Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• Conjugates, compositions and methods for localized delivery of therapeutic agents for treating a variety of disorders such as , proliferative diseases, autoimmune diseases, infectious diseases and inflammatory diseases.
• the conjugates which act as prodrugs, contain therapeutic agents and peptidic substrates that are cleaved by cell surface proteases to release therapeutic agents in the vicinity of the targeted cells.
• BACKGROUND Effective treatment of cancer and other proliferative diseases involves administration of chemotherapeutic agents, typically systemic administration.
• chemotherapeutic agents are cytotoxic agents that act by inhibiting proliferation or other metabolic processes, so that actively proliferating and growing cells will be targeted by the agent. Such targeting, however, is not highly specific, and the side-effects are often devastating.
• a goal in pharmacology is the design of specific agents that act with high specific activity on targeted cells or tissues.
• This aim is of particular importance, for example, in the design of agents for treatments of diseases, such as proliferative diseases, including neoplastic disease, and diseases of viral origin, in which the ratio of toxic dose to therapeutic dose is generally close to one and the dosage must be restricted. Numerous approaches to achieving this goal have been developed.
• conjugates that contain a targeting agent, such as an antibody and/or growth factor, and a therapeutic agent, that act on specific cells; the use of antisense technology that is targeted to specific genes and/or proteins; the use of genetic therapy to provide, for example, correct copies of defective genes or pharmaceutically active compounds, and the use of toxins that are relatively non-toxic unless delivered intracellularly.
• a targeting agent such as an antibody and/or growth factor
• a therapeutic agent that act on specific cells
• antisense technology that is targeted to specific genes and/or proteins
• genetic therapy to provide, for example, correct copies of defective genes or pharmaceutically active compounds
• toxins that are relatively non-toxic unless delivered intracellularly.
• the compounds are conjugates that contain a peptidic substrate for a cell surface protease, or a soluble, shed or released form thereof, and an agent that upon cleavage by the protease is a therapeutic agent or in a form that can be activated by the targeted cell or tissue or in the localter thereof.
• the agents include therapeutic agents, such as a cytotoxic agents, drugs, therapeutic nucleic acid moleulces, and diagnostic agents, such as labelled moieties and imaging agents.
• the cell surface proteases are proteases located at a cell surface and, include, but are not limited to, membrane-bound proteases such as membrane-bound serine proteases (SPs), including, for example, proteases designated MTSPs and endotheliases. Also contemplated are proteases that are located at the cell surface by virtue of a specific binding interaction with a receptor therefor. Included among such proteases is urokinase plasminogen activator (u-PA; see, e.g. , Hung (1 984) Adv. Exp. Med. Biol.172:281 -293; Cheng et al.
• u-PA urokinase plasminogen activator
• the conjugates contain one or more substrates for one or a plurality of cell surface proteases linked either directly or via a linker to a targeted agent, including a therapeutic agent, such as a cytotoxic agent.
• the conjugates provided herein contain the following components: (peptidic substrate),., (Iinker) q , and (targeted agent) t in which: at least one peptidic substrate moiety is linked with or without a linker (L) to at least one therapeutic agent, s is 1 or more and each substrate is the same or different, and is typically is between 1 and 6, generally 1 , 2 or 3; q is 0 or more as long as cell surface protease(s) cleaves the peptidic substrate(s) and releases active therapeutic agent or, releases the agent in a form that is converted by the cell, tissue or surrounding environment to an active form, q is 0 to t, generally 1 to 4; t is 1 or more, generally 1 or 2 and each targeted agent are the same or different; linker refers to any linker; and the targeted agent is any agent, typically a therapeutic agent, such as a cytotoxic agent, a nucleic acid, a diagnostic agent, such as an imaging agent or labeled moiety, or a drug,
• the therapeutic agents include any biologically active molecule. These agents include toxins, cytokines and lymphokines, growth factors, nucleic acid molecules, such as antisense nucleic acid, dsRNA, and DNA molecules.
• the therapeutic agents include those that are active intracellularly, such as cytotoxins, or extracellularly, such as modulators of the activity of extracellular receptors. When in the conjugates the therapeutic agents are substantially inactive, and when cleaved are released in active form or in a form that can be activated by the targeted cell or tissue or environment thereof.
• the conjugates for use in the methods and compositions provided herein can be represented by the formula: (peptideVflinkerJ q -ftherapeutic agent) t or a derivative thereof, where peptide' is a peptidic substrate for a cell surface protease; s is greater than or equal to 1 , or is 1 to 6, or is 1 or 2, or is 1 ; linker is any linker; q is greater than or equal to 0, or is 0 to 4, or is 0 or 1 ; the therapeutic agent is, for example, a cytotoxic agent, including, but not limited to, an anti-tumor, anti-angiogenic, anti-cancer, pro-apoptotic and anti-mitotic agents; and t is 1 or more, or is 1 or 2.
• the therapeutic agent is covalently attached, optionally via a linker L, to either the C-terminus or the N-terminus of the peptidic substrate.
• peptide' is a substrate for a cell surface protease whereby, upon action of the protease, the conjugate, which is substantially inactive, is cleaved at a point on the peptidic substrate chain to release a compound of the formula: (peptide a ) s -(linker) q -(therapeutic agent), or a derivative thereof, that exhibits therapeutic activity in vitro and/or in vivo.
• the therapeutic agent is, for example, a cytotoxic agent
• peptide 8 is a truncated version of peptide 1 resulting from cleavage at the P1 - , P1 ' bond.
• the conjugates can be used to target and deliver the targeted agents to specific cells, and hence can be used for the treatment any diseases that are associated with cells or tissues that express a cell surface protease, including cell-associated and cell-localized proteases.
• the cells on which or near which such proteases are expressed are not necessarily involved in the disease or disease process, but are present and can serve to present the protease, which cleaves the targeted conjugate.
• diseases associated with cells or tissues that express a cell surface protease include, but not limited to, proliferative diseases, autoimmune diseases, infectious diseases and inflammatory diseases.
• diseases include e, but are not limited to, rheumatoid arthritis, lupus, multiple sclerosis, psoriasis, diabetic retinopathies, other ocular disorders, including recurrence of pterygii, scarring excimer laser surgery and glaucoma filtering surgery, various disorders of the anterior eye, cardiovascular disorders, restenosis, chronic inflammatory diseases, wounds, circulatory disorders, crest syndromes, bacterial infections, viral diseases, includuing AIDS, dermatological disorders, and cancer, including solid neoplasms and vascular tumors, including, but are not limited to, lung, colon, esophageal, breast, ovarian and prostate cancers.
• the methods involve identifying cell-surface protease-associated disease by identifying a cell involved in the disease process or a cell in the vicinity of the cell involved in the disease process; and identifying a cell surface protease on the cell. Conjugates that target such proteases as provided herein can then be prepared. DESCRIPTION OF THE FIGURES
• Figure 1 shows exemplary doxorubicin conjugates provided herein and in vitro CT 50 (min) data for cleavage of the conjugates by MTSP1 .
• Figure 2 shows exemplary doxorubicin conjugates provided herein and in vitro CT 50 (min) data for cleavage of the conjugates by u-PA.
• Figure 3 shows exemplary taxol conjugates provided herein and in vitro CT 50 (min) data for cleavage of the conjugates by MTSP1 .
• Figure 4 shows exemplary taxol conjugates provided herein and in vitro CT 50 (min) data for cleavage of the conjugates by u-PA.
• Figure 5 shows exemplary doxorubicin and taxol conjugates provided herein and in vitro CT 50 (min) data for cleavage of the conjugates by ET1 (endotheliase 1 ).
• a targeted agent is any agent intended for targeted delivery and includes therapeutic agents and diagnostic agents and any other agent intended for targeted delivery.
• targeted delivery means delivery to a selected cell or tissue that expresses a protease that releases the targeted agent. Such delivery does not have to be exclusively to such selected cell or tissue, but must include it, and generally deliveries higher amounts to such selected cells or tissues. Delivery includes introduction into a cell or tissue or binding to the cell or tissue or release in the vicinity of the cell or tissue.
• a tumor induces production of proteases, receptors, co-factors or substrates asssociated with the stroma; delivery, thus, includes targeting such induced stromal activities, such as proteases, receptors and/or enzyme co-factors, in invading cells or cells in the tumor that is targeted.
• therapeutic index is the ratio of LD 50 /ED 50 .
• a therapeutic agent is any drug or other agent that is intended for delivery to a targeted cell or tissue, such as proliferating cells, including tumor cells and cells involved in a proliferative, typically an undesirable, response.
• Therapeutic agents include, but are not limited to, anticancer agents, anti-angiogenic agents, pro-apoptotic agents, anti-mitotic growth factors, cytokines, such as tumor necrosis factors and interleuk ⁇ ns, and cytotoxic agents and other such agents as described herein and known to those of skill in the art.
• Therapeutic agents include those that are active upon intemalization and also those that act extracellularly, such modulators of the activities of certain cell surface receptors, such as G proteins that transduce extracellular signals.
• an inactive therapeutic agent is a therapeutic agent that is conjugated to a peptide and thereby, either by virtue of conformational changes or size or other factors such as steric hinderance does not exhibit any or exhibits substantially reduced activity compared to the released active therapeutic agent.
• conjugated doxorubicin is not toxic to cells until it is released from the conjugate in a form that can enter the cell. Upon cleavage of the agent from the conjugate it is in active form or in a form that is further processed by one or a plurality of steps, including enzymatically or chemically, in or on the cell, into an active form.
• 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.
• the active therapeutic agent is by virtue of cleavage able to exhibit its intended activity, typically by entering the cell.
• the therapeutic agents When conjugated the therapeutic agents have reduced or no activity as therapeutic agents, and upon cleavage are released in the vicinity of a cell.
• an anti-cancer agent refers to any agents used in the treatment of cancer. These include any agents, when used alone or in combination with other compounds, that can alleviate, reduce, ameliorate, prevent, or place or maintain in a state of remission of clinical symptoms or diagnostic markers associated with neoplasm, tumor or cancer, and can be used in methods, combinations and compositions provided herein.
• Non-limiting examples of anti- neoplasm agents include anti-angiogenic agents, alkylating agents, antimetabolite, certain natural products, platinum coordination complexes, anthracenediones, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, certain hormones, antagonists and anti-cancer polysaccharides.
• substantially inactive with reference to the conjugated thereapeutic agent means at least 1 %, generally 10, 20, 30, 50, 60, 70, 80 or 90 or 100% inactive compared to the unconjugated therapeutic agent in a standard or art-recognized assays, such as in vitro or in vivo assays, that assess the therapeutic activity of the agent.
• a targeted cell or tissue refers to the cells or tissues that include cell surface proteases that cleave the conjugates.
• the cells or tissues can be involved in the disease or can be present at the disease loci or locus by virtue of participation in the disease process or merely serendipitously.
• angiogenesis is intended to broadly encompass the totality of processes directly or indirectly involved in the establishment and maintenance of new vasculature (neovascularization), including, but not limited to, neovascularization associated with tumors.
• anti-angiogenic treatment or agent refers to any therapeutic regimen and compound, that, when used alone or in combination with other treatment or compounds, can alleviate, reduce, ameliorate, prevent, or place or maintain in a state of remission, one or more clinical symptoms or diagnostic markers associated with undesired and/or uncontrolled angiogenesis.
• an anti-angiogenic agent refers to an agent that inhibits the establishment or maintenance of vasculature.
• agents include, but are not limited to, anti-tumor agents, and agents for treatments of other disorders associated with undesirable angiogenesis, such as diabetic retinopathies, hyperproliferative disorders and others.
• non-anti-angiogenic anti-tumor agents refer to anti-tumor agents that do not act primarily by inhibiting angiogenesis. Whether anti-tumor agents act primarily by inhibiting angiogenesis can be determined using the assays provided herein, or using other assays well known to those of skill in the art.
• undesired and/or uncontrolled angiogenesis refers to pathological angiogenesis wherein the influence of angiogenesis stimulators outweighs the influence of angiogenesis inhibitors.
• deficient angiogenesis refers to pathological angiogenesis associated with disorders where there is a defect in normal angiogenesis resulting in aberrant angiogenesis or an absence or substantial reduction in angiogenesis.
• a cell surface protease is any protease that is located on or at a cell surface and/or proteases that are located at the cell surface by virtue of a specific binding interaction with a receptor therefor, or that is localized at or near or associated with the cell surface.
• cell surface proteases contemplated herein include cell surface-associated proteases. It also includes all forms thereof that can be circulating or inside a cell. To be categorized as a cell surface protease, there must be at least one form thereof that is located (i.e. on the surfaces such as transmembrane protease or bound to receptor therefor) on the surface of a cell at some point in its cycle.
• Cell surface protease include serine proteases, such as, but are not limited to, the transmembrane serine protease (MTSPs) and endotheliases and urokinases.
• MTSPs transmembrane serine protease
• a serine protease refers to a diverse family of proteases in which a serine residue is involved in the hydrolysis of proteins or peptides.
• the serine residue can be part of the catalytic triad mechanism, which includes a serine, a histidine and an aspartic acid in the catalysis, or be part of the hydroxyl/e-amine or hydroxyl/ -amine catalytic dyad mechanism, which involves a serine and a lysine in the catalysis.
• SPs of mammalian, including human, origin are particularly interested in these SPs of mammalian, including human, origin.
• soluble and released forms of cell surface proteases are contemplated. Such forms include, for example, forms found in serum upon proteolytic degradation or other removal of the extracellular portion of membrane bound protease, and splice variants that do not include a transmembrane domain.
• the protease activity of cell surface proteases and proteases associated with cells can be exploited to provide a means to concentrate therapeutic agents, such as cytotoxic agents, near such cells by providing conjugates that are activated upon cleavage by such enzymes.
• Such conjugates upon the action of a cell surface protease or cell-associate protease, release the therapeutic agent, such as a cytotoxic agent, or a derivative thereof that can be converted to a therapeutic agent, locally at the site of action.
• the substrates are designed to be substrates of targeted proteases that are expressed or are active on the surfaces of cells, such as tumor cells or endothelial cells, involved in or present at the site(s) or locus or loci of the disease.
• targeted proteases that are expressed or are active on the surfaces of cells, such as tumor cells or endothelial cells, involved in or present at the site(s) or locus or loci of the disease.
• administration of the conjugates provided herein permits targeting of therapeutic agents to such cells.
• active therapeutic agents are released in the immediate vicinity of the targeted cells.
• specific profiles of some of the MTSPs are as follows.
• transmembrane serine protease refers to a family of transmembrane serine proteases that share common structural features as described herein (see, also Hooper et al. (2001 ) J. Biol. Chem.276:857-860).
• MTSP transmembrane serine protease encompasses all proteins encoded by the MTSP genes, including but are not limited to: MTSP1 , MTSP3, MTSP4, MTSP6, MTSP7, MTSP9, MTSP10, MTSP1 2, MTSP20, MTSP22 and MTSP25 or an equivalent molecule obtained from any other source or that has been prepared synthetically or that exhibits the same activity.
• MTSPs include, but are not limited to, corin, enteropeptidase, human airway trypsin-like protease (HAT), TMPRSS2 and TMPRSS4.
• HAT human airway trypsin-like protease
• TMPRSS2 TMPRSS4.
• the MTSPs described herein can be used to identify other MTSPs.
• Methods for isolating nucleic acid encoding other MTSPs including nucleic acid molecules encoding full-length molecules and splice variants and MTSPs from species, such as cows, sheep, goats, pigs, horses, primates, including chimpanzees and gorillas, rodents, dogs, cats and other species of interest, such as domesticated animals, farm and zoo animals are known to those of skill in the art and are outlined herein.
• nucleic acid molecules described herein can be used to obtain nucleic acid molecules encoding full-length MTSP polypeptides from human sources or from other species, such as by screening appropriate libraries using the nucleic acid molecules or selected primers or probes based thereon. Sequences of encoding nucleic acid molecules and the encoded amino acid sequences of exemplary MTSPs and/or domains thereof are set forth in SEQ ID Nos. 1 -45, 269-270 and 272-276. The term also encompasses MTSPs with amino acid substitutions that do not substantially alter activity of each member and also encompasses polyeptides encoded by splice variants thereof.
• MTSPs with amino acid substitutions such that the resulting polypeptide retains at least 1 %, 5 %, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the proteolytic activity of the unaltered polypeptide, and also encompasses MTSPs encoded by splice variants thereof and MTSPs encoded by allelic variants, such as single nucleotide polymorphisms (SNPs).
• SNPs single nucleotide polymorphisms
• Suitable substitutions including, although not necessarily, conservative substitutions of amino acids, are known to those of skill in this art and can be made without eliminating the biological activity, such as the catalytic activity, of the resulting molecule.
• MTSPs include those of animal, such as mammalian, including human, origin.
• protease domain of an MTSP refers to an extracellular protease domain of an MTSP that exhibits proteolytic activity and shares homology and structural features with the chymotrypsin/trypsin family protease domains. Hence it is at least the minimal portion of the domain that exhibits proteolytic activity as assessed by standard in vitro assays.
• Exemplary MTSP polypeptides are set forth in SEQ ID Nos. 1 -45, 269-270 and 272-276, and including smaller portions thereof that retain or exhibit protease activity.
• the protease domains vary in size and constitution, including insertions and deletions in surface loops. They retain conserved structure, including at least one of the active site triad, primary specificity pocket, oxyanion hole and/or other features of serine protease domains of proteases.
• the protease domain is a portion of a MTSP, as defined herein, and is homologous to a domain of other MTSPs.
• MTSPs include, MTSP1 , MTSP3, MTSP4, MTSP6, MTSP7, MTSP9, MTSP1 0, MTSP1 2, MTSP20, MTSP22 and MTSP25 (see SEQ ID Nos. 1 -1 9, 42-45, 269-270 and 272-276; see, also International PCT application No. WO 02/00860 (see SEQ ID Nos. 38 and 97 therein, which provide an MTSP12 variant); corin (SEQ ID Nos. 28 and 29), enteropeptidase (SEQ ID Nos. 30 and 31 ) human airway trypsin-like protease (HAT) (SEQ ID Nos.
• hepsin SEQ ID Nos. 34 and 35
• TMPRSS2 SEQ ID Nos. 36 and 37
• TMPRSS4 SEQ ID Nos. 38 and 39.
• the MTSPs protease domains share a high degree of amino acid sequence identity.
• the His, Asp and Ser residues necessary for activity are present in conserved motifs. In those that are activated by cleavage, the activation site, which results in the N-terminus of second chain in the two chain forms has a conserved motif and readily can be identified (see, e.g.
• the C 573 (SEQ ID NO. 45 is a free Cys in a single chain form of the protease domain.
• the protease also can be provided as a two chain molecule. Single chain and two chain forms are proteolytically active. A two chain form is produced by bonding, typically between the C 573 and a Cys outside the protease domain, such as Cys 296 . Upon activation cleavage the disulfide bond remains resulting in a two chain polypeptide.
• the size of chain "A" is a function the starting length of the polypeptide prior to activation cleavage between the R 462 and l 463 .
• Two chain forms include at least the protease domain a polypeptide from C 296 up to and including C 573 .
• a two-chain form of the protease domain refers to a two- chain form that is formed from a single chain form of the protease in which the Cys pairing between, e.g., a Cys outside the protease domain such as, for example Cys 573 (SEQ ID No. 45 for MTSP), which links the protease domain to the remainder of the polypeptide, the "A" chain.
• a two chain protease domain form refers to any form in which the "remainder of the polypeptide" , i.e. , "A" chain, is shortened and includes a Cys from outside the protease domain.
• the catalytically active domain of an MTSP refers to the protease domain.
• Reference to the protease domain of an MTSP generally refers to a single chain form of the protein. If the two-chain form or both forms is intended, it is so-specified.
• the zymogen form of each protein is a single chain, which is converted to the active two or multi chain form by activation cleavage.
• active form is meant a form active in vivo or in vitro.
• activation cleavage refers to the cleavage of the protease at the N-terminus of the protease domain (generally between an R and l or V in the full-length protein.
• the resulting polypeptide has two chains ("A" chain and the "B" chain, which is the protease domain of an MTSP). Cleavage can be effected by another protease or autocatalytically.
• the conjugates provided herein advantageously contain sites that are recognized by the active cell surface protease (or cell-associated protease) and are cleaved thereby to release active or an inactive prodrug form of a therapeutic agent.
• an MTSP1 includes at least one or all of or any combination of: a polypeptide encoded by the sequence of nucleotides set forth in SEQ ID No. 1 or 40; a polypeptide encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides set forth in SEQ ID No. 1 or 40; a polypeptide that comprises the sequence of amino acids set forth in SEQ ID No.
• polypeptide that comprises a sequence of amino acids having at least about 40%, 60%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% sequence identity with the sequence of amino acids set forth in SEQ ID No. 2 or 41 ; and/or a polypeptide encoded by a splice variant of the MTSP1 set forth in SEQ ID No. 1 or 40.
• the MTSP1 can be from any animal, particularly a mammal, and includes but is not limited to, humans, rodents, fowl, ruminants and other animals.
• the full length zymogen or two chain activated form is contemplated or any domain thereof, including the protease domain, which can be a two chain activated form, or a single chain form.
• MTSP1 also is referred to TADG-1 5 and matriptase. As described below, the protein originally designated matriptase appears to be an MTSP1 splice variant or processed product.
• an MTSP3 includes at least one or all of or any combination of: a polypeptide encoded by the sequence of nucleotides set forth in SEQ ID No. 3; a polypeptide encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides set forth in SEQ ID No. 3; a polypeptide that comprises the sequence of amino acids set forth as amino acids 205-437 of SEQ ID No.
• polypeptide that comprises a sequence of amino acids having at " least about 40%, 60%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence of amino acids set forth in SEQ ID No. 4; and/or a polypeptide encoded by a splice variant of the MTSP3 set forth in SEQ ID Nos. 3 and 4.
• the MTSP3 can be from any animal, particularly a mammal, and includes but are not limited to, humans, rodents, fowl, ruminants and other animals.
• the full length zymogen or two chain activated form is contemplated or any domain thereof, including the protease domain, which can be a two chain activated form, or a single chain form.
• an MTSP4 includes at least one or all of or any combination of: a polypeptide encoded by the sequence of nucleotides set forth in any of SEQ ID No. 5, 7 or 9; a polypeptide encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides set forth in any of SEQ ID Nos. 5, 7 or 9; a polypeptide that comprises the sequence of amino acids set forth in any of SEQ ID Nos.
• polypeptide that comprises a sequence of amino acids having at least about 40%, 60%, 70%, 75 %, 80%, 81 %, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%o, 90%, 91 %, 92%, 93% , 94%, 95%, 96%, 97 %, 98% or 99% sequence identity with the sequence of amino acids set forth in SEQ ID No. 6, 8 or 10; and/or a polypeptide encoded by a splice variant of the MTSP4s set forth in SEQ ID Nos. 7-10.
• the MTSP4 can be from any animal, particularly a mammal, and includes but are not limited to, humans, rodents, fowl, ruminants and other animals.
• the full length zymogen or two chain activated form is contemplated or any domain thereof, including the protease domain, which can be a two chain activated form, or a single chain form.
• an MTSP6 includes at least one or all of or any combination of: a polypeptide encoded by the sequence of nucleotides set forth in any of SEQ ID No. 1 1 ; a polypeptide encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides set forth in any of SEQ ID Nos. 1 1 ; a polypeptide that comprises the sequence of amino acids set forth in any of SEQ ID No.
• polypeptide that comprises a sequence of amino acids having at least about 40%, 60%, 70%, 75 %, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% sequence identity with the sequence of amino acids set forth in SEQ ID No. 1 2; and/or a polypeptide encoded by a splice variant of the MTSP6 set forth in SEQ ID No. 1 2.
• the MTSP6 can be from any animal, particularly a mammal, and includes but are not limited to, humans, rodents, fowl, ruminants and other animals.
• the full length zymogen or two chain activated form is contemplated or any domain thereof, including the protease domain, which can be a two chain activated form, or a single chain form.
• an MTSP7 includes at least one or all of or any combination of: a polypeptide encoded by the sequence of nucleotides set forth in SEQ ID No. 1 3; a polypeptide encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides set forth in SEQ ID No. 1 3; a polypeptide that comprises the sequence of amino acids set forth in SEQ ID No.
• the MTSP7 can be from any animal, particularly a mammal, and includes but are not limited to, humans, rodents, fowl, ruminants and other animals.
• an MTSP9 includes at least one or all of or any combination of: a polypeptide encoded by the sequence of nucleotides set forth in SEQ ID No. 1 7 or SEQ ID No. 42; a polypeptide encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides set forth in SEQ ID No. 1 7 or 42; a polypeptide that comprises the sequence of amino acids set forth in SEQ ID No.
• polypeptide that comprises a sequence of amino acids having at least about 40%, 60%, 70%, 75 %, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% sequence identity with the sequence of amino acids set forth in SEQ ID No. 1 8 or 270; and/or a polypeptide encoded by a splice variant of the MTSP9 set forth in SEQ ID No. 1 7.
• the MTSP9 can be from any animal, particularly a mammal, and includes but are not limited to, humans, rodents, fowl, ruminants and other animals.
• the full length zymogen or two chain activated form is contemplated or any domain thereof, including the protease domain, which can be a two chain activated form, or a single chain form.
• an MTSP1 0, whenever referenced herein, includes at least one or all of or any combination of: a polypeptide encoded by the sequence of nucleotides set forth in SEQ ID No.
• the MTSP10 can be from any animal, particularly a mammal, and includes but are not limited to, humans, rodents, fowl, ruminants and other animals.
• the full length zymogen or two chain activated form is contemplated or any domain thereof, including the protease domain, which can be a two chain activated form, or a single chain form.
• MTSP1 0 polypeptides including, but not limited to splice variants thereof, and nucleic acids encoding MTSPs, and domains, derivatives and analogs thereof are provided herein.
• Single chain protease domains that have an
• N-terminus functionally equivalent to that generated by activation of the zymogen form of MTSP10 are also provided.
• the cleavage site for the protease domain of MTSP10 is between amino acid R and amino acids I (R 1 IIGGT)
• an MTSP1 2 includes at least one or all of or any combination of: SEQ ID No. 1 9 and 20 a polypeptide encoded by the sequence of nucleotides set forth in SEQ ID No. 1 9 or by a sequence of nucleotides that includes nucleotides that encode the sequence of amino acids set forth in SEQ ID No. 20; a polypeptide encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides set forth in is set forth as SEQ ID No. 19; a polypeptide that includes the sequence of amino acids set forth in SEQ ID No. 20 or a catalytically active portion thereof; a polypeptide that includes a sequence of amino acids having at least about 40%, 60%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85 %, 86%,
• the MTSP1 2 polypeptide with the protease domains as indicated in SEQ ID Nos. 1 9 and 20, is provided.
• the polypeptide is a single or multi-chain polypeptide.
• a protease domain of an MTSP1 2, whenever referenced herein, includes at least one or all of or any combination of or a catalytically active portion of: a polypeptide that includes the sequence of amino acids set forth in SEQ ID No. 20 or a catalytically active portion thereof but that does not include the sequence of amino acids set forth in SEQ ID No. 271 ; a polypeptide that includes the sequence of amino acids set forth in SEQ ID No.
• polypeptide that includes a sequence of amino acids having at least about 60%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence of amino acids set forth in SEQ ID No.
• polypeptide that includes a sequence of amino acids having at least about 60%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence of amino acids of the polypeptides of a)-e); a polypeptide encoded by a splice variant of a sequence of nucleotides that encodes an MTSP1 2 of any of the above.
• the MTSP1 2 can be from any animal, particularly a mammal, and includes but are not limited to, humans, rodents, fowl, ruminants and other animals.
• the full-length zymogen or two-chain activated form is contemplated or any domain thereof, including the protease domain, which can be a two-chain activated form, or a single chain form.
• MTSP1 2 also includes the variant described International PCT application No. WO 02/00860 (see SEQ ID Nos. 38 and 97 therein).
• an MTSP20 includes at least one or all of or any combination of: a polypeptide encoded by the sequence of nucleotides set forth in SEQ ID No. 273; a polypeptide encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides set forth in SEQ ID No. 273; a polypeptide that comprises the sequence of amino acids set forth in SEQ ID No.
• a polypetide that comprises a sequence of amino acids having at least about 40%, 60%, 70%, 75 %, 80%, 81 %, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% sequence identity with the sequence of amino acids set forth in SEQ ID No. 274; and/or a polypeptide encoded by a splice variant of the MTSP20 encoded by the sequence of nucleotides that includes the sequence set forth in SEQ ID No. 273.
• the MTSP20 may be from any animal, particularly a mammal, and includes but are not limited to, humans, rodents, fowl, ruminants and other animals.
• the full length zymogen or two-chain activated form is contemplated or any domain thereof, including the protease domain, which can be a two-chain activated form, or a single chain form.
• an MTSP22 whenever referenced herein, includes at least one or all of or any combination of: a polypeptide encoded by the sequence of nucleotides set forth in SEQ ID No.
• the MTSP22 may be from any animal, particularly a mammal, and includes but are not limited to, humans, rodents, fowl, ruminants and other animals.
• the full length zymogen or two-chain activated form is contemplated or any domain thereof, including the protease domain, which can be a two-chain activated form, or a single chain form.
• an MTSP25 includes at least one or all of or any combination of: a polypeptide encoded by the sequence of nucleotides set forth in SEQ ID No. 269; a polypeptide encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides set forth in SEQ ID No. 269; a polypeptide that comprises the sequence of amino acids set forth in SEQ ID No.
• a polypetide that comprises a sequence of amino acids having at least about 40%, 60%, 70%, 75 %, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 % , 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% sequence identity with the sequence of amino acids set forth in SEQ ID No. 270; and/or a polypeptide encoded by a splice variant of the MTSP25 set forth in SEQ ID No. 269.
• the MTSP25 may be from any animal, particularly a mammal, and includes but are not limited to, humans, rodents, fowl, ruminants and other animals.
• the full length zymogen or two-chain activated form is contemplated or any domain thereof, including the protease domain, which can be a two-chain activated form, or a single chain form.
• a human protein is one encoded by nucleic acid present in the genome of a human, including all allelic variants and conservative variations as long as they are not variants found in other mammals.
• not substantially cleaved by plasmin or prostate specific antigen (PSA) means in comparable in vitro assays (under optimal conditions for each enzyme) in which the activity of a targeted cell surface membrane protease or catalytically active portion of the activity of the protease domain (or a catalytically active form thereof) of prostate specific antigen (PSA) or plasmin for cleavage of the conjugate is compared, the relative activity is greater than at least 2:1 , 3: 1 , 4: 1 , 5: 1 , 10: 1 , 50: 1 or 100: 1 .
• activity refers to the ratio k cat /K m , where k oat is the rate of catalytic turnover for a particular enzyme, and K m is the Michaelis constant for the binding of the substrate.
• a "nucleic acid encoding a protease domain or catalytically active portion of a MTSP" shall be construed as referring to a nucleic acid encoding only the recited single chain protease domain or active portion thereof, and not the other contiguous portions of the MTSP as a continuous sequence.
• a CUB domain is a motif that mediates protein-protein interactions in complement components C1 r/C1 s and has also been identified in various proteins involved in developmental processes.
• a zymogen is an enzymatically inactive protein (i.e, typically, but not necessarily, less than 1 % of active form) that is converted to a proteolytic enzyme by the action of an activator, including by autoactivation.
• Inactive means less active than the form those of skill in the art consider to be the active form of the enzyme.
• the ratio of activity of a zymogen to the activated form varies from enzyme-to-enzyme.
• disease or disorder refers to a pathological condition in an organism resulting from, e.g. , infection or genetic defect, and characterized by identifiable symptoms.
• the diseases contemplated for treatment herein are any for which a cell surface protease, including a cell-localized or cell-associated protease is asssociated with a targeted cell or tissue involved in the disease or disease process. Such association can be because the protease is involved in the disease or is serendipitously associated with cells involved with the disease. These diseases herein are called cell surface protease-associated diseases.
• a cellsurface protease is identified that is expressed on cells associated with the disorder, such as, for example, immune cells for treating inflammatory diseases, and virally infected cells for treating viral diseases.
• the conjugate is designed as described herein for cleavage by the selected protease.
• neoplasm refers to abnormal new growth, and thus means the same as tumor, which can be benign or malignant. Unlike hyperplasia, neoplastic proliferation persists even in the absence of the original stimulus.
• neoplastic disease refers to any disorder involving cancer, including tumor development, growth, metastasis and progression.
• cancer refers to a general term for diseases caused by any type of malignant tumor.
• malignant as applied to tumors, refers to primary tumors that have the capacity of metastasis with loss of growth control and positional control.
• endotheliase refers to a mammalian protein, including human protein, that has a transmembrane domain and is expressed or active on the surface of endothelial cells and includes a protease domain, particularly an extracellular protease domain, and is generally a serine protease (see, also U.S. application Serial No. 09/71 7,473 and International PCT application No. WO 01 /36604).
• endotheliase encompasses all proteins encoded by the endotheliase gene family, or an equivalent molecule obtained from any other source or that has been prepared synthetically or that exhibits the same activity.
• the endotheliase gene family are transmembrane proteases expressed or active in endothelial cells. These proteases include serine proteases. These include proteins that have these features and also include a protease domain that exhibits sequence homology to the endotheliases 1 and 2. Endotheliase 1 and 2, for example exhibit about 40% or 45% identity.
• Sequence homology means sequence identity along its length when aligned to maximize identity of at least about 25 %, 40%, 60%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97 %, 98 % or 99% or greater number of residues. Sequence homology also is assessed by determining whether the encoding sequences of nucleic acids hybridize under conditions of at least moderate, or for more closely related proteins, high stringency to the nucleic acid molecules provided herein or to those that encode the same proteins but differ in sequence by virtue of the degeneracy of the genetic code.
• endotheliases encompasses endotheliases with amino acid substitutions, including those set forth in Table 1 , such that the resulting polypeptide retains at least 1 %, 5 %, 10%, 20%, 30%, 40%, 50%, 60%>, 70%, 80%, 90% of the proteolytic activity of the unaltered polypeptide.
• Suitable substitutions of amino acids are known to those of skill in this art and can be made generally without altering the biological activity of the resulting molecule.
• those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g. , Watson et al.
• endotheliases Also included within the definition of "endotheliases", is the catalytically active fragment or shed forms of an endotheliase.
• an endotheliase 1 includes at least one or all of or any combination of: a polypeptide encoded by the sequence of nucleotides set forth in
• SEQ ID No. 21 a polypeptide encoded by a sequence of nucleotides that hybridizes under conditions of low, moderate or high stringency to the sequence of nucleotides set forth in SEQ ID No. 21 ; a polypeptide that comprises the sequence of amino acids set forth in SEQ ID No.
• polypeptide that comprises a sequence of amino acids having at least about 40%, 60%, 70%, 75 %, 80%, 81 %, 82%, 83%, 84%, 85 %, 86%, 87%, 88 %, 89%, 90%, 91 %, 92%, 93%, 94%, 95 %, 96% , 97%, 98 % or 99% sequence identity with the sequence of amino acids set forth in SEQ ID No. 22; 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.
• the endotheliase 1 can be from any animal, particularly a mammal, and includes but are not limited to, humans, rodents, fowl, ruminants and other animals.
• the full length zymogen or two chain activated form is contemplated or any domain thereof, including the protease domain, which can be a two chain activated form, or a single chain form.
• an endotheliase 2 whenever referenced herein, includes at least one or all of or any combination of: a polypeptide encoded by the sequence of nucleotides set forth in SEQ ID No.
• the endotheliase 2 can be from any animal, particularly a mammal, and includes but are not limited to, humans, rodents, fowl, ruminants and other animals.
• the full length zymogen or two chain activated form is contemplated or any domain thereof, including the protease domain, which can be a two chain activated form, or a single chain form.
• the protease domain of an endotheliase refers to the polypeptide portion of the endotheliase that is the extracellular portion that exhibits protease activity.
• the protease domain is a polypeptide that includes at least the minimum number of amino acids, generally more than 50 or 100, required for protease activity.
• Protease activity can be assessed empirically, such as by testing the polypeptide for its ability to act as a protease. Assays, such as those described in the EXAMPLES, with the exception that a known endotheliase substrate is employed in place of the test compounds, can be used to assess protease activity.
• proteases particularly serine proteases, have characteristic structures and sequences or motifs, the protease domain can be readily identified by such structure and sequence or motif.
• a portion of protease domain of endotheliase refers to a portion of protease domain of endotheliase that is located within or is the extracellular domain of an endotheliase and exhibits serine proteolytic activity. Hence, it is at least the minimal portion of the extracellular domain that exhibits proteolytic activity as assessed by standard assays.
• An exemplary protease domain of an endotheliase is set forth in SEQ ID No. 22 and as amino acids 321 - 688 and 321 -562 of SEQ ID Nos. 24 and 26, respectively. Smaller portions thereof that retain protease activity are contemplated.
• protease domains vary in size and constitution, including insertions and deletions in surface loops. Such domains exhibit conserved structure, including at least one structural feature, such as the active site triad, primary specificity pocket, oxyanion hole and/or other features of serine protease domains of proteases.
• the protease domain is a portion of an endotheliase, as defined herein, but is homologous in terms of structural features and retention of sequence of similarity or homology the protease domain of chymotrypsin or trypsin. As used herein, homologous means about greater than about 25%, 40%,
• sequence identity the number of conserved amino acids as determined by standard alignment algorithms programs, and used with default gap penalties established by each supplier. Also homology can be assessed by conserved nucleic acid sequence, which includes anything that hybridizes under at least low stringency conditions and encodes the domain. Similarly, nucleic acid sequence alignment programs are commercially available (DNAStar "MegAlign” program (Madison, WI) and the University of Wisconsin Genetics Computer Group (UWG) "Gap” program (Madison, WI)). Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.
• a polypeptide consists essentially of the protease domain means that the only endotheliase portion of the polypeptide is a protease domain or a catalytically active portion thereof.
• the polypeptide can optionally include additional non-endotheliase-derived sequences of amino acids.
• domain refers to a portion of a molecule, e.g. , proteins or nucleic acids, that is structurally and/or functionally distinct from other portions of the molecule.
• an active form of a protease refers to an enzyme that catalyzes hydrolysis of proteins or peptides. Reference to a protease includes the active and zymogen or other less active form.
• nucleic acids include DNA, RNA and analogs thereof, including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single or two stranded. When referring to probes or primers, optionally labeled, with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that their targets are statistically unique or of low copy number (typically less than 5, generally less than 3) for probing or priming a library. Generally a probe or primer contains at least 14, 1 6 or 30 contiguous of sequence complementary to or identical to a gene of interest. Probes and primers can be 1 0, 20, 30, 50, 1 00 or more nucleic acids long.
• nucleic acid encoding a fragment or portion of an endotheliase refers to a nucleic acid encoding only the recited fragment or portion of endotheliase protein, and not the other contiguous portions of the endotheliase as a continuous sequence.
• heterologous nucleic acid is nucleic acid that, if it is DNA encodes RNA, or, if RNA, encodes proteins that generally are not normally produced in vivo by the cell in which it is expressed or that mediates or encodes mediators that alter expression of endogenous nucleic acid, such as DNA, by affecting transcription, translation, or other regulatable biochemical processes or that is located in a different locus from its normal locus.
• Heterologous nucleic acid is generally not endogenous to the cell into which it is introduced, but has been obtained from another cell or prepared synthetically. Generally, although not necessarily, such nucleic acid encodes RNA and proteins that are not normally produced by the cell in which it is now expressed.
• Heterologous nucleic acid such as DNA
• foreign nucleic acid such as DNA
• Any nucleic acid, such as DNA, that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which is expressed is herein encompassed by heterologous nucleic acid; heterologous nucleic acid includes exogenously added nucleic acid that is also expressed endogenously.
• heterologous nucleic acid examples include, but are not limited to, nucleic acid that encodes traceable marker proteins, such as a protein that confers drug resistance, nucleic acid that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and nucleic acid, such as DNA, that encodes other types of proteins, such as antibodies, and RNA, such as RNA interference (RNAi) or other double-stranded RNA, and antisense RNA.
• RNAi RNA interference
• nucleic acid can be secreted or expressed on the surface of the cell in which the heterologous nucleic acid has been introduced.
• nucleic acid can be the the targeted agent, such as the therapeutic or diagnostic agent, in the conjugate.
• Nucleic acids include ds RNA use for RNA interference (RNAi) (see, e.g. Chuang et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 57:4985) which is employed to inhibit the expression of a targeted gene by generating loss-of -function.
• RNAi RNA interference
• Methods relating to the use of RNAi to silence genes in organisms including, mammals, C. elegans, Drosophila and plants, and humans are known (see, e.g., Fire et al. (1 998) Nature 357:806- 81 1 Fire (1 999) Trends Genet. 75:358-363; Sharp (2001 ) Genes Dev.
• RNAi RNA RNA cleavage site
• genetic therapy involves the transfer of heterologous nucleic acid, such as DNA, into certain cells, target cells, of a mammal, particularly a human, with a disorder or conditions for which such therapy is sought.
• the nucleic acid molecules are included in a conjugate linked via a cell surface protein cleavage site.
• the nucleic acid such as DNA
• the nucleic acid is introduced into the selected target cells in a manner such that the heterologous nucleic acid, such as DNA, is expressed and a therapeutic product encoded thereby is produced.
• the heterologous nucleic acid, such as DNA can in some manner mediate expression of DNA that encodes the therapeutic product, or it can encode a product, such as a peptide or RNA that in some manner mediates, directly or indirectly, expression of a therapeutic product.
• Genetic therapy can also be used to deliver nucleic acid encoding a gene product that replaces a defective gene or supplements a gene product produced by the mammal or the cell in which it is introduced.
• the introduced nucleic acid can encode a therapeutic compound, such as a growth factor inhibitor thereof, or a tumor necrosis factor or inhibitor thereof, such as a receptor therefor, that is not normally produced in the mammalian host or that is not produced in therapeutically effective amounts or at a therapeutically useful time.
• a therapeutic compound such as a growth factor inhibitor thereof, or a tumor necrosis factor or inhibitor thereof, such as a receptor therefor, that is not normally produced in the mammalian host or that is not produced in therapeutically effective amounts or at a therapeutically useful time.
• the heterologous nucleic acid, such as DNA, encoding the therapeutic product can be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof.
• Genetic therapy can also involve delivery of an inhibitor or repressor or other modulator of gene expression, such dsRNA or antisense or other nucleic acid molecule.
• the conjugates herein can be used to deliver a product, such as a nucleic
• a therapeutically effective product for gene therapy is a product that is encoded by heterologous nucleic acid, typically DNA, that, upon introduction of the nucleic acid into a host, a product is expressed that ameliorates or eliminates the symptoms, manifestations of an inherited or acquired disease or that cures the disease.
• heterologous nucleic acid typically DNA
• biologically active nucleic acid molecules such as RNAi and antisense.
• a sequence complementary to at least a portion of an RNA means a sequence having sufficient complementarily to be able to hybridize with the RNA, generally under moderate or high stringency conditions, forming a stable duplex; in the case of double-stranded SP antisense nucleic acids, a single strand of the duplex DNA (or dsRNA) can thus be tested, or triplex formation can be assayed.
• the ability to hybridize depends on the degree of complementarily and the length of the antisense nucleic acid.
• the longer the hybridizing nucleic acid the more base mismatches with a SP encoding RNA it can contain and still form a stable duplex (or triplex, as the case can be).
• One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
• Amino acid substitutions can be made or occur in any SPs and protease domains thereof.
• Amino acid substitutions include conservative substitutions, such as those set forth in Table 1 , which do not eliminate proteolytic activity.
• substitutions that alter properties of the proteins, such as removal of cleavage sites and other such sites are also contemplated; such substitutions are generally non-conservative, but can be readily effected by those of skill in the art.
• Suitable conservative substitutions of amino acids are known to those of skill in this art and can be made generally without altering the biological activity, for example enzymatic activity, of the resulting molecule.
• the catalytically active fragment of an SP particularly a single chain protease portion.
• Conservative amino acid substitutions are made, for example, in accordance with those set forth in TABLE 1 as follows: TABLE 1
• substitutions are also permissible and can be determined empirically or in accord with known conservative substitutions.
• one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
• Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs.
• the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
• the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
• the positively charged (basic) amino acids include arginine, lysine and histidine.
• the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
• amino acids which occur in the various amino acid sequences appearing herein, are identified according to their well-known, three- letter or one-letter abbreviations.
• the nucleotides, which occur in the various DNA fragments, are designated with the standard single-letter designations used routinely in the art.
• hR or hArg for homoarginine
• hY or hTyr for homotyrosine
• Cha for cyclohexylalanine
• Amf for 4-aminomethylphenylalanine
• DPL for 2-(4,6-dimethylpyrimidinyl)lysine
• (imidazolyl)K for N'-(2-imidazolyl)lysine
• Me2P03-Y for O-dimethylphosphotyrosine
• O-Me-Y for O-methyltyrosine
• TIC for tetrahydro-3-isoquinoline carboxylic acid
• MeL for 2-keto-3-amino-5-methyl- hexane
• DAP for 1 ,3-diaminopropane
• TFA trifluoroacetic acid
• AA for acetic acid.
• 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.
• a probe or primer based on a nucleotide sequence disclosed herein includes at least 10, 14, generally at least 1 6 or 30 or 100 contiguous sequence of nucleotides.
• antisense polynucleotides refer to synthetic sequences of nucleotide bases complementary to mRNA or the sense strand of double- stranded DNA. Admixture of sense and antisense polynucleotides under appropriate conditions leads to the binding of the two molecules, or hybridization. When these polynucleotides bind to (hybridize with) mRNA, inhibition of protein synthesis (translation) occurs.
• Antisense nucleic acid molecules typically contain a sufficient number of nucleotides to specifically bind to a target nucleic acid, generally at least 5 contiguous nucleotides, often at least 14 or 1 6 or 30 contiguous nucleotides or modified nucleotides complementary to the coding portion of a nucleic acid molecule that encodes a gene of interest, for example, nucleic acid encoding a single chain protease domain of an SP.
• an array refers to a collection of elements, such as antibodies, containing three or more members.
• An addressable array is one in which the members of the array are identifiable, typically by position on a solid phase support. Hence, in general the members of the array are immobilized on discrete identifiable loci on the surface of a solid phase.
• antibody refers to an immunoglobulin, whether natural or partially or wholly synthetically produced, including any derivative thereof that retains the specific binding ability of the antibody.
• antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain.
• Antibodies include members of any immunoglobulin claims, including IgG, IgM, IgA, IgD and IgE.
• antibody fragment refers to any derivative of an antibody that is less than full-length, retaining at least a portion of the full-length antibody's specific binding ability.
• antibody fragments include, but are not limited to, Fab, Fab', F(ab) 2 , single-chain Fvs (scFV), FV, dsFV diabody and Fd fragments.
• the fragment can include multiple chains linked together, such as by disulfide bridges.
• An antibody fragment generally contains at least about 50 amino acids and typically at least 200 amino acids.
• an Fv antibody fragment is composed of one variable heavy domain (V H ) and one variable light domain linked by noncovalent interactions.
• a dsFV refers to an Fv with an engineered intermolecular disulfide bond, which stabilizes the V H -V L pair.
• an F(ab) 2 fragment is an antibody fragment that results from digestion of an immunoglobulin with pepsin at pH 4.0-4.5; it can be recombinantly expressed to produce the equivalent fragment.
• Fab fragments are antibody fragments that result from digestion of an immunoglobulin with papain; they can be recombinantly expressed to produce the equivalent fragment.
• scFVs refer to antibody fragments that contain a variable light chain (V L ) and variable heavy chain (V H ) covalently connected by a polypeptide linker in any order.
• the linker is of a length such that the two variable domains are bridged without substantial interference.
• Exemplariy linkers include, but are not limited to, (Gly-Ser) n residues, which can include ome Glu or Lys residues dispersed throughout, for example, to increase solubility.
• humanized antibodies refer to antibodies that are modified to include human sequences of amino acids so that administration to a human does not provoke an immune response. Methods for preparation of such antibodies are known.
• the encoding nucleic acid in the hybridoma or other prokaryotic or eukaryotic cell, such as an E. coli or a CHO cell, that expresses the monoclonal antibody is altered by recombinant nucleic acid techniques to express an antibody in which the amino acid composition of the non-variable region is based on human antibodies.
• Computer programs have been designed to identify such non- variable regions.
• diabodies are dimeric scFV; diabodies typically have shorter peptide linkers than scFvs, and they generally dimerize.
• production by recombinant means by using recombinant DNA methods means the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA.
• the term assessing is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the activity of an SP, or a domain thereof, present in the sample, and also of obtaining an index, ratio, percentage, visual or other value indicative of the level of the activity. Assessment can be direct or indirect and the chemical species actually detected need not of course be the proteolysis product itself but can for example be a derivative thereof or some further substance.
• biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture.
• Biological activity thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures.
• Biological activities can be observed in in vitro systems designed to test or use such activities.
• fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
• an effective amount of a compound for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount can be administered as a single dosage or can be administered according to a regimen, whereby it is effective. The amount can cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Repeated administration can be required to achieve the desired amelioration of symptoms.
• equivalent when referring to two sequences of nucleic acids, means that the two sequences in question encode the same sequence of amino acids or equivalent proteins. When equivalent is used in referring to two proteins or peptides, it means that the two proteins or peptides have substantially the same amino acid sequence with amino acid substitutions (see, e.g.
• Complementary when referring to two nucleotide sequences, means that the two sequences of nucleotides are capable of hybridizing, typically with less than 25 %, often with less than 1 5%, or even less than 5% or with no mismatches between opposed nucleotides. Generally the two molecules hybridize under conditions of high stringency.
• a method for treating or preventing disease or disorder associated with undesired and/or uncontrolled angiogenesis means that the diseases or the symptoms associated with the undesired and/or uncontrolled angiogenesis are alleviated, reduced, ameliorated, prevented, placed in a state of remission, or maintained in a state of remission. It also means that the hallmarks of pathological angiogenesis are eliminated, reduced or prevented by the treatment.
• Non-limiting examples of the hallmarks of the pathological angiogenesis include uncontrolled degradation of the basement membrane and proximal extracellular matrix of the endothelial cells, migration, division, and organization of the endothelial cells into new functioning capillaries, and the persistence of such functioning capillaries.
• operatively linked or operationally associated refers to the functional relationship of DNA with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences.
• operative linkage of DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
• start codons or other sequences that can interfere with or reduce expression either at the level of transcription or translation.
• consensus ribosome binding sites see, e.g. , Kozak (1 991 ) J. Biol. Chem. 255: 1 9867-1 9870
• the desirability of (or need for) such modification can be empirically determined.
• 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 operatively linked.
• the promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter.
• the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences can be cis acting or can be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, can be constitutive or regulated. Exemplary promoters contemplated for use in prokaryotes include the bacteriophage T7 and T3 promoters.
• sample refers to anything which can contain an analyte for which an analyte assay is desired.
• the sample can be a biological sample, such as a biological fluid or a biological tissue.
• biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like.
• Biological tissues are aggregates of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s).
• hybridize under conditions of a specified stringency is used to describe the stability of hybrids formed between two single-stranded DNA fragments and refers to the conditions of ionic strength and temperature at which such hybrids are washed, following annealing under conditions of stringency less than or equal to that of the washing step.
• high, medium and low stringency encompass the following conditions or equivalent conditions thereto:
• medium stringency 0.2 x SSPE or SSC, 0.1 % SDS, 50 °C
• low stringency 1 .0 x SSPE or SSC, 0.1 % SDS, 50°C.
• Equivalent conditions refer to conditions that select for substantially the same percentage of mismatch in the resulting hybrids. Additions of ingredients, such as formamide, Ficoll, and Denhardt's solution affect parameters such as the temperature under which the hybridization should be conducted and the rate of the reaction. Thus, hybridization in 5 X SSC, in 20% formamide at 42° C is substantially the same as the conditions recited above hybridization under conditions of low stringency.
• the recipes for SSPE, SSC and Denhardt's and the preparation of deionized formamide are described, for example, in Sambrook et al. (1 989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapter 8; see, Sambrook et a/. , vol. 3, p. B.1 3, see, also, numerous catalogs that describe commonly used laboratory solutions). It is understood that equivalent stringencies can be achieved using alternative buffers, salts and temperatures.
• substantially identical or similar varies with the context as understood by those skilled in the relevant art and generally means at least 40, 60, 80, 90, 95 or 98% .
• substantially identical to a product means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.
• target cell refers to a cell that expresses a cell surface protease.
• test substance refers to a chemically defined compound (e.g. , organic molecules, inorganic molecules, organic/inorganic molecules, proteins, peptides, nucleic acids, oligonucleotides, lipids, polysaccharides, saccharides, or hybrids among these molecules such as glycoproteins, etc.) or mixtures of compounds (e.g., a library of test compounds, natural extracts or culture supernatants, etc.) whose effect on or interaction with a cell surface protein or cell surface-associated protein, or a domain thereof, is determined by the methods herein.
• a chemically defined compound e.g. , organic molecules, inorganic molecules, organic/inorganic molecules, proteins, peptides, nucleic acids, oligonucleotides, lipids, polysaccharides, saccharides, or hybrids among these molecules such as glycoproteins, etc.
• mixtures of compounds e.g., a library of test compounds, natural extracts or culture supernatants
• a therapeutic agent As used herein, the terms a therapeutic agent, therapeutic regimen, radioprotectant, chemotherapeutic mean conventional drugs and drug therapies, including vaccines, which are known to those skilled in the art. Radiotherapeutic agents are well known in the art.
• vector refers to discrete elements that are used to introduce heterologous DNA into cells for expression and/or replication thereof.
• the vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome.
• vectors that are artificial chromosomes such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.
• An expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments.
• an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA.
• Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
• chemically stable means that the compound is stable enough to be formulated for pharmaceutical use. Such chemical stability is well known to those of skill in the art and can be determined by well known routine methods. Whether a given compound is chemically stable enough to be formulated for pharmaceutical use depends on a number of factors including, but not limited to, the type of formulation and route of administration desired, the disease to be treated, and the method of preparing the pharmaceutical formulation.
• a "functional equivalent" of a side chain of an amino acid is a group or moiety that functions in substantially the same way as the naturally occurring side chain to achieve substantially the same result (e.g. , a substrate for a cell surface protease).
• side chain of arginine include, but are not limited to, homoarginine, guanidinoaminopropyl, guanidinoaminoethyl, (Me) 2 arginine side chain,
• x is 0 or 1 (see, e.g., Webb et al. ( 1 991 ) J. Org. Chem. 56:3009), or a conformationally constrained arginine side chain analog such as:
• d is an integer from 0 to 5, or 1 to 3; and W is N or CH; or a mono- or di- substituted N-alkyl derivative of the above groups, where alkyl is, in certain embodiments, lower alkyl, such as methyl.
• pharmaceutically acceptable derivatives of a compound include salts, esters, enol ethers, enol esters, acids, bases, solvates, hydrates or prodrugs thereof.
• Such derivatives can be readily prepared by those of skill in this art using known methods for such derivatization.
• the compounds produced can be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs.
• Pharmaceutically acceptable salts include, but are not limited to, amine salts, such as but not limited to N,N'- dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N- benzylphenethylamine, 1 -para-chlorobenzyl-2-pyrrolidin- 1 '-ylmethyl- benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxy- methyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as
• esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and boronic acids.
• Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecule, generally 1 to about 100, typically 1 to about 10, such as 1 to about 2, 3 or 4, solvent or water molecules.
• treatment means any manner in which one or more of the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein, such as use for treating cancer.
• a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound.
• the pharmaceutically active compound is modified such that the active compound is regenerated by metabolic processes.
• the prodrug can be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug.
• the conjugates provided herein can contain chiral centers. Such chiral centers can be of either the (R) or (S) configuration, or can be a mixture thereof.
• the compounds provided herein can be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures.
• amino acid residues such residues can be of either the L- or D-form.
• the configuration for naturally occurring amino acid residues is generally L. When not specified the residue is the L form.
• 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, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form.
• the conjugates provided herein are prodrugs because they include a therapeutic agent in an inactive form that is ultimately converted to an active form at the targeted cell or tissue or in the environment thereof.
• a biologically, pharmaceutically or therapeutically active form of a compound is released, or, a derivative that can be further metabolized into a biologically, pharmaceutically or therapeutically active form of a compound.
• substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry (MS), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not alter the physical and chemical properties, such as enzymatic and biological activities, of the substance for its intended purpose.
• TLC thin layer chromatography
• HPLC high performance liquid chromatography
• MS mass spectrometry
• a peptidic substrate includes peptides and molecules, such as peptide mimetics and peptides that include peptide bond surrogates.
• conventional terminology Schotter et al. (1 967) Biochem. Biophys. Res. Commun. 27: 1 57-1 62
• Pn...P3-P2-P1 i P1 '-P2'-P3'...Pn' is used to refer to specific subsites of a protease substrate: Pn...P3-P2-P1 i P1 '-P2'-P3'...Pn'.
• the scissile bond i.e., the cleavage site
• Positions N-terminal of that bond are referred to as unprimed positions.
• Subsites are then assigned a number based on their distance from the scissile bond.
• Amino acids (or amino acid surrogates) that form the scissile bond are assigned the number 1 , adjacent residues the number 2, and so on, counting away from the scissile bond.
• Each specific subsite of the substrate therefore, is uniquely identified by a number and the designation as primed or unprimed.
• a surrogate of a peptide bond is a divalent group that possesses similar steric and/or electronic characteristics to -C(0)NH-.
• alkyl, alkenyl and alkynyl carbon chains contain from 1 to 20 carbons, generally 1 to 1 6 carbons, and are straight or branched.
• Alkenyl carbon chains of from 2 to 20 carbons typically contain 1 to 8 double bonds, and the alkenyl carbon chains of 2 to 1 6 carbons and typically contain 1 to 5 double bonds.
• Alkynyl carbon chains of from 2 to 20 carbons typically contain 1 to 8 triple bonds, and the alkynyl carbon chains of 2 to 1 6 carbons and generally contain 1 to 5 triple bonds.
• alkyl, alkenyl and alkynyl groups herein include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl and isohexyl.
• the alkyl, alkenyl and alkynyl groups optionally can be substituted, with one or more groups, generally alkyl group substituents that are the same or different.
• lower alkyl, lower alkenyl, and lower alkynyl refer to carbon chains having less than about 6 carbons.
• alk(en)(yn)yl refers to an alkyl group containing at least one double bond and at least one triple bond.
• cycloalkyl refers to a saturated mono- or multicyclic ring system, typically 3 to 1 0 carbon atoms, such as, for example, 3 to 6 carbon atoms; cycloalkenyl and cycloalkynyl refer to mono- or multicyclic ring systems that respectively include at least one double bond and at least one triple bond. Cycloalkenyl and cycloalkynyl groups contain, for example, 3 to 10 carbon atoms, with cycloalkenyl groups generally containing 4 to 7 carbon atoms and cycloalkynyl groups that contain, for example 8 to 10 carbon atoms.
• ring systems of the cycloalkyl, cycloalkenyl and cycloalkynyl groups can be composed of one ring or two or more rings which can be joined together in a fused, bridged or spiro-connected fashion, and optionally can be substituted with one or more alkyl group substituents.
• Cycloalk(en)(yn)yl refers to a cycloalkyl group containing at least one double bond and at least one triple bond.
• substituted alkyl refers to alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl groups, respectively, that are substituted with one or more substituents, in certain embodiments one to three substituents, independently selected from alkyl, halo, haloalkyl, such as halo lower alkyl, pseudohalo, aryl, amino, dialkylamino, nitro, cyano, azido, alkylsulfinyl, alkylsulfonyl, alkylcarbonylamino, alkoxycarbonylamino, aminoimino, hydroxy, alkoxy, ary
• aryl refers to cyclic groups 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.
• aryl also refers to aryl-containing groups, including, but not limited to, aryloxy, arylthio, arylcarbonyl and arylamino groups.
• heteroaryl refers to a monocyclic or multicyclic aromatic ring system, generally about 5 to about 1 5 members where one or more, such as 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms.
• the heteroaryl group optionally can be fused to a benzene ring.
• heteroaryl groups include, for example, furyl, imidazolyl, pyrrolidinyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl and isoquinolinyl, with pyridyl, thienyl and quinolinyl as examples thereof.
• heteroaryl also refers to heteroaryl-containing groups, including, but not limited to, heteroaryloxy, heteroarylthio, heteroarylcarbonyl and heteroarylamino.
• heterocyclyl refers to a monocyclic or multicyclic non- aromatic ring system, such as systems of 3 to 10 members, for exmaple 4 to 7 members or 5 to 6 members, where one or more, such as 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and/or sulfur atoms.
• substituted aryl refers to aryl, heteroaryl and heterocyclyl groups, respectively, that are substituted with one or more substituents, in certain embodiments one to three substituents, independently selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl optionally substituted with 1 or more, such as 1 to 3, substituents selected from halo, halo alkyl and alkyl, aralkyl, heteroaralkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, alk(en)(yn)yl groups, halo, pseudohalo, cyano, hydroxy, haloalkyl and polyhaloalkyl, such as halo lower alkyl, especially trifluoromethyl, formyi, alkylcarbonyl, arylcarbony
• aralkyl refers to an alkyl group in which one of the hydrogen atoms of the alkyl is replaced by an aryl group.
• heteroarylkyl refers to an alkyl group in which one of the hydrogen atoms of the alkyl is replaced by a heteroaryl group.
• alkyl refers to saturated carbon chains that contain one or more carbons; the chains can be straight or branched or include cyclic portions or be cyclic.
• haloalkyl can include one or more of the same or different halogens.
• C ⁇ alkoxyphenyl can include one or more of the same or different alkoxy groups containing one, two or three carbons.
• halo refers to F, CI, Br or I.
• pseudohalides are compounds that behave substantially similar to halides. Such compounds can be used in the same manner and treated in the same manner as halides (X " , in which X is a halogen, such as CI or Br).
• Pseudohalides include, but are not limited to, cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethoxy, difluoromethoxy, dichloromethoxy and azide.
• haloalkyl refers to a lower alkyl radical in which one or more of the hydrogen atoms are replaced by halogen.
• groups include, but not limited to, chloromethyl, trifluoromethyl, 1 -chloro-2-fluoroethyl and the like.
• haloalkoxy refers to RO- in which R is a haloalkyl group.
• sulfinyl or “thionyl” refers to -S(O)-.
• sulfonyl or “sulfuryl” refers to -S(0) 2 -.
• sulfo refers to - S(0) 2 0-.
• Carboxy refers to a divalent radical, -C(0)0-.
• aminocarbonyl refers to -C(0)NH 2 .
• alkylaminocarbonyl refers to -C(0)NHR in which R is hydrogen or alkyl, such as, for example, lower alkyl.
• dialkylaminocarbonyl refers to -C(0)NR ' R in which R ' and R are independently selected from hydrogen or alkyl, such as, for example, lower alkyl;
• carboxamide refers to groups of formula -NR ' COR.
• diarylaminocarbonyl refers to -C(0)NRR' in which R and R' are independently selected from aryl, such as lower aryl, for example, phenyl.
• aralkylaminocarbonyl refers to -C(0)NRR' in which one of R and R' is aryl, such as, lower aryl, for example, phenyl, and the other of R and R' is alkyl, such as, for example, lower alkyl.
• arylaminocarbonyl refers to -C(0)NHR in which R is aryl, such as lower aryl, for example, phenyl.
• hydroxycarbonyl refers to -COOH.
• alkoxycarbonyl refers to -C(0)OR in which R is alkyl, such as lower alkyl.
• aryloxycarbonyl refers to -C(0)OR in which R is aryl, such lower aryl, for example phenyl.
• alkoxy and alkylthio refer to RO- and RS-, in which R is alkyl, such as, for example, lower alkyl.
• aryloxy and arylthio refer to RO- and RS-, in which R is aryl, such lower aryl, for example, phenyl.
• alkylene refers to a straight, branched or cyclic, such as, for example, straight or branched, divalent aliphatic hydrocarbon group, for example, having from 1 to about 20 carbon atoms such as 1 to 1 2 carbons, and for exmaple, is lower alkylene. There optionally can be inserted along the alkylene group one or more oxygen, sulphur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
• Exemplary alkylene groups include methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (— -(CH 2 ) 3 -), cyclohexylene (-C 6 H 10 -), methylenedioxy (-0-CH 2 -0-) and ethylenedioxy (-0-(CH 2 ) 2 -0-).
• the term "lower alkylene” refers to alkylene groups having 1 to 6 carbons.
• Exemplary alkylene groups are lower alkylene, such as, for example, alkylene of 1 to 3 carbon atoms.
• alkenylene groups are lower alkenylene, such as, for example, alkenylene of 3 to 4 carbon atoms.
• alkynylene refers to a straight, branched or cyclic, generally straight or branched, divalent aliphatic hydrocarbon group, such those having from 2 to about 20 carbon atoms and at least one triple bond, generally 1 to 1 2 carbons, such as, for example, lower alkynylene.
• Exemplary alkynylene groups include
• lower alkynylene refers to alkynylene groups having 2 to 6 carbons.
• Exemplary alkynylene groups are lower alkynylene, such as, for example, alkynylene of 3 to 4 carbon atoms.
• alk(en)(yn)ylene refers to a straight, branched or cyclic, generally straight or branched, divalent aliphatic hydrocarbon group, having, for example, from 2 to about 20 carbon atoms and at least one triple bond, and at least one double bond; typically 1 to 1 2 carbons, such as, for example, lower alk(en)(yn)ylene.
• the term "lower alk(en)(yn)ylene” refers to alk(en)(yn)ylene groups having up to 6 carbons.
• Exemplary alk(en)(yn)ylene groups are lower alk(en)(yn)ylene, such as, for example, alk(en)(yn)ylene of 4 carbon atoms.
• cycloalkylene refers to a divalent saturated mono- or multicyclic ring system, generally 3 to 10 carbon atoms, such as 3 to 6 carbon atoms; cycloalkenylene and cycloalkynylene refer to divalent mono- or multicyclic ring systems that respectively include at least one double bond and at least one triple bond. Cycloalkenylene and cycloalkynylene groups can contain 3 to 1 0 carbon atoms, with, for example, cycloalkenylene groups containing 4 to 7 carbon atoms and cycloalkynylene groups containing 8 to 10 carbon atoms.
• ring systems of the cycloalkylene, cycloalkenylene and cycloalkynylene groups can be composed of one ring or two or more rings that can be joined together in a fused, bridged or spiro-connected fashion.
• Cycloalk(en)(yn)ylene refers to a cycloalkylene group containing at least one double bond and at least one triple bond.
• substituted alkylene refers to alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene and cycloalkynylene groups, respectively, that are substituted with one or more substituents, in certain embodiments one to three substituents, independently selected from halo, haloalkyl, such as, for example, halo lower alkyl, aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylth ⁇ o, arylthio, aralkyloxy, aralkylthio, carboxy alkoxycarbonyl, oxo and cycloalkyl.
• arylene refers to a monocyclic or polycyclic, such as monocyclic, divalent aromatic group, for example, having from 5 to about 20 carbon atoms and at least one aromatic ring, such as 5 to 1 2 carbons, and, is, for example, lower arylene. There optionally can be inserted around the arylene group one or more oxygen, sulphur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
• Exemplary arylene groups include 1 ,2-, 1 ,3- and 1 ,4-phenylene.
• the term "lower arylene” refers to arylene groups having 5 or 6 carbons. Exemplary arylene groups are lower arylene.
• heteroarylene refers to a divalent monocyclic or multicyclic aromatic ring system, such as of about 5 to about 1 5 members where one or more, typically, for example, 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and/or sulfur atom(s).
• heterocyclylene refers to a divalent monocyclic or multicyclic non-aromatic ring system, generally of 3 to 1 0 members, such as, for example, 4 to 7 members or 5 to 6 members, where one or more, such as, for example, 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and/or sulfur atom(s).
• substituted arylene refers to arylene, heteroarylene and heterocyclylene groups, respectively, that are substituted with one or more substituents, in certain embodiments one to three substituents, independently selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl optionally substituted with 1 or more, such as 1 to 3, substituents selected from halo, halo alkyl and alkyl, aralkyl, heteroaralkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, alk(en)(yn)yl groups, halo, pseudohalo, cyano, hydroxy, haloalkyl and polyhaloalkyl, such as, halo lower alkyl, for example trifluoromethyl, formyl, alkylcarbonyl, aryl
• aralkylidene refers to an alkylidene group in which either R' or R" is an aryl group.
• Cycloalkylidene are those where R' and R" are linked to form a carbocyclic ring.
• Heterocyclylidene are those where at least one of R' and R" contain a heteroatom in the chain, and R' and R" are linked to form a heterocyclic ring.
• amido refers to the divalent group -C(0)NH-.
• Thioamido refers to the divalent group -C(S)NH-.
• Oxyamido refers to the divalent group -OC(0)NH-.
• Thiaamido refers to the divalent group -SC(0)NH-.
• Dithiaamido refers to the divalent group -SC(S)NH-.
• Ureido refers to the divalent group -HNC(0)NH-.
• Thioureido refers to the divalent group - HNC(S)NH-.
• “semicarbazide” refers to -NHC(0)NHNH-.
• “Carbazate” refers to the divalent group -OC(0)NHNH-.
• “Isothiocarbazate” refers to the divalent group -SC(0)NHNH-.
• Thiocarbazate refers to the divalent group - OC(S)NHNH-.
• “Sulfonylhydrazide” refers to the group -S0 2 NHNH-.
• “Hydrazide” refers to the divalent group -C(0)NHNH-.
• Hydrazinyl refers to the divalent group -NH-NH-.
• amino acid refers to ⁇ -amino acids which are racemic, or of either the D- or L-configuration.
• the designation "d” preceding an amino acid designation refers to the D-isomer of the amino acid.
• the designation "dl” preceding an amino acid designation refers to a mixture of the L- and D-isomers of the amino acid.
• HHT and CHT refer to hexahydrotyrosyl (also known as cyclohexyltyrosyl or p-hydroxycyclohexylalanyl), CHA is cyclohexylalanyl, Pyr and pyroGlu refer to pyroglutamic acid, Pip is pipecolinic acid, Sar is sarcosine, nLeu and Nle are norleucine, nVal is norvaline, Aib is 2-aminoisobutyric acid, Quat is (R)-Glu( ⁇ -(3-am ⁇ dinobenzyl)), and Abu and But are 2-aminobutyric acid.
• PEG represents a polyethylene glycol containing substituent having the designated number of ethyleneoxy subunits.
• PEG(2) represents:
• the cyclic moieties and heteroatom-containing cyclic moieties so defined include, but are not limited to:
• hydroxylated represents substitution on a substitutable carbon of the ring system being so described by a hydroxyl moiety.
• poly- hydroxylated represents substitution on two or more substitutable carbons of the ring system being so described by 2, 3 or 4 hydroxyl moieties.
• cotininyl represents the following structure:
• Quat 3 refers to:
• Quat 4 refers to:
• the conjugates herein are designed to target proteases that are located on cell surfaces, particularly tumor cells and cells involved in tumorigenic processes and angiogenesis and other proliferative processes.
• the conjugates described in detail below, contain a peptidic substrate for a selected targeted cell surface protease linked, either directly or via a linker, to a therapeutic agent, typically a cytotoxic agent, which is substantially inactive when in the conjugate.
• a therapeutic agent typically a cytotoxic agent, which is substantially inactive when in the conjugate.
• the therapeutic agent is released in a form that is active or that can be activated in the vicinity of the targeted cell or tissue to which it is delivered. As a result, active therapeutic agent accumulates at the targeted cells or tissue or in the targeted cells.
• the targeted protease is selected by identifying a protease that is located on a cell or tissue (or associated therewith) that is involved in the disease process or serendipitously present in the locale of cells or tissues involved in the disease or disease process, and, generally, is not located at all or present or active at lower levels, generally substantially lower levels, or exhibits altered activity or specificity, on many, if not all, other cells or tissues.
• the variety and numbers of non-targeted cells or tissues that expresss the active protease varies for particular proteases and diseases intended for treatment. Those of skill in the art will select a target based upon the disease, targeted agents and tolerable or acceptable levels of side-effects. The goal is to achieve enhanced therapeutic index compared with administration of the targeted agent by itself.
• the targeted protease may or may not be involved in the disease process and its expression can be serendiptous; for purposes herein its particular role or lack thereof is not important; it is the fact that it is active in the locale of targeted tissues or cells that is important.
• many of the cell surface proteases of interest herein are expressed or active on tumor cells or cells involved in the tumorigenic processes. Any method known to one of skill in the art for determining or detecting a tissue or cell expression profile can be used. For example, RNA blots composed of RNA from numerous tissues (e.g.
• a multiple tissue expression (MTE) array available from CLONTECH, Palo Alto, CA
• MTE multiple tissue expression
• probes based upon the nucleic acid sequence of the protease of interest to identify cells that express the protease.
• Northern analysis of the blots to test for expression also can be used.
• MTSPs type II membrane-bound serine proteases
• proteases that are located at the cell surface by virtue of a specific interaction with a cell surface protein.
• Urokinase plasminogen activator (u-PA) bound to urokinase plasminogen activator receptor (u-PAR) is exemplary of such proteases.
• Nucleic acid sequence information and expression profiles of exemplary MTSPs and endotheliases are as follows (see, also EXAMPLE 6).
• MTSPs Cell surface proteolysis is a mechanism for the generation of biologically active proteins that mediate a variety of cellular functions. These membrane- anchored proteins, include a disintegrin-like and metalloproteinase (ADAM) and membrane-type matrix metalloproteinase (MT-MMP). In addition to the MMPs, serine proteases have been implicated in neoplastic disease progression. Most serine proteases, which are either secreted enzymes or are sequestered in cytoplasmic storage organelles, have roles in blood coagulation, wound healing, digestion, immune responses and tumor invasion and metastasis.
• ADAM disintegrin-like and metalloproteinase
• MT-MMP membrane-type matrix metalloproteinase
• Transmembrane serine proteases appear to be involved in the etiology and pathogenesis of tumors. These enzymes are expressed in certain cancerous and tumor cells and in other cells associated with other proliferative disorders and other disease states, such as in inflammatory cells and and can be tissue or organ-specific. In mammals, more than 20 members of the family are known (see, Hooper et al. (2001) J. Biol. Chem.276:857-860, see, also U.S. application Serial No.09/776,191, filed February 2, 2001 and International PCT application No. PCT/US01/03471 ; see, also U.S.
• MTSP1 also called TADG-15 and matriptase, see SEQ ID Nos. 1 and 2; accession nos. AF133086/AF118224, AF04280022; Takeuchi et al. (1999) Proc. Natl. Acad. Sci. U.S.A.56:11054-1161; 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 45:420-429); hepsin (see, accession nos.
• TMPRSS4 see, Accession No. NM 016425; Wallrapp et al. (2000) Cancer 60:2602-2606. Also known MTSP3, MTSP4, MTSP6, MTSP7, MTSP9,
• proteases including transmembrane serine proteases, have been implicated in processes involved in neoplastic development and progression. While the precise role of these proteases has not been elaborated, serine proteases and inhibitors thereof are involved in the control of many intra- and extracellular physiological processes, including degradative actions in cancer cell invasion, metastatic spread, and neovascularization of tumors, that are involved in tumor progression. It is believed that proteases are involved in the degradation of extracellular matrix (ECM) and contribute to tissue remodeling, and are necessary for cancer invasion and metastasis. The activity and/or expression of some proteases have been shown to correlate with tumor progression and development, and also are shown to be active in specific cell types.
• ECM extracellular matrix
• MTSP1 membrane-type serine protease
• matriptase also called matriptase; see SEQ ID Nos. 1 and 2 from U.S. Patent No. 5,972,61 6; and GenBank Accession No. AF1 1 8224; (1 999) J. Biol. Chem. 274: 1 8231 -1 8236; U.S. Patent No. 5,792,61 6; see, also Takeuchi (1 999) Proc. Natl. Acad. Sci. U.S.A. 56: 1 1054-1 1 61 ) that is expressed in epithelial cancer and normal tissue (Takeucuhi et al. ( 1 999) Proc. Natl. Acad. Sci.
• Hepsin a cell surface serine protease identified in hepatoma cells, is overexpressed in ovarian cancer (Tanimoto et al. (1 997) Cancer Res. , 57:2884-7).
• the hepsin transcript appears to be abundant in carcinoma tissue and is almost never expressed in normal adult tissue, including normal ovary. It has been suggested that hepsin is frequently overexpressed in ovarian tumors and therefore can be a candidate protease in the invasive process and growth capacity of ovarian tumor cells.
• NES1 normal epithelial cell-specific 1
• MTSPs Each MTSP has a characteristic tissue expression profile; the MTSPs in particular, although not exclusively expressed or activated in tumors, exhibit characteristic tumor tissue expression or activation profiles.
• MTSPs can have different activity in a tumor cell from a non-tumor cell by virtue of a change in a substrate or cofactor therefor or other factor that would alter functional activity of the MTSP.
• each can serve as a diagnostic marker for particular tumors, by virtue of a level of activity and/or expression or function in a subject (i.e. a mammal, particularly a human) with neoplastic disease, compared to a subject or subjects that do not have the neoplastic disease.
• detection of activity (and/or expression) in a particular tissue can be indicative of neoplastic disease.
• they can serve as therapeutic targets, such as by administration of modulators of the activity thereof, or, as by administration of a prodrug specifically activated by one of the MTSPs.
• Each or any of the MTSPs can exhibit activity or expression levels or substrate specificities that differ in tumor cells from the levels in normal cells.
• tumor cells include, but are not limited to, colon, lung, prostate, breast, esophagous, pancreas, cervic, uterus, endometrium, and other solid tumors and in blood and lymphatic tumors.
• conjugates provided herein can be designed by selection of substrate specificity for treatment of any of such tumors and neoplastic conditions.
• Tissue expression profiles The following are exemplary tissue and gene (see also, EXAMPLE 8) profiles of some exemplary MTSPs. These profiles are not intended to define the full scope of expression or activation of these MTPSs, but demonstrate that MTSPs are expressed in tumors, and, hence there expression or activation or substrate specificity on the surface of tumor cells can be exploited in the methods herein and conjugates, designed in accord with the methods herein and as exemplified herein, that are cleaved by one or more of these MTSPs can be prepared and employed for treatment of neoplastic or other diseases or conditions or to target to cells that express these proteins on there surfaces.
• MTSP1 (matriptase)
• MTSP1 also called matriptase
• matriptase is a trypsin-like serine protease with broad spectrum cleavage activity and two potential regulatory modules. It was named “matriptase” based on its ability to degrade the extra-cellular matrix and its trypsin-like activity.
• MTSP1 When isolated from breast cancer cells (or T-47D cell conditioned medium), MTSP1 has been reported to be primarily in an uncomplexed form.
• MTSP1 has been isolated from human milk; when isolated from human milk, it was reported to be in one of two complexed forms, 95 kDa (the predominant form) and 1 10 kDa; uncomplexed MTSP1 was not detected (Liu, e al. ( 1 999) J. Biol. Chem. 21 '4: 1 8237 '-1 8242) . It has been proposed that MTSP1 exists as an uncomplexed protease when in its active state. In breast milk, it has been reported to exist in complex with a fragment of hepatocyte growth factor inhibitor-1 (HAI-1 ), a Kunitz-type serine protease inhibitor having activity against trypsin-like serine proteases.
• HAI-1 hepatocyte growth factor inhibitor-1
• Nucleic acids encoding the protein designed matriptase were cloned from T-47D human breast cancer cell-conditioned medium (Lin et al. (1 999) J. Biol. Chem. 274: 1 8231 -18236). Upon analysis of the cDNA, it was determined that the full length protease has 683 amino acids and contains three main structural regions: a serine protease domain near the carboxyl-terminal region, four tandem low-density lipoprotein receptor domains, and two tandem complement subcomponents C 1 r and C1 s (see SEQ ID No. 1 ). Studies to identify additional serine proteases made by cancer cells were done using PC-3 cells.
• MT-SP1 serine protease termed "MT-SP1 ” by the authors, reported to be a transmembrane protease was cloned (Takeuchi et al. ( 1 999) Proc. Natl. Acad. Sci. U.S.A. 56: 1 1054-1 1061 ). It was subsequently found that originally identified matriptase sequence is included in the translated sequence of the cDNA that encodes MTSP1 . The nucleic acid encoding the protein originally designated matriptase is a partial MTSP1 clone that lacks 51 6 of the coding nucleotides (Takeuchi, et a/. , J. Biol.
• matriptase is a variant form of MTSP1 .
• MTSP1 demonstrates trypsin-like protease activity and is a Type II transmembrane protein with an extracellular protease domain.
• PAR2 protease-activated receptor 2
• pro-HGF pro-hepatacyte growth factor
• sc-uPA single-chain urokinase-type plasminogen activator
• PAR2 functions in inflammation, cytoprotection and/or cell adhesion, while sc-uPa functions in tumor cell invasion and metastasis.
• HGF serves a growth and pro- angiogenic factor.
• SEQ ID Nos 1 and 2 An exemplary nucleotide sequence encoding a human MTSP1 is set forth in SEQ ID Nos 1 and 2. As previously noted SEQ ID No. 1 sets for an MTSP1 - encoding nucleic acid sequence. This sequence is the longer version and includes the protease domain, which is common to both variants.
• MTSP1 is expressed in breast, prostate and colorectal tumors. Hence conjugates with substrates therefor can be used for treatment of such tumors.
• the MTSP3 transcript was detected in lung carcinoma (LX-1 ), colon adenocarcinoma (CX-1 ), colon adenocarcinoma (GI-1 1 2) and ovarian carcinoma (GI-102). No apparent signal was detected in another form of lung carcinoma (GI-1 1 7), breast carcinoma (GI-101 ), pancreatic adenocarcinoma (GI-103) and prostatic adenocarcinoma (PC3) .
• MTSP4 The MTSP4 transcript, a DNA fragment encoding part of the LDL receptor domain and the protease domain was used to probe an RNA blot composed of 76 different human tissues (catalog number 7775-1 ; human multiple tissue expression (MTE) array; CLONTECH) . As in the northern analysis of gel blot, a very strong signal was observed in the liver.
• PCR of the MTSP4 transcript from cDNA libraries made from several human primary tumors xenografted in nude mice was performed using MTSP4-specific primers.
• the MTSP4 transcript was detected in breast carcinoma (GI-101 ), lung carcinoma (LX-1 ), colon adenocarcinoma (GI-1 1 2) and pancreatic adenocarcinoma (GI-103). No apparent signal was detected in another form of lung carcinoma (GI-1 1 7), colon adenocarcinoma (CX-1 ), ovarian carcinoma (GI-102). and prostatic adenocarcinoma (PC3).
• the MTSP4 transcript was also detected in LNCaP and PC-3 prostate cancer cell lines as well as in HT-1080 human fibrosarcoma cell line.
• MTSP6 MTSP6 is expressed at high levels in the colon. It also is expressd in the, stomach, trachea, mammary gland, thyroid gland, salivary gland, pituitary gland and pancreas. It is expressed at lower levels in other tissues (see EXAMPLE 6). MTSP6 also is expressed in several tumor cell lines including HeLa S3 > colorectal adenocarcinoma (SW480) > leukemia MOLT-4 > leukemia K-562.
• SW480 colorectal adenocarcinoma
• MOLT-4 leukemia K-562.
• the MTSP6 transcript was strongly detected in lung carcinoma (LX-1 ), moderately detected in pancreatic adenocarcinoma (GI-103), weakly detected in ovarian carcinoma (GI-102); and weakly detected in colon adenocarcinoma (GI-1 1 2 and CX-1 ), breast carcinoma (GI-101 ), lung carcinoma (GI-1 17) and prostatic adenocarcinoma (PC3).
• the MTSP6 transcript was also detected in breast cancer cell line MDA-MB-231 , prostate cancer cell line PC-3, but not in HT-1080 human fibrosarcoma cell line.
• MTSP6 also is expressed in mammary gland carcinoma cDNA (Clontech). MTSP6 also is over expressed in ovarian tumor cells.
• MTSP7 The MTSP7 transcript was 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, breast, cervical and other tumors.
• MTSP9 MTSP9 is, for example, expressed in esophageal tumor tissues, in lung carcinoma, in colorectal carcinoma, lymphoma, a cervical carcinoma (HeLaS3) and leukemia cell lines as well as in certain normal cells and tissues.
• MTSP9 also can be a marker for breast, prostate, cervical and colon cancer.
• MTSP9 is highly expressed in the esophagus and expressed at a low level in many other tissues.
• the MTSP9 transcript is found in kidney (adult and fetal), spleen (adult and fetal), placenta, liver (adult and fetal), thymus, peripheral blood leukocyte, lung (adult and fetal), pancreas, lymph node, bone marrow, trachea, uterus, prostate, testes, ovary and the gland organs (mammary, adrenal, thyroid, pituitary and salivary).
• MTSP9 also is expressed in esophagus tumor tissues, in a lung carcinoma and, at a lower level, in a colorectal carcinoma, lymphoma, a cervical carcinoma (HeLaS3) and leukemia cell lines.
• MTSP10 also is expressed in esophagus tumor tissues, in a lung carcinoma and, at a lower level, in a colorectal carcinoma, lymphoma, a cervical carcinoma (HeLaS3) and leukemia cell lines.
• MTSP10 for example, is expressed in esophageal tumor tissues, in lung carcinoma, prostate cancers, pancreatic and breast cancers and in cell lines as well as in certain normal cells and tissues (see e.g. , EXAMPLES for tissue- specific expression profile).
• the level of activated MTSP1 0 can be diagnostic of prostate, uterine, lung esophagus, or colon cancer or leukemia or other cancer.
• the expression and/or activation of MTSP10 on or in the vicinity of a cell or in a bodily fluid in a subject can be a marker for breast, prostate, lung, colon, esophageal and other cancers.
• MTSP10 transcript was detected in pancreas, lung and kidney.
• MTSP10 transcript was also detected in small intestine Marathon-Ready cDNA (Clontech). The MTSP10 transcript was detected in breast carcinoma (GI-1 01 ), lung carcinoma (LX-1 and GI-1 17), ovarian carcinoma (GI-1 02), and pancreatic adenocarcinoma (GI-103). The MTSP10 transcript was weakly detected in prostatic adenocarcinoma (PC3) . The MTSP10 transcript was also detected in CWR22R prostate tumor grown in nude mice. No apparent signal was detected in two forms of colon adenocarcinomas (GI-1 1 2 and CX-1 ).
• MTSP12 MTSP1 2 transcript was detected in pancreas, lung and kidney. MTSP1 2 transcript was also detected in small intestine Marathon-Ready cDNA (Clontech). The MTSP1 2 transcript was detected in breast carcinoma (GI-1 01 ), lung carcinoma (LX-1 and GI-1 1 7), ovarian carcinoma (GI-1 02), and pancreatic adenocarcinoma (GI-103). The MTSP1 2 transcript was weakly detected in prostatic adenocarcinoma (PC3). The MTSP1 2 transcript was also detected in CWR22R prostate tumor grown on nude mice. No apparent signal was detected in two forms of colon adenocarcinomas (GI-1 1 2 and CX-1 ).
• MTSP20 MTSP20 is expressed in the lung, colon, cervical tumors and in leukemic cells. It may also be expressed in breast, ovarian, pancreatic, prostate and in other tumors. MTSP20 transcript was detected in liver, lymph node, cerebellum, pancreas, prostate, uterus, testis, glands (adrenal, thyroid and salivary), thymus, kidney and spleen. Lower transcript level was found in lung, placenta, bladder, ovary, digestive system, circulatory system and other parts of the the brain.
• MTSP20 is also expressed in certain tumor cell lines including lung carcinoma (A51 9), colorectal carcinoma (SW480), lymphoma (Raji and Daudi), cervical carcinoma (HeLaS3) and leukemia (HL-60, K-562 and MOLT-4) cell lines.
• lung carcinoma A51 9
• colorectal carcinoma SW480
• lymphoma Raji and Daudi
• cervical carcinoma HeLaS3
• leukemia HL-60, K-562 and MOLT-4
• MTSP22 MTSP22 is expressed in the uterine tissue, thymus, adipose tissue, and lymph node. It may also be expressed in lung, stomach, uterine, breast, ovarian, prostate and in other tumors. MTSP22 transcript was detected in some uterus tissue samples, but not in their matched tumor samples. In one of 42 uterus samples, MTSP22 is expressed in tumor and its metastatic tissues, but not in the normal tissue counterpart. MTSP22 is also expressed in some stomach tumors and lung tumors, but not in their normal tissue counterparts. MTSP22 is also expressed in the normal tissue of a pancreas matched cDNA pair. MTSP22-encoding cDNA was detected in thymus, adipose tissue, and lymph node
• MTSP25 MTSP25 is expressed in breast, colon, uterine, ovarian, kidney, prostate, testicular cancer tissue. It may also be expressed in lung, stomach, prostate and in other tumors. MTSP25 transcript was expressed weakly in the lymph node. In the cancer profiling array analysis, MTSP25 is highly expressed in prostate samples (in normal and cancer samples). MTSP25 was highly expressed in a kidney tumor sample, but not in its normal tissue counterpart. MTSP25 was also expressed a breast cancer samples, but not in its normal tissue counterpart. MTSP25 was expressed in normal uterus samples, but not in their tumor counterparts. MTSP25 expression was also ovarian cancer samples.
• MTSP25 was also detected in one of the matched normal tissue counterparts. MTSP25 expression was also detected in tumor samples in colon cDNA pairs. PCR analysis revealed that MTSP25 cDNA was strongly detected in testis and mammary gland adenocarcinoma, weakly detected in brain, placenta, lung, spleen, prostate, small intestine, colon, and leukocyte, and very weakly detected in heart, liver and pancreas. 2. Endotheliases
• Endotheliases are a class of cell surface proteases that are expressed on cells, particularly endothelial cells, particularly those proliferating endothelial cells, which are involved in a variety of proliferative processes, including undesirable angiogenesis associated with tumor growth and metastasis, and with other hyperproliferative disorders, such as restenosis, scarring, diabetic retinopathies, diseases and disorders of the anterior eye (see, U.S. application Serial No. 09/71 7,473, filed November 20, 2000, and International PCT application No. PCT/US00/31 803).
• Endotheliases are particularly useful targets for delivery of therapeutic agents for treatment of any disorder involving aberrant angiogenesis.
• Endothelial cells play a key role in angiogenesis, which is is the generation of new blood vessels from parent microvessels.
• Angiogenesis plays a major role in the metastasis of cancer and in the pathology of a variety of other disorders.
• angiogenesis proceeds in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a
• Angiogenesis is highly regulated by a system of angiogenic stimulators and inhibitors.
• angiogenesis stimulators include certain growth factors, cytokines, proteins, peptides, carbohydrates and lipids (Norrby ( 1 997) APMIS 705:41 7-437); Polverini ( 995) Crit. Rev. Oral. Biol. Med. 6:230- 247).
• endogenous and exogenous angiogenesis inhibitors are known in the art (Jackson et al. (1 997) FASEB 7 7:457-465; Norrby (1 997) APMIS 705:41 7-437); and O'Reilly (1 997) Investigational New Drugs, 1 5 :5-1 3).
• Angiogenesis is essential for normal placental, embryonic, fetal and post- natal development and growth, but almost never occurs physiologically in adulthood except in very specific restricted situations.
• angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta.
• Angiogenesis in the adult is often associated with disease states.
• Persistent, unregulated angiogenesis occurs in a multiplicity of disease states, tumor metastasis and abnormal growth by endothelial cells and supports the pathological damage seen in these conditions.
• the diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic dependent or angiogenic associated diseases.
• angiogenesis is involved in the manifestation or progress of various diseases, for example, various inflammatory diseases, such as rheumatoid arthritis, psoriasis, diabetic retinopathies, certain ocular disorders, including recurrence of pterygii, scarring excimer laser surgery and glaucoma filtering surgery, various disorders of the anterior eye, cardiovascular disorders, chronic inflammatory diseases, wound repair, circulatory disorders, crest syndromes, dermatological disorders (see, e.g. , U.S. Patent Nos. 5,593,990, 5,629,327 and 5,71 2,291 ) and notably cancer, including solid neoplasms and vascular tumors.
• various inflammatory diseases such as rheumatoid arthritis, psoriasis, diabetic retinopathies, certain ocular disorders, including recurrence of pterygii, scarring excimer laser surgery and glaucoma filtering surgery, various disorders of the anterior eye, cardiovascular
• Angiogenesis is essential for the growth and persistence of solid tumors and their metastases. Repressing, eliminating or modulating this activity, should impact the etiology of these diseases and serve as a point of therapeutic intervention. In the disease state, prevention of angiogenesis could avert the damage caused by the invasion of the new microvascular system. Therapies directed at control of the angiogenic processes could lead to the abrogation or mitigation of these diseases. Hence there is a need to develop therapeutics that target angiogenesis and modulate, particularly, inhibit aberrant or uncontrolled angiogenesis.
• conjugates that contain endotheliase substrates can be used to deliver therapeutic agents for the treatment of diseases including, but are not limited to, rheumatoid arthritis, psoriasis, diabetic retinopathies, other ocular disorders, including recurrence of pterygii, scarring from excimer laser surgery and glaucoma filtering surgery, various disorders of the anterior eye, cardiovascular disorders, autoimmune diseases, chronic inflammatory diseases, wounds, circulatory disorders, crest syndromes, restenosis, psoriasis and other dermatological disorders (see, e.g. , U.S. Patent Nos. 5,593,990, 5,629,327 and 5,71 2,291 ) and notably cancer, including solid neoplasms and vascular tumors.
• diseases including, but are not limited to, rheumatoid arthritis, psoriasis, diabetic retinopathies, other ocular disorders, including recurrence of ptery
• Endotheliases 1 and 2 Exemplary of endotheliases are two different endotheliases and variant forms thereof designated endotheliase 1 and endotheliase 2 (see SEQ ID Nos. 21 -27. Other members of the family can be identified by probing for genes or searching libraries for genes that have sequence identity, particularly at least 40%, 60%, 80%, 90%, 95 %, 98 % or greater sequence identity to the protease domain of an endotheliase identified herein, or that hybridize under conditions of high stringency to the full-length of the nucleic acid encoding a protease domain of an endotheliase provided herein, and that are expressed on endothelial cells.
• an endotheliase can be identified by the methods, such by identifying ESTs or other nucleic acid fragments that have sequences similar to a protease and then using such fragments as probes to identify and select cDNA clones encoding full-length proteases or protease domains thereof, identifying those that have the characteristics of transmembrane proteins, and then determining the gene expression profile to identify those that are expressed on the surface of endothelial cells.
• Encoded proteins that have protease activity, that include a transmembrane domain and an extracellular domain, and that are expressed in endothelial cells are endotheliases.
• Endotheliase 1 Exemplary of the endotheliase are endotheliase 1 and endotheliase 2. These are expressed on endothelial cells. Exemplary of a full-length endotheliase 1 is one that includes the sequence of amino acids set forth in SEQ ID No. 42 (see. International PCT application No. WO 00/5006, which describes a gene it designates DESC1 that is expressed in squamous cell carcinomas and prostate tumors). As noted endotheliases are expressed on endothelial cells. A protease domain thereof is set forth in SEQ ID NO: 22.
• RNA blot composed of 76 different human tissues (catalog number 7775-1 ; human multiple tissue expression (MTE) array; CLONTECH, Palo Alto, CA). Significant expression was observed in the esophagus, with minor expression levels in the stomach, salivary gland, pancreas, prostate, bladder, trachea and uterus.
• Northern analysis using RNA blots confirmed that the expression was restricted to the esophagus. Two transcripts (approximately 1 .7 and 2 kb) were detected in the esophagus. Endotheliase 1 also is expressed in umbilical vein endothelial cells, PC3 and LnCAP cells.
• Endotheliase 2 and nucleic acids encoding endotheliase 2 Two splice variant forms of endotheliase 2 designated endotheliase 2-S and endotheliase 2-L are exemplified herein (see SEQ ID Nos. 23-26).
• the open reading frame of the nucleic acid encoding endotheliase 2-S (SEQ ID No. 23) is composed of 1 ,689 bp, which translates to a 562-amino acid protein (SEQ ID No. 24), while the ORF of endotheliase 2-L is composed of 2,067 bp (SEQ ID
• the nucleic acid encoding the protease domain of endotheliase 2-S is composed of 729 bp which translates to a 242-amino acid protein (amino acids 321 -562 of SEQ ID Nos. 23 and 24), while that of endotheliase 2-L is composed of 1 ,107 bp, which translates to a 368-amino acid protein (amino acids 321 -688 of SEQ ID Nos. 25 and 26).
• endotheliase 2 In addition to expression in endothelial cells, endotheliase 2 is expressed in placenta, pancreas, thyroid gland, liver and lung tissues. It also is expressed at lower levels in mammary gland, salivary gland, kidney, trachea, esophagus, appendix, heart and fetal lung. Endotheliase 2 also is expressed in several tumor cell lines and, hence, in certain tumors, including lung and colon, including breast carcinoma, lung carcinomas, colon adenocarcinomas, pancreatic adenocarcinoma (GI-103), and ovarian carcinoma. It has also been detected in prostate and fibrosarcoma cell lines.
• Conjugates that are substrates for proteases on the surfaces of cells, particularly serine proteases, including type II membrane-bound serine proteases, and endotheliases are provided. Any cell surface protease, including cell- associated or localized proteases, is contemplated herein. Generally proteases expressed at high levels in active forms in essential tissues are not ideal target candidates. The proteases include those that are expressed on relatively limited numbers of cells or that are expressed at high levels in cells, such as tumor cells and endothelial cells and immune cells, that are involved in disease states or are present in diseases states in the locale of cells involved in the disease states.
• endothelial cells by virtue of their role in angiogenesis are involved in numerous proliferative disorders; immune cells are involved in many disease processes including cancers and diseases and inflammatory disorders.
• Other cell surface proteases are expressed at higher levels in certain tumors than in normal cells. Whether or not such proteases have a role in the disorder their higher expression in cells involved in a disease state is sufficient for use for targeting therapeutic agents in the conjugates provided herein.
• the conjugates which contain a therapeutic agent, such as a cytotoxic agent, is activated upon cleavage by a cell surface protease, including cell- associated and cell-localized proteses.
• proteases are the MTSPs, such as, but not limited to, MTSP1 , MTSP3, MTsP4, MTSP6, MTSP7, MTSP9, MTSP10, MTSP1 2, MTSP20, MTSP22, MTSP25, urokinases and endotheliases.
• the conjugates targeted to such proteases are prodrugs in that the therapeutic agent is inactive as administered and is ultimately activated in the vicinity of the targeted cell or tissue.
• cell surface proteases such as transmembrane proteases, are the intended targets, any released, shed or soluble forms of the proteases and others also can be targeted.
• the conjugates which contain a therapeutic agent, such as a cytotoxic agent, are substantially inactive prior to action by a cell surface protease, a peptidic moiety that is a substrate for a targeted cell surface protease (i.e. , a peptidic substrate), and, optionally, a linker.
• the therapeutic agents in the conjugates are activated upon cleavage of the peptidic substrate of the conjugate by a cell surface protease.
• the therapeutic agents, such as cytotoxic agents are released as the free yagent, or, alternatively, are released coupled to the portion of the peptidic substrate (P1 -P2-P3-etc.
• cytotoxic agents in these forms, are released in the vicinity of cells that express the proteases. Activation is effected, in certain embodiments, because the therapeutic agent, such as cytotoxic agent, following action of the cell surface protease, can cross the cell membrane or otherwise interact with the cell or tissue and exhibit therapeutic activity. In other embodiments, any remaining peptidic moieties or amino acids can be cleaved from therapeutic agent to render it active.
• the conjugates act as prodrugs because the therapeutic agents when conjugated are substantially inactive. Upon cleavage by the targeted protease, the therapeutic agent is released either in active form or in a form that is activated by the targeted cell, tissue or surrounding environment.
• the targeted agent is a cytotoxic agent and the conjugates for use in the methods and compositions provided herein have the formula:
• peptide' is a peptidic substrate for a cell surface protease or a released, shed or otherwise unbound membrane protease, such as an MTSP; s is greater than or equal to 1 , or is 1 to 6, or is 1 or 2, or is 1 ; linker is any linker; q is greater than or equal to 0, or is 0 to 4, or is 0 or 1 ; the cytotoxic agent is an anti-tumor, anti-cancer or anti mitotic agent, including anti- antiangiogenic agents; and t is 1 or more, or is 1 or 2.
• the cytotoxic agent is covalently attached, optionally via a linker, to either the C- terminus or the N-terminus of the peptidic substrate.
• the therapeutic agent such as a cytotoxic agent
• the N-terminus optionally is capped.
• N-Terminal caps for use herein include, but are not limited to, acyl, sulfonyl and carbamoyl groups.
• the C-terminus is a carboxamide derivative.
• peptide' is a peptidic substrate for a cell surface protease or a soluble MTSP whereby, upon action of the protease, the conjugate, which is substantially inactive, is cleaved at the P1 -P1 ' bond to release a compound of the formula:
• the conjugates for use in the methods and compositions provided herein possess two therapeutic agents, such as cytotoxic agents, which are the same or different, linked to the C-terminus and the N- terminus, respectively, optionally via linkers linker 1 and linker 2 , of a peptidic substrate for cell surface protease or a soluble MTSP.
• the conjugates have the formula:
• peptide' is a peptidic substrate for a cell surface protease, or a soluble MTSP; s is greater than or equal to 1 , or is 1 to 6, or is 1 or 2, or is 1 ; linker 1 and linker 2 are each independently any linker and are the same or different; q and w are each independently greater than or equal to 0, or are 0 to 4, or are 0 or 1 ; the therapeutic agents, which are the same or different, are anti-tumor, anti-cancer or anti mitotic agents; and t and x are each independently 1 or more, or are 1 or 2.
• peptide' is a peptidic substrate for a cell surface protease or a soluble MTSP whereby, upon action of the protease, the conjugate, which is substantially inactive, is cleaved at a point on the peptidic chain to release two compounds of the formulae: (therapeutic agent 1 ) x -(linker 1 ) w -(peptide a1 ) s ; and (peptide a2 ) s -(linker 2 ) q -(therapeutic agent 2 ) t or derivatives thereof.
• the released therapeutic agents are active or are further activated by the cell, tissue or surrounding environment.
• peptide 31 and peptide 82 are N-terminal and C-terminal truncated portions, respectively, of peptide 1' resulting from cleavage at the PI -PI ' bond.
• the conjugates for use in the compositions and methods provided herein have formula I: X n -(P6) m -(P5) p -(P4) r (P3) r (P2) r P1 -(P1 ') u -(P2') k -(P3') r -(L) n -Z or a derivative thereof, where Z is a therapeutic agent; L is a linker; I, j, i, p and m are selected as follows: I is 0 or 1 ; when I is 0, j, i, p and m are 0; when I is 1 , j is 0 or 1 ; when j is 0, i, p and m are 0; when j
• the conjugates for use in the compositions and methods provided herein have formula II: Z-(L) n -(P6) m -(P5) p -(P4) r (P3) r (P2) r P1 -(P1 ') u -(P2') k -(P3') r -X c or a derivative thereof, where Z is a therapeutic agent; L is a linker; I, j, i, p and m are selected as follows:
• I is 0 or 1 ; when I is 0, j, i, p and m are 0; when I 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 a
• the P6 to P3' residues are linked by peptide bonds or peptide bond surrogates.
• the P6 to P3' portion of the conjugate is a peptidic substrate, as defined herein.
• the conjugates for use in the compositions and methods provided herein have formula III: Z 1 -(L 1 ) n -(P6) m -(P5) p -(P4) r (P3) r (P2),-P1 -(P1 ') u -(P2') k -(P3') r -(L 2 ) v -Z 2 or a derivative thereof, where Z and Z 2 are each therapeutic agents and are the same or different; L 1 and L 2 are each linkers and are the same or different; I, j, i, p and m are selected as follows: I is 0 or 1 ; when I is 0, j, i, p and m are 0; when I is 1 ,
• P6 to P3' are amino acid residues, as defined below.
• the P6 to P3' residues are linked by peptide bonds or peptide bond surrogates.
• the P6 to P3' portion of the conjugate is a peptidic substrate, as defined herein.
• the conjugates for use in the compositions and methods provided herein have formula IV: X n -(P6) m -(P5) p -(P4) i -(P3) J -(P2),-P1 -(P1 ') u -(P2') k -(P3') r -(P4') s -(L) n -Z or a derivative thereof, where Z is a therapeutic agent; L is a linker; I, j, i, p and m are selected as follows:
• I is 0 or 1 ; when I is 0, j, i, p and m are 0; when I 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 ; 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 0, s is 0 or 1 ; n is 0 or 1 ;
• the P6 to P4' residues are linked by peptide bonds or peptide bond surrogates.
• the P6 to P4' portion of the conjugate is a peptidic substrate, as defined herein.
• the conjugates for use in the compositions and methods provided herein have formula V:
• conjugates for use in the compositions and methods provided herein have formula VI:
• the peptidic substrates contemplated for use in the conjugates are substrates for the targeted cell surface protease or a soluble, shed or released form thereof, and contain a sufficient number of amino acid residues to render any therapeutic agent in the conjugate substantially inactive.
• the therapeutic agent is, for example, doxorubicin
• the conjugate is substantially inactive by virtue of the inability of the conjugated therapeutic agent to cross the cell membrane.
• the peptidic substrate contains at least 1 , 2, 3, 4 or 5 amino acid residues, and can contain up to nine or ten residues. Longer peptidic substrates can be used in the conjugates as long as upon cleavage, the resulting therapeutic agent or therapeutic agent-amino acid or -peptidic moiety conjugate exhibits the desired therapeutic effect in vivo and in vitro.
• exemplary peptidic substrates for use in the conjugates provided herein possess at least one amino acid (P1 ), two amino acids (P1 -PV), three amino acids (P2-P1 -PV) and typically contain four, five or six amino acid residues (P3-P2-P1 -P1 ', P4-P3-P2-P1 -P1 ' or P4-P3-P2-P1 -P1 '-P2'), where the
• P1 -P1 ' bond is the site of cleavage of cell surface protease, or a soluble, shed or released form thereof, including, but not limited to, a cell surface protease, such as a serine protease, including, for example, but not limited to, uPA bound to its receptor, MTSPs and endotheliases.
• the peptidic substrates optionally further possess a P5, P6 or P3' amino acid residue, and, in certain embodiments, possess P7, P8, P9, P10, P4', P5', P6' residues.
• the peptidic substrates for use in the conjugates provided herein are penta-, hexa-, hepta-, octa- and nona-peptidic substrates, and can contain 10, 1 1 , 1 2, 1 3, 14, 1 5 or more residues as long as, upon cleavage of the conjugate by the protease, the resulting therapeutic agent or therapeutic agent-amino acid or -peptidic moiety conjugate exhibits the desired therapeutic effect in vivo and in vitro.
• the peptidic substrates are conjugated to the therapeutic agent (or to a linker to which the therapeutic agent is linked) via the C-terminal residue (i.e.
• the peptidic substrates for example, can be straight chains, but can be cyclized or include cyclized portions.
• the peptidic substrate optionally possesses a cap, such as an acyl or carbamoyl cap at the N-terminus.
• the peptidic substrate further possess a terminal group, such as a carboxamide group, at the C-terminus.
• the conjugates can contain a plurality of peptidic substrates and a plurality of therapeutic agents. For example, in conjugates that contain two therapeutic agents, which are the same or different, conjugation to the therapeutic agent(s) or linker linked thereto can be via the C-terminal and N- terminal residues of the peptidic substrate.
• substrates can be designed based upon known specificities of other proteases. For example, the specificities of trypsin- like and trypsin family members can aid in design of possible substrates.
• substrate preferences for particular serine proteases see, e.g. , Harris et al. (2000) PNAS 97(14):77S4-7759) .
• Typical protocols for preparation of the conjugates can include the steps of: 1 ) identification of a targeted protease; 2) expression and assay development; 3) substrate selection, such as, for example, by testing chromogenic or fluorogenic substrates to identify those cleaved by a selected target protease, by use of substrate phage display to identify peptidic substrates cleaved by a targeted protease, by use of a natural protein or peptide substrate or a natural inhibitor of the protease, and by use of combinatorial libraries to identify substrates cleaved by a targeted protease; 4) synthesis of conjugates containing the identified substrate; and 5) biological evaluation thereof, including, but not limited to, in vitro assays, cell culture assays, biological assays, and in vivo animal models (see, e.g. , EXAMPLE 10).
• a conjugate can be designed by any methods known to those of skill in the art. The following provides an exemplary protocol. First, a series of commercially available chromogenic and fluorogenic peptidic substrates can be tested for cleavage by the protease of interest (see Examples for lists of exemplary chromogenic and fluorogenic substrates and the table below). The peptidic portion of these substrates occupies the unprimed binding sites of the protease while the reporter group is located on the primed side of the scissle bond. Effective conjugates can then be designed based on the structure of the substrates that are efficiently cleaved by the protease.
• the peptidic portion of these efficiently cleaved substrates can be used as the unprimed region of the conjugate, and Ser-therapeutic agent, such as a cytotoxic agent (e.g., doxorubicin), Ser-Leu-therapeutic agent or Ser-Ser-Leu- therapeutic agent can be used as the primed region of the conjugate.
• Ser-therapeutic agent such as a cytotoxic agent (e.g., doxorubicin)
• Ser-Leu-therapeutic agent or Ser-Ser-Leu- therapeutic agent can be used as the primed region of the conjugate.
• Cleavage of these conjugate prodrugs releases either Ser-therapeutic agent, Ser-Leu- therapeutic agent or Ser-Ser-Leu-therapeutic agent compounds.
• the Ser in the released Ser-therapeutic agent may be replaced by other amino acid residues including, but not limited to, Ala, hSer, Abu, Thr, Met, nLeu and Val.
• the amino acid residue conjugated 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, (hex)Gly, (allyl)Gly, (propargyl)Gly and (cyclopropyl)Ala.
• the amino acid residue conjugated 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.
• conjugate prodrug for a protease substrate is to use substrate phage display to elucidate optimal subsite occupancy for the protease. The resulting information can then be used to design the peptidic, unprimed portion of the conjugate.
• the primed region of the conjugate can be fixed as Ser-therapeutic agent, Ser-Leu- therapeutic agent or Ser-Ser-Leu-therapeutic agent.
• a third approach to design an effective prodrug conjugate involves the use of combinatorial fluorogenic substrate libraries to determine optimal residues for the unprimed region of a protease substrate. These selected sequences can then be used as the unprimed portion of the conjugate prodrug and, and Ser- therapeutic agent, (e.g., doxorubicin), Ser-Leu-therapeutic agent or Ser-Ser-Leu- therapeutic agent can be used as the primed region of the conjugate.
• Ser- therapeutic agent e.g., doxorubicin
• Ser-Leu-therapeutic agent e.g., Ser-Leu-therapeutic agent
• Ser-Ser-Leu- therapeutic agent can be used as the primed region of the conjugate.
• sequences including GSGR were based on or dervied from substrate phage display experiments using u-PA as the taret protease.
• seqeuence sequences in natural substrates or natural inhibitors of a protease target, such as uPA, including VSAR, PGR (from P3-P1 of plasminogen) and related sequences were used in design of u-PA-targetd conjugates.
• sequences from chromgenic substrates, such as D-HHT-Gly-Arg, and related sequences were used for design of ET-1 -targeted conjugates.
• a protease is incubated with a zymogen, such as plasminogen or trypsinogen, in the presence of a labelled known substrate, such as lys- plasminogen or Spec PL (for plasmin), for the zymogen. If protease activates the zymogen, the activated enzyme, such as plasmin and trypsin, will degrade the substrate, thereby changing the spectral properties of the substrate.
• a zymogen such as plasminogen or trypsinogen
• peptidic substrates for cleavage by proteases such as MTSP1 (or matriptase), endotheliase 1 and urokinase, and a general discussion of properties of the residues.
• proteases such as MTSP1 (or matriptase), endotheliase 1 and urokinase
• peptidic substrates for cleavage by other cell surface proteases, or a soluble, shed or released form thereof can be similarly designed by identifying peptidic substrates for the selected protease and then preparing conjugates that contain such peptidic substrates.
• Amino acid residues for use at the P1 position of the peptidic substrates for use in the conjugates provided herein include Arg, Arg surrogates and Lys.
• Arg surrogates include unnatural amino acids that possess a group or moiety that functions in substantially the same way as the naturally occurring side chain of arginine to achieve substantially the same result (e.g. , acting as the P1 residue in a substrate for a MTSP1 , urokinase or endotheliase) .
• Arg surrogates include, but are not limited to, ⁇ -amino acids that possess as the side chain any of the following: the side chain of homoarginine; guanidinoaminopropyl; guanidinoaminoethyl; (Me) 2 arginine side chain; (Et) 2 arginine side chain; (4- aminomethyDphenylmethyl; 4-amidinophenylmethyl; 4-guanidinophenyl- methyl; or the Arg surrogate is a conformationally constrained arginine analog such as:
• z is 0 or 1 (see, e.g. , Webb et al. (1 991 ) J. Org. Chem. 56:3009); or the side chain is a conformationally constrained arginine side chain analog such as:
• d is an integer from 0 to 5, or 1 to 3; and W is N or CH; or a mono- or di-substituted N-alkyl derivative of the above groups, where alkyl is, in certain embodiments, lower alkyl, such as, for example, methyl.
• the P1 residue is Arg. b.
• the P2 residue 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.
• the P2 residue is selected from Phe, Ser, Gly and Ala.
• the P2 residue is Ser or Ala.
• the P2 residue is Gly or Ala. c.
• the P3 Residue Amino acid residues for use at the P3 position of the conjugates provided herein include Arg, Lys, Gin, Quat, Arg surrogates, Ser, Thr, hSer, dSer, Pro, (hS)Gly, Tyr, 4,4-dimethylThr, Asn, Met(O 2 ), Quat 2 , Quat 3 , Quat 4 and Quat 5 .
• the P3 residue is selected from Arg, Lys, Gin, Quat and Arg surrogates.
• Arg surrogates include those described above for the P1 residue.
• the P3 residue is Gin or Ser. d.
• the P4 residue is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe, Val, N,N-dimethylGly, ⁇ -Ma, Cys(Me), Gin, t-butylGly and nVal.
• the P4 residue is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val.
• the P4 residue is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Phe or Val.
• the P4 residue is Arg or Gly. e.
• the peptidic substrates used in the conjugates contain a P5 and, optionally, a P6 residue.
• P5 residues include lie, Arg and Arg surrogates.
• P5 residues include Arg and Arg surrogates.
• Arg surrogates include those described above for the P1 residue.
• P6 residues include, for example, Leu, Val and Arg.
• P6 residues include, for example, Leu. f. The PI ' Residue
• the P1 ' residue of the conjugates provided herein is Gly, Ser, Ala, Leu, lie, d-lle, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, Thr or hSer.
• the PT residue of the conjugates provided herein is Gly, Ser, Ala, Leu, lie, d-lle, nLeu, Val, nVal, Aib, Abu, Met or 6- aminohexanoyl.
• the PT residue is Ser, Ala, hSer, Abu, Thr, Met, nLeu or Val.
• the PT residue is Gly or Ala.
• the PT residue is Ser, Ala or Gly.
• the PT residue is Leu, Abu, nLeu, nVal, CHA, hCHA, (hex)Gly, (allyl)Gly, (propargyl)Gly or (cyclopropyl)Ala.
• the PT residue is Ala, Ser, Gly, lie or d-lle. g. The P2' Residue
• the conjugates provided herein possess a P2' residue.
• P2' residues for use herein include, but are not limited to, Gly, Ser, Ala, Leu, lie, d-lle, nLeu, Val, nVal, Aib, Abu, Met, 6- aminohexanoyl, hCHA, CHA, hexylGly, allylGly and Phe.
• P2' residues for use herein include, but are not limited to, Gly, Ser, Ala, Leu, lie, d-lle, nLeu, Val, nVal, Aib, Abu, Met and 6- aminohexanoyl.
• the P2' residue is Ser, hSer, Abu, nLeu, nVal, CHA, hCHA, (allyl)Gly or (hexyl)Gly.
• the P2' residue is Gly or Ala.
• the P2' residue is Leu, Abu, nLeu, nVal, CHA, hCHA, (hex)Gly, (allyl)Gly, (propargyl)Gly or (cyclopropyl)Ala.
• the P2' residues are Ala, Gly, lie or d-lle. h.
• the peptidic substrates used in the conjugates provided herein include a P3' residue.
• P3' residues for use herein include, but are not limited to, Gly, Ser, Ala, Leu, lie, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA and allylGly.
• the P2' residue is Ser, hSer, Abu, nLeu, nVal, CHA, hCHA, (allyl)Gly or (hexyl)Gly.
• P3' residues for use herein include, but are not limited to, Gly, Ser, Ala, Leu, He, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl.
• the P3' residue is Gly or Ala.
• the P3' residue is Leu, Abu, nLeu, nVal, CHA, hCHA, (hex)Gly, (allyl)Gly, (propargyl)Gly or (cyclopropyl)Ala.
• the peptidic substrates used in the conjugates provided herein include a P4' residue.
• P4' residues for use herein include, but are not limited to, Gly, Ser, Ala, Leu, lie, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA and allylGly.
• P4' residues for use herein include, but are not limited to, Gly, Ser, Ala, Leu, Me, nLeu, Val, nVal, Aib, Abu, Met and 6- aminohexanoyl.
• the P4' residue is Gly or Ala.
• the P4' residue is Leu, Abu, nLeu, nVal, CHA, hCHA, (hex)Gly, (allyl)Gly, (propargyl)Gly or (cyclopropyl)Ala. In another embodiment, the P4' residue is Leu. j. Caps
• the N-terminus of the peptidic substrate optionally is capped with an acyl, sulfonyl or carbamoyl derivative.
• the cap is chosen, in certain embodiments, to increase the hydrophilicity of the conjugate.
• a non- hydrophilic N-terminal cap such as an acetyl group, can be used.
• the N-terminal amino acid is modified with a hydrophilic blocking group.
• a hydrophilic blocking group are chosen based upon the presence of hydrophilic functionality.
• Such blocking of the terminal amino group can also reduce or eliminate the enzymatic degradation of such peptidyl therapeutic agents by the action of exogenous amino peptidases which are present in the blood plasma of warm blooded animals.
• N-Terminal blocking groups that increase the hydrophilicity of the conjugates and therefore increase the aqueous solubility of the conjugates include, but are not limited to, hydroxylated alkanoyl, polyhydroxylated alkanoyl, polyethylene glycol, glycosylates, sugars and crown ethers. ln certain embodiments herein, the N-terminal blocking group is one of the following: a)
• 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 r 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 alkyl; d) substituted C,-C 6 alkyl wherein the substituent on the substituted C,-C 6 alkyl is selected from unsubstituted or substituted aryl, unsubstituted or substituted heterocyclyl, 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 combined to form -(CH 2 ) f - where one of the carbon atoms optional
• R 3 is selected from: hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclyl, C,-C 6 alkyl and C 3 -C 10 cycloalkyl;
• R 4 is selected from: unsubstituted or substituted aryl, unsubstituted or substituted heterocyclyl, C C 6 alkyl and C 3 -C 10 cycloalkyl; e is 0, 1 or 2; a is 1 , 2, 3 o r 4; b is zero or an integer between I and 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.
• R 1 and R 2 are each independently hydrogen, OH, C C 6 alkyl, C ⁇ -C 6 alkoxy, C ⁇ -C 6 aralkyl or aryl.
• a is 1 , 2, 3 or 4;
• b is 0 or an integer between 1 and 1 00; and
• c is 0 to 1 0, provided that if b is 0, c is 1 to 10.
• N-terminal cap (X ⁇ ) is hydrogen, or (i), (ii), (iii) or (iv) as follows: (i)
• R 1 and R 2 are each independently hydrogen, C,-C 6 alkyl and aryl; a is 1 , 2, 3 or 4; a' is 0, 1 , 2 or 3; b is 0 or an integer between 1 and 14; and c is 0 or 1 , provided that if b is 0, c is 1 .
• X n is R 30 0-C(0)-, R 31 R 32 N-C(O)-, R 33 (CH 2 ) k C(O)- or H-C(O)-; where k is an integer from 1 to 4, or is 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.
• X n is hydrogen, acetyl, hydroxyacetyl, 2,3-dihydroxypropionyl, 2,3,4-trihydroxybutanoyl, PEG( 1 ), PEG(2), PEG(4), PEG(6), PEG(14), PEG(1 5), PEG(1 6), PEG(1 7), PEG( 1 8) or PEG(1 9) .
• X n is hydrogen, acetyl, hydroxyacetyl, succinyl, quinyl, gallyl, 4-imidazolylacetyl, cotininyl, 3- phosphonylpropionyl, gulonyl, 4-phosphonylbutyryl, glutaryl, ethoxysquaryl or PEG(2) .
• X n is hydrogen, acetyl, -C(O)NH 2 , HOCH 2 CH 2 C(O)-, diaminopropanoyl, or NH 2 -(CH 2 ) 5 -C(O)-.
• X n is hydrogen, acetyl, succinyl, glutaryl, PEG(2) or malonyl.
• 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-methylenedioxyphenyIacetyl, 2- pyridylacetyl, phenoxyacetyl, phenylacetyl, methoxyacetyl, 2- methoxyethoxycarbonyl, 2-methoxyethoxyacetyl, 3-phenyl-2- hydroxypropanoyl, pent-4-yn
• the N-terminal cap (X n ) is acetyl, glutaryl, or related acyl, sulfonyl or carbamoyl derivatives.
• Capping groups include, but are not limited to, a simple N-acetyl residue through larger fragments that impact the overall physicochemical properties of the conjugate. Appropriate choice of the capping group allows delivery of either relatively hydrophilic or hydrophobic molecules to a target site.
• X n is acetyl.
• the C-terminus of the peptidic substrate is a carboxylic acid or a carboxamide derivative.
• Appropriate choice of the capping group allows delivery of either relatively hydrophilic or hydrophobic molecules to a target site.
• X c together with the carbonyl group to which it is attached, forms a carboxamide derivative of formula -C(O)NR d R e , where R d and R e are selected from (i) or (ii) as follows:
• R d and R e are each independently hydrogen, C.
• R d and R e are not hydrogen or C,-C 6 -alkyl
• R d and R e together form a -CH 2 CH 2 OCH 2 CH 2 - diradical; b is zero or an integer between I and 1 00; and c is 0 or 1 , provided that if b is zero, c is 1 .
• R d is hydrogen and R e is 2-hydroxyethyl.
• the conjugates optionally contain a linker (i.e. , L, L 1 or L 2 of formulae I, II and III) that covalently binds the peptidic substrate to the therapeutic agent.
• the linkers are any that result in a conjugate in which the peptidic portion is a substrate for a cell surface protease and the therapeutic agent is substantially inactive when n the conjugate and is released in active form or in a form subsequently activated by the cell, tissue or environment of the targeted tissue.
• the linker can include of carbohydrate, peptide, diamine, arylamine, and/or hydrocarbon core structures.
• Linkers are desirably synthetically accessible, provide shelf-stable products, and do not possess any intrinsic biological activity that interferes with the conjugates activity. They can add desirable properties such as increasing solubility or serving to aid in trafficking the cleaved therapeutic agent in the cell.
• some linkers will be enzymatically cleaved in vitro and in vivo, and fragment to release active therapeutic agent or activatable therapeutic agent.
• the linker is, for example, a sugar and/or a peptide, such the aminosugar daunosamine.
• linkers for use herein include, but are not limited to, a biscarbonyl alkyl diradical whereby an amine moiety on the therapeutic agent is connected with the linker unit to form an amide bond and the amino terminus of the peptidic substrate is connected with the other end of the linker unit also forming an amide bond.
• a diaminoalkyl diradical linker unit whereby a carbonyl moiety on the cytotoxic agent is covalently attached to one of the amines of the linker unit while the other amine of the linker unit is covalently attached to the C-terminus of the peptidic substrate, also can be useful.
• linker units which are stable to the physiological environment when not in the presence of a cell surface protease, but are cleavable upon the cleavage of the cell surface protease proteolytic cleavage site, are intended for use herein. Furthermore, linker units can be utilized that, upon cleavage of the cell surface protease proteolytic cleavage site, remain attached to the therapeutic agent but do not significantly decrease the therapeutic activity of such a post-cleavage therapeutic agent derivative when compared with an unmodified therapeutic agent.
• the linker is a diamine containing a cyclic alkyl moiety and, in certain embodiments, the diamine contains a bicycloalkylene moiety.
• diamine linkers include, but are not limited to, 1 ,4-bis(aminomethyl)cyclohexane, 1 ,4-bis(aminomethyI)- cycloheptane, 1 ,3-bis(aminomethyl)cycIopentane, 1 -amino-4-(amino- methyl)cyclohexane, 1 ,4-diaminocyclohexane and 1 ,4-bis(aminomethyl)- bicyclo[2.2.2]octane.
• linkers include 1 , -diaminoalkanes, including, but not limited to, 1 ,3-diaminopropane, and 1 , ⁇ /-dicarbonylalkanes, including, but not limited to, oxalic, malonic, succinic, glutaric, adipic and pivalic acids.
• linkers for use in the conjugates provided herein include self-eliminating linkers such as those of the following formulae:
• the therapeutic agents are any agents, including proteins and polypeptides, small molecules and other molecules that " possess or potentiate a desired biological activity.
• Such molecules include cytotoxic agents, such as, but are not limited to, a toxin such as abrin, ricin A, pseudomonas exotoxin, shiga toxin, diphtheria toxin and other such toxins and toxic portions and/or subunits or chains thereof; proteins such as, but not limited to, tumor necrosis factor, -interferon, -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin- 1 (IL-1 ), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor (G-CSF), erythropo
• IL-1 interleukin-1
• IL-2 interleuk
• therapeutic agents include, but are not limited to, anti- tumor, anti-angiogenic, pro-apoptotic, anti-cancer and anti-mitotic agents. These are conjugated, optionally via a linker, to a substrate, such as peptidic substrate, which is a substrate for the protease.
• cytotoxic agents that include, in general, but are not limited to, alkylating agents, toxins, antiproliferative agents and tubulin binding agents.
• Classes of cytotoxic agents for use herein include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, the maytansinoids, the epothilones, the taxanes and the podophyllotoxins.
• Exemplary members of those classes include, for example, doxorubicin, carminomycin, daunorubicin, aminopterin, methofrexate, methopterin, dichloro-methotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine, maytansinol, epothilone A or B, taxotere, taxol and the like.
• Such therapeutic agents include estramustine, cisplatin, combretastatin and analogs, and cyclophosphamide.
• estramustine cisplatin
• combretastatin and analogs cyclophosphamide.
• One skilled in the art can make chemical modifications to the desired therapeutic agent in order to make reactions of that compound more convenient for purposes of preparing the conjugates.
• R 12 is amino or hydroxy;
• R 7 is hydrogen or methyl;
• R 8 is hydrogen, fluoro, chloro, bromo or iodo;
• R 9 is hydroxy or a moiety which completes a salt of the carboxylic acid.
• R 11 is hydroxy, amino, C,-C 3 alkylamino, di(C C 3 alkyl)amino, C 4 -C 6 polymethylene amino, -NHCH 2 CH 2 CH 2 CH 2 NH-C(NH)NH 2 or -NHCH 2 CH 2 CH 2 S + (CH 3 ) 2 .
• R 13 is hydrogen or methyl
• R 14 is methyl or thienyl or a phosphate salt thereof.
• R 15 is H, CH 3 or CHO; and R 18 is H, and one of R 6 and R 7 is ethyl and the other is H or OH; when R 17 and R 18 are taken together with the carbons to which they are attached, they form an oxirane ring in which case R 16 is ethyl; and
• R 19 is hydrogen, alkyl)-CO, or chlorosubstituted (C C 3 alkyl)-CO.
• the conjugates provided herein where the therapeutic agent is the vinca alkaloid vinblastine include those of formula:
• peptidic substrate is as described above for formulae I and II;
• L is a linker such as -NH-(CH 2 ) U -T-(CH 2 ) U -NH-;
• R 1 and R 2 are independently hydrogen, OH, C C 6 alkyl, C r C 6 alkoxy, aralkyl and aryl;
• R 1 a is C r C 6 -alkyl, hydroxylated C 3 -C 8 -cycloalkyl, polyhydroxylated C 3 -C 8 -cycloalkyl, hydroxylated aryl, polyhydroxylated aryl or aryl,
• R 19 is hydrogen, (C r C 3 alkyl)-CO, or chlorosubstituted (C.,-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 between 1 and 100; c is 0 or 1 , provided that if b is zero, c is 1 ; g is 1 , 2 or 3; u is 0, 1 , 2 or 3; or a pharmaceutically acceptable derivative thereof.
• R 21 is a base of one of the formulae:
• R 22 is hydrogen, methyl, bromo, fluoro, chloro or iodo
• R 23 is -OH or -NH2
• R 2 is hydrogen, bromo, chloro or iodo.
• R a is -CH 3 , - CH 2 0H, -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-methoxyethyl amine;
• R 5 is -OH -OTHP or -H;
• R 6 is -OH or -H provided that R6 is not -OH when R 5 is -OH or -OTHP.
• Table 2 which follows, provides a number of anthracycline drugs and their generic or trivial names:
• a daunorubicin is an alternative name for daunomycin
• doxorubicin is an alternative name for adriamycin.
• the therapeutic agent when the therapeutic agent is doxorubicin, it is conjugated to the peptidic substrate via the amino group of the aminoglycoside moiety of doxorubicin. m. Maytansinol
• R is PhC(O) or t-BuOC(O).
• the peptidic substrate is conjugated to the secondary hydroxyl group of the cyclohexane moiety of taxol.
• Ribosome-inactivating proteins which include ricin, abrin and saporin, are plant proteins that catalytically inactivate eukaryotic ribosomes. RIPS inactivate ribosomes by interfering with the protein elongation step of protein synthesis.
• the RIP saporin hereinafter also referred to as SAP
• SAP has been shown to enzymatically inactivate 60S ribosomes by cleavage of the n-glycosidic bond of the adenine at position 4324 in the rat 28S ribosomal RNA (rRNA).
• Some RIPs such as the toxins abrin and ricin, contain two constituent chains: a cell-binding chain that mediates binding to cell surface receptors and internalization of the molecule; and an enzymatically active chain responsible for protein synthesis inhibitory activity.
• Such RIPs are type II RIPs.
• Other RIPs, such as the saporins are single chains and are designated type I RIPs. Because such RIPs lack a cell-binding chain, they are less toxic to whole cells than the RIPs that have two chains.
• Two chain RIPs are generally used for conjugation herein, unless a single chain is further conjugated to an agent, such as a growth factor that mediates binding and internalization.
• Saponaria officinalis Several structurally related RIP's have been isolated from seeds and leaves of the plant Saponaria officinalis (soapwort) .
• SAP-6 is the most active and abundant, representing 7% of total seed proteins.
• Saporin is very stable, has a high isoelectric point, does not contain carbohydrates, and is resistant to denaturing agents, such as sodium dodecyl sulfate (SDS), and a variety of proteases.
• SDS sodium dodecyl sulfate
• the amino acid sequences of several saporin-6 isoforms from seeds are known and there appear to be families of saporin RIPs differing in a few amino acid residues. Because saporin is a type I RIP, it does not possess a cell- binding chain.
• the conjugates provided herein are prepared by identifying suitable peptidic substrates for the targeted cell surface protease, or a soluble, shed or released form thereof, and forming a conjugate of the peptidic substrate(s) with a therapeutic agent(s).
• exemplary conjugates, containing peptidic substrates designed, for example, for cleavage by MTSP1 , endotheliase 1 and urokinase, are described.
• a cell surface protease including cell- associated and cell-localized proteases, or a soluble, shed or released form thereof, in or associated with a cell involved in a disease or other conditions of interest, or with a cell present in the vicinity of a cell or tissue involved in or associated with a disease or other condition of interest
• suitable peptidic substrates therefor can be empirically designed and then conjugated to therapeutic agents as exemplified herein.
• the conjugates for use in the compositions and methods provided herein include: Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 46);
• the conjugates are Ac-Leu-Arg-Ala-
• the conjugates for use herein include the following : pyroGlu-Pro-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 78);
• Benzoyl-Val-Gly-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 81 );
• H-D-lle-Pro-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 85);
• H-D-Pro-Phe-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 87); H-D-Val-Leu-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 88);
• Benzoyl-Pro-Phe-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 91 ); H-D-Phe-Pip-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 92);
• H-D-Nle-HHT-Lys-Ala-Ala-(therapeutic agent) (SEQ ID NO: 94);
• H-D-HHT-Ala-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 99);
• H-D-CHT-GIy-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 100);
• MeS0 2 -dPhe-Pro-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 101 ); ⁇ 5-Z-D-Lys-Pro-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 1 02); and
• conjugates for use in the compositions and methods provided herein include: Ac-Arg-Gln-Ser-Arg-Ala-Ala-(therapeutic agent) (SEQ ID NO: 104);
• the conjugates for use in the compositions and methods provided herein include:
• the conjugates are Ac-Leu-Arg-Ala- Quat-Gly-Arg-Ser-Leu-(therapeutic agent) (SEQ ID NO: 1 29); Ac-Leu-Arg-
• the conjugates are N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoe
• the conjugates for use herein include the following: pyroGlu-Pro-Arg-Ser-Leu-(therapeutic agent) (SEQ ID NO: 145);
• H-D-Pro-Phe-Arg-Ser-Leu-(therapeutic agent) (SEQ ID NO: 1 54);
• H-D-Phe-Pip-Arg-Ser-Leu-(therapeutic agent) (SEQ ID NO: 159); H-D-Val-Leu-Lys-Ser-Leu-(therapeutic agent) (SEQ ID NO: 1 60);
• H-D-Nle-HHT-Lys-Ser-Leu-(therapeutic agent) (SEQ ID NO: 1 61 );
• H-D-HHT-Ala-Arg-Ser-Leu-(therapeutic agent) (SEQ ID NO: 1 66);
• H-D-CHT-Gly-Arg-Ser-Leu-(therapeutic agent) (SEQ ID NO: 1 67);
• MeS0 2 -dPhe-Pro-Arg-Ser-Leu-(therapeutic agent) (SEQ ID NO: 168); -Z-D-Lys-Pro-Arg-Ser-Leu-(therapeutic agent) (SEQ ID NO: 1 69); and
• conjugates for use in the compositions and methods provided herein include:
• conjugates provided herein include:
• conjugates for use in the compositions and methods provided herein include:
• the conjugates are Ac-Leu-Arg-Ala-
• the conjugates for use herein include the following: pyroGlu-Pro-Arg-Ser-Ser-Leu-(therapeutic agent) (SEQ ID NO: 223);
• H-D-lle-Pro-Arg-Ser-Ser-Leu-(therapeutic agent) (SEQ ID NO: 230);
• H-D-Pro-Phe-Arg-Ser-Ser-Leu-(therapeutic agent) (SEQ ID NO: 232); H-D-Val-Leu-Arg-Ser-Ser ⁇ Leu-(therapeutic agent) (SEQ ID NO: 233);
• H-D-Nle-HHT-Lys-Ser-Ser-Leu-(therapeutic agent) SEQ ID NO: 239
• Pyr-Arg-Thr-Lys-Arg-Ser-Ser-Leu-(therapeutic agent) SEQ ID NO: 240
• H-D-HHT-Ala-Arg-Ser-Ser-Leu-(therapeutic agent) SEQ ID NO: 244
• H-D-CHT-Gly-Arg-Ser-Ser-Leu-(therapeutic agent) SEQ ID NO: 245)
• MeS0 2 -dPhe-Pro-Arg-Ser-Ser-Leu-(therapeutic agent) (SEQ ID NO: 246); -Z-D-Lys-Pro-Arg-Ser-Ser-Leu-(therapeutic agent) (SEQ ID NO: 247); and
• conjugates for use in the compositions and methods provided herein include:
• conjugates provided herein include:
• conjugates provided herein include:
• Ae-Gly-dSer-Ala-Arg-Ser-Ala-(therapeutic agent) (SEQ ID NO: 569);
• conjugates provided herein are selected from: Ac-R-Q-G-R-S-L-(therapeutic agent) (SEQ ID NO: 491 );
• MeOCO-Quat2-G-R-S-L-(therapeutic agent) (SEQ ID NO: 483);
• MeOCO-Quat3-G-R-S-L-(therapeutic agent) (SEQ ID NO: 484); MeOCO-Quat-G-R-S-L-(therapeutic agent) (SEQ ID NO: 485);
• MeOCO-Quat4-G-R-S-L-(therapeutic agent) (SEQ ID NO: 486);
• MeOCO-Quat5-G-R-S-L-(therapeutic agent) (SEQ ID NO: 487);
• MeOCO-Quat2-G-R-S-S-L-(therapeutic agent) (SEQ ID NO: 488);
• MeOCO-Quat4-G-R-S-L-(therapeutic agent) (SEQ ID NO: 489); MeOCO-Quat2-G-R-S-L-(therapeutic agent) (SEQ ID NO: 490);
• conjugates provided herein are selected from:
• MeOCO-T-G-R-S-nL-(therapeutic agent) (SEQ ID NO: 392);
• MeOEtCO-T-G-R-S-nL-(therapeutic agent) SEQ ID NO: 394
• MeO(EtO)2Ac-T-G-R-S-nL-(therapeutic agent) SEQ ID NO: 395
• MeOAc-T-G-R-S-nL-(therapeutic agent) (SEQ ID NO: 401);
• PhAc-T-G-R-S-nL-(therapeutic agent) (SEQ ID NO: 402);
• MeOEtOCO-T-G-R-S-nL-(therapeutic agent) (SEQ ID NO: 403); MeOEtOAc-T-G-R-S-nL-(therapeutic agent) (SEQ ID NO: 404);
• HOOCButa-T-G-R-S-nL-(therapeutic agent) (SEQ ID NO: 405);
• EtOCO-T-G-R-S-nL-(therapeutic agent) SEQ ID NO: 407;
• NapAc-T-G-R-S-nL-(therapeutic agent) (SEQ ID NO: 410); iBoc-T-G-R-S-nL-(therapeutic agent) (SEQ ID NO: 411);
• HOAc-T-G-R-S-nL-(therapeutic agent) (SEQ ID NO: 412);
• MeSucc-T-G-R-S-nL-(therapeutic agent) (SEQ ID NO: 413); N,N-diMeGly-T-G-R-S-nL-(therapeutic agent) (SEQ ID NO: 414);
• HCO-T-G-R-S-nL-(therapeutic agent) (SEQ ID NO: 416);

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