-Cha- (therapeutic agent) (SEQ ID NO: 379);
Ac-TGRS-hCha- (therapeutic agent) (SEQ ID NO: 380);
Ac-TGRT-Abu- (therapeutic) (SEQ ID NO: 381);
Ac-TGR-hS-nL- (therapeutic agent) (SEQ ID NO: 382);
Ac-TGR-Abu-nL- (therapeutic agent) (SEQ ID NO: 383);
Ac-TGR-Abu-nV- (therapeutic) (SEQ ID NO: 384);
Ac-TGF (Gn) -S-nL- (therapeutic agent) (SEQ ID NO: 385);
Ac-TGF (Gn) -S-Cha- (therapeutic agent) (SEQ ID NO: 386);
Ac-TGF (Gn) -Abu-nV- (therapeutic) (SEQ ID NO: 387);
Ac-TGK (alloc) -S-nL- (therapeutic agent) (SEQ ID NO: 388);
Ac-TGKS-nL- (therapeutic agent) (SEQ ID NO: 389);
Ac-TG-hR-S-nL- (therapeutic agent) (SEQ ID NO: 390);
Ac- (hS) GGRS-nL- (therapeutic agent) (SEQ ID NO: 391);
MeOCO-TGRS-nL- (therapeutic agent) (SEQ ID NO: 392);

PhSO ₂ -TGRS-nL- (therapeutic) (SEQ ID NO: 393);
MeOEtCO-TGRS-nL- (therapeutic) (SEQ ID NO: 394);
MeO (EtO) 2Ac-TGRS-nL- (therapeutic agent) (SEQ ID NO: 395);
4-oxo-pentanoyl-TGRS-nL- (therapeutic agent) (SEQ ID NO: 396);
3,4-methyldioxy PhAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 397);
2-pyridyl Ac-TGRS-nL- (therapeutic agent) (SEQ ID NO: 398);
PhOAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 399);
L-3-Ph lactyl-TGRS-nL- (therapeutic agent) (SEQ ID NO: 400);
MeOAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 401);
PhAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 402);
MeOEtOCO-TGRS-nL- (therapeutic agent) (SEQ ID NO: 403);
MeOEtOAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 404);
HOOCButa-TGRS-nL- (Therapeutic) (SEQ ID NO: 405);
ZTGRS-nL- (therapeutic agent) (SEQ ID NO: 406);
EtOCO-TGRS-nL- (therapeutic) (SEQ ID NO: 407);
βΑ-Τ-GRS-nL- (therapeutic agent) (SEQ ID NO: 408);
Pent-4-ynoyl-TGRS-nL- (therapeutic agent) (SEQ ID NO: 409);
NapAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 410);
iBoc-TGRS-nL- (therapeutic) (SEQ ID NO: 411);
HOAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 412);
MeSucc-TGRS-nL- (therapeutic) (SEQ ID NO: 413);
N, N-diMeGly-TGRS-nL- (therapeutic agent) (SEQ ID NO: 414);
Succ-TGRS-nL- (therapeutic agent) (SEQ ID NO: 415);
HCO-TGRS-nL- (therapeutic agent) (SEQ ID NO: 416);
Ac-TARS-nL- (therapeutic agent) (SEQ ID NO: 417);
Ac-TAF (Gn) -S-nL- (therapeutic agent) (SEQ ID NO: 418);
Ac-TAR-Abu-nV- (therapeutic agent) (SEQ ID NO: 419);
Ac-TARS-Abu- (therapeutic agent) (SEQ ID NO: 420);
Ac-TART-Abu- (therapeutic) (SEQ ID NO: 421);
Ac-TS (O-Me) -RS-nL- (therapeutic agent) (SEQ ID NO: 422);
Ac-T-hS-RS-nL- (therapeutic agent) (SEQ ID NO: 423);
Ac-T- (1-Me) HRS-nL- (therapeutic agent) (SEQ ID NO: 424);
Ac-T- (3-Me) HRS-nL- (therapeutic agent) (SEQ ID NO: 425);

Ac-THRS-nL- (therapeutic) (SEQ ID NO: 426);
Ac-T-Sar-RS-nL- (therapeutic agent) (SEQ ID NO: 427);
Ac-T-nV-RS-nL- (therapeutic agent) (SEQ ID NO: 428);
Ac-T-nL-RS-nL- (therapeutic agent) (SEQ ID NO: 429);
Ac-TARS-Cha- (therapeutic) (SEQ ID NO: 430);
Ac-T-Abu-RS-nL- (therapeutic agent) (SEQ ID NO: 431);
Ac-4,4 diMeThr-GRS-nL- (therapeutic agent) (SEQ ID NO: 432);
Ac-hS-GRS-nL- (therapeutic agent) (SEQ ID NO: 433);
Ac-hS-GR-hS-Cha- (therapeutic agent) (SEQ ID NO: 434);
Ac-hS-GRS-Cha- (therapeutic agent) (SEQ ID NO: 435);
Ac-hS-GRT-Cha- (therapeutic agent) (SEQ ID NO: 436);
Ac-hS-ARS-Cha- (therapeutic agent) (SEQ ID NO: 437);
Ac-NGRS-nL- (therapeutic agent) (SEQ ID NO: 438);
Ac-YGRSSL- (therapeutic agent) (SEQ ID NO: 439);
Ac-YGRS-Cha- (therapeutic agent) (SEQ ID NO: 440);
Ac-QGRSS-nL- (therapeutic) (SEQ ID NO: 441);
Ac-QGRSS-nV- (therapeutic) (SEQ ID NO: 442);
Ac-LRGSGRSA- (therapeutic agent) (SEQ ID NO: 573);
Ac-LRGSGRSL- (therapeutic agent) (SEQ ID NO: 342);
Ac-LRGSGRSL- (therapeutic agent) (SEQ ID NO: 343);
Ac-LRGSGRSS-nL- (therapeutic) (SEQ ID NO: 344);
Ac-LRGSGRSS-Cha- (Therapeutic) (SEQ ID NO: 345);
Ac-LRG-dS-ARSA- (therapeutic agent) (SEQ ID NO: 574);
Ac-LRGSARSSL- (therapeutic agent) (SEQ ID NO: 346);
Ac-LRGSARSL- (therapeutic agent) (SEQ ID NO: 347);
Ac-LRGSARSS-Cha- (therapeutic agent) (SEQ ID NO: 348);
Ac-LRGSARSS-nV- (therapeutic) (SEQ ID NO: 349);
Ac-LRGSARSS-nL- (therapeutic) (SEQ ID NO: 350);
Ac-VIVSGRAL- (therapeutic) (SEQ ID NO: 351);
Ac-VIVSARSL- (therapeutic agent) (SEQ ID NO: 352);
Ac-VIVSGRSSL- (therapeutic agent) (SEQ ID NO: 353);
Ac-VIVSARMA- (therapeutic agent) (SEQ ID NO: 354);
Ac-VIVSAR-nL-A- (therapeutic agent) (SEQ ID NO: 355);
Ac-VIVSARS-nL- (therapeutic agent) (SEQ ID NO: 356);
Ac-VIVSARS-Cha- (therapeutic agent) (SEQ ID NO: 357);
Ac-VIVSARS-Cha- (therapeutic) (SEQ ID NO: 358);

Ac-VIVSARSS-Cha- (therapeutic agent) (SEQ ID NO: 359);
Ac-RR- (Me) CPGRVV- (therapeutic agent) (SEQ ID NO: 360);
Ac-RR-nV-PARSL- (therapeutic) (SEQ ID NO: 361);
Ac-RG-dS-ARSA- (therapeutic) (SEQ ID NO: 309);
Ac-RGSGRSA- (therapeutic) (SEQ ID NO: 310);
Ac-RGSGRAL- (therapeutic agent) (SEQ ID NO: 311);
Ac-RGSGRSL- (therapeutic agent) (SEQ ID NO: 312);
Ac-RGSGR--S-nL- (therapeutic) (SEQ ID NO: 313);
Ac-RGSGRA-nL- (therapeutic) (SEQ ID NO: 314);
Ac-RGSGRSSL- (therapeutic agent) (SEQ ID NO: 315);
Ac-RGSGRS-Cha- (therapeutic) (SEQ ID NO: 316);
Ac-RGSGRSS-Cha- (therapeutic) (SEQ ID NO: 317);
Ac-RGSARS-Cha- (therapeutic) (SEQ ID NO: 318);
Ac-RGSARSS- (therapeutic agent) (SEQ ID NO: 319);
Ac-RGSARS-nV- (therapeutic) (SEQ ID NO: 320);
Ac-RGSARSS-nV-(therapeutic) (SEQ ID NO: 321);
Ac-RGSARSL- (therapeutic) (SEQ ID NO: 322);
Ac-R- (Me) CPGRVV- (therapeutic agent) (SEQ ID NO: 323);
Ac-R- (Me) CPGRVV- (therapeutic agent) (SEQ ID NO: 324);
Ac-RC (Me) -PGRSL- (therapeutic) (SEQ ID NO: 325);
Ac-RLPGRSL- (therapeutic agent) (SEQ ID NO: 326);
Ac-RVPGRSL- (therapeutic agent) (SEQ ID NO: 327);
Ac-RVPGRSL- (therapeutic agent) (SEQ ID NO: 328);
Ac-R-nL-PGRSL- (therapeutic agent) (SEQ ID NO: 329);
Ac-RG (tBu) -PARSL- (therapeutic agent) (SEQ ID NO: 330);
Ac-RLPARSL- (therapeutic agent) (SEQ ID NO: 331);
Ac-RVPARSL- (therapeutic agent) (SEQ ID NO: 332);
Ac-R-nL-PARSL- (therapeutic agent) (SEQ ID NO: 333);
Ac-IVSGRAL- (therapeutic agent) (SEQ ID NO: 334);
Ac-IVSGRSSL- (therapeutic agent) (SEQ ID NO: 335);
Ac-IVSGRASL- (therapeutic agent) (SEQ ID NO: 336);
Ac-IVSARMA- (therapeutic agent) (SEQ ID NO: 337);
Ac-IVSAR-nL-A- (therapeutic) (SEQ ID NO: 338);
Ac-IVSARSL- (therapeutic agent) (SEQ ID NO: 339);
Ac-IVSARS-nL- (therapeutic) (SEQ ID NO: 340);
Ac-IVSARSSL- (therapeutic) (SEQ ID NO: 341);
Ac-GSGRSA- (therapeutic agent) (SEQ ID NO: 585);
Ac-GSGRSL- (therapeutic agent) (SEQ ID NO: 277);
Ac-GSGRAL- (therapeutic) (SEQ ID NO: 278);
Ac-GSGRSSL- (therapeutic agent) (SEQ ID NO: 279);
Ac-GSGRL- (therapeutic agent) (SEQ ID NO: 280);
Ac-GSG- (4-guan) Phg-SL- (therapeutic) (SEQ ID NO: 281);
Ac-GSGRSS-Cha- (therapeutic) (SEQ ID NO: 282);
Ac-GSGRASL- (therapeutic) (SEQ ID NO: 283);
Ac-GSGRS-nL- (therapeutic) (SEQ ID NO: 284);
Ac-GTGRS-nL- (therapeutic agent) (SEQ ID NO: 285);

Succ-bA-TGRS-nL- (Therapeutic) (SEQ ID NO: 286);
Ac-GTGRS-hCha- (therapeutic agent) (SEQ ID NO: 287);
Ac-G-hS-GRS-nL- (therapeutic agent) (SEQ ID NO: 288);
Ac-G-dS-ARSA- (therapeutic agent) (SEQ ID NO: 289);
Ac-GSARSL- (therapeutic agent) (SEQ ID NO: 290);
Ac-GSARSS-Cha- (therapeutic agent) (SEQ ID NO: 291);
Ac-GSARSSL- (therapeutic) (SEQ ID NO: 292);
Ac-GSARASL- (therapeutic agent) (SEQ ID NO: 293);
Ac-VSGRSL- (therapeutic agent) (SEQ ID NO: 294);
Ac-VSGRAL- (therapeutic agent) (SEQ ID NO: 295);
Ac-VSGRASL- (therapeutic) (SEQ ID NO: 296);
Ac-VSGRSSL- (therapeutic agent) (SEQ ID NO: 297);
Ac-VSARMA- (therapeutic agent) (SEQ ID NO: 298);
Ac-VSAR-nL-A- (therapeutic agent) (SEQ ID NO: 299);
Ac-VSARS-nL- (therapeutic agent) (SEQ ID NO: 300);
Ac-VSARSL- (therapeutic agent) (SEQ ID NO: 301);
Ac- (Me) CPGRVV- (therapeutic agent) (SEQ ID NO: 302);
Ac- (Me) CPGRVV- (therapeutic agent) (SEQ ID NO: 303);
Ac-C (Me) -PGRAL- (therapeutic agent) (SEQ ID NO: 304);
Ac-C (Me) -PGRSL- (therapeutic agent) (SEQ ID NO: 305);
Ac-C (Me) -PARSL- (therapeutic) (SEQ ID NO: 306);
Ac-C (Me) -PARASL- (therapeutic agent) (SEQ ID NO: 307); and
Ac-G (tBu) -PGRSL- (therapeutic agent) (SEQ ID NO: 308);

In other embodiments, the conjugates provided herein are selected from:
Ac-QSRAA- (therapeutic agent) (SEQ ID NO: 552);
Ac-QSRSA- (therapeutic) (SEQ ID NO: 553);
Ac-QSRSG- (therapeutic) (SEQ ID NO: 554);
Ac-RSRAA- (therapeutic agent) (SEQ ID NO: 555);
Ac-RQSRAA- (therapeutic) (SEQ ID NO: 556);
Ac-RQSRSA- (therapeutic agent) (SEQ ID NO: 557); and
Ac-RQSRSAA- (therapeutic agent) (SEQ ID NO: 558);

In other embodiments, the conjugates provided herein are selected from:
Ac-RGSGRSA- (therapeutic agent) (SEQ ID NO: 559);
Ac-SGRAA- (therapeutic) (SEQ ID NO: 560);
Ac-SGRSA- (therapeutic agent) (SEQ ID NO: 561);
Ac-SGRSSA- (therapeutic) (SEQ ID NO: 562);
Ac-SGRASA- (therapeutic agent) (SEQ ID NO: 563);
Ac-SGRSG- (therapeutic agent) (SEQ ID NO: 564);
Ac-SGRSSG- (therapeutic) (SEQ ID NO: 565);
Ac-SGRSGA- (therapeutic) (SEQ ID NO: 566);
Ac-SGRSGG- (therapeutic agent) (SEQ ID NO: 567); and
Ac-GTGRSGG- (therapeutic agent) (SEQ ID NO: 568);

In other embodiments, the conjugates provided herein are selected from:
Ac-LRRQSRAA- (therapeutic agent) (SEQ ID NO: 597);
MeSO ₂ -dA (Chx) -Abu-RSL- (Therapeutic) (SEQ ID NO: 598);
Ac-RARSL- (therapeutic agent) (SEQ ID NO: 599);
Ac-dA (Chx) -Abu-RSL- (therapeutic) (SEQ ID NO: 600);
Ac-dA (Chx) -Abu-RSSL- (therapeutic) (SEQ ID NO: 601);
Ac-QGRSSL- (therapeutic agent) (SEQ ID NO: 602);
MeOCO-dhF-P (OH) -RSSL- (therapeutic) (SEQ ID NO: 603);
MeOCO-Quat4-GRSL- (therapeutic agent) (SEQ ID NO: 604);
Ac-dCha-P (OH) -RSSL- (therapeutic) (SEQ ID NO: 605);
Ac-dCha-Abu-RSSA- (therapeutic agent) (SEQ ID NO: 606);
MeOCO-Quat2-GRSL- (therapeutic agent) (SEQ ID NO: 607);
MeOCO-Quat3-GRSL- (therapeutic) (SEQ ID NO: 608); and
MeOCO-Quat-GRSL- (therapeutic) (SEQ ID NO: 609).

It is also understood that conjugates containing the above peptidic substrate moieties can be made by other capping groups instead of Ac (see, eg, the description herein for capping group ^Xn ). Therapeutic agents for use in the above conjugates include, but are not limited to, toxins such as abrin, ricin A, Pseudomonas aeruginosa exotoxin Shiga toxin, diphtheria toxin and other such toxins. Cytotoxic agents and their toxic moieties; tumor necrosis factor, interferons such as α-interferon, gamma-interferon, pro-blood coagulants such as tissue factor and tissue factor variants, pros such as FAS-ligand A protein such as an apoptotic agent, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; for example, lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), Interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GMCSF), granulocyte Biological response modifiers such as colony stimulating factor (G-CSF), fibroblast growth factor (FGF) and other growth factors, methotrexate drug group, anthracycline family drugs, vinca alkaloid drugs, mitomycin, bleomycin, cytosine Cytotoxic nucleotides including arabinoside and difluoronucleosides, pteridine family drugs, diynenes, taxanes and podophyllotoxins. All such conjugates are within the scope of this disclosure and can be made and used as disclosed herein.

  Thus, the conjugates provided herein include therapeutic agents such as doxorubicin, carminomycin, daunorubicin, detorubicin, idarubicin, epirubicin, esorubicin, THP, AD-32, aminopterin, methotrexate, methotterin, dichloromethotrexate Mitomycin C, porphyromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinbutastine, vincristine, leurosidine , Vindesine, leucine, taxol, estramustine, cisplatin, combretastatin and analogs, and cyclophosphamide, the book Those disclosed in Saisho is, but are not limited to those. In one embodiment, the therapeutic agent is doxorubicin. In another embodiment, the therapeutic agent is taxol.

Any conjugate corresponding to the above conjugate or any conjugate disclosed herein wherein P1 ′ and / or P2 ′ is Ile instead of Ala are within the scope of this disclosure, and It can be made and used as disclosed herein.
Any peptidic substrate formed by permutation and selection of amino acids from those indicated in the definitions above for the P group is contemplated.

D. Creation of conjugates Peptidic substrates of conjugates provided herein are synthesized from their constituent amino acids by conventional peptide synthesis techniques, such as by solid phase techniques. The peptidic substrate is then purified by reverse layer high performance liquid chromatography (HPLC).

  Peptide acids can be made from its constituent Fmoc-amino acids. Standard methods of peptide synthesis are disclosed, for example, in the following works: Synthesis Notes Section, NovaBiochem Catalog 2002/3, Schroeder et al., “Peptides”, Vol. 1, Academic Press 1965; Bodansky et al. al., “peptide synthesis”, Interscience Publishers, 1966; McOmie (ed.), “protecting groups in organic chemistry”, Plenum Press, 1973, Barany et al., “peptides: analysis, synthesis, biology”, 2, Chapter 1, Academic Press, 1990, and Stewart et al., “Solid Phase Peptide Synthesis, Second Edition”, Pierce Chemical Company, 1994. The disclosures of these references are hereby incorporated by reference.

  Pharmaceutically acceptable salts of the conjugates provided herein include conventional non-toxic salts of the conjugate, such as formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid: as well as acetic acid, propionic acid, succinic acid, Glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenyl-acetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumar And salts prepared from organic acids such as acids, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and trifluoroacetic acid.

  The conjugates provided herein, including peptidic moieties containing cell surface protease cleavage sites and therapeutic agents, can be similarly synthesized by techniques known to those skilled in the art. For example, a free amine moiety on a therapeutic agent can be conjugated to a peptidic substrate at the carboxy terminus such that an amide bond is formed. Similarly, an amide bond can be formed by conjugate coupling of the amine moiety of the peptidic substrate and the carboxyl moiety of the therapeutic agent. For these purposes, 2- (1H-benzotriazol-1-yl) -1,3,3-tetramethyl-uronium hexafluorophosphate (known as HBTU) and 1-hydroxybenzotriazole hydrate ( Known as HOBT), dicyclohexyl-carbodiimide (DCC), N-ethyl-N- (3-dimethylaminopropyl) -carbodiimide (EDC), diphenylphosphoryl azide (DPPA), benzotriazol-1-yl-oxy-tris- ( Reagents such as (dimethylamino) -phosphonium hexafluorophosphate (BOP) and the like can be utilized in combination or alone.

  In addition, the conjugate can be formed by a non-peptide bond between the cell surface protease cleavage site and the therapeutic agent. For example, a therapeutic agent can be conjugatedly attached to the carboxy terminus of the peptidic substrate via a hydroxyl moiety on the therapeutic agent, thereby forming an ester bond. For this purpose, reagents such as a combination of HBTU and HOBT, a combination of BOP and imidazole, a combination of DCC and DMAP, etc. can be used. Carboxylic acids can also be activated by forming nitrophenyl esters and the like and reacted in the presence of DBU (1,8-diazabicyclo [5,4,0] undes-7-ene).

  Alternatively, the conjugate can be formed by attaching a peptidic substrate to a therapeutic agent via a linker unit. Such linker units include, for example, an amine moiety on a therapeutic agent linked to a linker unit to form an amide bond, and the amino terminus of the peptidic substrate is linked to the other end of the linker unit to form an amide. Biscarbonylalkyl diradicals that also form bonds. Conversely, the carbonyl moiety on the cytotoxic agent is conjugated to one amine of the linker unit, while the other amine of the linker unit is conjugated to the C-terminus of the peptidic substrate. Also useful are linker units of diaminoalkyl diradicals. In the absence of a cell surface protease or its soluble, shed or released form, it is stable to the physiological environment, but the cell surface protease proteolytic site or its soluble, shed or released Other such linker units are also envisioned, which are cleavable by mold cleavage. Furthermore, cleavage of cell surface protease proteolytic sites remains attached to the therapeutic agent but does not significantly reduce the therapeutic activity of the therapeutic agent after such cleavage when compared to unmodified therapeutic agents. However, linker units can be used.

  Those skilled in the art need to protect various reactive functionalities on the starting compounds or intermediates while synthesizing the conjugates provided herein while the desired reaction takes place in other parts of the molecule. Understand what is possible. After the desired reaction is complete, or at any desired time, usually such protecting groups will be removed, for example by means of hydrolysis or hydrogenolysis. Such protection and deprotection is conventional in organic chemistry. Those of skill in the art may refer to “Protecting Groups in Organic Chemistry,” McOmie, ed., Plenum Press, NY, NY (1973), as teaching materials for protecting groups that may be useful in making the conjugates provided herein. And "Protecting Groups in Organic Synthesis", Greene, ed., John Wiley & Sons, NY, NY (1991).

By way of example only, useful amino-protecting groups include, for example, C ₁ -C ₁₀ alkanoyl groups such as formyl, acetyl, dichloroacetyl, propionyl, hexanoyl, 3,3-diethylhexanoyl, γ-chlorobutyryl and the like; -Butoxycarbonyl, benzyloxycarbonyl. C ₁ -C ₁₀ alkoxycarbonyl and C ₅ -C ₁₅ aryloxycarbonyl groups such as allyloxycarbonyl, 4-nitrobenzyloxycarbonyl, fluorenylmethyloxycarbonyl and cinnamoyloxycarbonyl; 2,2,2-trichloro Mention may be made of halo (C ₁ -C ₁₀ ) -alkoxycarbonyl such as ethoxycarbonyl; and C ₁ -C ₁₅ arylalkyl and alkenyl groups such as benzyl, phenethyl, allyl, trityl and the like. Other commonly used amino protecting groups are in the form of enamines made with β-keto-esters such as methyl acetoacetate or ethyl.

Useful carboxy-protecting groups include, for example, C ₁ -C ₁₀ alkyl groups such as methyl, tert-butyl, decyl; halo C ₁ -C ₁₀ such as 2,2,2-trichloroethyl and 2-iodoethyl. Alkyl; C ₅ -C ₁₅ arylalkyl such as benzyl, 4-methoxybenzyl, 4-nitrobenzyl, triphenylmethyl, diphenyl-methyl; C ₁ -C ₁₀ alkanoyloxy such as acetoxy-methyl, propionoxymethyl and the like Methyl; and tri- (C ₁ -C ₃ alkyl) silyl such as phenacyl, 4-halophenacyl, allyl, dimethylallyl, trimethylsilyl, β-p-toluenesulfonylethyl, β-p-nitrophenyl-thioethyl, 2,4 , 6-trimethylbenzyl, β-methylthioethyl, phthalimi Methyl, 2,4-dinitro - phenyl sulfenyl, 2-nitro-benzhydryl and related groups, may be mentioned.

  Similarly, useful hydroxy protecting groups include, for example, formyl, chloroacetyl, benzyl, benzhydryl, trityl, 4-nitrobenzyl, trimethylsilyl, phenacyl, tert-butyl, methoxymethyl, A tetrahydropyranyl group, a tert-butyl-dimethylsilyl group and the like may be mentioned.

  For peptidic substrate embodiments coupled to the anthracycline antibiotic doxorubicin, the following reaction scheme illustrates the synthesis of the conjugates provided herein.

Reaction formula I:

Reaction Formula II:

Scheme III:

Reaction Formula IV:

Reaction formula V:

Scheme VI, attachment of vinblastine illustrates the creation of a conjugate of the peptidic substrate provided herein and the vinca alkaloid cytotoxic agent vinblastine at the C-terminus of the peptidic substrate. The use of a 1,3-diaminopropane linker is merely exemplary; other linker units between the carbonyl of vinblastine and the C-terminus of the peptidic substrate are also envisioned (eg, (CH ₂ ) _u -T - (CH _{2) u).} The acyl azide starting material is made by reaction of vinblastine with hydrazine (60-65 ° C., MeOH) followed by reaction with HCl / DMF / isoamyl nitrite.

  In addition, Scheme VI illustrates the synthesis of conjugates in which the C4-hydroxy moiety is reacetylated after addition of a linker unit. A deacetylvinblastine conjugate is also effective, eliminating the step of protecting the primary amine of the linker shown in Scheme VI, and reacting its intermediate with acetic anhydride followed by deprotection of the amine (See, for example, International Patent Application Publication No. WO 98/10651). Conjugation of the peptidic substrate and vinblastine functional groups at other positions is readily accomplished by one of ordinary skill in the art to conjugate that is a substrate for cell surface proteases or soluble, shed or released forms thereof. Also expected to provide.

Reaction formula VI:

  Scheme VII illustrates the creation of specific conjugates utilized in the compositions and methods provided herein, wherein the peptidic substrate is combined with the vinca alkaloid cytotoxic agent vinblastine. Attachment of the N-terminal vinblastine of a peptidic substrate is illustrated (S.P. Kandukuri et al. (1985) J. Med. Chem. 28: 1079-1088).

  Also, the N-terminus of the peptidic substrate utilized in the compositions and methods provided herein is combined with a therapeutic agent such as a cytotoxic agent such as vinblastine while the C-terminus. It is understood that conjugates can be made that are simultaneously bound to other cytotoxic agents that are the same or different cytotoxic agents such as doxorubicin. Scheme VIII illustrates the synthesis of such multiple cytotoxic agent conjugates. Such multi-cytotoxic conjugates can provide advantages over conjugates containing only one cytotoxic agent.

Reaction formula VII:

Reaction Formula VII (continued):

Reaction formula VIII:

Scheme VIII (continued):

  For peptidic substrate embodiments in combination with desacetylvinblastine, the following Reaction Schemes IX and X illustrate the synthesis of the conjugates provided herein.

  Scheme IX includes a peptidic substrate provided herein and a vinca alkaloid cytotoxic agent vinblastine, wherein the oxygen bond of 4-desacetylvinblastine is at the C-terminus of the peptidic substrate. FIG. 4 illustrates the creation of the conjugate provided in FIG. While other reaction sequences may be useful in forming such conjugates, initial attachment of a single amino acid to 4-oxygen and subsequent attachment of the remaining peptidic substrate sequence to that amino acid is exemplary (See International Patent Application Publication No. WO 99/28345). It is also known that 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (ODHBT) can be used in place of HOAc in the final coupling step.

  Scheme X illustrates the creation of a peptidic substrate conjugate provided herein, where hydroxyalkanoyl acid is used as a linker between the vinca drug and the peptidic substrate.

Scheme IX:

Scheme IX (continued):

Reaction formula X:

Reaction formula X (continued):

  The taxol conjugate provided herein can be made by the general methods provided below. The creation of 7-Ala-taxol and 7-Gly-taxol is disclosed by Mathew et al. (1992) J. Med. Chem. 35: 145-151.

E. Formulation and Administration of Pharmaceutical Compositions The conjugates and compositions provided herein provide a higher level of cell surface compared to other cells or soluble, shed or released forms on the surface. Used to treat, prevent or ameliorate one or more symptoms of any disease or disorder that can be treated by targeting of cells or tissues expressing proteases, particularly serine proteases . These include proliferative diseases such as cancer, any disease associated with ectopic or excessive angiogenesis, autoimmune disorders, inflammatory diseases, and cell-related and cell-localized proteases Any other disease where cell surface proteases can be identified, including but not limited to.

  The pharmaceutical compositions provided herein are useful in the prevention, treatment, or amelioration of one or more symptoms of a disease or disorder associated with undesirable and / or uncontrolled angiogenesis or neovascularization. Wherein a therapeutically effective amount of one or more conjugates provided herein is included. Such diseases or disorders include solid neoplasia, including lung, colon, esophagus, breast, ovarian and prostate cancer; angiofibroma, angiolipoma, atherosclerosis, restenosis / reperfusion injury Vascular malformations and cardiovascular disorders, including but not limited to, arteriovenous malformations, hemangiomatosis and vascular adhesion, chondrogenesis with hematoma, hereditary hemorrhagic telangiectasia and Von Hipple Lindau syndrome Diabetes, hemophilia joint, inflammatory bowel disease, non-healing fracture, rapid progressive periodontal disease, juvenile periodontal disease, psoriasis, rheumatoid arthritis, venous stasis ulcer, granulation burn, hypertrophic scar, Chronic inflammatory disease and adhesive wound repair including, but not limited to, cirrhosis, bone radiation necrosis, postoperative adhesions, purulent granulomas and systemic sclerosis; circulation, including but not limited to Raynaud phenomenon Disability Crest syndrome; including, but not limited to, calcification, esophageal, bimotility, scleroderma and teaangiectasis; systemic vasculitis, scleroderma, gangrenous pyoderma, angiopathy, venous, arteries Skin disorders including, but not limited to, sexual ulcers, Sturge-Weber syndrome, wine-like blemishes, blue gum-like nevus syndrome, Klippel-Trenaunay-Weber syndrome and Osler-Weber-Rendu syndrome; Includes vascular diseases, corneal graft neovascularization, macular degeneration in the eye, neovascular glaucoma, trachoma, diabetic retinopathy, myopic degeneration, retinopathy of prematurity, posterior lens fiber hyperplasia and corneal neovascularization Includes, but is not limited to, eye disorders.

  The composition contains one or more conjugates provided herein. Conjugates can be, for example, solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs for oral administration, or sterile solutions or suspensions for parenteral administration. Liquids and suitable pharmaceutical preparations such as transdermal patch preparations and dry powder inhalers can be formulated. Typically, the conjugates described above use techniques and procedures well known in the art (see, for example, Ansel (1985), “Introduction to Pharmaceutical Preparations, Fourth Edition”, p. 126), Formulated into a pharmaceutical composition. Effective concentrations can be determined empirically using animal models, in vitro models, or test subjects.

  In the composition, an effective concentration of one or more conjugates or pharmaceutically acceptable derivatives thereof is mixed with a suitable pharmaceutical carrier or vehicle. The conjugate can be derivatized as the corresponding salt, ester, enol ether or ester, acid, base, solvate or hydrate, prior to formulation, as described above. The concentration of the conjugate in the composition treats, prevents or ameliorates one or more symptoms of a disease or disorder associated with undesirable and / or uncontrolled angiogenesis or neovascularization upon administration. It is effective to deliver the amount.

  Such diseases or disorders include solid neoplasia; hemangiofibroma, angiolipoma, atherosclerosis, restenosis / reperfusion injury, arteriovenous malformation, hemangiomatosis and vascular adhesion, cartilage with hematoma Vascular malformations and cardiovascular disorders, including but not limited to dysplasia, hereditary hemorrhagic telangiectasia and Von Hipple Lindau syndrome; diabetes, hemophilic joints, inflammatory bowel disease, non-healing Fractures, rapidly progressive periodontal disease, juvenile periodontal disease, psoriasis, rheumatoid arthritis, venous stasis ulcer, granuloburn, hypertrophic scar, cirrhosis, bone radiation necrosis, postoperative adhesions, purulent granulomas and systemic Chronic inflammatory disease and adhesive wound repair, including but not limited to sclerosis; circulatory disorders including but not limited to Raynaud phenomenon; calcification, esophageal, bimotility, sclerotia and teangiectasis Including, but not limited to, crest syndrome; systemic vasculitis, scleroderma, pyoderma gangrenosum, angiopathy, venous, arterial ulcer, Sturge-Weber syndrome, wine-like stain, blue gum Skin disorders including, but not limited to, like nevus syndrome, Klippel-Trenaunay-Weber syndrome and Osler-Weber-Rendu syndrome; and ocular neovascular diseases, corneal graft neovascularization, macular degeneration in the eye, new Ocular disorders, including but not limited to angiogenic glaucoma, trachoma, diabetic retinopathy, myopic degeneration, retinopathy of prematurity, posterior lens fiber hyperplasia and corneal neovascularization It is not limited to.

  The conjugates herein can be formulated into pharmaceutical compositions suitable for topical, local, intravenous and systemic application. An effective concentration of one or more conjugates is mixed with a suitable pharmaceutical carrier or vehicle. The concentration or amount of conjugate that is effective requires the delivery of an amount that, upon administration, ameliorates the symptoms or treats the disease. Typically, the composition is formulated for single dose administration. The therapeutically effective concentration and amount can be determined empirically by testing the conjugate in known in vitro and in vivo systems such as those described herein; then for humans or other animals The dose can then be extrapolated.

  In mixing or adding the conjugate to the vehicle, the resulting mixture can be a solution, suspension, emulsion or other such composition. The form of the resulting mixture depends on a number of factors, including the intended mode of administration and the solubility of the conjugate in the selected carrier or vehicle. The effective concentration is sufficient to ameliorate the symptoms of the disease, disorder or condition being treated and may be in vitro and / or in vivo, such as data from a mouse xenograft model or a rabbit eye model for the tumor It can be determined empirically based on the data. If necessary, pharmaceutically acceptable salts or other derivatives of the conjugates can be made.

  Suitable pharmaceutical carriers or vehicles for administration of the conjugates provided herein include any such carrier known to those of skill in the art to be suitable for a particular mode of administration. . In addition, the conjugate can be formulated as a single pharmaceutically active ingredient in the composition or combined with other active ingredients.

  The conjugate may be in liquid, semi-liquid or solid form by any suitable route, eg, orally, parenterally, intravenously, intradermally, subcutaneously or topically. And is formulated in a manner appropriate to the respective route of administration. Exemplary modes of administration depend on the application being treated. Dermatological and ophthalmic applications will typically be treated locally; whereas tumors and vascular proliferative disorders are typically systemic, intradermal or intramuscular modes of administration Will be treated.

  The conjugate is included in a pharmaceutically acceptable carrier in an amount sufficient to exhibit a therapeutically useful effect in the absence of adverse side effects that are detrimental to the patient being treated. It will be appreciated that the number and extent of side effects depends on the abnormality to which the conjugate is administered. For example, certain adverse and undesirable side effects that would not be tolerated when treating milder prognostic disorders are tolerated when treating fatal illnesses such as tumors.

  The concentration of the conjugate in the composition will depend on its absorption, inactivation and excretion rates, the dosage regimen and amount administered and other factors known to those skilled in the art.

  Typically, a therapeutically effective dose should produce a serum concentration of the active ingredient from about 0.1 ng / ml to about 50-100 μg / ml. The pharmaceutical composition typically has a dosage of about 0.01 mg to about 100-2000 mg of conjugate, depending on the conjugate selected to be adjusted for body surface area and / or body weight. Should be provided. Typically, a daily dose of about 0.05 to 0.5 mg / kg should be sufficient for intravenous or systemic treatment. Topical application for ophthalmic disorders should provide from about 1 ng to 100 μg, generally from about 1 μg to about 10 μg per single dose administration. It is understood that the amount to be administered is a function of the conjugate selected, the application being treated, and possibly side effects that would be tolerated. The dosage can be determined empirically using recognized models for each disorder.

  Typically, the composition is formulated for single dose administration. To formulate the composition, the weight of conjugate must be dissolved, suspended, dispersed in the selected vehicle at an effective concentration that reduces or ameliorates the disorder being treated. Mix. Suitable pharmaceutical carriers or vehicles for administering the conjugates provided herein include any such carrier known to those skilled in the art as appropriate for the particular mode of administration. It is done.

  In addition, the conjugate can be formulated as a single component in the composition or combined with other active ingredients. Liposomal suspensions, including liposomes targeted to tissues, particularly targeted to tumors, may also be suitable as pharmaceutically acceptable carriers. These are prepared according to methods known to those skilled in the art. For example, liposomal formulations can be prepared as described in US Pat. No. 4,522,811. Briefly, liposomes such as multilamellar vesicles (MLV) can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (7: 3 molar ratio) inside the flask. A solution of the conjugate provided herein in phosphate buffered saline (PBS) lacking a divalent cation is added and the flask is shaken until the lipid membrane is dispersed. The resulting vesicles are washed to remove unencapsulated conjugate, pelleted by centrifugation, and then resuspended in PBS.

  The conjugate is included in a pharmaceutically acceptable carrier in an amount sufficient to exhibit a therapeutically useful effect in the absence of undesirable side effects in the patient being treated. The therapeutically effective concentration was determined empirically by testing the conjugate in the in vitro and in vivo systems described herein (see, eg, Examples 3 and 4), and then human Extrapolated to dose.

  The concentration of the conjugate in the pharmaceutical composition will depend on the absorption, inactivation and excretion rates of the conjugate, the physicochemical properties of the conjugate, the dosage regimen and amount administered and other factors known to those skilled in the art. Will. For example, the amount delivered is to improve one or more symptoms of a disease or disorder associated with undesired and / or uncontrolled angiogenesis or neovascularization, as described herein. It is enough.

  Typically, a therapeutically effective dose should produce a serum concentration of the active ingredient from about 0.1 ng / ml to about 50-100 μg / ml. Typically, a pharmaceutical composition should provide a dosage of about 0.001 mg to about 2000 mg per kilogram of body weight per day. Pharmaceutical unit dosage forms are prepared to provide about 1 mg to about 1000 mg, generally about 10-500 mg of the essential active ingredient or combination of essential ingredients per unit dosage form.

  The conjugate can be administered at once or can be divided into a number of smaller doses for administration at time intervals. It is understood that the exact dosage and duration of treatment is a function of the disease to be treated and can be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data. . It should be noted that concentration and dose values can also vary with the severity of the abnormality to be alleviated. For any particular subject, the specific dosing regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or managing the administration. It should be further understood that, as well as the concentration ranges set forth herein, are exemplary only and are not intended to limit the scope or practice of the claimed composition. .

  Exemplary pharmaceutically acceptable derivatives include acids, bases, enol ethers and esters, salts, esters, hydrates, solvates and conjugate forms. The derivative is selected such that its pharmacokinetic properties are superior to the corresponding neutral conjugate.

  Thus, an effective concentration or amount of one or more of the conjugates described herein or a pharmaceutically acceptable derivative thereof is a suitable pharmaceutical carrier for systemic, local or local administration. Or mixed with a vehicle to form a pharmaceutical composition. The conjugate ameliorates or treats one or more symptoms of a disease or disorder associated with undesired and / or uncontrolled angiogenesis or neovascularization, as described herein. Contained in an amount effective to prevent or prevent. The concentration of the conjugate in the composition will depend on absorption, inactivation and excretion rates of the conjugate, dosage regimen and amount administered, the particular formulation, and other factors known to those of skill in the art.

  The composition is intended to be administered by any suitable route including oral, parenteral, rectal, topical and topical. For oral administration, capsules and tablets are generally used. The compositions are in liquid, semi-liquid or solid form and are formulated in a manner appropriate to the respective route of administration. Exemplary modes of administration include parenteral and oral modes of administration.

  Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application may be any of the following components: water for injection, saline solution, fatty oil, polyethylene glycol, glycerin, propylene glycol Or sterile diluents such as other synthetic solvents; antibacterial agents such as benzyl alcohol and methyl paraben; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); acetates Buffering agents such as citrate, phosphate; and isotonicity adjusting agents such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.

  In examples where the conjugate exhibits poor solubility, a method for dissolving the conjugate can be used. Such methods are known to those skilled in the art and include the use of a co-solvent such as dimethyl sulfoxide (DMSO), the use of a surfactant such as TWEEN®, or an aqueous sodium bicarbonate solution. Dissolution, but is not limited thereto. Conjugate derivatives can also be used to formulate effective pharmaceutical compositions.

  In mixing or adding the conjugate (s), the resulting mixture can be a solution, suspension, emulsion, or the like. The form of the resulting mixture depends on a number of factors, including the intended mode of administration and the solubility of the conjugate in the selected carrier or vehicle. The effective concentration is sufficient to ameliorate the symptoms of the disease, disorder or condition being treated and can be determined empirically.

  The pharmaceutical composition comprises a tablet, capsule, pill, powder, granule, sterile parenteral solution or a suitable amount of the conjugate or pharmaceutically acceptable derivative thereof for administration to humans and animals. Provided in single dosage forms, such as suspensions and oral solutions or suspensions, and oil-water emulsions. The conjugates and derivatives thereof are typically formulated and administered in single or multiple dose forms.

  As used herein, a single dosage form refers to physically discrete units suitable for humans and animals and individually packaged as is known in the art. Each single volume contains a predetermined amount of conjugate sufficient to produce the desired effect in association with the required pharmaceutical carrier, vehicle or diluent. Examples of single dosage forms include ampoules and injections and individually packaged tablets or capsules. A single dosage form can be administered in that fraction or multiple. A multiple dose form is a plurality of identical single dose forms packaged in a single container for administration in a separate single dose form. Examples of multiple dosage forms include vials, tablet or capsule bottles, or pint or gallon bottles. Thus, a multiple dose is a plurality of single doses that are not separated in the package.

  Compositions with conjugates: diluents such as lactose, sucrose, di-calcium phosphate or carboxymethyl cellulose; lubricants such as magnesium stearate, calcium stearate and talc; starch, gum acacia-gelatin Natural rubber, glucose, molasses, polyvinylpyrrolidine, cellulose and its derivatives, binders such as povidone, crospovidone and other such binders known to those skilled in the art. A pharmaceutically administrable liquid composition comprises, for example, a conjugate as defined above and any pharmaceutical auxiliaries, eg, a carrier such as water, saline, aqueous dextrose, glycerol, glycol, ethanol, etc. It can be prepared by dissolving, dispersing, or otherwise mixing, thereby forming a solution or suspension.

  If desired, the pharmaceutical composition to be administered comprises a wetting agent, emulsifier or solubilizer, pH buffering agent, etc., for example acetate, sodium citrate, cyclodextrin derivative, sorbitan monolaurate, triethanolamine sodium acetate, oleic acid Small amounts of non-toxic auxiliary substances such as triethanolamine and other such agents may also be included. Actual methods of preparing such formulations will be known or apparent to those skilled in the art; see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. The composition or formulation to be administered will, in any event, contain the amount of the conjugate in an amount sufficient to alleviate the symptoms of the subject being treated.

  Formulations or compositions containing the active ingredient in the range of 0.005% to 100% with the balance made from non-toxic carrier may be prepared. For oral administration, pharmaceutically acceptable non-toxic compositions include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talc, cellulose derivatives, croscarmellose sodium, glucose, sucrose, magnesium carbonate Alternatively, it is formed by incorporating any commonly used additive such as sodium saccharin. Such compositions include, but are not limited to, solutions, suspensions, tablets, capsules, powders and implants and microencapsulated delivery systems, as well as collagen Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polyacetic acid and others. Methods for preparing these compositions are known to those skilled in the art. The contemplated compositions may contain 0.001% to 100% active ingredient, such as 0.1 to 85%, such as 75% to 95%.

  The conjugate or pharmaceutically acceptable derivative can be prepared with a carrier that protects the conjugate against rapid elimination from the body, such as a sustained release formulation or coating. The composition may include other conjugates to achieve the desired combination of properties. The conjugates provided herein, or pharmaceutically acceptable derivatives thereof described herein, are also such as diseases or disorders associated with undesirable and / or uncontrolled angiogenesis or neovascularization. For therapeutic or prophylactic purposes, along with other agents known in the general art to be useful in treating one or more of the diseases or medical abnormalities referred to herein above Can be advantageously administered. It should be understood that such combination therapy constitutes a further aspect of the compositions and treatment methods provided herein.

1. Compositions for oral administration Oral dosage forms are either solid, gel or liquid. Solid dosage forms are tablets, capsules, granules and bulk powders. Oral tablet types include tableted chewable lozenges and tablets that can be enterically coated, sugar coated or film coated. Capsules can be hard or soft gelatin capsules, while granules and powders can be provided in a non-foaming or foaming form in combination with other ingredients known to those skilled in the art.

  In certain embodiments, the formulation is in the form of a solid dosage form, such as a capsule or tablet. Tablets, pills, capsules, troches and other dosage forms may contain, for example, any of the following ingredients or compounds of similar nature: binders; diluents; disintegrants; lubricants; slip agents; Agents; as well as flavoring agents.

  Examples of binders include microcrystalline cellulose, tragarant gum, dextrose solution, gum arabic gum, gelatin solution, sucrose and starch paste. Lubricants include talc, magnesium or calcium stearate, buffalo and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and di-calcium phosphate. Examples of slip agents include, but are not limited to, colloidal silicon dioxide. Disintegrants include croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose.

  Coloring agents include, for example, any approved guaranteed water soluble FD and C pigment, mixtures thereof; and water insoluble FD and C pigments suspended on alumina hydrate. Sweeteners include artificial sweeteners such as sucrose, lactose, mannitol and saccharin, and any number of spray-dried flavors. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of conjugates that produce a pleasant sensation, such as but not limited to peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Emetic coating agents include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalate. Examples of the film coating agent include hydroxyethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

  If oral administration is desired, the conjugate is provided in a composition that protects it from the acidic environment in the stomach. For example, the composition may be formulated with an enteric coating that retains its integrity in the stomach and releases the conjugate in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

  When the single dosage form is a capsule, in addition to the above types of materials, it may contain a liquid carrier such as a fatty oil. In addition, single dosage forms may contain various other substances that modify the physical form of the dosage unit, such as coatings of sugars and other enteric agents. The conjugate can also be administered as a component of elixirs, suspensions, syrups, wafers, sprinkles, chewing gums and the like. A syrup may contain, in addition to the conjugates, sucrose as a sweetening agent and certain preservatives, pigments and colorings and flavors.

  The conjugate can also be mixed with other active substances that do not impair the desired action, or with substances that assist the desired action, such as antacids, H2 blockers and diuretics. High concentrations up to about 98% by weight of the conjugate can be included.

  Pharmaceutically acceptable carriers contained in tablets are binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, and wetting agents. Enteric-coated tablets resist the action of gastric acid because of the enteric-coating and dissolve or disintegrate in the neutral or alkaline intestine. Sugar-coated tablets are tablet tablets to which different layers of pharmaceutically acceptable substances are applied. Film-coated tablets are tablet tablets that are coated with a polymer or other suitable coating agent. A multi-tablet tablet is a tablet tablet made by two or more tableting cycles utilizing the pharmaceutically acceptable substances mentioned above. Colorants can also be used in the above dosage forms. Used in tableted tablets, sugar-coated tablets, and multiple tableted tablets. Flavoring and sweetening agents are particularly useful in the formation of chewable tablets and lozenges.

  Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent formulations reconstituted from effervescent granules. Can be mentioned. Examples of the aqueous solution include elixirs and syrups. Emulsions are either oil-in-water or water-in-oil droplets.

  An elixir is a clear, sweetened hydroalcoholic formulation. Examples of the pharmaceutically acceptable carrier used in the elixir include a solvent. A syrup is a concentrated aqueous solution of a sugar, such as sucrose, and may contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed throughout the other liquid in the form of microspheres. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifiers and preservatives. The suspension uses pharmaceutically acceptable suspending agents and preservatives. Non-effervescent granules and pharmaceutically acceptable substances used to reconstitute into liquid oral dosage forms include diluents, sweeteners and wetting agents. Effervescent granules and pharmaceutically acceptable materials used to reconstitute into liquid oral dosage forms include sources of organic acids and carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.

  Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol. Examples of non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil. Examples of emulsifiers include surfactants such as gelatin, acacia, tragacanth, bentonite, and polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Diluents include lactose and sucrose. Sweetening agents include sweetening agents such as sucrose, syrup, glycerin and saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric acid and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Colorants include any approved guaranteed water soluble FD and C pigments, mixtures thereof. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of conjugates that create a pleasant taste sensation.

  For solid dosage forms, for example, solutions or suspensions in propylene carbonate, vegetable oils or triglycerides, for example, formulations thereof can be encapsulated in gelatin capsules. Such solutions and their preparation and their encapsulation are disclosed in US Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. For liquid dosage forms, for example, a solution in polyethylene glycol, for example, can be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier, such as water, and easily metered for administration.

  Alternatively, liquid or semi-solid oral formulations dissolve or disperse the conjugate or derivative thereof in vegetable oils, glycols, triglycerides, propylene glycol esters (eg, propylene carbonate) and other such carriers; These solutions or suspensions can be prepared by encapsulating them in hard or soft gelatin capsule shells. Other useful formulations include those set forth in US Pat. Nos. Re28,819 and 4,358,603. Briefly, such formulations include the conjugates provided herein, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, Dialkylated mono- or poly-alkylene glycols, including but not limited to polyethylene glycol-750-dimethyl ether, where 350, 550 and 750 refer to the approximate average molecular weight of polyethylene glycol, and butylated hydroxy Toluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarin, ethanolamine, lecithin, cephalin, ascorbic acid, phosphorus Examples include, but are not limited to, those containing golic acid, sorbitol, phosphoric acid, thiodipropionic acid and esters thereof, and one or more antioxidants such as dithiocarbamate.

  Other formulations include, but are not limited to, hydroalcoholic solutions containing pharmaceutically acceptable acetals. The alcohol used in these formulations is any pharmaceutically acceptable water-miscible solvent having one or more hydroxy groups, including but not limited to propylene glycol and ethanol. Acetals include, but are not limited to, di (lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.

  In all embodiments, tablet and capsule formulations may be coated as known to those skilled in the art to modify or sustain the dissolution of the conjugate. Thus, for example, they can be coated with conventional enteric coating agents such as phenyl salicylate, wax and cellulose acetate phthalate.

2. Injectables, Solutions and Emulsions Parenteral administration, generally characterized by injection, either subcutaneously, intramuscularly or intravenously, is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable additives are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical composition to be administered can also comprise wetting or emulsifying agents, pH buffering agents, stabilizing agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins, It may contain solubility enhancers, as well as small amounts of non-toxic auxiliary substances such as, for example, other such agents. Also contemplated herein is a slow-release or sustained-release system infusion (see, eg, US Pat. No. 3,710,795) such that a constant dosage level is maintained.

  Briefly, the conjugates provided herein include solid internal matrices such as polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized Nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymer, silicon rubber, polydimethylsiloxane, silicon carbonate copolymer, acrylic acid and methacrylic acid ester Dispersed in hydrophilic polymers such as hydrogels, collagen, cross-linked polyvinyl alcohol and cross-linked partially hydrolyzed polyvinyl acetate, etc., it is a polymer that is insoluble in body fluids Outer membrane, eg, polyester Rene, polypropylene, ethylene / propylene copolymer, ethylene / ethyl acrylate copolymer, ethylene / vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride Copolymer of vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber, epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / vinyl alcohol terpolymer, and ethylene / vinyl Surrounded by oxyethanol copolymer. The conjugate diffuses through the polymeric outer membrane at the rate limiting step of release. The percentage of conjugate contained in such parenteral compositions is highly dependent on the specific nature of the conjugate and the activity of the conjugate and the needs of the subject.

  Parenteral administration of the composition includes intravenous, subcutaneous and intramuscular administration. Formulations for parenteral administration include sterile ready-to-use solutions, sterile dry soluble products such as lyophilized powders, formulated with solvents immediately before use, including tablets for subcutaneous injection, injections Sterile suspensions ready for use, sterile dry insoluble products and sterile emulsions that are immediately combined with the vehicle just prior to use. The solution can be either aqueous or non-aqueous.

  If administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS), and thickening such as glucose, polyethylene glycol and polypropylene glycol and mixtures thereof. And a solution containing an agent and a solubilizer.

  Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspensions and dispersions, emulsifiers. Sequestering or chelating agents as well as other pharmaceutically acceptable substances.

  Examples of aqueous vehicles include sodium chloride injection, Ringer injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fatty oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Multiple times where the antibacterial agent at the bacteriostatic or fungistatic concentration comprises phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl P-hydroxybenzoate, thimerosal, benzalkonium chloride and benzethonium chloride Must be added to parenteral formulations packaged in dose containers. Isotonic agents include sodium chloride and dextrose. Buffering agents include phosphate and citrate. Examples of the antioxidant include sodium sulfite. Examples of local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Examples of the emulsifier include polysorbate 80 (TWEEN [registered trademark] 80). Examples of the metal ion sequestering or chelating agent include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

The concentration of the conjugate is adjusted so that the injection provides an amount sufficient to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as is known in the art.
Single volume parenteral formulations are packaged in ampoules, vials or syringes with needles. All formulations for parenteral administration must be sterile, as is known and practiced in the art.

  Illustratively, intravenous or intraarterial infusion of a sterile aqueous solution containing the conjugate is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing a conjugate that is injected as needed to produce the desired pharmacological effect.

  Injectables are designed for local and systemic administration. Typically, a therapeutically effective dose is at least about 0.1% w / w to about 90% w / w or more, generally to the tissue (s) being treated. In contrast, it is formulated to contain a conjugate concentration of 2% w / w or greater. The conjugate can be administered at once, or can be divided into a number of smaller doses for administration at time intervals. The exact dose and duration of treatment is a function of the tissue to be treated and can be empirically determined using known test protocols or extrapolated from in vivo or in vitro test data. It should be noted that the concentration and dosage values can also vary with the age of the individual being treated. In addition, for any particular subject, the specific dosing regimen is adjusted over time according to the individual's needs and the human judgment that governs the administration or administration of the composition. It is to be understood that the concentration ranges given herein are merely exemplary and are not intended to limit the scope or practice of the claimed formulation.

  The conjugate can be micronized or suspended in other suitable forms or derivatized to make a more soluble product. The form of the resulting mixture will depend on a number of factors, including the intended mode of administration and the solubility of the conjugate in the selected carrier or vehicle. The effective concentration is sufficient to ameliorate abnormal symptoms and can be determined empirically.

3. Lyophilized powders Of interest herein are also lyophilized powders that can be reconstituted for administration as solutions, emulsions and other mixtures. They can also be reconstituted and formulated as a solid or gel.

  A sterile, lyophilized powder is prepared by dissolving the conjugate provided herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. The solvent may contain an additive or other pharmacological component of the powder that improves stability or a reconstituted solution prepared from the powder. Additives that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents. The solvent may also contain citrate, sodium or potassium phosphate or other such buffers known to those skilled in the art, typically at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. In general, the resulting solution will be dispensed into vials for lyophilization. Each vial will contain a single dose (such as 10-1000 mg, such as 100-500 mg) or multiple doses of the conjugate. The lyophilized powder can be stored under appropriate conditions, such as at about 4 ° C. to room temperature.

  Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, typically about 1-50 mg of lyophilized powder, such as 5-35 mg or about 9-30 mg, is added per ml of sterile water or other suitable carrier. The exact amount will depend on the conjugate selected, the intended subject, and other parameters that can be determined empirically. Thus, the amount can be determined empirically.

4). Topical administration Topical mixtures are prepared as described for local and systemic administration. The resulting mixture can be a solution, suspension, emulsion, etc. and a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, pasta, foam May be formulated as an agent, aerosol, irrigation, spray, suppository, bandage, skin patch or any other suitable formulation for topical administration.

  Conjugates or pharmaceutically acceptable derivatives thereof are described in US Pat. No. 4,044,126, which describes aerosols for the delivery of steroids useful for the treatment of inhalation (eg, inflammatory diseases, particularly asthma). No. 4,414,209 and 4,364,923) can be formulated as an aerosol for topical application. These formulations for administration to the respiratory tract may be in the form of an aerosol or solution for a nebulizer or as an ultrafine powder for inhalation alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation will typically have a diameter of 50 microns or less, generally 10 microns or less.

Conjugates are for topical or topical application, such as for topical application to the mucosa as in the skin and eye, and for ocular or intracerebral or transdermal applications. Therefore, it can be formulated in the form of gels, creams, and lotions. Topical administration contemplates transdermal delivery and also administration to the eye or mucosa, or inhalation therapy. Nasal solutions alone or in combination with other pharmaceutically acceptable excipients can also be administered.
These solutions, particularly those intended for ophthalmic use, can be formulated as 0.01% -10% isotonic solutions, pH 5-7, with appropriate salts.

5. Compositions for other routes of administration Other routes of administration such as topical application, transdermal patches, and rectal administration are also contemplated herein.

  For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories, as used herein, refer to solid objects for insertion into the rectum that dissolve or soften at body temperature to release one or more conjugates. Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and drugs to raise the melting point. Examples of bases include cocoa butter (theobromine oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and suitable mixtures of mono-, di- and triglycerides of fatty acids. Various base combinations may be used. Agents for raising the melting point of suppositories include whale wax and wax. Rectal suppositories can be prepared either by the compressed method or by casting. The typical weight of a rectal suppository is about 2-3 gm.

  Tablets or capsules for rectal administration are manufactured by the same method using the same pharmaceutically acceptable substances as for formulations for oral administration.

6). Articles of Manufacture Conjugates or pharmaceutically acceptable derivatives are used herein for the treatment, prevention or amelioration of one or more symptoms associated with packaging materials, proliferative diseases or disorders. Provided conjugates or pharmaceutically acceptable derivatives thereof, as well as treating, preventing or ameliorating one or more symptoms associated with a proliferative disease or disorder wherein the conjugate or pharmaceutically acceptable derivative thereof is Can be packaged as a manufactured product containing a label indicating that it is used for the purpose.

  Articles of manufacture provided herein include packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those skilled in the art. See, for example, US Pat. Nos. 5,323,907, 5,052,558, and 5,033,252. Examples of pharmaceutical packaging materials include blister packaging, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and the intended mode of administration and treatment But are not limited thereto. The broad family of formulations of conjugates and compositions provided herein is a variety of treatments for any disorder where cell surface proteases or soluble, shed or released forms thereof are suggested. Intended as.

F. Assessment of the activity of the conjugate Standard physiological, pharmacological and biochemical procedures are used to test the conjugate and therapeutic activity with the action of cell surface proteases or their soluble, shed or released forms. Can be used to identify those possessing In vitro and in vivo assays that can be used to assess therapeutic activity, such as cytotoxicity, of the conjugate will depend on the therapeutic agent being tested.

  Exemplary assays are briefly discussed below with reference to cytotoxic conjugates (see also the examples). It will be appreciated that the particular activity assayed will depend on the therapeutic agent being bound.

1. In vitro assays The therapeutic activity, such as cytotoxicity, of the conjugates provided herein is any of those commonly used to assess therapeutic activity, such as cytotoxicity, of an unbound therapeutic agent. Can be assessed by these assays. A number of such assays are known, for example, the assay can use cells that express the targeted cell surface protease and the therapeutic activity of the therapeutic agent is evaluated. For example, cytotoxicity can be assessed by measuring cell viability or measuring cell proliferation, such as by incorporation of labeled nucleotides or other such labels. In general, activity is compared to cells that do not express the targeted protease.

  For example, the cell may be anything that expresses the targeted MTSP or endotheliase. Such cells can be selected by selecting cells that are known to express cell surface proteases, as discussed above, or by measuring cell expression profiles, or cell lines with various antibodies. Against protease activity in the presence of a labeled substrate, such as chromogenesis for the protease, in the presence or absence of a known inhibitor of the targeted protease, or in the presence or absence of a known inhibitor of the targeted protease It can be obtained by screening.

  Alternatively, a nucleic acid encoding the protease can be introduced into a cell line that does not express the protease and expressed there to create a cell line that expresses the protease of interest. The resulting recombinant cells can be used in cytotoxicity assays.

2. In Vivo Assays A number of animal models are known for assessing therapeutic activity. Any suitable in vivo model can be used. Mouse xenograft models and chicken embryo models are exemplary.

Chicken Embryo Model The CAM model (chick embryo outer membrane model; Ossowski (1988) J. Cell Biol. 107: 2437-2445) provides another method for assessing the inhibitory activity of test compounds. In the CAM model, tumor cells invade through the outer embryonic membrane, including CAM (tumor cells in the presence of some serine protease inhibitors result in less or no invasion of tumor cells through the membrane). . Thus, CAM assays are performed with CAM and tumor cells in the presence and absence of various concentrations of test compound. The degree of tumor cell invasion is measured under such conditions to provide an indication of the inhibitory activity of the compound. Compounds with inhibitory activity correlate with less tumor invasion.

  Thus, CAM assays are performed with CAM and tumor cells in the presence and absence of various concentrations of test compound. Compounds with activity correlate with changes in tumor invasion and / or tumor growth.

  For example, the ability of a cell surface protease to release a therapeutic agent such as a cytotoxic agent or the activity of a binding agent can be assessed using this model. If the therapeutic agent is released from the compound and it is an inhibitor, there will be less tumor invasion or tumor size reduction. If the therapeutic agent is inactive in the binder, there will be no effect on tumor invasion.

  The CAM model is also used in angiogenesis (ie, a standard assay for effects on the formation of new blood vessels (Brooks et al. (1991) Methods in Molecular Biology 129: 257-269)). According to this model, a filtration disc containing an angiogenesis inducer such as basic fibroblast growth factor (bFGF) is placed on the CAM. Cytokine diffusion into the CAM induces local angiogenesis, which can be measured in several ways, such as by counting the number of vascular bifurcations in the CAM just below the filtration disk. . The ability of the identified compounds to inhibit cytokine-induced angiogenesis can be tested using this model. The test compound can either be added to a filtration disc containing an angiogenesis inducer, placed directly on the membrane, or administered systemically. The degree of new angiogenesis in the presence and / or absence of the test compound can be compared using this model. Less new blood vessel formation in the presence of the test compound would be indicative of anti-angiogenic activity.

  This is referred to herein as 1) to assess the activity of a therapeutic agent in the conjugate; 2) to use a conjugate to assess the ability of a particular cell surface protease to release the therapeutic agent from the conjugate. Can be adapted for.

Mouse Xenograft Model In vivo activity can be assessed using recognized animal models, such as the well-known mouse xenograft model for anti-tumor activity (eg, Beitz et al. (1992) Cancer Research 52 : 227-230; Houghton et al. (1982) Cancer Res. 42: 535-539; Bogden et al. (1981) Cancer (Philadelphia) 48: 10-20; Hoogenhout et al. (1983) Int. J. Radiat Oncol., Biol. Phys. 9: 871-879; Stastny et al. (1993) Cancer Res. 53: 5740-5744). In vivo mouse solid tumor xenograft models are used in assays to test the ability of agents to inhibit tumor cell growth and / or spontaneous metastasis.

  For example, conjugates are evaluated for anti-tumor activity against any tumor subtype that expresses a targeted cell surface protease, such as an ovarian tumor, in a mouse tumor xenograft model. Nude mice are given one or more doses, such as four intravenous injections of the conjugate. Dosage substances are prepared by mixing the test substance with an appropriate volume, eg, PBS / 0.1% BSA, to achieve the desired dosage. Mouse IV injection (250-300 μl) into the tail vein is, for example, the day when the tumor cells are injected into the mouse is called day 1 and is like day 5, day 12, day 19, day 26 During the experimental period, mice are injected. The dose is fixed or standardized for body weight differences. Tumor volume is measured twice a week for a selected period.

  Female Balb / c nu / nu athymic mice (Roger Williams Hospital Animal Facility, Providence, RI), 8-12 weeks old are suitable mice. They should be maintained in a sterile environment and selected so that the weight ranges from about 25-30 grams the day before administration. Animals are maintained in an isolated room and handled under aseptic conditions. Diet and water are provided freely. Appropriate tumor cells can be obtained, for example, from the American Type Culture Collection (Rockville, MD) and grown in modified Eagle's medium supplemented with 10% fetal calf serum. For a selected number of days, such as 5 days prior to injection of test substance, mice receive a subcutaneous injection of tumor cells in the right posterior flank.

Vernier calipers are used to measure the size of each tumor. Maximum and minimum width measurements (mm) are performed at selected intervals, such as every two weeks, prior to test substance injection and during the experimental period. Tumor volume (mm ³ ) can be expressed, for example, by the formula:
Volume = [(width) ² (length) ² ] / 2
Is used to calculate.

G. Methods for identifying a protease to be targeted Also provided are methods for identifying a protease to which a conjugate for treatment of a disease should be targeted. The method involves identifying a cell-surface protease-related disease by identifying a cell involved in the disease process or a cell in the vicinity of a cell involved in the disease process. For example, if the disease is associated with a particular tumor, proteases are identified that are localized on cells located on or near that particular tumor. The cell surface protease on the cell for targeting and the substrate for it are then identified. Conjugates that target proteases as provided herein can then be made.

  The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

General Procedure for Making Peptide-Doxorubicin Conjugates Step A: Synthesis of Peptides on Wang Resin Using an ABI 431A peptide synthesizer from Perseptive Biosystems on preloaded Wang resin (0.25 mmol) Peptides were created automatically. ABI 431A uses HOBT, HBTU, DIEA activation. The synthesis of N-acetyl (or other amide) capped peptides involved the use of AcOH (or other respective carboxylic acid) during the final coupling step on ABI 431A. The other N-terminal caps were coupled manually using the following reagents: For carbamates and sulfonamides, the peptides were ROCOCl or RSO ₂ Cl and DIEA (4 eq, 1 eq, 1 mL, respectively) in DMF (3 mL). Time).

Step B: Cleavage of peptide from Wang resin
Cleavage of the peptide from the Wang resin, the resin _{TFA / H 2 O (95:} 5) accompanied by shaking for 45 minutes at 2 mL. The resin was removed by filtration and the filtrate was left for an additional hour. The solution was concentrated to a residue. The crude peptide was analyzed by analytical HPLC (System A). Typical purity of the crude peptide ranged from 80% to 95%. The peptide was purified by preparative HPLC (System B) using an appropriate gradient (typically 10-30%). The pure fraction was then lyophilized to give the desired peptide as a white solid. Typical yields were 20-50% with a purity of 96-99%.

Analytical HPLC conditions (System A)
Column: Chromolith RP-18e from EM science 4.6mm x 100mm
Gradient: 5 to 50% B in A in 6 minutes
Flow rate: 4 mL / min Solvent A: 0.1% TFA in water
Solvent B: 0.1% TFA in acetonitrile
Wavelength: 210nm, 280nm

Preparative HPLC conditions (System B)
Column: Ultro 120 5 C18Q 150 × 20 mm from Peeke Science
Gradient: 0-20% or 10-30% or 20-40% B in A in 40 minutes
Flow rate: 18 mL / min Solvent A: 0.1% TFA in water
Solvent B: Acetonitrile Wavelength: 214 nm

Step C: Coupling of Peptide Acid to Doxorubicin Peptide Acid (0.052 mmol, 1.2 eq), Doxorubicin Hydrochloride (0.043 mmol, 25 mg) and HATU (0.0604 mmol, 22.9 mg, 1.4 eq) To the mixture was added DMF (1 mL) followed by 2,6-lutidine (0.17 mmol, 20 μL, 4 eq). The mixture was mixed until a homogeneous solution was obtained. After 4-24 hours (monitored by HPLC system A), the reaction was diluted with water (9 mL) and directly purified by preparative HPLC (system D). The pure fraction was then lyophilized to give the doxorubicin conjugate of the desired peptide as a fluffy red solid. The quality of the final conjugate was assayed by analytical HPLC (System C) and mass spectrometry. Typical yields were 10-30% with 95-99% purity. (Note: When the peptide acid contains a histidine residue, it was replaced with DIEA as the base, reducing the reaction time to 1 hour).

  Deprotection of fluorenylmethyl ester of peptide doxorubicin conjugate: When free carboxylic acid is present in the conjugate, fluorenylmethyl ester can be used to couple the C-terminal of the peptide acid to doxorubicin. The carboxylic acid was protected, followed by removal of the fluorenyl group with 10% morpholine in DMF in 1 hour.

Analytical HPLC conditions (System C)
Column: Chromolith RP-18e from EM science 4.6mm x 100mm
Gradient: 5 to 50% B in A in 6 minutes
Flow rate: 4 mL / min Solvent A: 0.1% TFA in water
Solvent B: 0.1% TFA in acetonitrile
Wavelength: 210nm, 280nm
Example of retention time (min) Doxorubicin 4.05
Ac-Gly-Ser-Gly-Arg-Ser-nLeu-Dox 4.34
MeOCO-Thr-Gly-Arg-Ser-nLeu-Dox 4.39
PhSO2-Thr-Gly-Arg-Ser-nLeu-Dox 4.83
N, N-dimethylglycine-Thr-Gly-Arg-Ser-nLeu-Dox 4.27
Ac-Thr-Gly-Arg-Ser-nLeu-Dox 4.32.

Preparative HPLC conditions (System D)
Column: Ultro 120 5 C18Q 150 × 20 mm from Peeke Science
Gradient: 10-30% B in A over 40 minutes
Flow rate: 18 mL / min Solvent A: 0.1% acetic acid in water Solvent B: Acetonitrile Wavelength: 214 nm

Preparation of Ac-Gly-Ser-Gly-Arg-Ser-nLeu-Dox Step A: Manual synthesis of Ac-Gly-Ser (tBu) -Gly-Arg (Pbf) -Ser (tBu) -nLeu-Wang resin Nitrogen stirring And Fmoc-nL-Wang resin (nova-biochem, 3.3 grams, 0.9 mmol / g, 3 mmol) in DMF using 30 mL in a 250 mL fritted peptide synthesis vessel equipped with a vacuum assisted drainer. Pre-swelled for minutes. The peptides were then extended by repeating the following four-step procedure a total of 5 times for the following Fmoc amino acids: Fmoc-Ser (tBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH.

Repeated coupling procedure The resin was mixed with 20% piperidine in DMF (100 mL) for 5 minutes and then drained (repeat 3 times).
2. The resin was stirred with DMF (100 mL) for 30 seconds and then drained (repeat 3 times).
3. To a mixture of Fmoc-amino acid (12 mmol), HOBT (12 mmol, 4 eq, 1.622 g), TBTU (11.7 mmol, 3.9 eq, 3.757 g), DMF (10 mL) and NMP (90 mL) was added DIEA (90 mL). 12 mmol, 4 eq, 2.10 mL) was added. After pre-activation by stirring for 5 minutes, the solution was added to the synthesis vessel. The completion of the reaction was checked by the ninhydrin test and then drained. (If the ninhydrin test was blue, a double coupling (repeat step 3) was performed.)
4). The resin was stirred with DMF (100 mL) for 30 seconds and then drained (repeat 3 times).

The stretched resin (Fmoc-Gly-Ser (tBu) -Gly-Arg (Pbf) -Ser (tBu) -nLeu-Wang resin) was treated in steps 1 and 2 above to remove Fmoc. A solution of acetic anhydride (15 mmol, 5 eq, 1.42 mL), DIEA (15 mmol, 5 eq, 2.62 mL), DMF (10 mL) and NMP (90 mL) was added to the reaction vessel. After 1 hour, the resin was washed with DMF (100 mL, 3 times), CH ₂ Cl ₂ (100 mL, 3 times) and MeOH (100 mL, 3 times). The resin was dried under vacuum for 15 hours.

Step B: Preparation of Ac-Gly-Ser-Gly-Arg-Ser-nLeu-OH Ac-Gly-Ser (tBu) -Gly-Arg (Pbf) -Ser (tBu) -nLeu-Wang resin (3 mmol) To the above synthesis vessel was added TFA / H ₂ O (95: 5, 50 mL). After gentle stirring for 45 minutes, the cleavage solution was collected and the filtrate was left for an additional 90 minutes. The solution was concentrated to a residue. The crude peptide was analyzed by analytical HPLC (System A, RT = 1.73, purity = 90%). The residue was dissolved in water (50 mL) and hexane (10 mL) and mixed. The hexane layer was removed and nitrogen was blown into the aqueous layer to evaporate any remaining hexane. The crude peptide was purified by preparative HPLC (System E). The pure fractions were then lyophilized to give Ac-Gly-Ser-Gly-Arg-Ser-nLeu-OH (1.04 g, 1.68 mmol, 56%) as a white solid. Purity was assessed by analytical HPLC (System A, RT = 1.73 min, 97% purity) and structure was assessed by mass spectrometry (ions observed at 617.9).

Preparative HPLC conditions (System E)
Column: Waters Delta-Pak centrifugal compression column 15 μm, 100A
Gradient: 5-15% B in A in 40 minutes
Flow rate: 80 mL / min Solvent A: 0.1% acetic acid in water Solvent B: Acetonitrile Wavelength: 214 nm

Step C: Preparation of Ac-Gly-Ser-Gly-Arg-Ser-nLeu-Dox Ac-Gly-Ser-Gly-Arg-Ser-nLeu-OH (1.68 mmol, 1.04 g, 1.1 eq), DMF (40 mL) to a mixture of doxorubicin hydrochloride (1.53 mmol, 887.8 mg) and HATU (1.76 mmol, 669.6 mg, 1.15 eq), then 2,6-lutidine (6.12 mmol, 709 μL, 4 equivalents) was added. The solution was stirred for 18 hours. The reaction was diluted with water (100 mL), acidified with acetic acid (400 μL), purified in 3 batches and immediately on preparative HPLC (System E). Each red fraction was analyzed by analytical HPLC (System F). Subsequently, fractions with a purity of 95% or more were combined. The acetonitrile was removed under vacuum and the remaining solution was lyophilized to give Ac-Gly-Ser-Gly-Arg-Ser-nLeu-Dox (0.682 mmol, 780 mg, 45%) as a fluffy red solid. Purity was assessed by analytical HPLC (System F, RT = 3.51 min, 95% purity) and structure was assessed by mass spectrometry (ions observed at 1143.5).

Analytical HPLC conditions (System F)
Column: Chromolith RP-18e from EM science 4.6mm x 100mm
Gradient: 20-40% B in A in 6 minutes
Flow rate: 4 mL / min Solvent A: 0.1% TFA in water
Solvent B: 0.1% TFA in acetonitrile
Wavelength: 210nm, 280nm

Preparative HPLC conditions (System E)
Column: Waters Delta-Pak centrifugal compression column 15 μm, 100A
Gradient: 15-25% B in A in 40 minutes
Flow rate: 80 mL / min Solvent A: 0.1% acetic acid in water Solvent B: Acetonitrile Wavelength: 214 nm

General Method for Making Peptide-Taxol Conjugates Step A: Synthesis of Peptides on Wang Resin See Example 1, Step A Step B: Cleavage of Peptides from Wang Resin Example 1, Step B Please refer to

Step C: Coupling of Peptide Acid to 7-Gly-Taxol or 7-Ala-Taxol Peptide Acid (0.0121 mmol, 1.1 eq), 7-Gly-Taxol or 7-Ala-Taxol (0.011 mmol) And HATU (0.0154 mmol, 5.9 mg, 1.4 eq) were added DMF (0.3 mL), followed by 2,6-lutidine (0.044 mmol, 5.1 μL, 4 eq). The mixture was mixed until a homogeneous solution was obtained. After 4-24 hours (monitored by HPLC system H), the reaction was diluted with water (9 mL) and purified directly by preparative HPLC (System I). The pure fraction was then lyophilized to give the desired peptide taxol conjugate as a fluffy white solid. The quality of the final conjugate was assayed by analytical HPLC (System H) and mass spectrometry. Typical yields were 30-50% with 96-99% purity.

Analytical HPLC conditions (System H)
Column: Chromolith RP-18e from EM science 4.6mm x 100mm
Gradient: 5 to 90% B in A in 6 minutes
Flow rate: 4 mL / min Solvent A: 0.1% TFA in water
Solvent B: 0.1% TFA in acetonitrile
Wavelength: 210nm, 280nm

Example of retention time (min) Ac-Gln-Ser-Arg-Ala-Ala-Taxol 2.86
Ac-Gln-Ser-Arg-Ser-Ala-Ala-Taxol 2.79
Ac-Ser-Gly-Arg-Ala-Ser-Ala-Taxol 2.87
Ac-Arg-Ser-Arg-Ala-Ala-Taxol 2.80
Ac-Ser-Gly-Arg-Ser-Ser-Ala-Taxol 2.81

Preparative HPLC conditions (System I)
Column: Ultro 120 5 C18Q 150 × 20 mm from Peeke Scientific
Gradient: 20-45% B in A in 40 minutes
Flow rate: 18 mL / min Solvent A: 0.1% TFA in water
Solvent B: Acetonitrile Wavelength: 214 nm
~ 196

Creation of N-Ac-Arg-Gln-Ser-Arg-Ala-Ala-DOX

Step A: N-Ac-Arg-Gln-Ser-Arg-Ala-Ala-OH
The N-acetyl peptide substrate N-Ac-Arg-Gln-Ser-Arg-Ala-Ala-OH was synthesized in a peptide synthesis flask using the following general procedure. Starting with commercially available Fmoc-Ala-Wang resin (0.35 g, 0.84 mmol, Nova), standard Fmoc-deprotection with 20% piperidine followed by sequential iterative coupling-Fmoc deprotection strategy It was. In each coupling, a 3-fold excess (2.52 mmol) of Fmoc-Ala, Fmoc-Arg (Boc) ₂ , Fmoc-Ser (tBu), Fmoc-Gln (Trt) and Fmoc-Arg (Boc) ₂ was respectively added. used. Coupling was achieved with PyBOP (2.52 mmol) and DIEA (2.52 mmol) in DMF solvent. During each coupling cycle, the Fmoc protecting group was removed using 20% piperidine in DMF.

  After removal of the N-terminal Fmoc group, capping with acetic anhydride (1.43 mmol, 1.7 eq), DMAP (0.25 mmol, 0.3 eq) and DIEA (1.26 mmol, 1.5 eq) Gave the N-acetyl intermediate bound to. The protected peptide resin was treated with 50% TFA in methylene chloride for 30 minutes to cleave the Wang-resin, then Boc, and the Trt and t-Bu protecting groups were removed with 70% TFA in methylene chloride. Solvent and other volatile byproducts were removed under reduced pressure, and the crude product was dissolved in water and lyophilized to give the title compound as an almost colorless, amorphous solid. Mass spectrometry confirmed the desired molecular weight. HPLC analysis indicated that the product was approximately 95% pure. The peptide carboxylic acid intermediate can be triturated in a solvent or further purified by preparative HPLC if desired.

Step B: N-Ac-Arg-Gln-Ser-Arg-Ala-Ala-DOX
The intermediate from Step A (20 mg, 0.027 mmol) was dissolved in dry DMF (0.8 mL) and stirred at room temperature under a nitrogen atmosphere. To this solution was added doxorubicin hydrochloride (15.6 mg, 0.027 mmol), EDC (6.8 mg, 0.035 mmol), HOAt (4.8 mg, 0.035 mmol) and 2,6-lutidine (7.3 μL, 0. 0.06 mmol) was added. Stirring was continued until the end of the coupling while monitoring by analytical HPLC (System J, see below). The solution was filtered, the crude product, C18 _RP-HPLC; was purified by _{(A = 0.1% AcOH / H} 2 O B = from CH 3 CN, gradient elution 100% 60 minutes until 60% A) . Homogeneous product fractions (assessed by HPLC, system J) were pooled and lyophilized to give the title compound as a pale red solid.

HPLC conditions (System J)
Column: Phenomenex 15cm # 00F-3033-E0, C18
Eluent: gradient from 95: 5 (A: B) to 25:75 (A: B) in 20 minutes
A = 0.1% TFA / H ₂ O, B = 0.1% TFA / acetonitrile Flow rate: 1 mL / min Wavelength: 210 nm, 280 nm
Retention time: doxorubicin = 8.89 minutes
N-Ac-Arg-Gln-Ser-Arg-Ala-Ala-DOX = 8.4 min

Physical properties:
Molecular _{formula: C 55 H 78 N 14 O} 20
Molecular weight: 1255.3
Low resolution mass spectrometry: 628.2 (M + 2/2)

  Table 2 displays data for additional peptidic substrate-doxorubicin conjugates. These conjugates were made from the appropriate amino acid precursor as made by the general procedure described in Example 4.

Table 2

Measurement of time to 50% cleavage of the doxorubicin / peptidic substrate conjugate by the recombinant protease domain of MTSP1 A 1 mM stock solution was made for each peptidic substrate conjugate in double distilled water. The conjugate 100 μM was then added in 29.2 mM Tris, pH 8.4, 29.2 mM imidazole, 217 mM NaCl, to the recombinantly produced active single stranded protease domain of MTSP1 (nucleotide 1865 of SEQ ID NO: 1). A cleavage reaction was performed where it mixed with residues 615-855) 1 or 10 nM in SEQ ID NO: 2 encoded by 2582. The final reaction volume was 200 μL. These reactions were incubated at 37 ° C. in a water bath. At times ranging from 2 to 128 minutes, 20 μL of sample was removed and enzyme activity was stopped by adding trifluoroacetic acid to 0.33%. The amount of hydrolysis in each sample was measured by reverse layer HPLC. The product peak area was then divided by the sum of the substrate and product peak areas to calculate the percent hydrolysis. The percentage of unhydrolyzed substrate was plotted against the logarithm of the reaction time and the plot was fitted to a sigmoidal curve using Graphpad Inc. (San Diego, Calif.) Prism software, with 50% of each substrate being cleaved. The time was measured.

The results for the specific conjugates provided herein are shown for FIG. 1 (conditions: 12 mM Tris (hydroxymethyl) aminomethane, pH 8.0, 25 mM NaCl, 0.5 mM CaCl ₂ at 100 ° M conjugate at 37 ° C. 1 nM MTSP1; the reaction was stopped with 0.33% trifluoroacetic acid).

In vitro assay of conjugate cytotoxicity The conjugate cytotoxicity can also be tested to confirm that the conjugate acts as a prodrug. The conjugates are tested against cell lines that are known to be killed by unmodified cytotoxic agents using the Alamar Blue assay. Cells such as LNCaP cells (The American Type Culture Collection (Rockville, Maryland)) that express cell surface proteases, such as MTSP1 or endopeptidase, are 1 × 10 ⁴ cells / well (0.1 mL / well). Seeds in a 96-well plate at a density of A plate containing only medium is used as a control. Cells are incubated at 37 ° C. for 3 days and 20 μL of Alamar Blue is added to the assay well. After 7 hours of incubation, cell death is measured using an EL-310 plate reader at 570 and 600 nm. Cell mortality values are expressed as a percentage decrease in cell number relative to the media control.

In vivo efficacy of conjugates Tumor cells are trypsinized, resuspended in growth medium and centrifuged at 200 xg for 6 minutes. Cells are resuspended in serum free α-MEM and counted. The appropriate volume of this solution containing the desired number of cells is then transferred to a conical centrifuge tube, centrifuged as before, and resuspended in an appropriate volume of a 1: 1 cold mixture of α-MEM-Matrigel. It becomes cloudy. The suspension is kept on ice until the animals are inoculated.

10 week old male nude mice are used. Mice are weighed individually and grouped with a weight difference of 2 grams or less between individual mice within each group (n = 10 per group). On day 1, mice are inoculated subcutaneously with tumor cell lines. Each mouse is inoculated with 0.5 mL of 0.5 × 10 ⁶ to 10 ⁸ tumor cells / mL, for example, in a 60% solution of ice-cold Matrigel and a-MEM. The conjugate administration was then started 24 hours later. Vehicle treated mice are injected with 5% dextrose in water. At the end of a predetermined time, such as from 18 days to 2 months or longer, mice are sacrificed and tumor size and volume or other parameters are measured. Tumor size and volume or other parameters for mice treated with the conjugate are compared to vehicle treated mice to determine the effectiveness of the conjugate.

After inoculation with tumor cells, mice are treated under one of three protocols:
Protocol A
One day after cell inoculation, the animals contain 1 to 100, or 3 to 50, or 7 to 7, containing 7.2 or 17.9 μg / kg of test conjugate, unmodified cytotoxic agent or vehicle control (sterile water). Dosing 22 μg / kg. The dose of conjugate and cytotoxic agent is initially a maximum non-lethal dose, but can be subsequently titrated lower. The same dose is administered for 5 days at 24 hour intervals. At the end of 5.5 weeks or other suitable period, mice are sacrificed and any tumor present is weighed. Animals are weighed at the beginning and end of the assay.

Protocol B
14-15 days after cell inoculation, the animals are given 1-100, or 3-50, containing 7.2 or 17.9 μg / kg of test conjugate, unmodified cytotoxic agent or vehicle control (sterile water), Alternatively, 7-22 μg / kg is administered. The dose of conjugate and cytotoxic agent is initially a maximum non-lethal dose, but can be subsequently titrated lower. The same dose is administered for 5 days at 24 hour intervals. At the end of 5.5 weeks or other suitable period, mice are sacrificed and any tumor present is weighed. Animals are weighed at the start and end of the assay.

Protocol C
One day after cell inoculation, the animals contain 1 to 100, or 3 to 50, or 7 to 7, containing 7.2 or 17.9 μg / kg of test conjugate, unmodified cytotoxic agent or vehicle control (sterile water). 22 μg / kg is administered by intraperitoneal administration. The dose of conjugate and cytotoxic agent is initially a maximum non-lethal dose, but can be subsequently titrated lower. The same dose is administered at 7-day intervals for 5 weeks. At the end of 5.5 weeks or other suitable period, mice are sacrificed and any tumor present is weighed. Animals are weighed at the start and end of the assay.

Example MTSP Gene Expression Profiles and Domain Organization MTSP1 Gene Expression Profiles in Normal Tissues, Cancer Cells and Cancer Tissues To obtain information on MTSP1 tissue distribution and gene expression levels, encoding nucleic acids DNA inserts from the Pichia pastoris expression vector, pPIC9K-MTSP1, including, from 76 different human tissues (Catalog Number 77775-1; Human Multiple Tissue Expression (MTE) Array; CLONTECH, Palo Alto, CA) Blots containing RNA were probed. Significant expression was observed in the colon (ascending, transverse and descending), rectum, trachea, esophagus and duodenum. Moderate expression levels were observed in the jejunum, ileum, ileocecum, stomach, prostate, pituitary gland, appendix, kidney, lung, placenta, pancreas, thyroid, salivary gland, mammary gland, fetal kidney and fetal lung. Lower expression levels were observed in spleen, thymus, peripheral vascular leukocytes, lymph nodes, bone marrow, bladder, uterus, liver, adrenal gland, fetal heart, fetal liver, fetal spleen and fetal thymus.

  Significant amounts of MTSP1 transcript were also detected in colorectal adenocarcinoma cell line (SW480), Burkitt lymphoma cell line (Daudi) and leukemia cell line (HL-60). RT-PCR of MTSP1 transcripts in several human primary tumors xenografted in athymic nude mice was performed using gene specific primers. High levels of MTSP1 transcript were detected in colon adenocarcinoma (CX-1) and pancreatic adenocarcinoma (GI-103). Moderate levels were observed in other colon adenocarcinoma (GI-112), ovarian carcinoma (GI-102), lung carcinoma (LX-1) and breast carcinoma (GI-101). Another lung carcinoma (GI-117) expressed low levels of MTSP1 transcript. Similar RT-PCR was performed to detect the presence of MTSP1 transcripts in PC-3 and LNCaP cell lines. Both cell lines expressed significant amounts of MTSP1 transcript. MTSP1 is also a marker for ovarian cancer.

Gene Expression Profile of Serine Protease MTSP3 in Normal and Tumor Tissues To obtain information about the tissue distribution of MTSP3 transcripts, 76 different human tissues (Cat. No. 7775) were used with DNA inserts encoding the MTSP3 protease domain. -1; Human multiple tissue expression (MTE) array; CLONTECH, Palo Alto, CA) RNA blots were probed. The observed expression pattern is as follows: decreasing order of signal level: trachea = colon (descending) = esophagus> colon (ascending)> colon (transverse) = rectum>ileum>jejunum>jejunum>gallbladder>ileocecum>stomach>kidney> It was an appendix. It is also expressed in lower amounts in fetal kidney and in the two tumor cell lines, HelaS3 and leukemia cell line K-562. Northern analysis using RNA blots (Catalog Nos. 7780-1, 7765-1 and 7782-1; human 12-lane, human muscle and human digestive system multiple tissue Northern (MTN) blot; CLONTECH) shows the highest expression in the colon In addition, it was found to be detected moderately in the esophagus, small intestine, gallbladder and kidney and in lower amounts in the stomach and rectum. A single transcript of approximately 2 kb was detected.

  Amplification of MTSP3 transcripts in several human primary tumors xenografted into mice was performed using gene specific primers. MTSP3 transcripts were detected in lung carcinoma (LX-1), colon adenocarcinoma (CX-1), colon adenocarcinoma (GI-112) and ovarian carcinoma (GI-102). No obvious signal was detected in the other forms of lung carcinoma (GI-117), breast carcinoma (GI-101), pancreatic adenocarcinoma (GI-103) and prostate adenocarcinoma (PC3).

Gene expression profile of MTSP4 in normal and tumor tissues 76 different human tissues using DNA fragments encoding portions of the LDL receptor domain and the protease domain to obtain information on the gene expression profile of the MTSP4 transcript An RNA blot consisting of (Cat. No. 77775-1; human multiple tissue expression (MTE) array; CLONTECH) was probed. A very strong signal was observed in the liver, as in Northern analysis of gel blots. Signals in other tissues (in decreasing order of signal level): fetal liver> heart = kidney = adrenal = testis = fetal heart and kidney = skeletal muscle = gallbladder = placenta> brain = spinal cord = colon = stomach = spleen = lymph node = Bone marrow = trachea = uterus = pancreas = salivary gland = mammary gland = lung. MTSP4 also has several tumors including HeLaS3 = leukemia K-562 = Burkitt lymphoma (Raji and Daudi) = colon adenocarcinoma (SW480)> lung carcinoma (A549) = leukemia MOLT-4 = leukemia HL-60 It is expressed in lower amounts in cell lines.

  PCR of MTSP4 transcripts from a cDNA library made from several human primary tumors xenografted in nude mice (Human Tumor Multiple Tissue cDNA Panel, Catalog No. K1522-1, CLONTECH) was performed using MTSP4-specific primers. It carried out using. MTSP4 transcripts were detected in breast carcinoma (GI-101), lung carcinoma (LX-1), colon adenocarcinoma (GI-112) and pancreatic adenocarcinoma (GI-103). No obvious signal is detected in another form of lung carcinoma (GI-117), colon adenocarcinoma (CX-1), ovarian carcinoma (GI-102) and prostate adenocarcinoma (PC3) It was. MTSP4 transcripts were also detected in LNCaP and PC-3 prostate cancer cell lines and in the HT-1080 human fibrosarcoma cell line.

Gene expression profile of MTSP6 in normal and tumor tissues To obtain information on the gene expression profile of MTSP6 transcript, a 495 bp DNA fragment obtained from PCR reaction with primers Ch17-NSP-3 and NSP-4AS was used. RNA blots consisting of a number of different human tissues (Cat. No. 77775-1; human multiple tissue expression (MTE) array; CLONTECH) were probed. The strongest signal was observed in the duodenum. Signals in other tissues (in decreasing order of signal level): stomach> trachea = mammary gland = thyroid = salivary gland = pituitary gland = pancreas>kidney>lung> jejunum = ileum = ileocecum = appendix = fetal kidney> fetal lung Was observed. A very weak signal can also be detected in some other tissues. MTSP6 is also expressed in several tumor cell lines including HeLaS3> colon adenocarcinoma (SW480)> leukemia MOLT-4> leukemia K-562.

  PCR analysis of MTSP6 transcripts from cDNA libraries made from several human primary tumors xenografted in nude mice (Human Tumor Multiple Tissue cDNA Panel, Catalog No. K1522-1, CLONTECH) Performed with primers (Ch17-NSP-3 and Ch17-NSP2AS). MTSP6 transcripts are detected strongly in lung carcinoma (LX-1), moderately in pancreatic adenocarcinoma (GI-103), and weakly detected in ovarian carcinoma (GI-102); and colon adenocarcinoma (GI -112 and CX-1), breast carcinoma (GI-101), lung carcinoma (GI-117) and prostate adenocarcinoma (PC3) were detected very weakly. The MTSP6 transcript was also detected in the breast cancer cell line MDA-MB-231, prostate cancer cell line PC-3, but not in the HT-180 human fibrosarcoma cell line. MTSP6 is also expressed in mammary carcinoma cDNA (Clontech).

MTSP9 gene expression profile in normal tumor tissues and cell lines To obtain a gene expression profile of the MTSP9 transcript, MTSP9 cDNA fragments obtained from human pancreas were used to obtain 76 different human tissues (human multiple tissue expression ( MOT) arrays; Clontech, Palo Alto; Cat. No. 77775-1). Point blots consisting of RNA extracted were probed. The analysis results indicate that MTSP9 is highly expressed in the trachea and is expressed at low levels in many other tissues. MTSP9 transcripts are found in kidney (adult and fetus), spleen (adult and fetus), placenta, liver (adult and fetus), thymus, peripheral blood leukocytes, lung (adult and fetus), pancreas, lymph node, bone marrow, trachea, It is found in the uterus, prostate, esophagus, testis, ovary and glandular organs (breastline, adrenal gland, thyroid, pituitary and salivary lines). MTSP9 is also a tumor esophageal tissue, in lung carcinoma (A549 cell line), and at low levels, colorectal carcinoma (SW480), lymphoma (Raji and Daudi), cervical carcinoma (HelaS3) and leukemia ( HL-60, K-562 and MOLT-4).

Gene expression profile of MTSP10 in normal and tumor tissues To obtain information on gene expression of MTSP10 transcripts, MTSP10-specific primers were used to determine the number of human adult tissues (Clontech, Cat. # K1420-1). PCR analysis was performed on a cDNA panel made from the cDNA panel. MTSP10 transcript was detected in pancreas, lung and kidney. The MTSP10 transcript was also detected in the small intestine Marathon-Ready cDNA (Clontech). PCR of MTSP10 transcripts from a cDNA library made from several human primary tumors xenografted in nude mice (Human Tumor Multiple Tissue cDNA Panel, Catalog No. K1522-1, CLONTECH) was also performed.

  MTSP10 transcripts were detected in breast carcinoma (GI-101), lung carcinoma (LX-1 and GI-117), ovarian carcinoma (GI-102) and pancreatic adenocarcinoma (GI-103). The MTSP10 transcript can also be weakly detected in prostate cancer (PC-3). No obvious signal was detected in the two forms of colon adenocarcinoma (GI-112 and CX-1). MTSP10 transcripts were also detected in CWR22R prostate tumors grown on nude mice.

Domain organization and gene expression profiles of MTSP12 in normal and tumor tissues Domain organization of MTSP12PD1, -PD2 and -PD3 and homology to other serine proteases Translated MTSP12-PD1, -PD2 and -PD3 nucleotides Analysis of the sequence and protein domain shows that these three serine proteases are in close proximity. The sequence is as follows: MTSP12-PD1 followed by MTSP12-PD2 is found at the N-terminus and MTSP12-PD3 is found at the C-terminus. MTSP12-PD1 and -PD2 respectively to protease activation cleavage site (MTSP12-PD1 and _{-PD2 ··· R 236 ↓ I 237 VGGMEAS} ··· and _{··· R 537 ↓ V 538 VGGFGAA ···} [ Where ↓ indicates a protease-activated cleavage site]) and catalytic triad residues in three highly conserved regions of its catalytic domain (His ₂₇₇ , As and Ser _{421 for} MTSP12-PD1; His for MTSP12-PD2) ₅₇₈ , As and Ser ₇₂₁ ), contains a trypsin-like serine protease domain (aa236-aa465 and aa537-aa765 for MTSP12-PD1 and -PD2, respectively). MTSP12-PD3 contains serine protease domain (aa861~aa1087); it has a protease activation cleavage site _{(··· R 860 ↓ I 861 VGGSAAG} ···), and has a catalytic _{His 902} and As However, it has Ala ₁₀₄₃ instead of the conserved catalytic serine found in serine proteases.

Several domains, are found upstream of MTSP12-PD1 serine protease domain, which are the transmembrane domain (aa28~aa50), SEA [sea urchins semen protein (s ea urchin sperm protein) - enterokinase (e nterokinase) - including agrin a (a grin)] domain (aa51~aa170) and LDLa [low-density lipoprotein receptor class a (l ow d ensity l ipoprotein receptor class a)] domain (aa187~aa225). There are five possible N-linked glycosylation sites (N ₁₁₆ SS, N ₅₈₁ HT, N ₆₇₂ AT, N ₆₉₇ FS and N ₈₂₀ ST). In the protease domain of MTSP12-PD1, there is one unpaired cysteine (C ₃₄₆ ) in the single-chain form of the protease domain, and the following Cys pairing is observed: C ₂₆₂ -C ₂₇₈ ; C _{360- C427} ; _{C417- C446} ; _{C392- C406} . In the protease domain of MTSP12-PD2, there is one unpaired cysteine (C ₆₄₆ ) in the single-chain form of the protease domain and the following Cys pairing is observed: C ₅₆₃ -C ₅₇₉ ; C _{660- C727} ; _{C692- C706} ; _{C717- C746} . In the protease domain of MTSP12-PD3, there is one unpaired cysteine (C ₉₆₉ ) in the single-chain form of the protease domain and the following Cys pairing is observed: C ₈₈₇ -C ₉₀₃ ; C _{983- C1049} ; _{C1014- C1028} ; _{C1039- C1068} .

  The alignment (blastp; http://www.ncbi.nlm.nih.gov/BLAST) of the protein sequences of the respective MTSP12-PD1, MTSP12-PD2 and MTSP12-PD3 against known serine proteases deposited in public databases is 45%, 45% and 48% identity for matriptase, 44%, 43% and 41% identity for DESC1 / endothelase 1, 44 for prostamine (AB030036) %, 43% and 48% identity, 43%, 39% and 39% identity for spinesin (TMPRSS5; NM_030770) and 40%, 38% and 38% for malapsin (NM_031948) Identity. The clone is approximately at the nucleotide and encoded protein level relative to the clone and encoded one described and provided in International PCT Application No. WO 02/00860 (see SEQ ID NOs: 38 and 97 therein). Has 93% homology. The encoded protein described in the PCT application, however, differs from the sequence shown in SEQ ID NO: 271 between amino acids Leu373 and Val374 of SEQ ID NO: 20 and another extended amino acid sequence of amino acids Ala48 of SEQ ID NO: 20. And lacks amino acids 91-124 of SEQ ID NO: 20. The proteins provided in International PCT Application No. WO02 / 00860 can be used in the methods provided herein.

Gene and tissue expression profiles of MTSP12 To obtain information on the tissue distribution profiles of MTSP12PD1, -PD2 and -PD3 transcripts, three cDNA probes were generated. The data show that MTSP12PD1, -PD2 and -PD3 transcripts are expressed at low levels in the majority of 76 tissues and cell lines, but at higher levels in lymph nodes and testis.

  To compare the expression profiles of MTSP12PD1, -PD2, and -PD3 in a range of normal human and matched tumor tissues, a matched tumor / normal expression array (Cat # 7480) consisting of 68 paired cDNA samples from individual patients. -1; http://www.clontech.com). The results show that MTSP12PD1, -PD2 and -PD3 transcripts are expressed at low levels in a number of normal tissues including breast, uterus, colon, ovary, lung, kidney and rectum but are not differential in any of the matched tumors It is not expressed. It is also at low levels, Hela (cervical carcinoma), Daudi (Burkitt lymphoma), K562 (chronic myeloid leukemia), HL-60 (promyelocytic leukemia), G361 (malignant melanoma), A549 (lung cancer) Tumors), MOLT-4 (lymphoblastic leukemia), SW480 (colorectal adenocarcinoma) and Raji (Burkitt lymphoma).

  Several SMART® 5 ′ made from normal breast, normal testis, normal prostate, prostate cancer cell line and breast cancer cell line for the presence of MTSP12PD1, -PD2 and -PD3 transcripts -The RACE cDNA library (catalog number K1811-1: http://www.clontech.com) was analyzed by RT-PCR using two sets of gene specific primers. MTSP12-PD2 and -PD3 transcripts were detected in normal prostate, PC-3, LNCaP, normal breast, MDA-MB-231, MDA-MB-361, MDA-MB-453 and DU4475, but normal Higher levels were observed in the breast and MDA-MB-231. MTSP12-PD1 transcripts were detected in the same tissues and cell lines except that higher levels were observed in normal breast, MDA-MB-231 and DU4475.

Gene expression profile of MTSP20 in normal tumor tissues and cell lines To obtain information on gene expression of MTSP20 transcripts, we used 76 different human tissues (human multiple tissue expression) using MTSP20 cDNA fragments obtained from human lung tissue. Point blots consisting of RNA extracted from (MTE) arrays; Clontech, Palo Alto, CA; catalog number 7775-1) were probed. The results indicate that RNA encoding MTSP20 is expressed in various tissues. MTSP20 transcripts are found in the liver, lymph nodes, cervix, pancreas, prostate, uterus, testis, glands (adrenal, thyroid and salivary glands), thymus, kidney and spleen. Lower transcript levels can be found in the lung, placenta, gallbladder, ovary, digestive system, circulatory system and other parts of the brain. MTSP20 transcripts also include lung carcinoma (A519), colorectal carcinoma (SW480), lymphoma (Raji and Daudi), cervical carcinoma (HeLaS3) and leukemia (HL-60, K562 and MOLT-4). It is expressed in specific tumor cell lines.

MTSP22 Gene Expression Profile in Normal Tumor Tissues and Cell Lines MTSP22 is expressed in uterine tissue, thymus, adipose tissue and lymph nodes. It is also expressed in the lung, stomach, uterus, breast, ovary, prostate and other tumors. To obtain information on the gene expression profile of the MTSP22 transcript, cDNA fragments encoding the entire serine protease domain were used to analyze 72 different human tissues (human multiple tissue expression (MTE) array; Clontech, Palo Alto, CA; Point blots consisting of RNA extracted from catalog number 7776-1) and normalization from 241 tumors and corresponding normal tissues from individual patients (cancer profiling array, Clontech, catalog number 7874-1) A point blot consisting of the prepared cDNA was probed. The results of MTE analysis indicated that the MTSP22 transcript is primarily expressed in the esophagus. In tumor profiling array analysis, MTSP22 is highly expressed in 3 out of 42 normal uterine tissue samples but not in those matched tumor samples.

In one of 42 uterine samples, MTSP22 is expressed in the tumor and its metastatic tissue but not in the normal tissue counterpart. MTSP22 is also expressed in 2 of 17 gastric tumors and 2 of 21 lung tumors, but not in their normal tissue counterparts. MTSP22 is also expressed in normal tissues of only pancreatic matched cDNA pairs. PCR analysis was also performed using commercially available cDNA panels from several human adult tissues (Clontech, catalog # K1420-1 and K14220-2) and primary tumors (Clontech catalog # K1522-1) and several Marathon- This was carried out using Ready cDNA (Clontech).
MTSP22 cDNA was detected in thymus, adipose tissue, and lymph nodes. Homology of MTSP22 to serine protease domain and other proteases

  Sequence analysis of the translated MTSP22 protease domain sequence shows that MTSP22 is a catalytic triad of residues at the amino terminus of the domain and three highly conserved regions (histidine, aspartate and serine). Was found to contain a trypsin-like serine protease domain characterized by the presence of. Alignment of the protein sequence with the sequence of endothelinase 1 (same as the serine protease DESC1 protein; GenBank accession number AF064819) indicated that the two proteins share 50% sequence identity in their protease domains.

MTSP25 Gene Expression Profile in Normal Tumor Tissues and Cell Lines MTSP25 is expressed in breast, colon, uterus, ovary, kidney, prostate, and testicular cancer tissues. It can also be expressed in lung, stomach, prostate and other tumors. To obtain information about the gene expression profile of the MTSP25 transcript, a 369 bp DNA fragment containing the MTSP25 protease domain sequence (obtained from the PCR reaction) was used to generate 72 different human tissues (human multiple tissue expression (MTE) array). Dot blot consisting of RNA extracted from Clontech, Palo Alto, CA; catalog number 7776-1) and corresponding normal tissue from 241 tumors and individual patients (cancer profiling array, Clontech, catalog number); A point blot consisting of normalized cDNA from 7874-1) was probed. The results of MTE analysis indicate that the MTSP25 transcript is weakly expressed in esophageal lymph nodes. In cancer profiling array analysis, MTSP25 is highly expressed in all four prostate samples (normal and cancer samples). In one of the 20 kidney cDNA pairs, MTSP25 is highly expressed in tumor samples but not in its normal tissue counterpart. MTSP25 is also expressed in one of the 50 breast cancer samples but not in its normal tissue counterpart.

  MTSP25 is also expressed in 3 of 42 normal uterine samples, but not in their tumor counterparts. MTSP25 is also detected in 3 of 14 ovarian cancer samples. In these three samples, MTSP25 expression was also detected in one of the matched normal tissue counterparts. MTSP25 expression was also detected in 5 tumor samples in 34 colon cDNA pairs.

  PCR analysis was also performed using commercially available cDNA panels from several human adult tissues (Clontech catalog # K1420-1 and K1420-2) and several Marathon-Ready cDNAs (Clontech). MTSP25 cDNA was strongly detected in testis and mammary gland cancer, weakly detected in brain, placenta, lung, spleen, prostate, small intestine, colon and leukocytes, and very weakly detected in heart, liver and pancreas.

Conjugates made according to the procedures of Examples 1-4 with routine and minor modifications of procedures such as using different Fmoc-amino acid building blocks are as follows:
Ac-RQGRSL- (Dox) (SEQ ID NO: 491);
Ac-RQGRSSL- (Dox) (SEQ ID NO: 492);
Ac-RQGRS-nL- (Dox) (SEQ ID NO: 493);
Ac-RQGRS-nV- (Dox) (SEQ ID NO: 494);
Ac-RQGRSF- (Dox) (SEQ ID NO: 495);
Ac-RQGRAL- (Dox) (SEQ ID NO: 496);
Ac-RQGRAL- (Dox) (SEQ ID NO: 497);
Ac-RQGRA-nL- (Dox) (SEQ ID NO: 498);
Ac-RQGRA-nL- (Dox) (SEQ ID NO: 499);
Ac-RQGRA-nV- (Dox) (SEQ ID NO: 500);
Ac-RQGRA-Cha- (Dox) (SEQ ID NO: 501);
Ac-RQGRAF- (Dox) (SEQ ID NO: 502);
Ac-RNGRSL- (Dox) (SEQ ID NO: 503);
Ac-RNGRA-nL- (Dox) (SEQ ID NO: 504);
Ac-RQARSL- (Dox) (SEQ ID NO: 505);

Ac-RQARS-nL- (Dox) (SEQ ID NO: 506);
Ac-RQARS-nV- (Dox) (SEQ ID NO: 507);
Ac-RQAAS-Cha- (Dox) (SEQ ID NO: 508);
Ac-RQARSS-Cha- (Dox) (SEQ ID NO: 509);
Ac-RQART-nL- (Dox) (SEQ ID NO: 510);
Ac-RQARAL- (Dox) (SEQ ID NO: 511);
Ac-RQARA-nL- (Dox) (SEQ ID NO: 513);
Ac-RQARA-nV- (Dox) (SEQ ID NO: 514);
Ac-RQARA-Cha- (Dox) (SEQ ID NO: 515);
Ac-RQSRAA- (Dox) (SEQ ID NO: 516);
Ac-RQSRA- (Dox) (SEQ ID NO: 517);
Ac-RQSRA-nL- (Dox) (SEQ ID NO: 518);
Ac-RQSRAL- (Dox) (SEQ ID NO: 519);
Ac-RQSRA-nV- (Dox) (SEQ ID NO: 520);
Ac-RQSRA-Cha- (Dox) (SEQ ID NO: 521);
Ac-RQSRSSL- (Dox) (SEQ ID NO: 522);
Ac-RQSRSL- (Dox) (SEQ ID NO: 523);
Ac-RQSRS-dnL- (Dox) (SEQ ID NO: 524);
Ac-RQSRS-nL- (Dox) (SEQ ID NO: 525);
Ac-RQSRS-nV- (Dox) (SEQ ID NO: 526);
Ac-RQSRS-allyl G- (Dox) (SEQ ID NO: 527);
Ac-RQSRS-Cha- (Dox) (SEQ ID NO: 528);
Ac-RQSRT-nL- (Dox) (SEQ ID NO: 529);
Ac-RQTRSSL- (Dox) (SEQ ID NO: 530);
Ac-RQTRSL- (Dox) (SEQ ID NO: 531);

Ac-RNSRS-nL- (Dox) (SEQ ID NO: 532);
Ac-RQFRSL- (Dox) (SEQ ID NO: 533);
Ac-RQFRS-nL- (Dox) (SEQ ID NO: 534);
Ac-RQFRS-nV- (Dox) (SEQ ID NO: 535);
Ac-RQFRS-nL- (Dox) (SEQ ID NO: 536);
Ac-RQFRS-Cha- (Dox) (SEQ ID NO: 537);
Ac-RQFRAL- (Dox) (SEQ ID NO: 538);
Ac-RQFRA-nL- (Dox) (SEQ ID NO: 539);
Ac-RQFRA-nV- (Dox) (SEQ ID NO: 540);
Ac-RQFRA-Cha- (Dox) (SEQ ID NO: 541);
Ac-QSRSS-nL- (Dox) (SEQ ID NO: 542);
MeOCO-Quat2-GRSL-NH2 (SEQ ID NO: 483);
MeOCO-Quat3-GRSL-NH2 (SEQ ID NO: 484);
MeOCO-Quat-GRSL-NH2 (SEQ ID NO: 485);
MeOCO-Quat4-GRSL-NH2 (SEQ ID NO: 486);
MeOCO-Quat5-GRSL-NH2 (SEQ ID NO: 487);
MeOCO-Quat2-GRSSL-NH2 (SEQ ID NO: 488);
MeOCO-Quat4-GRSL- (Dox) (SEQ ID NO: 489);
MeOCO-Quat2-GRSL- (Dox) (SEQ ID NO: 490);
Ac-QGRSL- (Dox) (SEQ ID NO: 445);
Ac-QGRSSL- (Dox) (SEQ ID NO: 446);
Ac-QGRASL- (Dox) (SEQ ID NO: 447);
Ac-NGRSSL- (Dox) (SEQ ID NO: 448);
Ac-QGRSS-nL- (Dox) (SEQ ID NO: 449);
Ac-QGRSS-nV- (Dox) (SEQ ID NO: 450);

Ac-QGRSS-Cha- (Dox) (SEQ ID NO: 451);
Ac-QGRSS-allyl G- (Dox) (SEQ ID NO: 452);
Ac-QGRSS-allyl G- (Dox) (SEQ ID NO: 453);
Ac-QARSL- (Dox) (SEQ ID NO: 454);
Ac-QARSSL- (Dox) (SEQ ID NO: 455);
Ac-QSRSL- (Dox) (SEQ ID NO: 456);
Ac-QSRSS-nV- (Dox) (SEQ ID NO: 457);
Ac-QSRSS-Cha- (Dox) (SEQ ID NO: 458);
Ac-QSRSSL- (Dox) (SEQ ID NO: 459);
Ac-QTRSSL- (Dox) (SEQ ID NO: 460);
Ac-Q-Aib-RSS-Cha- (Dox) (SEQ ID NO: 461);
Ac-Q-Aib -RSSL- (Dox) (SEQ ID NO: 462);
Ac-Q-Abu-RSS-Cha- (Dox) (SEQ ID NO: 463);
Ac-Q-Abu-RSSL- (Dox) (SEQ ID NO: 464);
Ac-Q-Cha-RSS-Cha- (Dox) (SEQ ID NO: 465);
Ac-QFRSL- (Dox) (SEQ ID NO: 466);
Ac-QFRSSL- (Dox) (SEQ ID NO: 467);
Ac-QYRSSL- (Dox) (SEQ ID NO: 468);
Ac-RGRSL- (Dox) (SEQ ID NO: 469);

Ac-RGRSSL- (Dox) (SEQ ID NO: 470);
Ac-RGRSS-Cha- (Dox) (SEQ ID NO: 471);
Ac-RGRS-Cha- (Dox) (SEQ ID NO: 472);
Ac-RARSL- (Dox) (SEQ ID NO: 473);
Ac-RARSSL- (Dox) (SEQ ID NO: 474);
Ac-RSRSL- (Dox) (SEQ ID NO: 475);
Ac-RSRSSL- (Dox) (SEQ ID NO: 476);
Ac-RSRS-Cha- (Dox) (SEQ ID NO: 477);
Ac-RSRSS-Cha- (Dox) (SEQ ID NO: 478);
Ac-RFRSL- (Dox) (SEQ ID NO: 479);
Ac-RFRS-Cha- (Dox) (SEQ ID NO: 480);
Ac-YGRSSL- (Dox) (SEQ ID NO: 481);
Ac-M (O2) -SRSL- (Dox) (SEQ ID NO: 482);
Ac-RRQSRAA- (Dox) (SEQ ID NO: 105);
Ac-RRQSRI- (Dox) (SEQ ID NO: 610);
Ac-RRQSRSSL- (Dox) (SEQ ID NO: 543);
Ac-RRQSRSL- (Dox) (SEQ ID NO: 544);
Ac-RGSGRSL- (Dox) (SEQ ID NO: 545);
Ac-RGSGR--S-nL- (Dox) (SEQ ID NO: 546);

Ac-RGSGRA-nL- (Dox) (SEQ ID NO: 547);
Ac-RGSGRSSL- (Dox) (SEQ ID NO: 548);
Ac-IVSGRASL- (Dox) (SEQ ID NO: 549);
Ac-RRQSRA- (Dox) (SEQ ID NO: 108);
Ac-RRQSRI- (Dox) (SEQ ID NO: 111);
Ac-LRRQSRAA- (Dox) (SEQ ID NO: 106);
Ac-LRRQSRGG- (Dox) (SEQ ID NO: 109);
Ac-LRRQSRA- (Dox) (SEQ ID NO: 110);
Ac-LRRQSRAI- (Dox) (SEQ ID NO: 112);
Ac-LRRQSRAI- (Dox) (SEQ ID NO: 611);
Ac-LRRQSRSSL- (Dox) (SEQ ID NO: 550);
Ac-LRRQSRSL- (Dox) (SEQ ID NO: 551);
Ac-SGRSL- (Dox) (SEQ ID NO: 362);
Ac-SGRSSL- (Dox) (SEQ ID NO: 363);
Ac-SGRSSSL- (Dox) (SEQ ID NO: 364);
Ac-SGRS-nL- (Dox) (SEQ ID NO: 365);
Ac-SGRS-nV- (Dox) (SEQ ID NO: 366); Isomer 1
Ac-SGRS-nV- (Dox) (SEQ ID NO: 367); Isomer 2
Ac-SGRSG (hex)-(Dox) (SEQ ID NO: 368);
Ac-SGRS-Cha- (Dox) (SEQ ID NO: 369);
Ac-SGRS-hCha- (Dox) (SEQ ID NO: 370);
Ac-SARSL- (Dox) (SEQ ID NO: 371);
Ac-SARSSL- (Dox) (SEQ ID NO: 372);
Ac-SSRS-nL- (Dox) (SEQ ID NO: 373);
Ac-TGRS-Abu- (Dox) (SEQ ID NO: 374);
Ac-TGRSL- (Dox) (SEQ ID NO: 375);

Ac-TGRS-nV- (Dox) (SEQ ID NO: 376);
Ac-TGRS-nL- (Dox) (SEQ ID NO: 377);
Ac-TGRSG (hex)-(Dox) (SEQ ID NO: 378);
Ac-TGRS-Cha- (Dox) (SEQ ID NO: 379);
Ac-TGRS-hCha- (Dox) (SEQ ID NO: 380);
Ac-TGRT-Abu- (Dox) (SEQ ID NO: 381);
Ac-TGR-hS-nL- (Dox) (SEQ ID NO: 382);
Ac-TGR-Abu-nL- (Dox) (SEQ ID NO: 383);
Ac-TGR-Abu-nV- (Dox) (SEQ ID NO: 384);
Ac-TGF (Gn) -S-nL- (Dox) (SEQ ID NO: 385);
Ac-TGF (Gn) -S-Cha- (Dox) (SEQ ID NO: 386);
Ac-TGF (Gn) -Abu-nV- (Dox) (SEQ ID NO: 387);
Ac-TGK (alloc) -S-nL- (Dox) (SEQ ID NO: 388);
Ac-TGKS-nL- (Dox) (SEQ ID NO: 389);
Ac-TG-hR-S-nL- (Dox) (SEQ ID NO: 390);
Ac- (hS) GGRS-nL- (Dox) (SEQ ID NO: 391);
MeOCO-TGRS-nL- (Dox) (SEQ ID NO: 392);
PhSO ₂ -TGRS-nL- (Dox) (SEQ ID NO: 393);
MeOEtCO-TGRS-nL- (Dox) (SEQ ID NO: 394);
MeO (EtO) 2Ac-TGRS-nL- (Dox) (SEQ ID NO: 395);
4-Oxo-pentanoyl-TGRS-nL- (Dox) (SEQ ID NO: 396);
3,4-methyldioxy PhAc-TGRS-nL- (Dox) (SEQ ID NO: 397);
2-pyridyl Ac-TGRS-nL- (Dox) (SEQ ID NO: 398);
PhOAc-TGRS-nL- (Dox) (SEQ ID NO: 399);
L-3-Ph lactyl-TGRS-nL- (Dox) (SEQ ID NO: 400);
MeOAc-TGRS-nL- (Dox) (SEQ ID NO: 401);
PhAc-TGRS-nL- (Dox) (SEQ ID NO: 402);

MeOEtOCO-TGRS-nL- (Dox) (SEQ ID NO: 403);
MeOEtOAc-TGRS-nL- (Dox) (SEQ ID NO: 404);
HOOCButa-TGRS-nL- (Dox) (SEQ ID NO: 405);
ZTGRS-nL- (Dox) (SEQ ID NO: 406);
EtOCO-TGRS-nL- (Dox) (SEQ ID NO: 407);
βΑ-Τ-GRS-nL- (Dox) (SEQ ID NO: 408);
Pent-4-ynoyl-TGRS-nL- (Dox) (SEQ ID NO: 409);
NapAc-TGRS-nL- (Dox) (SEQ ID NO: 410);
iBoc-TGRS-nL- (Dox) (SEQ ID NO: 411);
HOAc-TGRS-nL- (Dox) (SEQ ID NO: 412);
MeSucc-TGRS-nL- (Dox) (SEQ ID NO: 413);
N, N-diMeGly-TGRS-nL- (Dox) (SEQ ID NO: 414);
Succ-TGRS-nL- (Dox) (SEQ ID NO: 415);
HCO-TGRS-nL- (Dox) (SEQ ID NO: 416);
Ac-TARS-nL- (Dox) (SEQ ID NO: 417);
Ac-TAF (Gn) -S-nL- (Dox) (SEQ ID NO: 418);
Ac-TAR-Abu-nV- (Dox) (SEQ ID NO: 419);
Ac-TARS-Abu- (Dox) (SEQ ID NO: 420);
Ac-TART-Abu- (Dox) (SEQ ID NO: 421);
Ac-TS (O-Me) -RS-nL- (Dox) (SEQ ID NO: 422);
Ac-T-hS-RS-nL- (Dox) (SEQ ID NO: 423);
Ac-T- (1-Me) HRS-nL- (Dox) (SEQ ID NO: 424);
Ac-T- (3-Me) HRS-nL- (Dox) (SEQ ID NO: 425);
Ac-THRS-nL- (Dox) (SEQ ID NO: 426);
Ac-T-Sar-RS-nL- (Dox) (SEQ ID NO: 427);
Ac-T-nV-RS-nL- (Dox) (SEQ ID NO: 428);
Ac-T-nL-RS-nL- (Dox) (SEQ ID NO: 429);
Ac-TARS-Cha- (Dox) (SEQ ID NO: 430);
Ac-T-Abu-RS-nL- (Dox) (SEQ ID NO: 431);
Ac-4,4 diMeThr-GRS-nL- (Dox) (SEQ ID NO: 432);
Ac-hS-GRS-nL- (Dox) (SEQ ID NO: 433);
Ac-hS-GR-hS-Cha- (Dox) (SEQ ID NO: 434);

Ac-hS-GRS-Cha- (Dox) (SEQ ID NO: 435);
Ac-hS-GRT-Cha- (Dox) (SEQ ID NO: 436);
Ac-hS-ARS-Cha- (Dox) (SEQ ID NO: 437);
Ac-NGRS-nL- (Dox) (SEQ ID NO: 438);
Ac-YGRSSL- (Dox) (SEQ ID NO: 439);
Ac-YGRS-Cha- (Dox) (SEQ ID NO: 440);
Ac-QGRSS-nL- (Dox) (SEQ ID NO: 441);
Ac-QGRSS-nV- (Dox) (SEQ ID NO: 442);
Ac-LRGSGRSA- (Dox) (SEQ ID NO: 573);
Ac-LRGSGRSL- (Dox) (SEQ ID NO: 342);
Ac-LRGSGRSL- (Dox) (SEQ ID NO: 343);
Ac-LRGSGRSS-nL- (Dox) (SEQ ID NO: 344);
Ac-LRGSGRSS-Cha- (Dox) (SEQ ID NO: 345);
Ac-LRG-dS-ARSA- (Dox) (SEQ ID NO: 574);
Ac-LRGSARSSL- (Dox) (SEQ ID NO: 346);
Ac-LRGSARSL- (Dox) (SEQ ID NO: 347);
Ac-LRGSARSS-Cha- (Dox) (SEQ ID NO: 348);
Ac-LRGSARSS-nV- (Dox) (SEQ ID NO: 349);
Ac-LRGSARSS-nL- (Dox) (SEQ ID NO: 350);
Ac-VIVSGRAL- (Dox) (SEQ ID NO: 351);
Ac-VIVSARSL- (Dox) (SEQ ID NO: 352);
Ac-VIVSGRSSL- (Dox) (SEQ ID NO: 353);
Ac-VIVSARMA- (Dox) (SEQ ID NO: 354);
Ac-VIVSAR-nL-A- (Dox) (SEQ ID NO: 355);
Ac-VIVSARS-nL- (Dox) (SEQ ID NO: 356);
Ac-VIVSARS-Cha- (Dox) (SEQ ID NO: 357);

Ac-VIVSARS-Cha- (Dox) (SEQ ID NO: 358);
Ac-VIVSARSS-Cha- (Dox) (SEQ ID NO: 359);
Ac-RR- (Me) CPGRVV- (Dox) (SEQ ID NO: 360);
Ac-RR-nV-PARSL- (Dox) (SEQ ID NO: 361);
Ac-RG-dS-ARSA- (Dox) (SEQ ID NO: 309);
Ac-RGSGRSA- (Dox) (SEQ ID NO: 310);
Ac-RGSGRAL- (Dox) (SEQ ID NO: 311);
Ac-RGSGRSL- (Dox) (SEQ ID NO: 312);
Ac-RGSGR--S-nL- (Dox) (SEQ ID NO: 313);
Ac-RGSGRA-nL- (Dox) (SEQ ID NO: 314);
Ac-RGSGRSSL- (Dox) (SEQ ID NO: 315);
Ac-RGSGRS-Cha- (Dox) (SEQ ID NO: 316);
Ac-RGSGRSS-Cha- (Dox) (SEQ ID NO: 317);
Ac-RGSARS-Cha- (Dox) (SEQ ID NO: 318);
Ac-RGSARSS- (Dox) (SEQ ID NO: 319);
Ac-RGSARS-nV- (Dox) (SEQ ID NO: 320);
Ac-RGSARSS-nV-(Dox) (SEQ ID NO: 321);
Ac-RGSARSL- (Dox) (SEQ ID NO: 322);
Ac-R- (Me) CPGRVV- (Dox) (SEQ ID NO: 323);
Ac-R- (Me) CPGRVV- (Dox) (SEQ ID NO: 324);
Ac-RC (Me) -PGRSL- (Dox) (SEQ ID NO: 325);
Ac-RLPGRSL- (Dox) (SEQ ID NO: 326);
Ac-RVPGRSL- (Dox) (SEQ ID NO: 327);
Ac-RVPGRSL- (Dox) (SEQ ID NO: 328);
Ac-R-nL-PGRSL- (Dox) (SEQ ID NO: 329);
Ac-RG (tBu) -PARSL- (Dox) (SEQ ID NO: 330);
Ac-RLPARSL- (Dox) (SEQ ID NO: 331);
Ac-RVPARSL- (Dox) (SEQ ID NO: 332);
Ac-R-nL-PARSL- (Dox) (SEQ ID NO: 333);
Ac-IVSGRAL- (Dox) (SEQ ID NO: 334);
Ac-IVSGRSSL- (Dox) (SEQ ID NO: 335);
Ac-IVSGRASL- (Dox) (SEQ ID NO: 336);

Ac-IVSARMA- (Dox) (SEQ ID NO: 337);
Ac-IVSAR-nL-A- (Dox) (SEQ ID NO: 338);
Ac-IVSARSL- (Dox) (SEQ ID NO: 339);
Ac-IVSARS-nL- (Dox) (SEQ ID NO: 340);
Ac-IVSARSSL- (Dox) (SEQ ID NO: 341);
Ac-GSGRSA- (Dox) (SEQ ID NO: 585);
Ac-GSGRSL- (Dox) (SEQ ID NO: 277);
Ac-GSGRAL- (Dox) (SEQ ID NO: 278);
Ac-GSGRSSL- (Dox) (SEQ ID NO: 279);
Ac-GSGRL- (Dox) (SEQ ID NO: 280);
Ac-GSG- (4-guan) Phg-SL-NH2 (SEQ ID NO: 281);
Ac-GSGRSS-Cha- (Dox) (SEQ ID NO: 282);
Ac-GSGRASL- (Dox) (SEQ ID NO: 283);
Ac-GSGRS-nL- (Dox) (SEQ ID NO: 284);
Ac-GTGRS-nL- (Dox) (SEQ ID NO: 285);
Succ-bA-TGRS-nL- (Dox) (SEQ ID NO: 286);
Ac-GTGRS-hCha- (Dox) (SEQ ID NO: 287);
Ac-G-hS-GRS-nL- (Dox) (SEQ ID NO: 288);
Ac-G-dS-ARSA- (Dox) (SEQ ID NO: 289);
Ac-GSARSL- (Dox) (SEQ ID NO: 290);
Ac-GSARSS-Cha- (Dox) (SEQ ID NO: 291);
Ac-GSARSSL- (Dox) (SEQ ID NO: 292);
Ac-GSARASL- (Dox) (SEQ ID NO: 293);
Ac-VSGRSL- (Dox) (SEQ ID NO: 294);
Ac-VSGRAL- (Dox) (SEQ ID NO: 295);
Ac-VSGRASL- (Dox) (SEQ ID NO: 296);
Ac-VSGRSSL- (Dox) (SEQ ID NO: 297);
Ac-VSARMA- (Dox) (SEQ ID NO: 298);
Ac-VSAR-nL-A- (Dox) (SEQ ID NO: 299);
Ac-VSARS-nL- (Dox) (SEQ ID NO: 300);
Ac-VSARSL- (Dox) (SEQ ID NO: 301);
Ac- (Me) CPGRVV- (Dox) (SEQ ID NO: 302);
Ac- (Me) CPGRVV- (Dox) (SEQ ID NO: 303);
Ac-C (Me) -PGRAL- (Dox) (SEQ ID NO: 304);
Ac-C (Me) -PGRSL- (Dox) (SEQ ID NO: 305);
Ac-C (Me) -PARSL- (Dox) (SEQ ID NO: 306);
Ac-C (Me) -PARASL- (Dox) (SEQ ID NO: 307);
Ac-G (tBu) -PGRSL- (Dox) (SEQ ID NO: 308);

Ac-QSRAA- (taxol) (SEQ ID NO: 552);
Ac-QSRSA- (taxol) (SEQ ID NO: 553);
Ac-QSRSG- (taxol) (SEQ ID NO: 554);
Ac-RSRAA- (taxol) (SEQ ID NO: 555);
Ac-RQSRAA- (taxol) (SEQ ID NO: 556);
Ac-RQSRSA- (taxol) (SEQ ID NO: 557);
Ac-RQSRSAA- (taxol) (SEQ ID NO: 558);
Ac-RGSGRSA- (taxol) (SEQ ID NO: 559);
Ac-SGRAA- (taxol) (SEQ ID NO: 560);
Ac-SGRSA- (taxol) (SEQ ID NO: 561);
Ac-SGRSSA- (taxol) (SEQ ID NO: 562);
Ac-SGRASA- (taxol) (SEQ ID NO: 563);
Ac-SGRSG- (taxol) (SEQ ID NO: 564);
Ac-SGRSSG- (taxol) (SEQ ID NO: 565);
Ac-SGRSGA- (taxol) (SEQ ID NO: 566);
Ac-SGRSGG- (taxol) (SEQ ID NO: 567);
Ac-GTGRSGG- (taxol) (SEQ ID NO: 568);
Ac-LRRQSRAA- (Dox) (SEQ ID NO: 597);
MeSO ₂ -dA (Chx) -Abu-RSL- (Dox) (SEQ ID NO: 598);
Ac-RARSL- (Dox) (SEQ ID NO: 599);
Ac-dA (Chx) -Abu-RSL- (Dox) (SEQ ID NO: 600);
Ac-dA (Chx) -Abu-RSSL- (Dox) (SEQ ID NO: 601);
Ac-QGRSSL- (Dox) (SEQ ID NO: 602);
MeOCO-dhF-P (OH) -RSSL- (Dox) (SEQ ID NO: 603);
MeOCO-Quat4-GRSL- (Dox) (SEQ ID NO: 604);
As-dCha-P (OH) -RSSL- (Dox) (SEQ ID NO: 605);
Ac-dCha-Abu-RSSA- (taxol) (SEQ ID NO: 606);
MeOCO-Quat2-GRSL-NH2 (SEQ ID NO: 607);
MeOCO-Quat3-GRSL-NH2 (SEQ ID NO: 608); and
MeOCO-Quat-GRSL-NH2 (SEQ ID NO: 609)
Is mentioned.

Pharmacokinetic studies in untreated and tumor-bearing mice of doses that are metabolized to the conjugate and doxorubicin and leucine-doxorubicin. Twelve week old nude mice were used. Tumor cells for transplantation were generated by one of the following three protocols.

Protocol A Tumor Cells Collected from Tissue Culture Tumor cells are trypsin digested, resuspended in growth medium and centrifuged for 6 minutes at 200 × g. Cells are resuspended in serum free medium and counted. The appropriate volume of solution containing the desired number of cells is then transferred to a conical centrifuge tube and centrifuged as before, into an appropriate volume of a 1: 1 cold mixture of cells: matrigel in phenol-free medium. Resuspend. Each mouse is inoculated subcutaneously or orthotopically with 0.2 to 0.5 mL containing 1 × 10 ⁶ to 1 × 10 ⁷ tumor cells.

Protocol B Tumor cell suspension Established tumors (200-1000 mm ³ ) are excised from mice, weighed and rinsed in tumor cell growth medium. Tumors are passed through a steel cell dissociation sieve. Rinse cells with growth medium through sieve. The cells are centrifuged at 200 × g for 6 minutes and resuspended in an appropriate volume of a 1: 1 cold mixture of cells: matrigel. Each mouse is inoculated subcutaneously or orthotopically with 0.2-0.5 mL of tumor cells.

Protocol C Tumor Fragment Alternatively, an approximately 800 mm ³ minute tumor is excised from the mouse, rinsed in tumor cell growth medium and cut into 1-2 mm ³ fragments. Each fragment is inoculated subcutaneously or orthotopically using a trocar.

Pharmacokinetic studies Untreated or tumor-bearing mice are individually weighed and grouped. Mice are administered 1-100 μmol / kg intraperitoneally or intravenously, containing 30 μmol / kg, 25 μmol / kg or 21.5 μmol / kg of the test conjugate. Mice are sacrificed at given time points between 5 minutes and 24 hours after compound administration. Blood is collected in a syringe containing a protease inhibitor such as EDTA, AEBSF, aprotinin, leupeptin, bestatin, pepstanin A or E64 and transferred to a heparinized blood collection tube. Plasma is created by centrifugation. Tumors are collected and ground in liquid nitrogen. The obtained tumor powder is stored at -80 ° C. Tumor powder and plasma are extracted and the parent test conjugate and its product containing leucine-doxorubicin (or norleucine-doxorubicin etc.) and doxorubicin are analyzed.

  See the delivery of toxins to tumor cells, and also look at the levels of parent conjugates and toxins (dox and nor-Leu dox) in plasma

Results For example, test conjugates (Ac-Gly-Ser-Gly-Arg-Ser-nLeu-Dox 21.5 μmol / kg (see Example 2)) were administered intraperitoneally (IP) or intravenously. Administered to (IV) naive and tumor bearing (TB) mice. One hour after dosing, plasma and tumor tissue were collected from the mice. The concentrations of test conjugate and its product were compared. The results show that the conjugate does not enter the tumor and the norleu dox and dox-μM concentrations in the tumor at 1 hour after the single injection (both IP and IV). There were low levels of dox and nor-leu dox.

Extraction, Chromatography LC / MS conditions Plasma: Plasma samples are made using acetonitrile protein precipitation. Standard curves were constructed from the addition of standard compounds to 0.1 mL or 0.05 mL volumes of plasma on 5-20 μL volumes of ice. The standard curve ranges from 10 ng / mL to 1 μg / mL or 100 ng to 4 μg / mL of the standard compound. Immediately after the standard addition, acetonitrile is added to precipitate the protein. Frozen plasma samples were thawed on ice to make test plasma samples. An aliquot was added directly to acetonitrile. After sample preparation, the sample is mixed using vortex mixing. The precipitate was pelleted using centrifugation.

  The supernatant was dried using vacuum centrifugation. Samples were reconstituted with 30% acetonitrile-70% (0.01 M ammonium acetate with 0.1% formic acid). For LC-MS analysis, 0.01 mL of sample was injected. HPLC conditions were 30 × 2.1 mm Zorbax SB C18 HPLC column, 20% acetonitrile to 80% (10 mM ammonium acetate-0.1% formic acid) to 50% acetonitrile to 50% in 1 minute at 0.3 mL / min. Linear gradient up to (10 mM ammonium acetate-0.1% formic acid). Detection was done with a triple quadruple mass spectrometer with electrospray ionization. Doxorubicin was monitored using m / z transitions 544.1-396.8. Leucine-doxorubicin was monitored using 657.2-242.8. Exemplary parent conjugates were monitored using 1555.9-1555.9. Scanning LC-MS and fluorescence detection were used to identify cleavage products other than doxorubicin or leucine-doxorubicin (or such as norleucine-doxorubicin) in plasma.

  Tumor: Immediately after resection from the mouse, the tumor for analysis is weighed and placed in a mortar containing liquid nitrogen. Place the mortar on a dry ice bed and break the tumor into a fine powder, adding additional liquid nitrogen as needed to avoid thawing. When a uniform tumor powder is achieved, the remaining liquid nitrogen is distilled off. Quantitatively transfer the tumor powder to a 15 ml conical tube that has been pre-cooled and placed on dry ice. Samples are stored at -70 ° C until analysis. Tumor powder is thawed on ice and vortex mixed with 0.01 M ammonium acetate at a concentration of 1 gram tumor / mL ammonium acetate solution to form a slurry. A 0.1 mL aliquot of the tumor slurry is precipitated with 0.5 mL of acetonitrile. The supernatant is separated from the precipitated solid and then evaporated using vacuum centrifugation. Quantification of doxorubicin, leucine-doxorubicin (or norleucine-doxorubicin, etc.) by referencing a standard curve constructed from adding measured amounts of standard compounds (doxorubicin, leucine-doxorubicin, etc.) to the control tumor slurry. To achieve.

  A typical standard curve ranges from 1 ng to 200 ng of compound per aliquot of the tumor slurry. After the unknown samples and standards are processed and dried, the residue is reconstituted in 0.15 mL of 30% acetonitrile-70% (0.01 M ammonium acetate + 0.1% formic acid). 10 μL of the solution is injected into the liquid chromatography-mass spectrometry system. HPLC conditions were 30 × 2.1 mm Zorbax SB C18 HPLC column, 20% acetonitrile-80% (10 mM ammonium acetate—0.1% formic acid) to 50% acetonitrile-50% (0.3 mL / min in 1 minute). Linear gradient up to 10 mM ammonium acetate-0.1% formic acid). Detection was done with a triple quadruple mass spectrometer with electrospray ionization. Doxorubicin was monitored using m / z transitions 544.1-396.8. Leucine-doxorubicin was monitored using 657.2-242.8.

  Since variations will be apparent to those skilled in the art, it is intended that the invention be limited only by the scope of the appended claims.

[Sequence Listing]

Claims (148)

  A conjugate comprising a therapeutic agent and a peptidic substrate optionally linked via a linker, wherein the peptidic substrate is proteolytically degraded by a cell surface protease or its soluble, shed or released form. A conjugate that is cleaved to release the therapeutic agent, where the conjugate is not substantially cleaved by plasmin or prostate specific antigen (PSA).   2. The conjugate of claim 1 wherein the released therapeutic agent is active.   2. The conjugate of claim 1, wherein cleavage releases the therapeutic agent in a form that requires further processing for activation. The conjugate of claim 1, wherein the components are: (peptidic substrate) _S , (linker) _q and (targeted drug) _t ;
[Wherein at least one peptidic substrate moiety is attached to at least one therapeutic agent with or without a linker, s is 1 to 6, q is 0 to t, and t is 1 to 6. Wherein the cell surface protease that cleaves the peptidic substrate (s) results in the transfer of the therapeutic agent to the cell.   2. A conjugate according to claim 1, wherein the peptidic substrate comprises one amino acid or more, wherein the resulting therapeutic agent is active upon proteolytic cleavage of the conjugate. Alternatively, a conjugate that is in an active form upon further processing.   The conjugate according to claim 1, wherein the cell surface protease is a serine protease.   The conjugate according to claim 1, wherein the cell surface protease is a type II transmembrane serine protease (MTSP) or endotheliase.   The conjugate according to claim 1, wherein the cell surface protease is endotheliase 1, endotheliase 2, MTSP1, MTSP3, MTSP4, MTSP6, MTSP7, MTSP9, MTSP10, MTSP12, MTSP20, MTSP22, MTSP25, A conjugate selected from choline, enterokinase, human airway trypsin-like protease (HAT), TMPRSS2, hepsin, and TMPRSS4. The conjugate of claim 1, wherein the cell surface protease is
SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 29, 31, 33, 35, 37, 39, 41, 43, 45, 270, 272, A polypeptide comprising the sequence of an amino acid set forth in any of 274 and 276;
SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44, 269, 273 and A polypeptide encoded by a nucleotide sequence that hybridizes under high stringency conditions to the nucleotide sequence set forth in any of 275;
SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 29, 31, 33, 35, 37, 39, 41, 43, 45, 270, 272, A polypeptide comprising a sequence of amino acids having at least about 40% sequence identity to the sequence of amino acids set forth in any of 274 and 276; and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, Encoded by a splice variant of the nucleotide sequence shown in any of 18, 20, 22, 24, 26, 29, 31, 33, 35, 37, 39, 41, 43, 45, 270, 272, 274 and 276 A conjugate comprising a polypeptide selected from the group consisting of:   2. The conjugate according to claim 1, wherein the therapeutic agent is a toxin, small organic molecule, nucleic acid, protein therapeutic agent, cytokine or growth factor.   The conjugate according to claim 1, wherein the therapeutic agent is an anticancer agent.   The conjugate of claim 1, wherein the therapeutic agent is an anti-angiogenic agent.   The conjugate according to claim 1, wherein the therapeutic agent is abrin, ricin A, Pseudomonas aeruginosa exotoxin Shiga toxin, diphtheria toxin, tumor necrosis factor, α-interferon, γ-interferon, nerve growth factor, tissue Factors and tissue factor variants, FAS-ligand platelet derived growth factor, tissue plasminogen activator, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL- 6) selected from granulocyte macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), nerve growth factor, fibroblast growth factor (FGF), and epidermal growth factor , Conjugate.   2. A conjugate according to claim 1, wherein the therapeutic agent is selected from alkylating agents, toxins, antiproliferative agents, pro-apoptotic agents, pro-coagulants, cytotoxic nucleosides and tubulin binding agents. body. A conjugate according to claim 1, wherein the therapeutic agent is a drug of the following class:
a) drugs of the anthracycline family,
b) Vinca alkaloid drug,
c) mitomycin,
d) bleomycin,
e) cytotoxic nucleosides,
f) pteridine family of drugs,
g) Diinen,
h) Estramustine,
i) cyclophosphamide,
j) taxanes,
k) podophyllotoxin,
l) maytansinoids,
m) an epothilone, and n) a conjugate selected from combretastatin and analogs or pharmaceutically acceptable derivatives thereof. A conjugate according to claim 1, wherein the therapeutic agent is a drug of the following class:
a) doxorubicin,
b) carminomycin,
c) daunorubicin,
d) aminopterin,
e) methotrexate,
f) Metopterin,
g) Dichloromethotrexate,
h) Mitomycin C,
i) porphyromycin,
j) 5-fluorouracil,
k) 6-mercaptopurine,
l) cytosine arabinoside,
m) podophyllotoxin,
n) etoposide o) etoposide phosphate p) melphalan,
q) Vinblastine,
r) vincristine,
s) Leurocidin,
t) vindesine,
u) Estramustine,
v) cisplatin,
w) cyclophosphamide x) taxol,
y) Leurocin,
z) 4-desacetylvinblastine,
aa) Epothilone B,
bb) Taxotere,
cc) maytansinol,
dd) epothilone A, and ee) combretastatin and analogs,
Or a conjugate selected from pharmaceutically acceptable derivatives thereof.   The conjugate of claim 1, further comprising a linker between the therapeutic agent and the peptidic substrate.   18. A conjugate according to claim 17, wherein the linker comprises a carbohydrate, peptide, and / or hydrocarbon nucleus. 18. A conjugate according to claim 17, wherein the linker is:
A biscarbonylalkyl diradical wherein the amine moiety on the therapeutic agent is linked to a linker unit to form an amide bond and the amino terminus of the peptidic substrate is linked to the other end of the linker unit to also form an amide bond; or therapy A diaminoalkyl diradical in which the carbonyl moiety on the drug is conjugated to one of the amines of the linker moiety, while the other amine of the linker unit is covalently bonded to the C-terminus of the peptidic substrate. Linker unit; or the following formula:

[Wherein A is NH or O; D is N (H or alkyl) or O; R ²⁵ is H, alkyl, cycloalkyl, cycloalkylalkyl, aryl, such as 1-3 Heteroaryl optionally substituted with one or more substituents selected from halo, haloalkyl and alkyl, aralkyl, heteroaralkyl, alkenyl containing 1-2 double bonds, 1-2 triple bonds Including alkynyl, alk (en) (in) yl groups, halo, pseudohalo, cyano, hydroxy, haloalkyl and polyhaloalkyl such as halo lower alkyl, especially trifluoromethyl, formyl, alkylcarbonyl, such as 1 to One or more such as three, eg, selected from halo, haloalkyl and alkyl Arylcarbonyl, heteroarylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminoimino, alkoxycarbonylamino, aryloxycarbonylamino, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl optionally substituted with groups , Diarylaminocarbonyl, aralkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, amino, alkylamino, dialkylamino, Arylamino, alkylarylamino, alkylcarbonylamino , Arylcarbonylamino, azide, nitro, mercapto, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl and arylamino Sulfonyl; and y is an integer from 1 to 3]
Self-removing linker:
A conjugate comprising   18. A conjugate according to claim 17, wherein the linker is a diamine containing a cyclic alkylene moiety.   18. A conjugate according to claim 17, wherein the diamine contains a bicycloalkylene moiety.   18. A conjugate according to claim 17, wherein the linker is 1,4-bis (aminomethyl) cyclohexane, 1,4-bis (aminomethyl) cycloheptane, 1,3-bis (aminomethyl) cyclopentane. A conjugate selected from 1-amino-4- (aminomethyl) cyclohexane, 1,4-diaminocyclohexane and 1,4-bis (aminomethyl) bicyclo [2.2.2] octane.   18. A conjugate according to claim 17, wherein the linker is a 1, ω-diaminoalkane.   18. A conjugate according to claim 17, wherein the linker is 1,3-diaminopropane.   18. A conjugate according to claim 17, wherein the linker is 1, ω-dicarbonylalkane.   26. The conjugate according to claim 25, wherein the linker is selected from oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid and pivalic acid.   The conjugate according to claim 1, wherein the peptidic substrate comprises P1, which is an arbitrary amino acid.   28. The conjugate according to claim 27, wherein P1 is a naturally occurring amino acid.   28. The conjugate according to claim 27, wherein P1 is an amino acid having an aromatic, branched or branched aromatic side chain.   2. The conjugate according to claim 1, wherein the peptidic substrate comprises P1, wherein P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr. A conjugate according to claim 1, wherein:
The peptidic substrate comprises a P1-P1 ′ bond;
P1-P1 ′ bond is the site of cleavage by cell surface proteases;
P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr; and P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu , Met or 6-aminohexanoyl,
Conjugate.   The conjugate of claim 1, wherein the peptidic substrate comprises P1, wherein P1 is Arg, Lys or Arg surrogate.   2. The conjugate of claim 1 further comprising a P2 residue selected from Phe, Ser, Gly and Ala.   2. The conjugate according to claim 1 further comprising a P3 residue selected from Arg, Lys, Gln, Ser, Quat and Arg surrogates.   2. The conjugate according to claim 1, further comprising a P4 residue selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val.   The conjugate of claim 1, further comprising a P5 residue selected from Arg and Arg surrogates.   2. The conjugate of claim 1 further comprising a P6 residue selected from Leu, Ile, and Val.   The binding of claim 1, further comprising a P2 'residue selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. body.   The conjugate of claim 1, further comprising a P3 'residue selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. A conjugate according to claim 1, wherein:
The peptidic substrate has the formula:
P4-P3-P2-P1-P1 ′ [where:
P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr;
P2 is selected from Phe, Ser, Gly and Ala;
P3 is selected from Arg, Lys, Gln, Quat and Arg surrogates;
P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val; and P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val , NVal, Aib, Abu, Met or 6-aminohexanoyl]
A conjugate comprising a 5-mer having 41. A conjugate according to claim 40, wherein:
One or more P5 or P2 ′ amino acid residues [where:
P5 is Arg or an Arg surrogate; and P2 ′ is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl] A conjugate optionally further comprising: 42. A conjugate according to claim 41, wherein:
If the peptidic substrate comprises a P5 amino acid residue, the peptidic substrate optionally further comprises a P6 amino acid residue selected from Leu, Ile, and Val; and if the peptidic substrate comprises a P2 ′ amino acid residue If included, the peptidic substrate is any P3 ′ amino acid residue selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl The conjugate further comprising:   2. A conjugate according to claim 1, wherein the therapeutic agent is attached to the C-terminus of the peptidic substrate directly or via a linker. The conjugate of claim 1, wherein:
A conjugate wherein the peptidic substrate comprises a cap at the N-terminus.   The conjugate according to claim 1, wherein the cap is a hydrophilic protective group.   2. A conjugate according to claim 1 wherein the cap is an acyl, sulfonyl or carbamoyl derivative.   46. A conjugate according to claim 45, wherein the protecting group is selected from alkanoyl hydroxide, polyhydroxy alkanoyl, polyethylene glycol, glycosylates, sugars and crown ethers. Formula I:
_{^{X n - (P6) m -}} (P5) p - (P4) i - (P3) j - (P2) l -P1- (P1 ') u - (P2') k - (P3 ') r - (L ) _n -Z
44. A conjugate according to claim 43 comprising a derivative thereof, wherein:
Z is a therapeutic agent;
L is a linker;
l, j, i, p and m are selected as follows:
l is 0 or 1; when l is 0, j, i, p and m are 0; when l is 1, j is 0 or 1; when j is 0, i , P and m are 0; when j is 1, i is 0 or 1; when i is 0, p and m are 0; when i is 1, p is 0 or When p is 0, m is 0; when p is 1, m is 0 or 1;
u, k and r are selected as follows:
u is 0 or 1; when u is 0, k and r are 0; when u is 1, k is 0 or 1; when k is 0, r is 0 When r is 0, r is 0 or 1;
n is 0 or 1;
X ⁿ is hydrogen or an acyl, sulfonyl or carbamoyl cap;
P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr;
P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl;
P2 is selected from Phe, Ser, Gly and Ala;
P3 is selected from Arg, Lys, Gln, Ser, Quat and Arg surrogates;
P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val;
P5 is selected from Arg and Arg surrogates;
P6 is selected from Leu, Ile, and Val;
P2 ′ is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl; and P3 ′ is Gly, Ser, Ala, Leu, Selected from Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl,
Conjugate.   49. The conjugate of claim 48, wherein P1 is Arg, Lys or Arg surrogate.   2. A conjugate according to claim 1, wherein the therapeutic agent is attached directly or via a linker to the N-terminus of the peptidic substrate. 51. The conjugate according to claim 50, wherein:
A conjugate, wherein the C-terminus of the peptidic substrate is a carboxylic acid or a carboxamide derivative. Formula II:
_{Z- (L) n - (P6} ) m - (P5) p - (P4) i - (P3) j - (P2) l -P1- (P1 ') u - (P2') k - (P3 ') _r -X ^c
Or a conjugate thereof according to claim 50, wherein the conjugate comprises:
Z is a therapeutic agent;
L is a linker;
l, j, i, p, and m are selected as follows:
l is 0 or 1; when l is 0, j, i, p and m are 0; when l is 1, j is 0 or 1; when j is 0, i , P and m are 0; when j is 1, i is 0 or 1; when i is 0, p and m are 0; when i is 1, p is 0 or When p is 0, m is 0; when p is 1, m is 0 or 1;
u, k and r are selected as follows:
u is 0 or 1; when u is 0, k and r are 0; when u is 1, k is 0 or 1; when k is 0, r is 0 When r is 0, r is 0 or 1;
n is 0 or 1;
^Xc together with the carbonyl group of the amino acid residue to which it is attached forms a carboxylic acid or carboxamide group;
P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr;
P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl;
P2 is selected from Phe, Ser, Gly and Ala;
P3 is selected from Arg, Lys, Gln, Ser, Quat and Arg surrogates;
P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val;
P5 is selected from Arg and Arg surrogates;
P6 is selected from Leu, Ile, and Val;
P2 ′ is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl; and P3 ′ is Gly, Ser, Ala, Leu, Selected from Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl,
Conjugate.   53. The conjugate of claim 52, wherein P1 is Arg, Lys or Arg surrogate. 2. A conjugate according to claim 1, wherein the first therapeutic agent is attached to the N-terminus of the peptidic substrate, optionally via a linker; and is the same as the first therapeutic agent or A conjugate wherein a different, second therapeutic agent is optionally attached to the C-terminus of the peptidic substrate via a second linker, which is the same as or different from the first linker. Formula III:
_{^{Z 1 - (L 1) n}} - (P6) m - (P5) p - (P4) i - (P3) j - (P2) l -P1- (P1 ') u - (P2') k - (P3 ') _r- (L ² ) _v -Z ²
55. A conjugate according to claim 54, comprising a derivative thereof, wherein:
Z ¹ and Z ² are each therapeutic agents and may be the same or different;
L ¹ and L ² are each a linker, and may be the same or different;
l, j, i, p, and m are selected as follows:
l is 0 or 1; when l is 0, j, i, p and m are 0; when l is 1, j is 0 or 1; when j is 0, i , P and m are 0; when j is 1, i is 0 or 1; when i is 0, p and m are 0; when i is 1, p is 0 or When p is 0, m is 0; when p is 1, m is 0 or 1;
u, k and r are selected as follows:
u is 0 or 1; when u is 0, k and r are 0; when u is 1, k is 0 or 1; when k is 0, r is 0 When r is 0, r is 0 or 1;
n and v are each independently 0 or 1;
P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr;
P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl;
P2 is selected from Phe, Ser, Gly and Ala;
P3 is selected from Arg, Lys, Gln, Ser, Quat and Arg surrogates;
P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val;
P5 is selected from Arg and Arg surrogates;
P6 is selected from Leu, Ile, and Val;
P2 ′ is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl; and P3 ′ is Gly, Ser, Ala, Leu, Selected from Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl,
Conjugate.   56. The conjugate of claim 55, wherein P1 is Arg, Lys or Arg surrogate. A conjugate according to any one of claims 1 to 57,
Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 46);
Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 47);
Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 48);
Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 49);
Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic agent) (SEQ ID NO: 50);
Ac-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (Therapeutic) (SEQ ID NO: 51);
Ac-Pro-Arg-Phe-Lys-Ile-Ile- (Therapeutic) (SEQ ID NO: 52);
Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 53);
Ac-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 54);
Ac-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 55);
Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 56);
Ac-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 57);
Ac-Pro-Arg-Phe-Arg-Ile-Ile- (Therapeutic) (SEQ ID NO: 58);
Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 59);
Ac-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 60);
Ac-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 61);
Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 62);
Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 63);
Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 64);
Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 65);
Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 66);
Ac-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (Therapeutic) (SEQ ID NO: 67);
Ac-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 68);
Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 69);
Ac-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 70);
Ac-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 71);
Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 72);
Ac-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 73);
Ac-Pro-Arg-Phe-Arg-Ile-Ile- (Therapeutic) (SEQ ID NO: 74);
Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 75);
Ac-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 76); and
Ac-Ser-Arg-Ser-Arg-Ala-Ala- (Therapeutic) (SEQ ID NO: 77)
Pyro Glu-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 78);
CH ₃ SO ₂ -D-HHT-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 79);
Np-tosyl-Gly-Pro-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 80);
Benzoyl-Val-Gly-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 81);
CH ₃ SO ₂ -D-HHT-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 82);
N-α-ZD-Arg-Gly-Arg-Ala-Ala- (therapeutic agent) where Z is benzyloxycarbonyl (SEQ ID NO: 83);
Pyro Glu-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 84);
HD-Ile-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 85);
Cbo-L- (γ) Glu (α-t-BuO) -Gly-Arg-Ala-Ala- (therapeutic agent) where Cbo is carbobenzoxy (SEQ ID NO: 86);
HD-Pro-Phe-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 87);
HD-Val-Leu-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 88);
Bz-Ile-Glu (γ-OH) -Gly-Arg-Ala-Ala- (therapeutic agent) where Bz is benzoyl (SEQ ID NO: 89);
Bz-Ile-Glu (γ-OMe) -Gly-Arg-Ala-Ala- (Therapeutic) (SEQ ID NO: 90);
Bz-Pro-Phe-Arg-Ala-Ala- (Therapeutic) (SEQ ID NO: 91);
HD-Phe-Pip-Arg-Ala-Ala- (Therapeutic) (SEQ ID NO: 92);
HD-Val-Leu-Lys-Ala-Ala- (therapeutic) (SEQ ID NO: 93);
HD-Nle-HHT-Lys-Ala-Ala- (therapeutic) (SEQ ID NO: 94);
Pyr-Arg-Thr-Lys-Arg-Ala-Ala- (Therapeutic) (SEQ ID NO: 95);
H-Arg-Gln-Arg-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 96);
Boc-Gln-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 97);
Z-Arg-Arg-Ala-Ala- (Therapeutic) (SEQ ID NO: 98);
HD-HHT-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 99);
HD-CHT-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 100);
MeSO ₂ -dPhe-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 101);
δ-ZD-Lys-Pro-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 102);
CH ₃ SO ₂ -D-CHA-But-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 103);
Ac-Arg-Gln-Ser-Arg-Ala-Ala- (therapeutic agent) (SEQ ID NO: 104);
Ac-Arg-Arg-Gln-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 105);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 106);
Ac-Arg-Gln-Ser-Arg-Ala- (therapeutic) (SEQ ID NO: 107);
Ac-Arg-Arg-Gln-Ser-Arg-Ala- (therapeutic agent) (SEQ ID NO: 108);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Gly-Gly- (therapeutic) (SEQ ID NO: 109);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ala- (therapeutic) (SEQ ID NO: 110);
Ac-Arg-Arg-Gln-Ser-Arg-Ile- (Therapeutic) (SEQ ID NO: 111);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ala-Ile- (therapeutic agent) (SEQ ID NO: 112);
Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 113);
Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 114);
Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 115);
Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 116);
Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 117);
Ac-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 118);
Ac-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 119);
Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 120);
Ac-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 121);
Ac-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 122);
Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 123);
Ac-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 124);
Ac-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 125);
Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 126);
Ac-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 127);
Ac-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 128);
Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 129);
Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 130);
Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 131);
Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 132);
Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 133);
Ac-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 134);
Ac-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 135);
Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 136);
Ac-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 137);
Ac-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 138);
Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 139);
Ac-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 140);
Ac-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 141);
Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 142);
Ac-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 143);
Ac-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 144);
Pyro Glu-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 145);
CH ₃ SO ₂ -D-HHT-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 146);
Np-tosyl-Gly-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 147);
Benzoyl-Val-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 148);
CH ₃ SO ₂ -D-HHT-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 149);
N-α-ZD-Arg-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 150) (Z = benzyloxycarbonyl);
Pyro Glu-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 151);
HD-Ile-Pro-Arg-Ser-Leu- (Therapeutic) (SEQ ID NO: 152);
Cbo-L- (γ) Glu (α-t-BuO) -Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 153) (Cbo = carbobenzoxy);
HD-Pro-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 154);
HD-Val-Leu-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 155);
Bz-Ile-Glu (γ-OH) -Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 156) (Bz = benzoyl);
Bz-Ile-Glu (γ-OMe) -Gly-Arg-Ser-Leu- (Therapeutic) (SEQ ID NO: 157);
Benzoyl-Pro-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 158);
HD-Phe-Pip-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 159);
HD-Val-Leu-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 160);
HD-Nle-HHT-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 161);
Pyr-Arg-Thr-Lys-Arg-Ser-Leu- (Therapeutic) (SEQ ID NO: 162);
H-Arg-Gln-Arg-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 163);
Boc-Gln-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 164);
Z-Arg-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 165);
HD-HHT-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 166);
HD-CHT-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 167);
MeSO ₂ -dPhe-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 168);
δ-ZD-Lys-Pro-Arg-Ser-Leu- (therapeutic agent) (SEQ ID NO: 169);
CH ₃ SO ₂ -D-CHA-But-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 170);
Ac-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 171);
Ac-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 172);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 173);
Ac-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 174);
Ac-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 175);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 176);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 177);
Ac-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 178);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 179);
Ac-Arg-Gln-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 180);
Ac-Arg-Gln-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 181);
Ac-Arg-Gln-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 182);
Ac-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 183);
Ac-Arg-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 184);
Ac-Arg-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 185);
Ac-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 186);
Ac-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 187);
Ac-Gln-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 188);
Ac-Gln-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 189);
Ac-Gln-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 190).
Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 191);
Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 192);
Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 193);
Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 194);
Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 195);
Ac-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 196);
Ac-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 197);
Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 198);
Ac-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 199);
Ac-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 200);
Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 201);
Ac-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 202);
Ac-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 203);
Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic agent) (SEQ ID NO: 204);
Ac-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 205);
Ac-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 206);
Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 207);
Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 208);
Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 209);
Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 210);
Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 211);
Ac-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 212);
Ac-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 213);
Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 214);
Ac-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 215);
Ac-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 216);
Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 217);
Ac-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 218);
Ac-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 219);
Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 220);
Ac-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 221);
Ac-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 222);
Pyro Glu-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 223);
CH ₃ SO ₂ -D-HHT-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 224);
Np-tosyl-Gly-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 225);
Benzoyl-Val-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 226);
CH ₃ SO ₂ -D-HHT-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 227);
N-α-ZD-Arg-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 228) (Z = benzyloxycarbonyl);
Pyro Glu-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 229);
HD-Ile-Pro-Arg-Ser-Ser-Leu- (Therapeutic) (SEQ ID NO: 230);
Cbo-L- (γ) Glu (α-t-BuO) -Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 231) (Cbo = carbobenzoxy);
HD-Pro-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 232);
HD-Val-Leu-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 233);
Bz-Ile-Glu (γ-OH) -Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 234) (Bz = benzoyl);
Bz-Ile-Glu (γ-OMe) -Gly-Arg-Ser-Ser-Leu- (Therapeutic) (SEQ ID NO: 235);
Benzoyl-Pro-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 236);
HD-Phe-Pip-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 237);
HD-Val-Leu-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 238);
HD-Nle-HHT-Lys-Ser-Ser-Leu- (Therapeutic) (SEQ ID NO: 239);
Pyr-Arg-Thr-Lys-Arg-Ser-Ser-Leu- (Therapeutic) (SEQ ID NO: 240);
H-Arg-Gln-Arg-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 241);
Boc-Gln-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 242);
Z-Arg-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 243);
HD-HHT-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 244);
HD-CHT-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 245);
MeSO ₂ -dPhe-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 246);
δ-ZD-Lys-Pro-Arg-Ser-Ser-Leu- (therapeutic agent) (SEQ ID NO: 247);
CH ₃ SO ₂ -D-CHA-But-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 248);
Ac-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 249);
Ac-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 250);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 251);
Ac-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 252);
Ac-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic agent) (SEQ ID NO: 253);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 254);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 255);
Ac-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 256);
Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 257);
Ac-Arg-Gln-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 258);
Ac-Arg-Gln-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 259);
Ac-Arg-Gln-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 260);
Ac-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 261);
Ac-Arg-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 262);
Ac-Arg-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 263);
Ac-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 264);
Ac-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 265);
Ac-Gln-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 266);
Ac-Gln-Ala-Arg-Ser-Ser-Leu- (therapeutic agent) (SEQ ID NO: 267); and
Ac-Gln-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 268)
A conjugate selected from.   36. A conjugate according to claim 35, wherein P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Phe and Val. 36. The conjugate according to claim 35, wherein:
A conjugate wherein P2, P3 and / or P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val. 36. The conjugate according to claim 35, wherein:
A conjugate wherein P2, P3 and / or P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Tyr, Glu, Leu, Phe and Val; and P1 is any amino acid.   61. The conjugate according to claim 60, wherein P1 is a naturally occurring amino acid.   61. The conjugate according to claim 60, wherein P1 is an amino acid having an aromatic, branched or branched aromatic side chain.   61. The conjugate according to claim 60, wherein P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr.   61. The conjugate according to claim 60, wherein P1 is Arg, Lys or Arg surrogate.   2. A conjugate according to claim 1, wherein the protease is localized on the cell surface thanks to specific binding interaction with its receptor.   66. The conjugate of claim 65, wherein the cell surface protease is urokinase plasminogen activator (u-PA) bound to urokinase plasminogen activator receptor (u-PAR).   The conjugate according to claim 1, wherein the compound is represented by the formula P6-P5-P4-P3-P2-P1-P1'-P2'-P3 '[wherein P1, P2, P3, P4, P5, P6, P1. A conjugate comprising a peptidic substrate wherein 'and P2' are each selected from the residues shown in FIGS. 1 and 2, and P6, P5, P4, P2 'and P3' are optional]. 66. The conjugate of claim 65, wherein:
P6 is optional and selected from L, V, R;
P5 is optional and selected from R, I, L;
P4 is optional and is selected from G, C, V;
P3 is selected from S, dS, P, A or G;
P2 is selected from A or G;
P1 is R;
P1 ′ is S, V, M or nL;
P2 ′ is optional and selected from S, L, A or V; and P3 ′ is optional and L,
Conjugate.   A conjugate selected from those shown in FIGS. 1-5, wherein the therapeutic agent doxorubicin (Dox) or taxol (Tax) is optionally replaced with any therapeutic agent.   66. The conjugate according to claim 65, wherein the therapeutic agent is a toxin, small organic molecule, nucleic acid, protein therapeutic agent, cytokine or growth factor.   66. The conjugate of claim 65, wherein the therapeutic agent is an anticancer agent.   66. The conjugate of claim 65, wherein the therapeutic agent is an anti-angiogenic agent.   66. The conjugate according to claim 65, wherein the therapeutic agent is abrin, ricin A, Pseudomonas aeruginosa exotoxin Shiga toxin, diphtheria toxin, tumor necrosis factor, α-interferon, γ-interferon, nerve growth factor, tissue Factors and tissue factor variants, FAS-ligand platelet derived growth factor, tissue plasminogen activator, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL- 6) selected from granulocyte macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), nerve growth factor, fibroblast growth factor (FGF), and epidermal growth factor , Conjugate.   66. The conjugate according to claim 65, wherein the therapeutic agent is selected from alkylating agents, toxins, antiproliferative agents, pro-apoptotic agents, pro-coagulants, cytotoxic nucleosides and tubulin binding agents. body. 66. The conjugate according to claim 65, wherein the therapeutic agent is a drug of the following class:
a) drugs of the anthracycline family,
b) Vinca alkaloid drug,
c) mitomycin,
d) bleomycin,
e) cytotoxic nucleosides,
f) pteridine family of drugs,
g) Diinen,
h) Estramustine,
i) cyclophosphamide,
j) taxanes,
k) podophyllotoxin,
l) maytansinoids,
m) an epothilone, and n) a conjugate selected from combretastatin and analogs or pharmaceutically acceptable derivatives thereof. 66. The conjugate according to claim 65, wherein the therapeutic agent is a drug of the following class:
a) doxorubicin,
b) carminomycin,
c) daunorubicin,
d) aminopterin,
e) methotrexate,
f) Metopterin,
g) Dichloromethotrexate,
h) Mitomycin C,
i) porphyromycin,
j) 5-fluorouracil,
k) 6-mercaptopurine,
l) cytosine arabinoside,
m) podophyllotoxin,
n) etoposide o) etoposide phosphate p) melphalan,
q) Vinblastine,
r) vincristine,
s) Leurocidin,
t) vindesine,
u) Estramustine,
v) cisplatin,
w) cyclophosphamide x) taxol,
y) Leurocin,
z) 4-desacetylvinblastine,
aa) Epothilone B,
bb) Taxotere,
cc) maytansinol,
dd) epothilone A, and ee) combretastatin and analogs,
Or a conjugate selected from pharmaceutically acceptable derivatives thereof.   66. The conjugate of claim 65, further comprising a linker between the therapeutic agent and the peptidic substrate.   66. The conjugate of claim 65, wherein the linker comprises a carbohydrate, peptide, and / or hydrocarbon nucleus. 78. The conjugate of claim 77, wherein the linker is:
A biscarbonylalkyl diradical wherein the amine moiety on the therapeutic agent is linked to a linker unit to form an amide bond and the amino terminus of the peptidic substrate is linked to the other end of the linker unit to also form an amide bond; or therapy A diaminoalkyl diradical in which the carbonyl moiety on the drug is conjugated to one of the amines of the linker moiety, while the other amine of the linker unit is covalently bonded to the C-terminus of the peptidic substrate. Linker unit; or the following formula:

[Wherein A is NH or O; D is N (H or alkyl) or O; R ²⁵ is H, alkyl, cycloalkyl, cycloalkylalkyl, aryl, such as 1-3 Heteroaryl optionally substituted with one or more substituents selected from halo, haloalkyl and alkyl, aralkyl, heteroaralkyl, alkenyl containing 1-2 double bonds, 1-2 triple bonds Including alkynyl, alk (en) (in) yl groups, halo, pseudohalo, cyano, hydroxy, haloalkyl and polyhaloalkyl such as halo lower alkyl, especially trifluoromethyl, formyl, alkylcarbonyl, such as 1 to One or more such as three, eg, selected from halo, haloalkyl and alkyl Arylcarbonyl, heteroarylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminoimino, alkoxycarbonylamino, aryloxycarbonylamino, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl optionally substituted with groups , Diarylaminocarbonyl, aralkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, amino, alkylamino, dialkylamino, Arylamino, alkylarylamino, alkylcarbonylamino , Arylcarbonylamino, azide, nitro, mercapto, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl and arylamino Sulfonyl; and y is an integer from 1 to 3]
Self-removing linker:
A conjugate comprising   78. The conjugate of claim 77, wherein the linker is a diamine containing a cyclic alkylene moiety.   78. The conjugate of claim 77, wherein the diamine contains a bicycloalkylene moiety.   78. The conjugate of claim 77, wherein the linker is 1,4-bis (aminomethyl) cyclohexane, 1,4-bis (aminomethyl) cycloheptane, 1,3-bis (aminomethyl) cyclopentane. A conjugate selected from 1-amino-4- (aminomethyl) cyclohexane, 1,4-diaminocyclohexane and 1,4-bis (aminomethyl) bicyclo [2.2.2] octane.   78. The conjugate of claim 77, wherein the linker is a 1, [omega] -diaminoalkane.   78. The conjugate of claim 77, wherein the linker is 1,3-diaminopropane.   78. The conjugate of claim 77, wherein the linker is 1, ω-dicarbonylalkane.   78. The conjugate according to claim 77, wherein the linker is selected from oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid and pivalic acid. 31. A conjugate according to any of claims 1-30, wherein:
The peptidic substrate comprises a P1-P1 ′ bond;
P1-P1 ′ bond is the site of cleavage by cell surface proteases;
P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr; and P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu , Met or 6-aminohexanoyl,
Conjugate.   A conjugate according to any of claims 1 to 32, comprising Phe, Ser, Gly, Ala, Ser (OMe), hSer, 1-methyl His, 3-methyl His, His, nVal, nLeu, Abu, (hS) A conjugate further comprising a P2 residue selected from Gly, Thr, Aib, CHA and Tyr. 34. The conjugate according to claim 1, wherein Arg, Lys, Gln, Quat, Arg proxy, Ser, Thr, hSer, dSer, Pro, (hS) Gly, Tyr, 4,4- A conjugate further comprising a P3 residue selected from dimethyl Thr, Asn, Met (O ₂ ), Quat ² , Quat ³ , Quat ⁴ and Quat ⁵ .   35. The conjugate according to claim 1, wherein Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe, Val, N, N-dimethylGly, β- A conjugate further comprising a P4 residue selected from Ala, Cys (Me), Gln, t-butyl Gly and nVal. 36. A conjugate according to any of claims 1-35, optionally further comprising P5 selected from Ile, Arg or Arg surrogate.
Further comprising a conjugate.   37. The conjugate according to any of claims 1-36, further comprising a P6 residue selected from Val, Leu, Ile, and Val.   38. The conjugate according to any one of claims 1 to 37, comprising Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, hCHA. A conjugate further comprising a P2 ′ residue selected from CHA, hexyl Gly, allyl Gly and Phe.   39. The conjugate according to any one of claims 1 to 38, comprising Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA and allyl Gly. A conjugate further comprising a selected P3 ′ residue.   40. The conjugate according to any one of claims 1 to 39, comprising Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA and allyl Gly. A conjugate further comprising a selected P4 ′ residue.   The conjugate according to any one of claims 1 to 39, wherein P4 'is Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. Conjugate.   40. The conjugate according to any one of claims 1 to 39, wherein P4 'is Leu. 40. A conjugate according to any of claims 1-39, wherein:
The peptidic substrate has the formula:
P4-P3-P2-P1-P1 ′ [where:
P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr;
P2 is selected from Phe, Ser, Gly, Ala, Ser (OMe), hSer, 1-methylHis, 3-methylHis, His, nVal, nLeu, Abu, (hS) Gly, Thr, Aib, CHA and Tyr ;
P3 is Arg, Lys, Gln, Quat, Arg proxy, Ser, Thr, hSer, dSer, Pro, (hS) Gly, Tyr, 4,4-dimethylThr, Asn, Met (O ₂ ), Quat ² , Quat ³ , selected from Quat ⁴ and Quat ⁵ ;
P4 is Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe, Val, N, N-dimethylGly, β-Ala, Cys (Me), Gln, t-butylGly and nVal And P1 ′ is Gly, Ser, hSer, Thr, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl]
Including a 5-mer having
Conjugate. 41. A conjugate according to claim 40, wherein:
One or more P5 or P2 ′ amino acid residues [where:
P5 is Ile, Arg or Arg surrogate; and P2 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, hCHA, Optionally selected from CHA, hexyl Gly, allyl Gly and Phe]
Conjugate. 42. A conjugate according to claim 41, wherein:
If the peptidic substrate comprises a P5 amino acid residue, the peptidic substrate optionally further comprises a P6 amino acid residue selected from Arg, Leu, Ile, and Val; and if the peptidic substrate comprises a P2 ′ amino acid residue If the group contains a peptide substrate, the P3 ′ amino acid residue selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA and allyl Gly And if the peptidic substrate contains a P3 ′ amino acid residue, the peptidic substrate is Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-amino. Further comprising a P4 ′ amino acid residue selected from hexanoyl, CHA and allyl Gly ,
Conjugate. Formula IV:
_{^{X n - (P6) m -}} (P5) p - (P4) i - (P3) j - (P2) l -P1- (P1 ') u - (P2') k - (P3 ') r - (P4 ') _s- (L) _n -Z
Or a conjugate according to any of claims 43 to 47, comprising a derivative thereof, wherein:
Z is a therapeutic agent;
L is a linker;
l, j, i, p and m are selected as follows:
l is 0 or 1; when l is 0, j, i, p and m are 0; when l is 1, j is 0 or 1; when j is 0, i , P and m are 0; when j is 1, i is 0 or 1; when i is 0, p and m are 0; when i is 1, p is 0 or When p is 0, m is 0; when p is 1, m is 0 or 1;
u, k and r are selected as follows:
u is 0 or 1; when u is 0, k, r and s are 0; when u is 1, k is 0 or 1; when k is 0, r and s Is 0; when k is 1, r is 0 or 1; when r is 0, s is 0; when r is 1, s is 0 or 1;
n is 0 or 1;
X ⁿ is hydrogen or an acyl, sulfonyl or carbamoyl cap;
P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr;
P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl;
P2 is selected from Phe, Ser, Gly and Ala;
P3 is selected from Arg, Lys, Gln, Ser, Quat and Arg surrogates;
P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val;
P5 is selected from Arg and Arg surrogates;
P6 is selected from Leu, Ile, and Val;
P2 ′ is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl;
P3 ′ is selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl; and P4 ′ is Gly, Ser, Ala, Leu, Ile, nLeu, Selected from Val, nVal, Aib, Abu, Met and 6-aminohexanoyl,
Conjugate. Formula V:
_{Z- (L) n - (P6} ) m - (P5) p - (P4) i - (P3) j - (P2) l -P1- (P1 ') u - (P2') k - (P3 ') _r − (P4 ′) _s −X ^c
52. A conjugate according to claim 50 or claim 51 having a derivative thereof, wherein:
Z is a therapeutic agent;
L is a linker;
l, j, i, p and m are selected as follows:
l is 0 or 1; when l is 0, j, i, p and m are 0; when l is 1, j is 0 or 1; when j is 0, i , P and m are 0; when j is 1, i is 0 or 1; when i is 0, p and m are 0; when i is 1, p is 0 or When p is 0, m is 0; when p is 1, m is 0 or 1;
u, k and r are selected as follows:
u is 0 or 1; when u is 0, k, r and s are 0; when u is 1, k is 0 or 1; when k is 0, r and s Is 0; when k is 1, r is 0 or 1; when r is 0, s is 0; when r is 1, s is 0 or 1;
n is 0 or 1;
^Xc together with the carbonyl group of the amino acid residue to which it is attached forms a carboxylic acid or carboxamide group;
P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr;
P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl;
P2 is selected from Phe, Ser, Gly and Ala;
P3 is selected from Arg, Lys, Gln, Ser, Quat and Arg surrogates;
P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val;
P5 is selected from Arg and Arg surrogates;
P6 is selected from Leu, Ile, and Val;
P2 ′ is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl;
P3 ′ is selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl; and P4 ′ is Gly, Ser, Ala, Leu, Ile, nLeu, Selected from Val, nVal, Aib, Abu, Met and 6-aminohexanoyl,
Conjugate. Formula VI:
_{^{Z 1 - (L 1) n}} - (P6) m - (P5) p - (P4) i - (P3) j - (P2) l -P1- (P1 ') u - (P2') k - (P3 ') _r- (P4') _s- (L ² ) _v -Z ²
55. A conjugate according to claim 54, comprising a derivative thereof, wherein:
Z ¹ and Z ² are each therapeutic agents and may be the same or different;
L ¹ and L ² are each a linker, and may be the same or different;
l, j, i, p, and m are selected as follows:
l is 0 or 1; when l is 0, j, i, p and m are 0; when l is 1, j is 0 or 1; when j is 0, i , P and m are 0; when j is 1, i is 0 or 1; when i is 0, p and m are 0; when i is 1, p is 0 or When p is 0, m is 0; when p is 1, m is 0 or 1;
u, k and r are selected as follows:
u is 0 or 1; when u is 0, k, r and s are 0; when u is 1, k is 0 or 1; when k is 0, r and s Is 0; when k is 1, r is 0 or 1; when r is 0, s is 0; when r is 1, s is 0 or 1;
n and v are each independently 0 or 1;
P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr;
P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl;
P2 is selected from Phe, Ser, Gly and Ala;
P3 is selected from Arg, Lys, Gln, Ser, Quat and Arg surrogates;
P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val;
P5 is selected from Arg and Arg surrogates;
P6 is selected from Leu, Ile, and Val;
P2 ′ is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl;
P3 ′ is selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl; and P4 ′ is Gly, Ser, Ala, Leu, Ile, nLeu, Selected from Val, nVal, Aib, Abu, Met and 6-aminohexanoyl,
Conjugate. A conjugate according to any one of claims 1 to 49,
Ac-RQGRSL- (therapeutic agent) (SEQ ID NO: 491);
Ac-RQGRSSL- (therapeutic agent) (SEQ ID NO: 492);
Ac-RQGRS-nL- (therapeutic) (SEQ ID NO: 493);
Ac-RQGRS-nV- (therapeutic) (SEQ ID NO: 494);
Ac-RQGRSF- (therapeutic agent) (SEQ ID NO: 495);
Ac-RQGRAL- (therapeutic) (SEQ ID NO: 496);
Ac-RQGRAL- (therapeutic agent) (SEQ ID NO: 497);
Ac-RQGRA-nL- (therapeutic agent) (SEQ ID NO: 498);
Ac-RQGRA-nL- (therapeutic agent) (SEQ ID NO: 499);
Ac-RQGRA-nV- (therapeutic agent) (SEQ ID NO: 500);
Ac-RQGRA-Cha- (therapeutic) (SEQ ID NO: 501);
Ac-RQGRAF- (therapeutic agent) (SEQ ID NO: 502);
Ac-RNGRSL- (therapeutic agent) (SEQ ID NO: 503);
Ac-RNGRA-nL- (therapeutic agent) (SEQ ID NO: 504);
Ac-RQARSL- (therapeutic agent) (SEQ ID NO: 505);
Ac-RQARS-nL- (therapeutic) (SEQ ID NO: 506);
Ac-RQARS-nV- (therapeutic) (SEQ ID NO: 507);
Ac-RQAAS-Cha- (therapeutic) (SEQ ID NO: 508);
Ac-RQARSS-Cha- (therapeutic) (SEQ ID NO: 509);
Ac-RQART-nL- (therapeutic agent) (SEQ ID NO: 510);
Ac-RQARAL- (therapeutic agent) (SEQ ID NO: 511);
Ac-RQARA-nL- (therapeutic) (SEQ ID NO: 512);
Ac-RQARA-nV- (therapeutic) (SEQ ID NO: 513);
Ac-RQARA-Cha- (therapeutic) (SEQ ID NO: 514);
Ac-RQSRAA- (therapeutic) (SEQ ID NO: 515);
Ac-RQSRA- (therapeutic agent) (SEQ ID NO: 516);
Ac-RQSRA-nL- (therapeutic) (SEQ ID NO: 517);
Ac-RQSRAL- (therapeutic agent) (SEQ ID NO: 518);
Ac-RQSRA-nV- (therapeutic) (SEQ ID NO: 519);
Ac-RQSRA-Cha- (therapeutic) (SEQ ID NO: 520);
Ac-RQSRSSL- (therapeutic) (SEQ ID NO: 521);
Ac-RQSRSL- (therapeutic agent) (SEQ ID NO: 522);
Ac-RQSRS-nL- (therapeutic) (SEQ ID NO: 523);
Ac-RQSRS-nL- (therapeutic agent) (SEQ ID NO: 524);
Ac-RQSRS-nV- (therapeutic agent) (SEQ ID NO: 525);
Ac-RQSRS-allyl G- (therapeutic) (SEQ ID NO: 526);
Ac-RQSRS-Cha- (therapeutic) (SEQ ID NO: 527);
Ac-RQSRT-nL- (therapeutic) (SEQ ID NO: 528);
Ac-RQTRSSL- (therapeutic) (SEQ ID NO: 529);
Ac-RQTRSL- (therapeutic agent) (SEQ ID NO: 530);
Ac-RNSRS-nL- (therapeutic agent) (SEQ ID NO: 531);
Ac-RQFRSL- (therapeutic agent) (SEQ ID NO: 532);
Ac-RQFRS-nL- (therapeutic) (SEQ ID NO: 534);
Ac-RQFRS-nV- (therapeutic) (SEQ ID NO: 535);
Ac-RQFRS-nL- (therapeutic) (SEQ ID NO: 536);
Ac-RQFRS-Cha- (therapeutic) (SEQ ID NO: 537);
Ac-RQFRAL- (therapeutic agent) (SEQ ID NO: 538);
Ac-RQFRA-nL- (therapeutic agent) (SEQ ID NO: 539);
Ac-RQFRA-nV- (therapeutic) (SEQ ID NO: 540);
Ac-RQFRA-Cha- (therapeutic) (SEQ ID NO: 541);
Ac-QSRSS-nL- (therapeutic agent) (SEQ ID NO: 542);
MeOCO-Quat2-GRSL- (therapeutic) (SEQ ID NO: 483);
MeOCO-Quat3-GRSL- (therapeutic) (SEQ ID NO: 484);
MeOCO-Quat-GRSL- (therapeutic) (SEQ ID NO: 485);
MeOCO-Quat4-GRSL- (therapeutic agent) (SEQ ID NO: 486);
MeOCO-Quat5-GRSL- (therapeutic agent) (SEQ ID NO: 487);
MeOCO-Quat2-GRSSL- (therapeutic) (SEQ ID NO: 488);
MeOCO-Quat4-GRSL- (therapeutic agent) (SEQ ID NO: 489);
MeOCO-Quat2-GRSL- (therapeutic agent) (SEQ ID NO: 490);
Ac-QGRSL- (therapeutic agent) (SEQ ID NO: 445);
Ac-QGRSSL- (therapeutic agent) (SEQ ID NO: 446);
Ac-QGRASL- (therapeutic agent) (SEQ ID NO: 447);
Ac-NGRSSL- (therapeutic agent) (SEQ ID NO: 448);
Ac-QGRSS-nL- (therapeutic) (SEQ ID NO: 449);
Ac-QGRSS-nV- (therapeutic) (SEQ ID NO: 450);
Ac-QGRSS-Cha- (therapeutic) (SEQ ID NO: 451);
Ac-QGRSS-allyl G- (therapeutic) (SEQ ID NO: 452);
Ac-QGRSS-allyl G- (therapeutic) (SEQ ID NO: 453);
Ac-QARSL- (therapeutic agent) (SEQ ID NO: 454);
Ac-QARSSL- (therapeutic agent) (SEQ ID NO: 455);
Ac-QSRSL- (therapeutic agent) (SEQ ID NO: 456);
Ac-QSRSS-nV- (therapeutic) (SEQ ID NO: 457);
Ac-QSRSS-Cha- (therapeutic) (SEQ ID NO: 458);
Ac-QSRSSL- (therapeutic agent) (SEQ ID NO: 459);
Ac-QTRSSL- (therapeutic agent) (SEQ ID NO: 460);
Ac-Q-Aib-RSS-Cha- (Therapeutic) (SEQ ID NO: 461);
Ac-Q-Aib-RSSL- (therapeutic) (SEQ ID NO: 462);
Ac-Q-Abu-RSS-Cha- (therapeutic agent) (SEQ ID NO: 463);
Ac-Q-Abu-RSSL- (therapeutic) (SEQ ID NO: 464);
Ac-Q-Cha-RSS-Cha- (therapeutic agent) (SEQ ID NO: 465);
Ac-QFRSL- (therapeutic agent) (SEQ ID NO: 466);
Ac-QFRSSL- (therapeutic agent) (SEQ ID NO: 467);
Ac-QYRSSL- (therapeutic) (SEQ ID NO: 468);
Ac-RGRSL- (therapeutic agent) (SEQ ID NO: 469);
Ac-RGRSSL- (therapeutic agent) (SEQ ID NO: 470);
Ac-RGRSS-Cha- (therapeutic) (SEQ ID NO: 471);
Ac-RGRS-Cha- (therapeutic) (SEQ ID NO: 472);
Ac-RARSL- (therapeutic agent) (SEQ ID NO: 473);
Ac-RARSSL- (therapeutic agent) (SEQ ID NO: 474);
Ac-RSRSL- (therapeutic agent) (SEQ ID NO: 475);
Ac-RSRSSL- (therapeutic agent) (SEQ ID NO: 476);
Ac-RSRS-Cha- (therapeutic agent) (SEQ ID NO: 477);
Ac-RSRSS-Cha- (therapeutic) (SEQ ID NO: 478);
Ac-RFRSL- (therapeutic agent) (SEQ ID NO: 479);
Ac-RFRS-Cha- (therapeutic) (SEQ ID NO: 480);
Ac-YGRSSL- (therapeutic agent) (SEQ ID NO: 481);
Ac-M (O2) -SRSL- (therapeutic) (SEQ ID NO: 482);
Ac-RRQSRAA- (therapeutic agent) (SEQ ID NO: 105);
Ac-RRQSRI- (therapeutic agent) (SEQ ID NO: 610);
Ac-RRQSRSSL- (therapeutic agent) (SEQ ID NO: 543);
Ac-RRQSRSL- (therapeutic agent) (SEQ ID NO: 544);
Ac-RGSGRSL- (therapeutic agent) (SEQ ID NO: 545);
Ac-RGSGR--S-nL- (therapeutic agent) (SEQ ID NO: 546);
Ac-RGSGRA-nL- (therapeutic) (SEQ ID NO: 547);
Ac-RGSGRSSL- (therapeutic agent) (SEQ ID NO: 548);
Ac-IVSGRASL- (therapeutic agent) (SEQ ID NO: 549);
Ac-RRQSRA- (therapeutic agent) (SEQ ID NO: 108);
Ac-RRQSRI- (therapeutic agent) (SEQ ID NO: 111);
Ac-LRRQSRAA- (therapeutic agent) (SEQ ID NO: 106);
Ac-LRRQSRGG- (therapeutic agent) (SEQ ID NO: 109);
Ac-LRRQSRA- (therapeutic agent) (SEQ ID NO: 110);
Ac-LRRQSRAI- (therapeutic agent) (SEQ ID NO: 112);
Ac-LRRQSRAI- (therapeutic) (SEQ ID NO: 611);
Ac-LRRQSRSSL- (therapeutic agent) (SEQ ID NO: 550);
Ac-LRRQSRSL- (therapeutic agent) (SEQ ID NO: 551);
Ac-SGRSL- (therapeutic agent) (SEQ ID NO: 362);
Ac-SGRSSL- (therapeutic agent) (SEQ ID NO: 363);
Ac-SGRSSSL- (therapeutic) (SEQ ID NO: 364);
Ac-SGRS-nL- (therapeutic agent) (SEQ ID NO: 365);
Ac-SGRS-nV- (therapeutic) (SEQ ID NO: 366); isomer 1
Ac-SGRS-nV- (therapeutic) (SEQ ID NO: 367); isomer 2
Ac-SGRSG (hex)-(therapeutic) (SEQ ID NO: 368);
Ac-SGRS-Cha- (therapeutic) (SEQ ID NO: 369);
Ac-SGRS-hCha- (therapeutic) (SEQ ID NO: 370);
Ac-SARSL- (therapeutic agent) (SEQ ID NO: 371);
Ac-SARSSL- (therapeutic) (SEQ ID NO: 372);
Ac-SSRS-nL- (therapeutic) (SEQ ID NO: 373);
Ac-TGRS-Abu- (therapeutic agent) (SEQ ID NO: 374);
Ac-TGRSL- (therapeutic agent) (SEQ ID NO: 375);
Ac-TGRS-nV- (therapeutic agent) (SEQ ID NO: 376);
Ac-TGRS-nL- (therapeutic agent) (SEQ ID NO: 377);
Ac-TGRSG (hex)-(therapeutic agent) (SEQ ID NO: 378);
Ac-TGRS-Cha- (therapeutic agent) (SEQ ID NO: 379);
Ac-TGRS-hCha- (therapeutic agent) (SEQ ID NO: 380);
Ac-TGRT-Abu- (therapeutic) (SEQ ID NO: 381);
Ac-TGR-hS-nL- (therapeutic agent) (SEQ ID NO: 382);
Ac-TGR-Abu-nL- (therapeutic agent) (SEQ ID NO: 383);
Ac-TGR-Abu-nV- (therapeutic) (SEQ ID NO: 384);
Ac-TGF (Gn) -S-nL- (therapeutic agent) (SEQ ID NO: 385);
Ac-TGF (Gn) -S-Cha- (therapeutic agent) (SEQ ID NO: 386);
Ac-TGF (Gn) -Abu-nV- (therapeutic) (SEQ ID NO: 387);
Ac-TGK (alloc) -S-nL- (therapeutic agent) (SEQ ID NO: 388);
Ac-TGKS-nL- (therapeutic agent) (SEQ ID NO: 389);
Ac-TG-hR-S-nL- (therapeutic agent) (SEQ ID NO: 390);
Ac- (hS) GGRS-nL- (therapeutic agent) (SEQ ID NO: 391);
MeOCO-TGRS-nL- (therapeutic agent) (SEQ ID NO: 392);
PhSO ₂ -TGRS-nL- (therapeutic) (SEQ ID NO: 393);
MeOEtCO-TGRS-nL- (therapeutic) (SEQ ID NO: 394);
MeO (EtO) 2Ac-TGRS-nL- (therapeutic agent) (SEQ ID NO: 395);
4-oxo-pentanoyl-TGRS-nL- (therapeutic agent) (SEQ ID NO: 396);
3,4-methyldioxy PhAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 397);
2-pyridyl Ac-TGRS-nL- (therapeutic agent) (SEQ ID NO: 398);
PhOAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 399);
L-3-Ph lactyl-TGRS-nL- (therapeutic agent) (SEQ ID NO: 400);
MeOAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 401);
PhAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 402);
MeOEtOCO-TGRS-nL- (therapeutic agent) (SEQ ID NO: 403);
MeOEtOAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 404);
HOOCButa-TGRS-nL- (Therapeutic) (SEQ ID NO: 405);
ZTGRS-nL- (therapeutic agent) (SEQ ID NO: 406);
EtOCO-TGRS-nL- (therapeutic) (SEQ ID NO: 407);
βΑ-Τ-GRS-nL- (therapeutic agent) (SEQ ID NO: 408);
Penta-4-inoyl-TGRS-nL- (therapeutic agent) (SEQ ID NO: 409);
NapAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 410);
iBoc-TGRS-nL- (therapeutic) (SEQ ID NO: 411);
HOAc-TGRS-nL- (therapeutic agent) (SEQ ID NO: 412);
MeSucc-TGRS-nL- (therapeutic) (SEQ ID NO: 413);
N, N-diMeGly-TGRS-nL- (therapeutic agent) (SEQ ID NO: 414);
Succ-TGRS-nL- (therapeutic agent) (SEQ ID NO: 415);
HCO-TGRS-nL- (therapeutic agent) (SEQ ID NO: 416);
Ac-TARS-nL- (therapeutic agent) (SEQ ID NO: 417);
Ac-TAF (Gn) -S-nL- (therapeutic agent) (SEQ ID NO: 418);
Ac-TAR-Abu-nV- (therapeutic agent) (SEQ ID NO: 419);
Ac-TARS-Abu- (therapeutic agent) (SEQ ID NO: 420);
Ac-TART-Abu- (therapeutic) (SEQ ID NO: 421);
Ac-TS (O-Me) -RS-nL- (therapeutic agent) (SEQ ID NO: 422);
Ac-T-hS-RS-nL- (therapeutic agent) (SEQ ID NO: 423);
Ac-T- (1-Me) HRS-nL- (therapeutic agent) (SEQ ID NO: 424);
Ac-T- (3-Me) HRS-nL- (therapeutic) (SEQ ID NO: 425);
Ac-THRS-nL- (therapeutic) (SEQ ID NO: 426);
Ac-T-Sar-RS-nL- (therapeutic agent) (SEQ ID NO: 427);
Ac-T-nV-RS-nL- (therapeutic agent) (SEQ ID NO: 428);
Ac-T-nL-RS-nL- (therapeutic agent) (SEQ ID NO: 429);
Ac-TARS-Cha- (therapeutic) (SEQ ID NO: 430);
Ac-T-Abu-RS-nL- (therapeutic agent) (SEQ ID NO: 431);
Ac-4,4 diMeThr-GRS-nL- (therapeutic agent) (SEQ ID NO: 432);
Ac-hS-GRS-nL- (therapeutic agent) (SEQ ID NO: 433);
Ac-hS-GR-hS-Cha- (therapeutic agent) (SEQ ID NO: 434);
Ac-hS-GRS-Cha- (therapeutic agent) (SEQ ID NO: 435);
Ac-hS-GRT-Cha- (therapeutic agent) (SEQ ID NO: 436);
Ac-hS-ARS-Cha- (therapeutic agent) (SEQ ID NO: 437);
Ac-NGRS-nL- (therapeutic agent) (SEQ ID NO: 438);
Ac-YGRSSL- (therapeutic agent) (SEQ ID NO: 439);
Ac-YGRS-Cha- (therapeutic agent) (SEQ ID NO: 440);
Ac-QGRSS-nL- (therapeutic) (SEQ ID NO: 441);
Ac-QGRSS-nV- (therapeutic) (SEQ ID NO: 442);
Ac-LRGSGRSA- (therapeutic agent) (SEQ ID NO: 573);
Ac-LRGSGRSL- (therapeutic agent) (SEQ ID NO: 342);
Ac-LRGSGRSL- (therapeutic agent) (SEQ ID NO: 343);
Ac-LRGSGRSS-nL- (therapeutic) (SEQ ID NO: 344);
Ac-LRGSGRSS-Cha- (Therapeutic) (SEQ ID NO: 345);
Ac-LRG-dS-ARSA- (therapeutic agent) (SEQ ID NO: 574);
Ac-LRGSARSSL- (therapeutic agent) (SEQ ID NO: 346);
Ac-LRGSARSL- (therapeutic agent) (SEQ ID NO: 347);
Ac-LRGSARSS-Cha- (therapeutic agent) (SEQ ID NO: 348);
Ac-LRGSARSS-nV- (therapeutic) (SEQ ID NO: 349);
Ac-LRGSARSS-nL- (therapeutic) (SEQ ID NO: 350);
Ac-VIVSGRAL- (therapeutic) (SEQ ID NO: 351);
Ac-VIVSARSL- (therapeutic agent) (SEQ ID NO: 352);
Ac-VIVSGRSSL- (therapeutic agent) (SEQ ID NO: 353);
Ac-VIVSARMA- (therapeutic agent) (SEQ ID NO: 354);
Ac-VIVSAR-nL-A- (therapeutic agent) (SEQ ID NO: 355);
Ac-VIVSARS-nL- (therapeutic agent) (SEQ ID NO: 356);
Ac-VIVSARS-Cha- (therapeutic agent) (SEQ ID NO: 357);
Ac-VIVSARS-Cha- (therapeutic) (SEQ ID NO: 358);
Ac-VIVSARSS-Cha- (therapeutic agent) (SEQ ID NO: 359);
Ac-RR- (Me) CPGRVV- (therapeutic agent) (SEQ ID NO: 360);
Ac-RR-nV-PARSL- (therapeutic) (SEQ ID NO: 361);
Ac-RG-dS-ARSA- (therapeutic) (SEQ ID NO: 309);
Ac-RGSGRSA- (therapeutic) (SEQ ID NO: 310);
Ac-RGSGRAL- (therapeutic agent) (SEQ ID NO: 311);
Ac-RGSGRSL- (therapeutic agent) (SEQ ID NO: 312);
Ac-RGSGR--S-nL- (therapeutic) (SEQ ID NO: 313);
Ac-RGSGRA-nL- (therapeutic) (SEQ ID NO: 314);
Ac-RGSGRSSL- (therapeutic agent) (SEQ ID NO: 315);
Ac-RGSGRS-Cha- (therapeutic) (SEQ ID NO: 316);
Ac-RGSGRSS-Cha- (therapeutic) (SEQ ID NO: 317);
Ac-RGSARS-Cha- (therapeutic) (SEQ ID NO: 318);
Ac-RGSARSS- (therapeutic agent) (SEQ ID NO: 319);
Ac-RGSARS-nV- (therapeutic) (SEQ ID NO: 320);
Ac-RGSARSS-nV-(therapeutic) (SEQ ID NO: 321);
Ac-RGSARSL- (therapeutic) (SEQ ID NO: 322);
Ac-R- (Me) CPGRVV- (therapeutic agent) (SEQ ID NO: 323);
Ac-R- (Me) CPGRVV- (therapeutic agent) (SEQ ID NO: 324);
Ac-RC (Me) -PGRSL- (therapeutic) (SEQ ID NO: 325);
Ac-RLPGRSL- (therapeutic agent) (SEQ ID NO: 326);
Ac-RVPGRSL- (therapeutic agent) (SEQ ID NO: 327);
Ac-RVPGRSL- (therapeutic agent) (SEQ ID NO: 328);
Ac-R-nL-PGRSL- (therapeutic agent) (SEQ ID NO: 329);
Ac-RG (tBu) -PARSL- (therapeutic agent) (SEQ ID NO: 330);
Ac-RLPARSL- (therapeutic agent) (SEQ ID NO: 331);
Ac-RVPARSL- (therapeutic agent) (SEQ ID NO: 332);
Ac-R-nL-PARSL- (therapeutic agent) (SEQ ID NO: 333);
Ac-IVSGRAL- (therapeutic agent) (SEQ ID NO: 334);
Ac-IVSGRSSL- (therapeutic agent) (SEQ ID NO: 335);
Ac-IVSGRASL- (therapeutic agent) (SEQ ID NO: 336);
Ac-IVSARMA- (therapeutic agent) (SEQ ID NO: 337);
Ac-IVSAR-nL-A- (therapeutic) (SEQ ID NO: 338);
Ac-IVSARSL- (therapeutic agent) (SEQ ID NO: 339);
Ac-IVSARS-nL- (therapeutic) (SEQ ID NO: 340);
Ac-IVSARSSL- (therapeutic) (SEQ ID NO: 341);
Ac-GSGRSA- (therapeutic agent) (SEQ ID NO: 585);
Ac-GSGRSL- (therapeutic agent) (SEQ ID NO: 277);
Ac-GSGRAL- (therapeutic) (SEQ ID NO: 278);
Ac-GSGRSSL- (therapeutic agent) (SEQ ID NO: 279);
Ac-GSGRL- (therapeutic agent) (SEQ ID NO: 280);
Ac-GSG- (4-guan) Phg-SL- (therapeutic) (SEQ ID NO: 281);
Ac-GSGRSS-Cha- (therapeutic) (SEQ ID NO: 282);
Ac-GSGRASL- (therapeutic) (SEQ ID NO: 283);
Ac-GSGRS-nL- (therapeutic) (SEQ ID NO: 284);
Ac-GTGRS-nL- (therapeutic agent) (SEQ ID NO: 285);
Succ-bA-TGRS-nL- (Therapeutic) (SEQ ID NO: 286);
Ac-GTGRS-hCha- (therapeutic agent) (SEQ ID NO: 287);
Ac-G-hS-GRS-nL- (therapeutic agent) (SEQ ID NO: 288);
Ac-G-dS-ARSA- (therapeutic agent) (SEQ ID NO: 289);
Ac-GSARSL- (therapeutic agent) (SEQ ID NO: 290);
Ac-GSARSS-Cha- (therapeutic agent) (SEQ ID NO: 291);
Ac-GSARSSL- (therapeutic) (SEQ ID NO: 292);
Ac-GSARASL- (therapeutic agent) (SEQ ID NO: 293);
Ac-VSGRSL- (therapeutic agent) (SEQ ID NO: 294);
Ac-VSGRAL- (therapeutic agent) (SEQ ID NO: 295);
Ac-VSGRASL- (therapeutic) (SEQ ID NO: 296);
Ac-VSGRSSL- (therapeutic agent) (SEQ ID NO: 297);
Ac-VSARMA- (therapeutic agent) (SEQ ID NO: 298);
Ac-VSAR-nL-A- (therapeutic agent) (SEQ ID NO: 299);
Ac-VSARS-nL- (therapeutic agent) (SEQ ID NO: 300);
Ac-VSARSL- (therapeutic agent) (SEQ ID NO: 301);
Ac- (Me) CPGRVV- (therapeutic agent) (SEQ ID NO: 302);
Ac- (Me) CPGRVV- (therapeutic agent) (SEQ ID NO: 303);
Ac-C (Me) -PGRAL- (therapeutic agent) (SEQ ID NO: 304);
Ac-C (Me) -PGRSL- (therapeutic agent) (SEQ ID NO: 305);
Ac-C (Me) -PARSL- (therapeutic) (SEQ ID NO: 306);
Ac-C (Me) -PARASL- (therapeutic agent) (SEQ ID NO: 307);
Ac-G (tBu) -PGRSL- (therapeutic agent) (SEQ ID NO: 308);
Ac-QSRAA- (therapeutic agent) (SEQ ID NO: 552);
Ac-QSRSA- (therapeutic) (SEQ ID NO: 553);
Ac-QSRSG- (therapeutic) (SEQ ID NO: 554);
Ac-RSRAA- (therapeutic agent) (SEQ ID NO: 555);
Ac-RQSRAA- (therapeutic) (SEQ ID NO: 556);
Ac-RQSRSA- (therapeutic) (SEQ ID NO: 557);
Ac-RQSRSAA- (therapeutic agent) (SEQ ID NO: 558);
Ac-RGSGRSA- (therapeutic agent) (SEQ ID NO: 559);
Ac-SGRAA- (therapeutic) (SEQ ID NO: 560);
Ac-SGRSA- (therapeutic agent) (SEQ ID NO: 561);
Ac-SGRSSA- (therapeutic) (SEQ ID NO: 562);
Ac-SGRASA- (therapeutic agent) (SEQ ID NO: 563);
Ac-SGRSG- (therapeutic agent) (SEQ ID NO: 564);
Ac-SGRSSG- (therapeutic) (SEQ ID NO: 565);
Ac-SGRSGA- (therapeutic) (SEQ ID NO: 566);
Ac-SGRSGG- (therapeutic agent) (SEQ ID NO: 567);
Ac-GTGRSGG- (therapeutic agent) (SEQ ID NO: 568);
Ac-GSGRSGG- (Therapeutic) (SEQ ID NO: 243)
Ac-LRRQSRAA- (therapeutic agent) (SEQ ID NO: 597);
MeSO ₂ -dA (Chx) -Abu-RSL- (Therapeutic) (SEQ ID NO: 598);
Ac-RARSL- (therapeutic agent) (SEQ ID NO: 599);
Ac-dA (Chx) -Abu-RSL- (therapeutic) (SEQ ID NO: 600);
Ac-dA (Chx) -Abu-RSSL- (therapeutic) (SEQ ID NO: 601);
Ac-QGRSSL- (therapeutic agent) (SEQ ID NO: 602);
MeOCO-dhF-P (OH) -RSSL- (therapeutic) (SEQ ID NO: 603);
MeOCO-Quat4-GRSL- (therapeutic agent) (SEQ ID NO: 604);
Ac-dCha-P (OH) -RSSL- (therapeutic) (SEQ ID NO: 605);
Ac-dCha-Abu-RSSA- (therapeutic agent) (SEQ ID NO: 606);
MeOCO-Quat2-GRSL- (therapeutic agent) (SEQ ID NO: 607);
MeOCO-Quat3-GRSL- (therapeutic) (SEQ ID NO: 608); and
MeOCO-Quat-GRSL- (Therapeutic) (SEQ ID NO: 609)
A conjugate selected from.   57. The conjugate according to any of claims 35 to 56, wherein P4 is Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe, Val, N, N-dimethyl. A conjugate selected from Gly, β-Ala, Cys (Me), Gln, t-butyl Gly and nVal.   67. A conjugate according to claim 1 or 66, wherein the formulas P6-P5-P4-P3-P2-P1-P1'-P2'-P3'-P4 '[wherein P1, P2, P3, P4, Each of P5, P6, P1 ′ and P2 ′ is selected from the residues shown in FIGS. 1 and 2, and P6, P5, P4, P2 ′, P3 ′ and P4 ′ are optional]. Conjugate. 68. The conjugate of claim 67, wherein:
P6 is optional and selected from L, V, R;
P5 is optional and selected from R, I, L;
P4 is optional and is selected from G, C, V;
P3 is selected from S, dS, P, A or G;
P2 is selected from A or G;
P1 is R;
P1 ′ is S, V, M or nL;
P2 ′ is optional and is selected from S, L, A or V;
P3 ′ is optional and L; and P4 ′ is optional and L,
Conjugate.   A conjugate comprising a therapeutic agent and a nucleic acid substrate linked thereto via a peptidic linker, wherein the peptidic linker is proteolytically cleaved by a cell surface protease or its soluble, shed or released form A conjugate that releases a therapeutic agent, wherein the conjugate is not substantially cleaved by plasmin or prostate specific antigen (PSA).   109. The conjugate of claim 108, wherein the nucleic acid is DNA.   109. The conjugate of claim 108, wherein the nucleic acid is RNA.   109. The conjugate of claim 108, wherein the nucleic acid is double stranded RNA. 68. The conjugate of claim 67, wherein:
P6 is optional and selected from L, V, R;
P5 is optional and selected from R, I, L;
P4 is optional and is selected from G, C, V;
P3 is selected from S, dS, P, A or G;
P2 is selected from A or G;
P1 is R;
P1 ′ is T, Abu, hS, nV or A;
P2 ′ is optional and is selected from S, L, A or V;
P3 ′ is optional and is L, nL, nV, G (hexyl), G (allyl), CHA, hCHA or Abu; and P4 ′ is optional and is L, nL, nV, G (hexyl), G (allyl), CHA, hCHA or Abu,
Conjugate. 68. The conjugate of claim 67, wherein:
P6 is optional and selected from L, V, R;
P5 is optional and selected from R, I, L;
P4 is optional and is selected from G, C, V;
P3 is selected from S, dS, P, A or G;
P2 is selected from A or G;
P1 is R;
P1 ′ is S, G or A;
P2 ′ is optional and is selected from G or A;
P3 ′ is optional and is L, nL, nV, G (hexyl), G (allyl), CHA, hCHA or Abu; and P4 ′ is optional and is L, nL, nV, G (hexyl), G (allyl), CHA, hCHA or Abu,
Conjugate.   114. The conjugate according to any of claims 1-113, wherein the therapeutic agent is taxol.   114. The conjugate according to any one of claims 1-113, wherein the therapeutic agent is doxorubicin.   114. A method of treating a disease, comprising administering to the subject a conjugate according to any of claims 1-113, wherein the disease is a cell surface protease related disease.   117. The method of claim 116, wherein the disease is selected from the group consisting of autoimmune diseases, inflammatory diseases, infectious diseases and endocrine diseases.   117. The method of claim 116, wherein the disease is a proliferative disease.   A method of treating a cell surface protease related disease comprising a therapeutic agent and a peptidic substrate optionally linked thereto via a linker, wherein the peptidic substrate is a cell surface protease or its soluble, released or shed Administering a conjugate that is proteolytically cleaved by the form to release the therapeutic agent, to a subject exhibiting symptoms of a cell surface protease-related disorder.   120. The method of claim 119, wherein the disease is selected from the group consisting of autoimmune diseases, inflammatory diseases, infectious diseases and endocrine diseases.   120. The method of claim 119, wherein the disease is a proliferative disease.   120. The method according to any one of claims 114 to 119, wherein the subject is a mammal.   121. The method of claim 120, wherein the mammal is a human.   122. The method of claim 118 or 121, wherein the disease is cancer.   122. The method according to claim 118 or 121, wherein the disease is selected from ocular disease, cardiovascular disease, chronic inflammatory disease, wound, circulatory system disease, dermatological disease and cancer.   122. The method of claim 118 or 121, wherein the disease is rheumatoid arthritis, psoriasis, diabetic retinopathy, recurrence of pterygium, scar from excimer laser surgery, scar from glaucoma filtration surgery, pre-macular degeneration A method selected from ocular, CREST syndrome, solid neoplasia and vascular tumor.   122. The method of claim 118 or 121, wherein the disease is selected from lung cancer, colon cancer, pancreatic cancer, esophageal cancer, breast cancer, ovarian cancer, prostate cancer, melanoma and Kaposi sarcoma. ,Method.   128. The method according to any of claims 116-127, wherein the therapeutic agent is taxol.   128. The method according to any of claims 116-127, wherein the therapeutic agent is doxorubicin.   114. A pharmaceutical composition comprising the conjugate according to any of claims 1-113 or a pharmaceutically acceptable derivative thereof in a pharmaceutically acceptable carrier.   134. The pharmaceutical composition of claim 130, formulated for single dose administration.   An article of manufacture that is contained within a packaging material that is used to treat, prevent or ameliorate one or more symptoms associated with the packaging material, a cell surface protease-related disease or disorder. 114. A conjugate according to any of 1-113 or a pharmaceutically acceptable derivative thereof, and one or more of the conjugates or pharmaceutically acceptable derivatives thereof associated with a cell surface protease-related disease or disorder An article of manufacture that includes a label indicating that it is used to treat, prevent or ameliorate symptoms.   114. A conjugate according to any of claims 1-113 when used for the treatment of cell surface protease related diseases.   134. The conjugate of claim 133, wherein the disease is a proliferative disease.   135. The conjugate according to claim 134, wherein the proliferative disease is cancer.   135. A conjugate according to claim 134, wherein the proliferative disease is selected from eye diseases, cardiovascular diseases, chronic inflammatory diseases, wounds, circulatory system diseases, dermatological diseases and cancer. .   135. The conjugate of claim 134, wherein the proliferative disorder is rheumatoid arthritis, psoriasis, diabetic retinopathy, pterygium recurrence, scar from excimer laser surgery, scar from glaucoma filtration surgery, macular degeneration A conjugate selected from anterior eye, CREST syndrome, solid neoplasia and vascular tumor.   135. The conjugate according to claim 134, wherein the proliferative disease is selected from lung cancer, colon cancer, pancreatic cancer, esophageal cancer, breast cancer, ovarian cancer, prostate cancer, melanoma and Kaposi's sarcoma. The conjugate.   114. Use of a conjugate according to any of claims 1-113 for the preparation of a medicament for use in the treatment of surface protease related diseases.   140. Use according to claim 139, wherein the disease is a proliferative disease.   143. Use according to claim 140, wherein the proliferative disorder is cancer.   141. Use according to claim 140, wherein the proliferative disease is selected from ocular diseases, cardiovascular diseases, chronic inflammatory diseases, wounds, circulatory system diseases, dermatological diseases and cancer.   142. Use according to claim 140, wherein the proliferative disease is rheumatoid arthritis, psoriasis, diabetic retinopathy, recurrence of pterygium, scar from excimer laser surgery, scar from glaucoma filtration surgery, pre-macular degeneration Use, selected from eye, CREST syndrome, solid neoplasia and vascular tumor.   142. Use according to claim 140, wherein the proliferative disease is selected from lung cancer, colon cancer, pancreatic cancer, esophageal cancer, breast cancer, ovarian cancer, prostate cancer, melanoma and Kaposi's sarcoma ,use. 114. A method of making a conjugate according to any of claims 1-113, comprising:
a) synthesizing a peptidic substrate;
b) optionally capping the peptidic substrate on either the N-terminus or the C-terminus;
c) optionally linking the uncapped end of the peptidic substrate to a linker;
d) Coupling the peptidic substrate to a therapeutic agent, optionally via a linker, to form a conjugate; and e) Optionally deprotecting the conjugate if protected:
Including a method.   145. The method of claim 145, wherein prior to step a), the method comprises identifying a peptidic substrate for a protease. A method,
a) selecting a disease;
b) identifying a cell involved in the disease process or a cell in the vicinity of a cell involved in the disease process; and c) identifying a cell surface protease on the cell, thereby targeting the target conjugate for treating the disease Identifying proteases:
Including the method.   148. The method of claim 147, further comprising creating a conjugate that targets a protease.

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