JP2009510166A - Latency procytotoxins and their uses - Google Patents

Abstract

  Disclosed are procytotoxins comprising a latency-related peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency-related peptide and the cytotoxic peptide. Also disclosed are compositions and methods for selectively destroying target cells. Also disclosed are compositions and methods for treating cancer in patients. Also disclosed are compositions and methods that provide latency to cytotoxins.

Description

  The present invention relates to methods and compositions for selectively destroying target cells. In particular, the present invention relates to methods for producing and using procytotoxins that can be specifically converted to cytotoxins by target cells, such that the target cells are destroyed by activated cytotoxins.

  Current methods of tumor treatment, particularly cancer treatment, are still not optimal and often involve severe complications. In fact, almost all known treatments have serious side effects, most often due to lack of specificity and integrity in the destruction or removal of tumor or cancer cells.

For example, surgery is a common procedure for removing cancer cells from a patient, but the removal is often incomplete and impairs the patient's appearance or prevents normal body function. Similarly, chemotherapy and radiation therapy often indiscriminately destroy normal cells and produce undesirable side effects, but many cancer cells remain unaffected. Chemotherapeutic agents, particularly antimetabolites, are effective to varying degrees against cancer cells that are continuously undergoing mitosis or are ready for mitosis, but are in the stationary phase (G ₀ ). It is not effective against cancer cells.

  Cancer treatment is effective if cancer cells can be removed from the patient's body as completely as possible. To achieve this goal, continuous or continuous doses are administered to the patient. Since most available chemotherapeutic agents are also toxic to normal cells, the dose of cytotoxic agent is adjusted to an acceptable range that achieves maximum destruction of malignant cells, and the rate of tumor regrowth is Must be between doses that do not exceed kill. Therefore, in order to achieve high efficiency with reduced side effects, chemotherapeutic agents must have high target cell specificity and high target cytotoxicity or potential. Despite many studies on cancer treatment, currently available treatments are ineffective in a significant number of cases.

  Therefore, there is a need for improved cancer treatments and many methods that are independent of the cell cycle of cancer cells. The present invention fulfills these needs and provides other related advantages.

  The present invention relates to methods and compositions for selectively destroying target cells. Specifically, the present invention provides a procytotoxin (a cytotoxin is maintained in an inactive form) that can be specifically converted to a cytotoxin by the target cell so that the target cell is destroyed by the activated cytotoxin. It relates to methods of manufacture and use.

  Accordingly, it is an object of the present invention to overcome some or all of the above-mentioned drawbacks associated with treating cancer, particularly with conventional therapies. To this end, compositions and methods for producing procytotoxins are described. Typically, a procytotoxin is provided that consists of a cytotoxic agent linked by a peptide bond to, for example, a latency associated peptide (LAP), the peptide bond being sensitive to cleavage by a tumor-specific protease Is done. Therefore, this procytotoxin can be expressed as VYX, where V is a cytotoxic agent, Y is a proteolytic cleavage site, and Y is LAP. The cytotoxic agent may be, for example, a cytotoxic peptide. The peptide bond may be a proteolytic cleavage site. The procytotoxin of the present invention may have a target molecule bound to the N-terminus and / or C-terminus of the cytotoxic peptide.

  In order to enhance its therapeutic effectiveness, the cytotoxin is modified according to the present invention so that the cytotoxin is in a non-toxic form known as procytotoxin. Procytotoxin is a cytotoxic agent that can be rendered non-toxic by providing a protective layer around the cytotoxic agent, and is hereinafter referred to as “caging”. LAP can provide a protective “cage” around the pharmaceutically active agent, thereby shielding the active agent and preventing or blocking its interaction with other molecules on the cell surface or molecules important for its activity. To do. The procytotoxin may be a cytotoxic peptide that can be rendered non-toxic by sterically inhibiting the formation of conformations that confer charge neutralization and / or peptide toxicity. Specifically, the procytotoxin may comprise a cytotoxic peptide linked to LAP by a peptide bond, said peptide bond being sensitive to cleavage by a tumor specific protease. This LAP prevents cytotoxic peptides from forming an active conformation. For example, when procytotoxin contacts a target cell, a tumor-specific protease, such as a protease present in the tumor cell membrane or extracellular matrix, cleaves the peptide bond between LAP and cytotoxin, thereby causing a cytotoxic peptide Makes it possible to form a pore conformation and destroy the cell membrane. Accordingly, a method for producing a procytotoxin comprising modifying a cytotoxic peptide to include LAP is described.

  Also provided is a pharmaceutical composition comprising the disclosed procytotoxin and a pharmaceutically suitable excipient.

  Furthermore, a method for selectively destroying target cells is presented. The disclosed methods typically involve contacting a target cell with a procytotoxin having a cytotoxic agent bound to LAP by a peptide bond, the peptide bond being sensitive to cleavage by a target-specific protease. is there. The target cell may be a microcirculation system surrounding the cancer cell and a cell contained in the cancer cell.

  Further provided are methods of treating a patient's cancer. In general, this method involves administering to a patient a therapeutically effective amount of procytotoxin. In certain embodiments, the procytotoxin may be based on amoebopore, melittin or a cytolytic peptide derived therefrom.

  Further methods are provided by conferring latency on cytotoxins. In general, the method involves covalently coupling a latency-related peptide and proteolytic cleavage site with a cytotoxin, wherein the latency-related peptide and proteolytic cleavage site provide latency to the cytotoxin.

FIG. 1 shows the detection of hLAP-MMP2-melittin in transfected CHO cells. Western blot using 30 μg of LAP CHO (transfected with hLAP-MMP2-melittin) and CHO (untransfected) lysates (searching for μg / μl throughout) followed by SDS PAGE followed by hLAP antiserum Was analyzed. 50 ng hLAP was used as a positive control.
FIG. 2 shows the induction of LAP-MMP2-melittin production by methotrexate in transfected CHO cells. CHO cells were transfected with LAP-MMP2-melittin plasmid DNA in the presence of normal transfection medium. After transfection, the cells were cultured in a medium containing 10% FBS and 50 nM MTX. The amount of FBS was gradually reduced to 1% with the medium. The amount of MTX in the medium was then increased to induce the cells to produce protein. Lysates were obtained from cells at various concentrations of MTX and analyzed by Western blot using SDS PAGE followed by hLAP antiserum. For each MTX concentration, approximately 30 μg of lysate was analyzed and 50 ng hLAP (Western blot positive control) was analyzed.
FIG. 3 shows analysis of lysates and supernatants from CHO cells transfected with hLAP-MMP2-melittin under non-denaturing conditions. The hLAP fusion protein (hLAP-MMP2-melittin) present in the medium was concentrated before analysis by affinity chromatography. 30 μg of fusion protein lysate was analyzed and 50 ng hLAP was used as a positive control. All samples were analyzed by Western blot using Native PAGE followed by hLAP antiserum.
FIG. 4 shows an analysis of partially purified hLAP-MMP2-melittin fusion protein. Purified hLAP-MMP2-melittin fusion protein was analyzed by Western blot using SDS PAGE followed by hLAP antiserum. The fusion protein was partially purified by size exclusion chromatography on a Waters HPLC system.

  Before disclosing and describing the compounds, compositions, products, devices and / or methods of the present invention, unless otherwise specified, they are not limited to, or specifically specified by, specific synthetic methods or specific recombinant biotechnology methods. It should be understood that it is not limited to a particular reagent unless it is not, and of course may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

Definitions As used in the specification and claims, the singular forms “a”, “an”, “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, “a pharmaceutical carrier” includes a mixture of two or more such carriers and the like.

  Ranges may be indicated herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, using the antecedent “about,” it will be understood that the particular value forms another embodiment. Furthermore, it will be understood that the endpoints of each range are both significant with respect to the other endpoints and independently of the other endpoints. It will also be appreciated that there are a number of values disclosed herein, and that each value is also disclosed herein as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Also, when a value is disclosed, there is also a possible range between that value “less than or equal to”, “greater than or equal to that value” and values, as will be appreciated by those skilled in the art. It will also be understood that it is disclosed. For example, if the value “10” is disclosed, “less than or equal to 10” as well as “greater than or equal to 10” are also disclosed. It will also be appreciated that the data throughout this specification is provided in a number of different formats, and this data indicates the endpoint and origin, and the range of any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, to 10 and 15 greater than 10 and 15, equal to or greater than 10 and 15, less than 10 and 15 It will be understood that equals or less than and equals 10 and 15 is considered to be disclosed between 10 and 15 as well.

  In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

  “As desired” or “as desired” means that the described event or situation may or may not occur, and that the description includes examples where the event or situation occurs or does not occur.

  A “primer” is a subset of probes that can support manipulation by one type of enzyme and can hybridize to one target nucleic acid so that manipulation by the enzyme can occur. Primers can be produced by any combination of nucleotides or nucleotide derivatives or analogs available in the art that do not prevent enzymatic manipulation.

  A “probe” is a molecule that can interact with a target nucleic acid, typically in a sequence-specific manner, for example by hybridization. Nucleic acid hybridization is well understood in the art and described herein. Typically, probes can be made with any combination of nucleotides or nucleotide derivatives or analogs available in the art.

  Unless otherwise specified by the context, the term “contacting the procytotoxin with the target cell” means that the protease expressed by the target cell or present in the extracellular matrix cleaves the proteolytic cleavage site of the procytotoxin. Capable of releasing LAP from the cytotoxin, thereby allowing the cytotoxin to become active, leading the procytotoxin and the target cell sufficiently close to each other to perform their function in the target cell Means.

  With respect to the terminology used herein, those skilled in the art will understand that a “standard” peptide bond is formed between the α-carboxyl group of one amino acid and the α-amino group of the next amino acid in the peptide chain, and the peptide sequence is It will be appreciated that it is read from its amino terminus to its carboxyl terminus.

  Unless otherwise specified by context, the term “RGD” not only means the peptide sequence Arg-Gly-Asp, but this generally refers to the class of minimal or core peptide sequences that mediate specific interactions with integrins. To do. Thus, “RGD target sequence” encompasses all types of integrin binding regions.

  Unless otherwise specified by context, the term “NGR” means not only the peptide sequence Asn-Gly-Arg, but also the minimal or core that mediates specific interactions with integrin signal sequences found on cells of the vasculature. Generally refers to the class of peptide sequences. For example, certain NGR target sequences can be used to bind specific integrins and can be used as useful target molecules for vascular endothelial cells and other cells of the neo-vasculature. Thus, “NGR target sequence” encompasses all types of integrin binding regions.

  Throughout this application, various documents are referenced. The disclosures of these publications are hereby incorporated herein by reference in their entirety in order to more fully describe the state of the art in the technical field of the present invention. The disclosed references are also individually and specifically incorporated as references in this specification with respect to the substances contained in the references mentioned in the text on which the references depend.

Compositions Disclosed are the components used to prepare the disclosed compositions as well as the compositions themselves to be used within the scope of the methods disclosed herein. Where these and other substances are disclosed herein and combinations, subsets, interactions, groups, etc. of these substances are disclosed, specific references to each of the various individual and collective combinations and these Although the permutation of the compounds may not be specifically disclosed, it will be understood that each is specifically contemplated and described herein. For example, when a particular procytotoxin is disclosed and discussed and a number of modifications that can be produced on a number of molecules containing the procytotoxin are discussed, all combinations and permutations of procytotoxins and differences are specifically indicated. Modifications that are possible unless specifically noted are specifically contemplated. Thus, if a class of molecules A, B and C is disclosed, as well as examples of classes D, E and F and combinations A to D are disclosed, each is individually Each of which is individually or collectively intended meaning combinations, A to E, A to F, B to D, B to E, B to F, C to D, C to E, and C-F is considered disclosed. Likewise, all subsets or combinations of these are also disclosed. Thus, for example, the subgroups A-E, B-F, and C-E are considered disclosed. This concept applies to all aspects of this application and includes, but is not limited to, process steps for making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it will be understood that each of these additional steps may be performed in any particular embodiment or combination of disclosed method embodiments.

  Methods and compositions for selectively destroying certain target cells are disclosed. In particular, the present invention relates to methods of making and using procytotoxins that can be converted to cytotoxins at the site of tumor cells such that the tumor cells are destroyed by activated cytotoxins.

  Accordingly, it is an object of the present invention to overcome some or all of the above-mentioned drawbacks associated with treating cancer, particularly with conventional therapies. To this end, compositions and methods for producing procytotoxins are described.

  Typically, a procytotoxin is provided that is composed of a cytotoxic agent linked by a peptide bond to a latency associated peptide (LAP), the peptide bond being sensitive to cleavage by a tumor specific protease. Thus, procytotoxin can be represented as VYX, where V is a cytotoxic peptide, Y is a proteolytic cleavage site, and Y is LAP. The cytotoxic agent may be a cytotoxic peptide. This peptide bond may be a proteolytic cleavage site. The procytotoxin of the present invention may have a target molecule bound to the N-terminus and / or C-terminus of this cytotoxic peptide.

  The cytotoxic peptide may be a cytolytic peptide. Cytotoxins include, but are not limited to, type I bacterial exotoxins, type II bacterial exotoxins, type III bacterial exotoxins, Ae I, sea anemone cytolysin, erolidine, amatoxin, entamoeba dispar Derived from amoebapore, amoebapore homolog, brevinin-1E, brevinin-2E, barbatridine, enterococcus faecalis cytolysin, delta hemolysin, diphtheria toxin, Vibrio cholerae (Vibrio cholerae) Cytolysin, Equinatoxin, Aeromonas hydrophila enterotoxin, Et Esculentin, granulysin, Vibrio parahaemolyticus hemolysin, Streptococcus intermedius, intermediary lysinovirus, intermedilinovirus Actinobacillus actinomycetemcomitans leukotoxin, magainin, melittin, cell membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment, neokyotorphin fragment 1 2, Neo-Kyotorphine fragment 3, Neo-Kyotorphin fragment 4, NK-lysine, paradaxin, perforin, clostridial perfringens perfringolysin O, theta toxin, farollicin, farotoxin, streptolysin And pore-forming cell-lytic peptide analogs and pore-forming cell-lytic peptide derivatives.

  For example, type I bacterial exotoxins can be used as cytotoxins. In addition, type I bacterial exotoxins can be fused to tumor cell cell membranes or target regions of the tumor microenvironment.

  Type II bacterial exotoxins can also be used as cytotoxins. Furthermore, the type III bacterial exotoxin can retain its own transmembrane region or the toxin can be fused with a different region such as TAT.

  An example of a procytotoxin has the following structure: (1) Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-IIe-Ser-Trp-Ile-Lys-Arg-Lys -Arg-Gln-Gln- [Y]-(X), and (2) Gly-Phe-Ile-Ala-Thr-Leu-Cys-Thr-Lys-Val-Leu-Asp-Phe-Gly-Ile-Asp -Lys-Leu-Ile-Gln-Leu-Ile-Glu-Asp-Lys- [Y]-(X), (X) is LAP, [Y] is proteolytic that can be oriented in either direction It is a sex cleavage site.

  This LAP may be, for example, a precursor peptide of TGFβ-1, 1.2, 2, 3, 4 or 5. The LAP may also be a precursor peptide of human TGFβ-1, 2, or 3 (SEQ ID NOs: 23 to 25). The LAP may also be a precursor peptide of chicken TGFβ-4 (SEQ ID NO: 26). The LAP may also be a precursor peptide of frog TGFβ-5 (SEQ ID NO: 27).

  The proteolytic cleavage site may be an MMP cleavage site, a PSA cleavage site or a GGH cleavage site. For example, the MMP proteolytic cleavage site may be an MMP 1, 2, 3, 7, 8, 9, or MT-1-MMP cleavage site. The MMP proteolytic cleavage site may be any MMP proteolytic cleavage site listed in SEQ ID NOs: 28-100.

  In addition, the proteolytic cleavage site may be a PSA cleavage site having the sequence described by Volkel et al. (Engineering of human clotting factor X variants activated by prostate specific antigen, Molecular Biotechnology, January 2005, Vol. 29, No. 1, pp. 19-30 (12)).

  Procytotoxins can further include target molecules. For example, the target molecule can interact, bind, attach, combine, join, and link to or to the target cell or tissue. More particularly, the target molecule can be a neovascular target sequence of an anti-fibronectin ED-B antibody. The target molecule can be an RGD or NGR target sequence as described below. Any molecule that can target a specific tissue can be used as a target molecule for the procytotoxin of the present invention. For example, target molecules include human epithelial cell mucin (Muc-1; a 20 amino acid core repeat of Muc-1 glycoprotein is present on breast and pancreatic cancer cells), Ha-ras oncogene product, p53, cancer Fetal antigen (CEA), raf oncogene product, gp100 / pmel17, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, BAGE, GAGE, tyrosinase, gp75, Melan-A / Mart-1, gp100, HER2 / neu, EBV-LMP1 & 2, HPV-F4, 6, 7, prostate specific antigen (PSA), HPV-16, MUM, α-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, ras cancer Gene product, HPV E7, Wilm's tumor antigen-1 It may be an antibody that interacts with antigen-1), telomerase, melanoma ganglioside, or a simple transmembrane sequence.

Procytotoxins can also neutralize charge in addition to containing steric inhibitors of cytotoxin activity. For example, [Gly-Ile-Gly-Ala-Val-Leu-Lys (R) -Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys (R) -Arg-Lys (R) -Arg-Gln-Gln-Gly-Ala-Ile-Gly-Gln-Pro]-(X), wherein (X) is a peptide of this peptide, as described herein LAP that binds to the peptide shown in parentheses by binding, this peptide shown in parentheses can be oriented in either direction, the peptide bond is sensitive to cleavage by tumor-specific proteases, and R is an adjacent lysine unmodified ε- amino group of residue, [ε-γ] -Glu, [ε-γ] -Glu- [α-γ] - (Glu) 1-3, [ε-α] - (Phe _{1-3, [ε-α] -} (Tyr) 1-3, [ε-α] - (Trp) 1-3, [ε-α] - (Lys) 1-3 and [ε-α] - ( Arg) independently selected from the group consisting of _1-3 , [ε-γ] represents the peptide bond between the epsilon amino group of lysine and the gamma carboxyl group of glutamic acid adjacent thereto, and [α-γ] Indicates the peptide bond between the α-amino group of one glutamic acid and the gamma-carboxyl group of the second glutamic acid, [ε-α] indicates the peptide bond between the epsilon amino acid of lysine and the α-carboxyl group of the indicated amino acid, and The subscript number indicates that an additional number of designated amino acids can be joined first by normal peptide bonds.

Procytotoxin To enhance the therapeutic utility of cytotoxin, cytotoxin is modified according to the present invention to make it a non-toxic form known as procytotoxin. In one embodiment of the invention, the procytotoxin is a cytotoxic peptide that can be rendered nontoxic by sterically inhibiting charge neutralization and / or formation of a conformation that renders the peptide toxic. Alternatively, the procytotoxin may be a cytotoxic peptide that can be rendered non-toxic by providing a protective layer or shell (“caging”) around the cytotoxic peptide. This masks the cytotoxic peptide and prevents or prevents its interaction with other molecules on the cell surface or molecules important for its activity. Specifically, the procytotoxin comprises a cytotoxic peptide linked to LAP by a peptide bond, the peptide bond being sensitive to cleavage by a tumor specific protease. LAP prevents cytotoxic peptides from forming an active conformation. For example, when procytotoxin contacts target cells, tumor-specific proteases cleave the peptide bond between LAP and cytotoxin, thereby causing the cytotoxic peptide to form a pore conformation and destroy the cell membrane. Enable. Accordingly, a method for producing a procytotoxin comprising modifying a cytotoxic peptide to include LAP is also described.

  A number of modification methods are available and their adaptability will depend on the structural properties of the cytotoxin. For example, LAP can be added to either the N-terminus and / or C-terminus of the cytotoxic peptide so as to prevent the cytotoxic peptide from forming a conformation that renders the peptide toxic, thus making the cytotoxic peptide non-toxic Make it sex. LAP also forms a protective shell around the cytotoxic peptide (“caging”), thereby shielding the cytotoxic peptide and preventing its interaction with other molecules on the cell surface or molecules important for its activity. Alternatively, LAP can be added to either the N-terminus and / or the C-terminus of the cytotoxic peptide to prevent or block. LAP can also sterically block the formation of the cytotoxin α-helix structure. Furthermore, cytotoxic peptides can be modified to contain negatively charged amino acids, thereby preventing the formation of toxic pore conformation.

  The procytotoxin of the present invention includes a latency-related peptide and a cytotoxic agent, and a proteolytic cleavage site is provided between the latency-related peptide and the cytotoxic agent. In certain embodiments, the cytotoxic agent may be a cytotoxic peptide or a cytolytic peptide as described above. In other embodiments, the cytotoxic agent may be melittin, a melittin analog, or a melittin derivative.

  For example, the procytotoxin of the present invention has the following structure: Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser -Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln- [Y]-(X), where (X) is LAP and [Y] can be oriented in either direction A cleavage site. The LAP may be selected from the group consisting of TGFβ-1, 2, 3, 4 or 5 precursor peptides. The LAP can also be a precursor peptide of human TGFβ-1, 2, or 3 (SEQ ID NOs: 23-25). The LAP may also be a precursor peptide of chicken TGFβ-4 (SEQ ID NO: 26). The LAP can also be a precursor peptide of frog TGFβ-5 (SEQ ID NO: 27).

  This proteolytic cleavage site may be an MMP cleavage site, a PSA cleavage site or a GGH cleavage site. For example, the MMP proteolytic cleavage site can be an MMP 1, 2, 3, 7, 8, 9 or 10 cleavage site. In one embodiment, the MMP cleavage site is an MMP2 cleavage site. Cleavage with a target-specific protease in [Y] can yield melittin peptides with a few additional amino acids that do not prevent pore formation at the C-terminus.

  Also contemplated in the present invention are target molecules that add another selectivity criterion. This target molecule can direct procytotoxin into or near the cell, as well as acting as a LAP to maintain the cytolytic peptide in an inactive conformation. The target molecule of the present invention can be selected from the group consisting of a signal sequence or an antibody. In certain embodiments, the target molecule may be an RGD target sequence.

  It will therefore be appreciated that charge determinants are also important in addition to or instead of steric determinants. If the positive charge of the pore-forming peptide is included in its cytolytic activity, removing / neutralizing the charge makes the peptide non-toxic. Even this partial neutralization of the positive charge is considered effective. Thus, in addition to removing / neutralizing the positive charge of the pore-forming peptide, it is also disclosed herein to disrupt the alpha-helical structure of cytotoxin by sterically blocking the formation of the structure. Is done. Thus, even if neutralization is not achieved, some of the modifications described below will result in steric changes that inactivate the toxin to create a protoxin.

  The procytotoxic peptides of the present invention can be chemically synthesized by standard solid phase methods using protection, deprotection and cleavage techniques and reagents appropriate for each specific amino acid or peptide. A combination of manual and automated (eg APPLIED BIOSSYSTEM.430A) solid phase methods can be used for the synthesis of the novel peptides of the invention. For background on solid phase methods, see Andreu et al. (1983) Proc. Natl. Acad. Sci USA 80: 6475-6479; Andreu et al. (1985) Biochemistry 24: 1683-1688; Fink et al. (June 1989) Int. J. et al. Peptide Protein Res. 33: 412-421; Fink et al. (1989) J. MoI. Biol. Chem. 264: 6260-6267, each of which is incorporated herein by reference.

  The in vivo stability of the procytotoxin of the present invention can be improved by adding a D amino acid to either the N-terminus or C-terminus that does not have a γ-bound glutamate residue. Some in vivo instabilities may be advantageous as they may reduce the risk of possible adverse side effects that can occur when procytotoxin is converted to cytotoxin. This treatment is particularly useful with the products of the invention used under parenteral or oral conditions where these products are subject to hydrolysis by naturally occurring enzymes before performing the desired physiological function. is there.

Cytotoxic peptides Many natural and synthetic cytotoxic peptides are known in the art. Some are useful as therapeutic agents against pathogenic bacteria and other types of microorganisms, which can be isolated from insects, frogs and other animals. Specific examples include alamethicins, atacins, bactenecins, cecropine (see Table 1, cecropin A, B and C amino acid sequences), and Bacillus thuringiensis CytA. And CytB, defensins, enterocin L50 (pediocin L50), lantibiotics, magainin, PGLa, protegrins, protegrins Including.

Cytolytic peptide In certain embodiments, the cytotoxic peptide is a cytolytic peptide. Cytolytic peptides, also known as pore-forming or channel-forming peptides, typically disrupt cell membranes as soon as they contact, causing cell lysis and cell death. Many naturally occurring cytolytic peptides from microorganisms, insects and higher animals are generally known. They are often called hemolysin because they lyse red blood cells and other eukaryotic cells. These toxins are type I bacterial exotoxins, type II bacterial exotoxins, type III bacterial exotoxins, Ae I, and other cytolysins of sea anemones, erolysin, amatoxin, amoeba pores from enthamoeba disper, amoeba pore homologs Brevinin-1E, Brevinin-2E, Barbatridine, Enterococcus faecalis cytolysin, Delta hemolysin, Diphtheria toxin, Vibrio cholerae ertor cell lysin, Equinatoxin, Aeromonas hydrophila enterotoxin, Esculentin, Granulidine, Vibrio parahaemoliticus hemolysin, Streptococcus intermedius intermedilysin, Lentiviral cytolytic peptide, Actinobacillus actinomycetemcomi Tansu's leukotoxin, magainin, melittin, cell membrane-related lymphotoxin, methoenkephalin, neokyotolphine and neokyotolphine fragment (1-4), NK-lysine, paradaxin, perforin (especially its amino terminus), Clostridium perfringens Perfringolysin O (PFO or sheetoxin), faroricin, farotoxin, streptricin. Some hemolysins such as melittin are also known to kill bacteria. Other cytolytic peptides include D, L-α-amino acid cyclic peptides. Fernandez-Lopez et al., Nature 412: 452-455 (2001).

  For example, type I bacterial exotoxins can be used as cytotoxins. In addition, type I bacterial exotoxins can be fused to tumor cell cell membranes or target regions of the tumor microenvironment.

  Type II bacterial exotoxins can also be used as cytotoxins. Furthermore, the type III bacterial exotoxin can retain its own transmembrane region or the toxin can be fused with a different region such as TAT.

Pore-forming cytolytic peptide The cytolytic peptide may be a pore-forming or channel-forming cytolytic peptide. Many cytolytic peptides are pore-forming toxins that belong to a group of cytotoxins that bind to cell membranes either non-specifically or to specific receptors to form discrete-sized transmembrane pores . Most toxic pore-forming peptides take advantage of features common to their cytolytic activity. For example, a large number of these toxins are inserted in the target cell membrane with the toxin monomer bound, then aggregated around the center to form a barrel side plate and pour water into the “barrel-stave”. ) "Let the cells lyse through the mechanism. This pore causes a rapid and irreversible electrolyte imbalance in the target cell leading to cell destruction.

  Most pore-forming peptides that act in both mammalian and bacterial cells require an amphipathic alpha helix structure and a net positive charge for their cytolytic activity. The strong electrostatic interaction between the cationic portion of the peptide and the lipid head group weakens the cell membrane and facilitates the insertion of the hydrophobic α-helix peptide. Thus, the cytotoxic peptides described in the present invention can be cytolytic and linear α-helix peptides. In general, these cytotoxic peptides, in their natural form, have a net positive charge that is responsible for their cytolytic activity.

Melittin According to one embodiment of the present invention, the cytolytic peptide can be melittin or an analog or derivative thereof. Melittin is isolated from bee venom and is an amphipathic alpha helix of 26 amino acids (Blondelle et al., (1991) Biochemistry 30: 4671-4678; Dempsey et al., 5 (1991) FEBS Lett. 281: 240-244). The amino acid sequence of melittin is shown in Table 1 and SEQ ID NO: 6. Residues 1-20 are mostly hydrophobic and residues 21-26 are hydrophilic and basic. Melittin has antibiotic activity, but in mammals it is lytic to leukocytes, erythrocytes and various other cells. Also, compounds similar to melittin within the scope of the present invention include bumblebee bombolitin (17 amino acid amphipathic α-helix), bee venom mastparan (14 amino acid amphipathic α-helix). And the wasp venom crabolin (13 amino acid amphiphilic α-helix) (Argiolas A. and Pisano JJ, 1985, J. Biol. Chem. 260, 1437-1444).

Amoebapore Another pore-forming peptide according to the present invention may be amoebapore, a 77-residue pore-forming peptide derived from Entoameba historica (Young et al., (1982) J. Exp. Med. 156: 1677. Lynch et al., (1982) EMBO J 7: 801; Young and Cohn, (1985) J. Cell Biol. 29: 299; Rosenberg et al., (1989) Molec. Biochem. Parasit 33: 237; Jansson et al., (1994). Science 263: 1440). It has four alpha helices from approximately amino acids 1-21, 25-36, 40-63 and 67-77, conventionally referred to as helices 1, 2, 3, and 4, respectively.

Amoebapore peptides are stabilized by three disulfide bonds and contain four predominantly amphiphilic α-helix structures. The third amphipathic helix structure (helix 3) possesses cytolytic activity similar to the wild type peptide. Synthetic peptides based on this third amphipathic α-helix sequence have recently been shown to have cytolytic activity against nucleated cells at high concentrations (10-100 μM) (Leippe et al. (1994) Proc. Natl. Acad.Sci.USA 91: 2602). Thus, in one embodiment, cytotoxin, Table _{1: NH 2 -Gly-Phe-} Ile-Ala-Thr-Leu-Cys-Thr-Lys-Val-Leu-Asp-Phe-Gly-Ile-Asp- Amoebapore derivatives listed in Lys-Leu-Ile-Gln-Leu-Ile-Glu-Asp-Lys-COOH.

Modifications and derivatives It is readily appreciated that the cytolytic peptides can be modified or derivatized to produce analogs and derivatives that retain cytolytic activity or even show enhanced cytolytic activity. For example, Andorra et al. Disclose that a shortened amoebapore analog has enhanced antibacterial and cytolytic activity (FEBS Letters, 385 (1996), pages 96-100, the entire disclosure of which is hereby incorporated by reference. Incorporated into the book).

  In designing such analogs or derivatives, one of ordinary skill in the art will be informed by the above review of amphipathic α-helix structure and net positive charge linked to cytolytic activity. Thus, those skilled in the art have designed cytotoxin analogs, i.e., non-naturally occurring forms that are virtually unknown based on the homology observed with natural proteins and known structures and properties, as cytolytic peptides according to the present invention. Can be used.

  In accordance with the present invention, modifications and derivatizations include, but are not limited to, substitutions, additions or deletions that provide functionally equivalent molecules (functions are evaluated by the examples below). obtain). Analogs and derivatives can also be made through modification of the side chains of amino acid residues of cytotoxic peptides. For example, the analog or derivative can enhance or increase functional activity as compared to a native protein or polypeptide.

  Also dimerization, truncation, diastereoisomers (D amino acid-incorporated analogs) (Shai et al. (1996) J. Biol. Chem. 271-7305-7308), and combinations thereof, are alternative treatments for cytotoxin peptides. It may also be used for the production of pharmaceutical forms, derivatives and analogs. See, for example, Shai et al. (1996) J. MoI. Biol. Chem. 271-7305-7308 disclose the effect of sequence and structural variation on the cytolytic activity of melittin, the entire disclosure of which is incorporated herein by reference.

Sequence similarity A homologue of a peptide of the invention is provided. As mentioned herein, it will be understood that the use of the terms homology and identity means the same as similarity. Thus, for example, when the term homology is used between two non-natural sequences, this does not necessarily indicate an evolutionary relationship between these two sequences, but rather the similarity between the sequences or It will be understood that the relationship is seen. Many of the methods for measuring homology between two evolutionarily related molecules are any two or more nucleic acids or proteins for measuring sequence similarity regardless of whether they are evolutionarily related Usually applied to.

  In general, one method of defining any known variants and derivatives or potential occurrences of the genes and proteins disclosed herein is that variants and derivatives with respect to homology to specific known sequences It will be understood that this is due to the definition of This identity of the specific sequences disclosed herein has also been stated elsewhere in the specification. In general, the gene and protein variants disclosed herein are typically at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, relative to the described or native sequence. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology. One skilled in the art will readily understand how to determine the homology of two nucleic acids, such as two proteins or genes. For example, this homology can be calculated after aligning the two sequences so that the homology is at its highest level.

  Other methods of calculating homology can also be performed with published algorithms. The optimal alignment of sequences for comparison is described by Smith and Waterman Adv. Appl. Math. 2: 482 (1981), according to the Needleman and Wunsch, J. et al. MoL Biol. 48: 443 (1970) by the homology alignment algorithm of Pearson and Lipman, Proc. Natl. Acad. Sci. U. S. A. 85: 2444 (1988) search for similarity method, these algorithms (Wisconsin Genetics Software Package, GAP, BESTFIT, FASTA, and TFSTA, Genetics Comp. 75 ), Or by inspection.

  The same type of homology is described, for example, in Zuker, M .; Science 244: 48-52, 1989, Jaeger et al., Proc. Natl. Acad. Sci. USA 86: 7706-7710, 1989, Jaeger et al., Methods Enzymol. 183: 281-306, 1989, which can be obtained for nucleic acids by at least the substances associated with nucleic acid alignments are incorporated herein by reference. Either method can typically be used, and in some cases the results of these various methods may vary, but if at least one of these methods finds identity, this It will be understood by one of ordinary skill in the art that the sequences are said to have the described identity and are disclosed herein.

  For example, as used herein, a sequence listed as having a certain percent homology to other sequences may have the listed homology calculated by any one or more of the above calculations. The sequence which has. For example, using the Zuker calculation method, if the first sequence is calculated to have 80 percent homology to the second sequence, the first sequence may be calculated as any of the other calculation methods, Even if the sequence does not have 80 percent homology to the second sequence, the first sequence has 80 percent homology to the second sequence as defined herein. As another example, even if the first sequence is 80 relative to the second sequence, as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation method, or any other calculation method. If the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation and both the Pearson and Lipman calculation methods, One sequence has 80 percent homology to the second sequence, as defined herein. As yet another example, using each calculation method (although different calculation methods often result in different calculated percentages of homology), the first sequence is 80 percent relative to the second sequence. The first sequence has 80 percent homology to the second sequence as defined herein.

Nucleic acids There are a variety of molecules disclosed herein that are based on nucleic acids, including, for example, nucleic acids encoding melittin and other proteins disclosed herein, as well as various functional nucleic acids. The disclosed nucleic acids consist of, for example, nucleotides, nucleotide analogs or nucleotide substituents. These non-limiting examples and other molecules are described herein. For example, if the vector is expressed in cells, it will be appreciated that the expressed mRNA typically consists of A, C, G and U. Similarly, for example, when an antisense molecule is introduced into a cell or cellular environment via exogenous delivery, the antisense molecule consists of a nucleotide analog that reduces degradation of the antisense molecule in the cellular environment. It will be appreciated that it is advantageous.

Nucleotides and related molecules A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate and sugar moieties to create internucleoside linkages. The base portion of this nucleotide is adenine-9-yl (A), cytosine-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymin-1-yl ( T). The sugar moiety of the nucleotide is ribose or deoxyribose. The phosphate portion of the nucleotide is a pentavalent phosphate. Non-limiting examples of nucleotides include 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).

  Nucleotide analogs are nucleotides that contain some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, and 2-aminoadenine and modification of sugar or phosphate moieties. Including.

  Nucleotide substituents are molecules that have functional properties similar to nucleotides but do not contain a phosphate moiety, such as peptide nucleic acids (PNA). Nucleotide substituents are molecules that recognize nucleic acids in a Watson-Crick or Hoogsteen manner but are linked together through moieties other than the phosphate moiety. Nucleotide substituents can adapt to a double helix type structure when interacting with the appropriate target nucleic acid.

  For example, other types of molecules (conjugates) can be conjugated to nucleotides or nucleotide analogs to enhance cellular uptake. The conjugate can be chemically conjugated to a nucleotide or nucleotide analog. Such conjugates include, but are not limited to, lipid moieties such as cholesterol moieties. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),

  A Watson-Crick interaction is an interaction at the Watson-Crick face of at least one nucleotide, nucleotide analog, or nucleotide substituent. The Watson-Crick face of a nucleotide, nucleotide analogue, or nucleotide substituent is a purine-based nucleotide, nucleotide analogue, or nucleotide substituent, C2, N1, and C6 positions and a pyrimidine-based nucleotide, nucleotide analogue, or Includes C2, N3, C4 positions of nucleotide substituents.

  Hoogsteen interaction is an interaction that occurs at the Hoogsten surface of nucleotides or nucleotide analogs exposed in the major groove of double-stranded DNA. The Hoogsteen face contains reactive groups (NH2 or O) at the N7 and C6 positions of purine nucleotides.

There are various sequences associated with, for example, melittin and other proteins disclosed herein in Genbank, and these and other sequences are herein described in their entirety and therein. The entirety of each included subsequence is hereby incorporated by reference.

  Various sequences are provided herein and these and others can be found in Genbank (www.pubmed.gov). One skilled in the art will understand how to determine sequence differences and differences and how to tailor compositions and methods that relate a particular sequence to other related sequences. Primers and / or probes can be designed for any sequence disclosed herein and provided with information well known in the art.

Peptides and proteins There are numerous variants, modifications or derivatives of the disclosed procytotoxin proteins disclosed and contemplated herein. Protein variants and derivatives are well understood by those of skill in the art and may include amino acid sequence modifications. For example, amino acid sequence modifications are typically classified into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and / or carboxyl terminal fusions as well as intrasequence insertions of one or more amino acid residues. The insertion is usually a smaller insertion, for example of the order of 1 to 4 residues, than insertion of an amino-terminal or carboxyl-terminal fusion. Immunogenic fusion protein derivatives, such as those described in the examples, are immunogenic to target sequences by cross-linking in vitro or by culturing recombinant cells transformed with DNA encoding this fusion. Made by fusing a polypeptide large enough to give Deletions are characterized by the deletion of one or more amino acid residues from the protein sequence. Typically, no more than about 2 to 6 residues are deleted at any site in the protein molecule. These variants are usually prepared by site-directed mutagenesis of the nucleotide of the DNA encoding the protein, thereby producing the DNA encoding the variant and then expressing the DNA in recombinant cell culture. . Techniques for making substitution mutants at predetermined sites in DNA having a known sequence, such as M13 primer mutagenesis and PCR mutagenesis, are well known. Amino acid substitutions are typically one residue, but can occur at many different sites simultaneously, insertions are usually on the order of about 1 to 10 amino acid residues, and deletions are about 1 to 30 residues. Range. Deletions or insertions can be made in adjacent pairs, ie, deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at the final construct. Mutations should not place the sequence out of reading frame and preferably do not create complementary regions that can lead to secondary mRNA structure. A substitution variant is a variant in which at least one residue is deleted and a different residue is inserted at that position. Such substitutions are generally made according to Tables 2 and 3 below and are called conservative substitutions.

  The essential change in functional or immunological identity is achieved by selecting a less conservative substitution than in Table 3, ie (a) the structure of the polypeptide backbone of the region to be substituted, eg a sheet or a helix The conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the effect of its substitution on maintaining the side chain size results from selecting residues that differ significantly. In general, substitutions that are thought to produce the greatest changes in protein properties include (a) hydrophilic residues such as seryl or threonyl, and hydrophobic residues such as leucyl, isoleucyl, phenylalanyl, valyl or alanyl. (B) replaces cysteine or proline with (d) and replaces other residues with (c), (c) residues with positively charged side chains, eg lysyl, arginyl Or histidyl (with), negatively charged residues such as glutamyl or aspartyl substituted with (d), or (d) residues with large side chains such as phenylalanine Non-residues such as glycine (in this case), in which case (e) for sulfation and / or glycosylation It substituted by increasing the number of positions.

  For example, the replacement of one amino acid residue with another amino acid residue that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution is the replacement of one hydrophobic residue with another hydrophobic residue or the replacement of one polar residue with another polar residue. This substitution includes combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included in the mosaic polypeptides provided herein.

  Substitution or deletion mutagenesis can be used to insert sites for N-glycosylation (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr). Also, deletion of cysteine or other unstable residues may be preferred. Deletion or substitution of a potential proteolytic site, such as Arg, is achieved, for example, by deleting one of the basic residues or replacing one residue with a glutaminyl or histidyl residue.

  Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl and aspartyl residues after translation. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of o-amino groups of lysine, arginine, and histidine side chains (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco 79-86 [1983]), acetylation of N-terminal amines and, in some cases, C-terminal carboxyl amidation.

  It will be appreciated that one method of defining variants and derivatives of the proteins disclosed herein is by defining variants and derivatives for homology / identity to specific known sequences. For example, SEQ ID NO: 6 shows the specific sequence of melittin and SEQ ID NO: 14 shows the specific sequence of the amoebapore protein. Specifically, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, at least for the specified sequence. Disclosed are variants of these and other proteins disclosed herein with 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology. In one embodiment, this variant carries only one tryptophan residue at position 19 of the disclosed melittin sequence. One skilled in the art will readily understand how to determine the homology of two proteins. For example, this homology can be calculated after aligning the two sequences so that the homology is at its highest level.

  Other methods of calculating homology can also be performed with published algorithms. The optimal alignment of sequences for comparison is described by Smith and Waterman Adv. Appl. Math. 2: 482 (1981), according to the Needleman and Wunsch, J. et al. MoL Biol. 48: 443 (1970) by the homology alignment algorithm of Pearson and Lipman, Proc. Natl. Acad. Sci. U. S. A. 85: 2444 (1988), these algorithms (Wisconsin Genetics Software Package GAP, BESTFIT, FASTA, and TFASTA, Genetics Computer Group, 575 Science Dr., Mad. Or by inspection.

  The same type of homology is described, for example, in Zuker, M .; Science 244: 48-52, 1989, Jaeger et al., Proc. Natl. Acad. Sci. USA 86: 7706-7710, 1989, Jaeger et al., Methods Enzymol. 183: 281-306, 1989, which can be obtained for nucleic acids by at least the substances associated with nucleic acid alignments are incorporated herein by reference.

  Conservative mutations and homology descriptions include at least 70% homology to a particular sequence, and the variants combine with each other in any combination, such as embodiments that are conservative mutants. It will be understood that it can.

  It will be appreciated that as the specification discusses various proteins and protein sequences, nucleic acids capable of encoding these protein sequences are also disclosed. This includes all degenerate sequences associated with a specific protein sequence, ie one specific protein sequence including degenerate nucleic acids encoding the disclosed variants and derivatives of this protein sequence and sequences encoding all nucleic acids. All nucleic acids having Thus, each particular nucleic acid sequence may not be written herein, but any sequence is disclosed and described herein through a protein sequence that is effectively disclosed. It will be understood. For example, one of the many nucleic acid sequences that can encode the protein sequence shown in SEQ ID NO: 6 is shown in SEQ ID NO: 5. Another nucleic acid sequence encoding the same protein sequence shown in SEQ ID NO: 14 is shown in SEQ ID NO: 13. The amino acid sequence does not dictate which particular DNA sequence encodes the protein in an organism, but if a particular variant of a disclosed protein is disclosed herein, that protein results It will also be appreciated that known nucleic acid sequences that encode the protein in a particular procytotoxin are also known and disclosed herein.

  It will be appreciated that there are numerous amino acid and peptide analogs that can be incorporated into the disclosed compositions. For example, there are a number of D amino acids or amino acids with different functional substituents, and the amino acids are shown in Tables 2 and 3. Inverse stereoisomers of naturally occurring peptides as well as stereoisomers of peptide analogs are disclosed. By designing a genetic construct that binds tRNA molecules to selected amino acids and inserts analog amino acids into peptide chains in a site-specific manner, for example using amber codons, these amino acids can be easily incorporated into polypeptide chains (Thorson et al., Methods in Molec. Biol. 77: 43-73 (1991), Zoller, Current Opinion in Biotechnology, 3: 348-354 (1992); Iba, Biotechnology & Genetic 97: ~ 216 (1995); Cahill et al., TIBS, 14 (10): 400-403 (1989); Benner, TIB Tech, 12: 158 ~ 163 (1994); Ibba and Hennecke, Bio / technology, 12: 678-682 (1994); all of which are hereby incorporated by reference for at least the substances associated with amino acid analogs).

Molecules similar to peptides can be made, but are not linked via natural peptide bonds. For example, binding of the amino acids or amino acid _{analogues, CH 2 NH -, - CH} 2 S -, - CH 2 -CH 2 -, - CH = CH- ( cis and _{trans), - COCH 2 -, -} CH (OH) CH ₂ -, and -CHH is ₂ SO- may include (these and others, Spatola, A.F.in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B.Weinstein ed, Marcel Dekker, New York, p. 267 (1983); Spatola, AF, Vega Data (March 1983), Volume 1, No. 3, Peptide Backbone Modifications (general reviews); Morley, Trends Pharm Sci (1980) pp.463~468; Hudson, D et _{al., Int J Pept Prot Res 14:} 177~185 (1979) (- CH 2 NH-, CH 2 CH 2 -); Spatola et al, Life _{Sci 38: 1243~1249 (1986) (} - CH H 2 -S); Hann J.Chem.Soc Perkin Trans.I 307~314 (1982) (- CH-CH-, cis and trans); Almquist et al., J _{.Med.Chem.23: 1392~1398 (1980) (-} COCH 2 -); Jennings-White et al., Tetrahedron Lett 23: 2533 (1982 ) (- COCH 2 -); Szelke et al, European App n, EP 45665 CA (1982) : 97: 39405 (1982) (- CH (OH) CH 2 -); Holladay et al., Tetrahedron.Lett 24: 4401~4404 (1983) (- C (OH) CH 2 -) And Hruby Life Sci 31: 189-199 (1982) (—CH ₂ —S—), each of which is incorporated herein by reference). A particularly preferred non-peptidic bond is —CH ₂ NH—. It will be appreciated that peptide analogs can have more than one atom between the bonding atoms, such as β-alanine, γ-aminobutyric acid, and the like.

  Amino acid analogs and analogs and peptide analogs have enhanced or desirable properties such as economic manufacturing, greater chemical stability, improved pharmacological properties (half-life, absorption, efficacy, efficacy, etc.), specificity modifications ( For example, it often has a wide range of biological activity), reduced antigenicity, and the like.

  Since D amino acids are not recognized by peptidases and the like, D amino acids can be used to produce more stable peptides. Systematic substitution of one or more amino acids in the consensus sequence using the same type of D-amino acid (eg, D-lysine instead of L-lysine) can be used to generate more stable peptides. . Cysteine residues can be used to cyclize or attach two or more peptides to each other. This can be beneficial for constraining the peptide to a specific conformation. (Rizo and Giersch Ann. Rev. Biochem. 61: 387 (1992), incorporated herein by reference).

Latency-related peptide (LAP)
Functions / Mechanisms Latency-related peptides (LAPs) according to the present invention are of interest by sterically inhibiting charge neutralization and / or formation of conformations that render the peptides toxic when attached to cytolytic peptides. Inactivate cytotoxins.

  The size of LAP varies. As long as the amino acids in the LAP cage produce charge neutralization and / or steric constraints of the lytic peptide's intramembrane insertion and organization into the pore, the peptide is a suitable LAP.

  For example, the LAP can be a precursor region of TGFβ or a sequence that is substantially identical thereto. The LAP can also be a phage or a phage particle.

  TGFβ can be synthesized as a dimeric potential cytokine composed of an amino acid terminal latency-related protein (TGFβ-LAP) and a TGFβ cytokine active at its COOH terminus (Roberts and Sporn, Peptide Growth Factors and their Receptors; Sporn, MB and Roberts, AB, Springer-Verlag, 419-472 (1996); Roth-Eicorn et al., Hepatology, 28 1588-1596 (1998)). The precursor peptide can contain a signal peptide (residues 1-29) required for protein secretion, which is derivatized through the Golgi and processed by proteolytic cleavage to be glycosylated. The TGFβ-LAP region can be separated from TGFβ by proteolytic cleavage with arginine (277-278). Mature TGFβ occurs at alanine 279. In addition to maintaining TGFβ in a potential form, TGFβ-LAP contains important residues necessary for interaction with other molecules. Recently, mutations in the TGFβ-LAP region have been associated with autosomal dominant Kamrach-Engelmann disease (Janssens et al., Nature Genetics, 26, 273: 275 (2000). Cysteines 224 and 226 are two TGFβ-LAP proteins. These cysteine to serine mutations “activate” the molecule (Sanderson et al., Proc. Natl. Acad. Sci. USA, 92, 2572-2576 (1995). Brunner et al., Mol. Endocrinol. 6, 1691-1700 (1992); Brunner et al., J. Biol. Chem, 264, 13660-13664 (1989)). (Munger et al., Mol. Biol. Of the Cell, 9, 2627-2638 (1998; Derynck R, TIBS, 19, 548-553 (1994))) Nucleic acid encoding TGFβ U.S. Pat. No. 5,801,231, which is hereby incorporated by reference for the disclosure of nucleic acid sequences encoding TGFβ-LAP.

  In most cell types examined, including cell types of mesenchymal, epithelial and endothelial origin, TGFβ is a TGFβ subunit covalently linked to a potential TGFβ-binding protein (LTBP) and a TGFβ-LAP propeptide dimer Secreted in the potential form of the body. LTBP is also required for TGFβ secretion and folding (Miyazano et al., EMBO J. 10, 1091-11101 (1991); Miyazano et al., J. Biol. Chem. 267, 5668-5675 (1992); Eklov et al. Cancer Res. 53, 3193-3197 (1993)). Cysteine 33 is important for disulfide bridging with the third eight cysteine-rich repeats of a potential TGFβ binding protein (LTBP) (Saharinen et al., The EMBO Journal, 15, 245-253 (1996)). Modification of LTBP by, for example, thrombospondin (Schultz et al., The Journal of Biological Chemistry, 269, 26783-26788 (1994); Crawford et al., Cell, 93, 1159-1170 (1998)); transglutaminase (Nunes et al., June et al. Cell, Biol.136, 1151-1163 (1997); Kojima et al., The Journal of Cell Biology, 121, 439-4. 48 (1993)); MMP9 and MMP2 (Yu and Stamenkovic, Genes and Dev, 14, 163-176 (2000)) may release the active portion of TGFβ from potential complexes.

  The LAP of the present invention is a precursor region of TGFβ, for example, a precursor peptide of TGFβ-1, 2, or 3 (derived from human) (Delynck et al., Nature, 316, 701-705 (1985); De Martin et al., EMBO J. et al. Hans et al., Proc. Natl. Acad. Sci. 85, 79-82 (1988); Delynck et al., EMBO J. 7, 3737-3743 (1988); Ten Dyke et al., Proc. Natl. Acad.Sci.USA, 85, 4715-4719 (1988)), TGFβ-4 (derived from chicken) (Jakowlew et al., Mol. Endocrinol. 2, 1186-1195 (1988)), or TGFβ-5 (derived from Xenopus laevis) ) (Konda ah, et al., it may include J.Biol.Chem.265,1089~1093 (1990)). The term “precursor region” is defined as a sequence that encodes a precursor peptide and does not include a sequence encoding an active TGFβ protein.

  In one embodiment, the amino acid sequence of LAP is determined using the default parameters of the BLAST computer program provided by HGMP (Human Genome Mapping Project) (Atschul et al., J. Mol. Biol. 215, 403-410 (1990) It can have at least 50% identity to the precursor region of TGFβ1, 2, 3, 4 or 5 at the amino acid level (Roberts and Sorn, Peptide Growth Factors and the Tear Receptors: Sporn, MB and Roberts, AB, Springer-Verlag, Chapter 8, 422 (1996)) In other embodiments, the LAP is at the nucleic acid or amino acid level at TGFβ1, 2, 3, 4 Or at least 60%, 70%, 80%, 90%, or 95% identity to 5 LAPs In another embodiment, the LAP is TGFβ1, 2, 3 It can have up to 99% or more than 99% identity at the nucleic acid or amino acid level for 4 or 5 LAPs.

  This LAP can be selected from the LAPs of SEQ ID NOs: 23-27.

  The LAP may contain at least 2, such as at least 4, 6, 8, 10, or 20 cysteine residues for disulfide bond formation.

  LAP can provide a protective “cage” around the pharmaceutically active agent, thereby shielding the active agent and preventing its interaction with other molecules on the cell surface or molecules important for its activity. Or stop.

  This LAP uses the sequence of amino acids encoded by nucleotides 1-831 of SEQ ID NO: 1 or nucleotides 103-933 of SEQ ID NO: 3, or the default parameters of the BLAST computer program provided by HGMP for this LAP At least 50%, 60%, 70%, 80%. Sequences with 90%, 95% or 99% identity can be included.

  This LAP uses the sequence of amino acids encoded by nucleotides 1-831 of SEQ ID NO: 9 or nucleotides 97-928 of SEQ ID NO: 11 or the default parameters of the BLAST computer program provided by HGMP for this LAP At least 50%, 60%, 70%, 80%. Sequences with 90%, 95% or 99% identity can be included.

Site The LAP can be added to the N-terminal or C-terminal of the cytotoxic peptide or both the N-terminal and C-terminal. For example, the cytotoxic peptide can be a cytolytic peptide and the LAP can be added to the C-terminus of the cytolytic peptide. Furthermore, as discussed herein, the LAP may further include a target molecule.

  Furthermore, a charge neutralizing component can be added to the procytotoxin, in which case the procytotoxin is further neutralized in addition to the steric determinants.

For example, [Gly-Ile-Gly-Ala-Val-Leu-Lys (R) -Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys (R) -Arg-Lys (R) -Arg-Gln-Gln-Gly-Ala-Ile-Gly-Gln-Pro]-(X) is provided, wherein (X) is a peptide bond described herein Is a LAP that binds to a peptide marked in parentheses via, wherein the peptide marked in brackets can be oriented in either direction, the peptide bond is sensitive to cleavage by a tumor-specific protease, and R is Unmodified ε-amino group of adjacent lysine residue, [ε-γ] -Glu, [ε-γ] -Glu- [α-γ]-(Glu) _1-3 , [ε-α]- ( _{he) 1-3, [ε-α} ] - (Tyr) 1-3, [ε-α] - (Trp) 1-3, [ε-α] - (Lys) 1-3 and [ε-α] -(Arg) _1-3, independently selected from the group consisting of _1-3 , [ε-γ] represents the gamma carboxyl group peptide bond of glutamic acid adjacent to the epsilon amino group of lysine, and [α-γ] is The peptide bond between the α-amino group of the first glutamic acid and the gamma-carboxyl group of the second glutamic acid is shown, [ε-α] shows the peptide bond between the epsilon amino acid of lysine and the α-carboxyl group of the indicated amino acid, And the subscript number indicates that an additional number of designated amino acids can be linked first by normal peptide bonds.

Target Specific Protease Function / Mechanism / Site The target specific protease described herein is an enzyme associated with a target cell that cleaves the peptide bond of a protein. The target cell described herein can be any cell for which cell lysis is desired. For example, the target cells can be cancer cells, bacterial cells, nerve cells, bone marrow cells, prostate cells, or vascular endothelial cells of the tumor neovasculature.

  The specific protease targeted by the present invention can typically be expressed in a tumor, but need not be expressed exclusively in a tumor. Thus, the targeted specific protease can be preferentially expressed in tumor cells when compared to normal cells. Furthermore, the specific protease targeted may be a protease expressed by a non-tumorigenic target cell such as a bacterial cell, but nevertheless preferentially expressed in the target cell when compared to a non-target cell. The Accordingly, the procytotoxins described herein are further useful as antimicrobial or antimicrobial agents. The procytotoxins described herein are also more useful for lysing any desired target cell as long as the specific protease to be targeted is present in the target cell.

Matrix metalloprotease (MMP)
In certain embodiments, the specific protease targeted may be a matrix metalloprotease (MMP). For example, the specific protease targeted can be MMP2. MMPs involve angiogenesis as well as neovascularization. Thus, targeting the tumor as well as the vasculature supplying newly formed blood vessels can help eliminate blood flow to the tumor and also prevent tumor shaping.

  For example, in the presence of a target-specific protease such as MMP, a cytolytic peptide bound to LAP by the sequence of amino acids recognized by MMP can be selectively released from LAP. This cytotoxic peptide is then free to form its active conformation and lyse the target cells.

  Other targeted specific proteases suitable for the present invention include the proteases described in Coams et al., Chem Biol, 5 (9): 475-88 (1988), PSMA for PSA and gamma-glutamyl hydrolase (GGH). It is.

Prostate specific antigen (PSA)
Another reactive peptide substrate suitable for the present invention includes those described in Coombs et al., Chem Biol, 5 (9): 475-88 (1988) for prostate specific antigen (PSA). PSA is a product of the KLK3 gene and is expressed in prostate and other tissues. Although it is expressed in healthy cells, abnormally high levels of PSA in the blood are found in men with prostate disorders and breast cancer, including prostate cancer. PSA is an important tumor marker used for the diagnosis and monitoring of both prostate and breast cancer.

  PSA is a 33 kd protein consisting of a single chain glycoprotein of 237 amino acid residues, 4 carbohydrate side chains, and multiple disulfide bonds. PSA is a homologue of the kallikrein family of proteases. To distinguish PSA from human glandular kallikrein-2 (hK-2), which is another prostate cancer marker with 80% homology, PSA is sometimes referred to as human glandular kallikrein-3 (hK-3). is there. Human glandular kallikrein-1 (hK-1) is a kallikrein that is found primarily in pancreatic and kidney tissues but shows 73% and 84% homology with PSA. The complete gene encoding PSA was sequenced and located on chromosome 19. PSA can be found in prostate epithelial cells and in semen.

  Also, continuous analysis of human glandular kallikrein-3 gene expression and EST expression profiles showed that human glandular kallikrein-3 gene upregulation in female genital (ovarian and uterine) and gastrointestinal (stomach, colon, esophagus and pancreatic) cancers showed that. Significant down-regulation was observed in breast and brain tumors with respect to their normal counterparts. (Diamandis et al., The Urological Clinics of North America: Prostate-Specific Antigen: The Best Prostatic Tumor Marker (1997)). Currently, PSA is considered the most useful tumor marker due to its tissue specificity and is widely used for prostate cancer screening, diagnosis and management.

The 5 'untranslated region of the gene encoding PSA contains regulatory elements, two of which are androgen response elements (ARE I and ARE II) and the other is a potent enhancer (Schuur). Et al., J Biol Chem (1996), Creutjens et al., Mol Endocrinol (1997)). PSA gene transcription in the prostate is known to be regulated by androgens through the action of AR (Riegman et al., Mol Endocrinol (1991); Schuur et al., J Biol Chem (1996); Creutjens et al., Mol Endocrinol (1996). 1997); and Young et al., Cancer Res (1991)). In seminal plasma where PSA is present in very large amounts (about 1-2 g / liter), the role of PSA is proteolytic cleavage of the sperm motility suppressor seminogelin and semen liquefaction post liquefaction (Malm J et al., Scan J Clin Lab Invest Suppl (1995) and McCorack et al., Urology (1995)). However, insulin-like growth factor binding protein 3 (IGFBP-3), protein C inhibitor, transforming growth factor β (TGF-β), PTH-related peptide (Cramer et al., J Urol (1996)), and putative vasoactivity Other substrates of PSA have been proposed, including unknown precursor proteins that release sex peptides (Fichner et al., J Urol (1996)). In male serum, PSA exists as a complex of α ₁ -antichymotrypsin (PSA-ACT) and α ₂ -macroglobulin (PSA-A2M) and as free PSA (Stenman et al., Cancer Res (1991) and Christenson et al., Eur J Biochem (1990)).

Prostate specific cell membrane antigen (PSMA)
Other targeted specific proteases can be type II transmembrane proteins such as prostate specific cell membrane antigen (PSMA). Prostate cancer cells have been previously shown to overexpress a type II transmembrane protein, a prostate-specific cell membrane antigen. PSMA has an intracellular epitope that is immunoreactive with the 7E11C5 immunoglobulin G monoclonal antibody (Horosczewicz et al. (1983), NLCAP model of human prosthetic carcinoma, Cancer Res. 43: 1809-1818). In prostate cancer patients, PSMA is highly expressed in the malignant prostate epithelium, but only slightly in the normal prostate and to a lesser extent in the benign prostatic hypertrophic epithelia. (Pinto et al., (1999) Prostate specific membrane antigen, a unique glutamate carboxypeptidase: a review of recent findings, The Prostate J.1: 15~26; Wright et al., (1995) Expression of Prostate Specific Membrane Antigen (PSMA) in normal benign and maligant prosthetic tissues, Urol.Oncol.1: 18-28; Lopes et al., (1990) Immunohistochemical and pharmacological charactarization of the site. specific immunoconjugate CYT-356 derived from antiprostate monoclonal antibody 7E11-C5, Cancer Res.50: 623~6428; Troyer et al., Detection and characterization of the prostate-specific membrane antigen (PSMA) in tissue extracts and body fluids, Int.J. Cancer 62: 552-558).

  The proteolytic region of PSMA is located outside the cell surface. After reaching the target cell, the procytotoxin of the present invention does not need to be internalized by the target cell, and its γ-glutamic acid residue (s) are cleaved and removed. Thus, procytotoxins can be activated on target cells that are exactly the desired site of action, as long as a similar mechanism exists to remove LAP. This direct effect on target cells enhances the cell killing effect of prostate cancer. The activated cytolytic peptide is immediately inserted into the target cell, leaving little opportunity to act on adjacent off-target cells. It is possible that one of the peptide ends has already been inserted into the cell membrane before the peptide is actually activated by PSMA. Furthermore, as described above, after cell lysis of the target cell, the cytolytic peptide remains adsorbed on the cell membrane debris of the target cell, so that the activated toxin is neutralized by the cell membrane and leaks out of the target cell. Does not harm.

Gamma glutamyl hydrolase (GGH)
Gamma glutamyl hydrolase (GGH) is an intracellular enzyme. In certain types of cancer, GGH can be secreted into the tumor microenvironment.

  GGH (EC 3.4.19.9) is a central enzyme in the metabolism of folyl and antifolyl poly-gamma-glutamic acid (McGuire, JJ and Coward, JK. (1984) In Folates and Pterins (Blakely, RL and Benkovic, SL, ed.), Volume 1, pages 135-190, John Wiley & Sons, Inc., NY). Folic acid is required as a cofactor for several enzymes in the de novo biosynthesis of DNA precursors and some amino acids and is essential for normal cell growth and replication. Antifolates such as methotrexate (MTX) have been a traditional cancer treatment for almost 40 years (Chabner et al., J. Clin. Invest. 76, 907-912). When folic acid or antifolates are transferred to the cells, they can undergo phorylpolyglutamate synthetase (FPGS) (EC 6.3.2.17) via sequential addition of glutamic acid. Converted to gamma glutamic acid or antiphoryl polygamma glutamic acid. These polygamma glutamates are retained in the cell and are generally better substrates or inhibitors than the corresponding monoglutamate for most folate-dependent enzymes (Galivan, J. (1980) Mol. Pharmacol. 17, 105- 110; Schirch, V. and Strong, WB (1989) Arch.Biochem.Biophys.269, 371-380; Rhee, MS, Lindau-Shepard, B., Chave, KJ, Galivan. J. and Ryan, TJ (1998) Mol Pharmacol 53, 1040-1046).

  GH catalyzes the removal of gamma-bound polyglutamate chains and converts its substrate to folyl or antiphoryl-mono-gamma glutamic acid, which is less well retained, thus reducing the overall effectiveness of folic acid and antifolates Tied to The role of FPGS and GH activity in regulating the degree of polyglutamylation of folic acid or antifolate is well documented. Increased GH activity (Rhee, MS, Wang, Y., Nair, MG and Galivan, J. (1993) Cancer Res, 53, 2227-2230; Yao, R., Rhee, MS And Galivan, J. (1995) Mol. Pharmacol 48, 505-511) or decreased FPGS activity (Pizzorno et al., Cancer Res, 55, 566-573; Rumberger et al., Cancer Commun. 2, 305-310; McCloskey et al. , J. Biol Chem. 266, 6181-6187) with in vitro resistance to antifolates such as MTX, 10-propargyl-5,8-dideazafolic acid, and 5,10-dideazatetrahydrofolic acid. It was.

  Increased GH activity in vivo has been reported to result in intrinsic resistance to MTX in acute myeloid leukemia (Rots et al., Blood 93, 1677-1683). Recently, the ratio of FPGS / GH activity has been used as an indicator of MTX polyglutamylation in acute lymphocytic leukemia (Longo et al., Oncol. Res. 9, 259-263).

Proteolytic cleavage site The proteolytic cleavage site may comprise any protease specific cleavage site. Proteolytic cleavage sites can include, but are not limited to, matrix metalloprotease (MMP) cleavage sites, PSA cleavage sites, PSMA cleavage sites, or GGH cleavage sites. (Zhang et al., J. Mol Biol. 289, 1239-1251 (1999); (Voth et al., Molecular and Biochemical Parasitology, 93, 31-41 (1998); Yosioka et al., Folia Pharmacolica 35, 397-35, 1107). Tort et al., Advances in Parasitology, 43, 161-266 (1999); McKerrow, International Journal for Parasitology, 29, 833-837 (1999); Young et al., International 86, 99, Journal 86, 1999 Coombs and M ttram, Parasitology, 114,61~80 (1997)).

  The MMP cleavage site can comprise any amino acid sequence that is cleavable by MMP. The amino acid sequence of the MMP cleavage site is represented by nucleotides 832 to 852 of the procytotoxin shown in SEQ ID NO: 1 (based on melittin) or nucleotides 832 to 852 of the procytotoxin shown in SEQ ID NO: 9 (based on amoebopore). Or at least 50%, 60%, 70%, 80% using the default parameters of the BLAST computer program provided by HGMP for the sequence of the MMP cleavage site. It can be encoded by a nucleotide sequence having 90%, 95% or 99% identity. For example, a nucleic acid sequence encoding an MMP cleavage site can include the minimum number of residues required for recognition and cleavage by the MMP.

  The MMP cleavage site may contain several amino acid residues that are recognizable by MMP. Furthermore, the amino acids at the MMP site can be linked by one or more peptide bonds that can be proteolytically cleaved by the MMP. MMPs that can cleave this MMP site include, but are not limited to, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10 or MT1-MMP (Yu and Stamenkovic, Genes and Dev. 14, 163-176 (2000); Nagase and Fields, Biopolymers, 40, 399-416 (1996); Massova et al., J. Mol. Model. 3, 17-30 (1997); Vu and Werb, Genes and Dev. 2213-2133 (2000)). The sequences of the protein cleavage sites of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 and MMP10 are shown in SEQ ID NOs: 28-100.

  In one embodiment, the proteolytic cleavage site of the present invention is cleaved at the site of the tumor and at the site of tumor neovascularization. For example, the proteolytic cleavage site of the present invention is an MMP cleavage site, such as any one or more of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 or MMP10 shown in SEQ ID NOs: 28-100. In certain embodiments, the cleavage site is an MMP2 cleavage site.

  For example, a cytotoxic peptide of the invention conjugated to LAP by the amino acid sequence recognized by MMP can be selectively released from LAP in the presence of a tumor specific protease such as MMP. This cytotoxic peptide is then released to form the active conformation of the peptide and destroy target cells.

Target molecules Selective delivery of therapeutic agents to cancer cells in the body is a new area of research where the targets of cancer-specific biomarkers are being intensively studied. (E. Mastrobattista, GA Koning and G. Storm, “Immonoliposomes for the Targeted Delivery of Antidrug Drugs”, Adv Drug Delivery. “Drug Delivery Via Folate Receptor”, Adv Drug Delivery Reviews 2000, 41: 147-62; SP Vyas and V. Sihordun-NigandiSand ------------ ug Delivery Reviews 2000,43: 101~64). For folate receptors (E. Mastrobastista, GA Koning and G. Storm, “Immoliposomes for the Targeted Delivery of Antigen Drugs. 27. Drug Delivery. , “Targeted Drug Delivery Via Receptor”, Adv Drug Delivery Reviews 2000, 41: 147-62) CA-125, (E. Mastrobatista, GA Ostom, G.A. Tumor Drugs ", Adv Drug Delivery Reviews 1999, 40: 103-27), and HER2 / neu antigen (DB Kirpotin, JW Park, K. Hong, S. Zalipsky, W.L. Li,). P. Carter, C. C. Benz, and D. Papahadjopoulos, “Sterically Stabilized Anti-HER2 Immunoposomes: Design and Targeting to Human Breast Cancer”. Mediating targets have been described.

  The target molecules of the present invention have selective features that provide a means of adding selectivity. This target molecule directs procytotoxin to the target cell, where it becomes toxic and selectively lyses its target. Furthermore, this target molecule can act as a LAP of the cytolytic peptide. In this regard, the target molecule can neutralize the charge or sterically inhibit pore formation of the cytolytic peptide. The target molecule can be added to the N-terminus or C-terminus, or both.

  In certain embodiments, the target molecule is an antibody. For example, the antibody can be an anti-fibronectin ED-B antibody, thereby directing procytotoxin to the extracellular matrix associated with neovascularization.

  A target molecule that targets the neovasculature can also be added to the LAP. Molecules that target the neovasculature can be readily identified by screening phage display libraries. Any such peptide is considered a suitable target molecule of the present invention.

  Target molecules that specifically bind to integrins are one class of signal sequences that can be found on cells of the vasculature. These peptides have a signal sequence based on Arg-Gly-Asp (RGD). Thus, sequences that bind specific integrins can function as useful target molecules for vascular endothelial cells and other cells of the neovasculature. For example, the target molecule can be an RGD target sequence.

  Non-structural spacers can be characteristic of the target molecule. Such spacers typically include glycine and / or proline residues. The length of these spacers can range from about 1 to about 5 amino acids. In addition, it is often preferred to physically constrain the target molecule by cyclization that usually results in increased binding. This is usually achieved by a pair of cysteine residues located on the sides of the RGD core, spaced from about 4 amino acids from each other (with only RGD in between) by 10 amino acids. For example, the cysteine residue pairs located on the sides of the RGD core can be 7 amino acid apart from each other.

Thus, a typical target molecule has the following structure:
-XRGDYX-
Wherein X is 0 to 5 amino acids, Y is 1 or 2 amino acids, and is selected from cysteine, serine, threonine and methionine. In particularly preferred embodiments, X consists of glycine residues, optionally containing at least one, typically one or two free thiol or amine-containing amino acids and / or one hydrophobic amino acid. It is out. Thiol-containing residues include methionine and cysteine, amine-containing residues include lysine and (at least one more) arginine, and hydrophobic residues include leucine, isoleucine, alanine and phenylalanine.

  Target molecules that specifically bind to integrins are one class of signal sequences that can be found on cells of the vasculature. These peptides have a signal sequence based on Asn-Gly-Arg (NGR). Thus, sequences that bind specific integrins can function as useful target molecules for vascular endothelial cells and other cells of the neovasculature. For example, the target molecule can be an NGR target sequence.

  Non-structural spacers can be characteristic of the target molecule. Such spacers typically include glycine and / or proline residues. The length of these spacers can range from about 1 to about 5 amino acids. Furthermore, it is often preferable to physically constrain the target molecule, usually by cyclization that results in increased binding. This is usually accomplished by a pair of cysteine residues located on the sides of the NGR core, separated from each other by about 10 amino acids (with only NGR in between). For example, the cysteine residue pairs located on the sides of the NGR core can be 7 amino acid apart from each other.

Thus, a typical target molecule has the following structure:
-XNGRYX-
Wherein X is 0 to 5 amino acids, Y is 1 or 2 amino acids, and is selected from cysteine, serine, threonine and methionine. In particularly preferred embodiments, X consists of glycine residues, optionally containing at least one, typically one or two free thiol or amine-containing amino acids and / or one hydrophobic amino acid. It is out. Thiol-containing residues include methionine and cysteine, amine-containing residues include lysine and (at least one more) arginine, and hydrophobic residues include leucine, isoleucine, alanine and phenylalanine.

  The target molecule may be a molecule that interacts with a tumor antigen. This tumor antigen includes human epithelial cell mucin (Muc-1; a 20 amino acid core repeat for the Muc-1 glycoprotein present on breast and pancreatic cancer cells), Ha-ras oncogene product, p53, carcinoembryonic antigen (CEA), raf oncogene product, gp100 / pme117, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, BAGE, GAGE, tyrosinase, gp75, Melan-A / Mart-1, gp100, HER2 / Neu, EBV-LMP1 & 2, HPV-F4, 6, 7, prostate specific antigen (PSA), HPV-16, MUM, α-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, ras oncogene product, HPV E7, Wilm tumor antigen-1, telomerase, and melano It can be selected from a list consisting of Maganglioside.

  The target molecule may be an aptamer or target specific antibody. Traditionally, identification of biomarkers and development of antibodies against their specific targets has been a difficult and time consuming process that does not always provide the best results for a particular application. For example, despite the identification of many cancer-related biomarkers, only a few of these show promising results for cancer screening and prognosis, and only these biomarker combinations are the best in cancer and normal tissues It has been recognized that enabling identification can be true.

  A number of reviews have been written about the implementation and products in the in vitro selection of aptamers. (Conrad, RC, L. Giber et al. (1996). “In vitro selection of nucleic acid aptamers that bind proteins”, Methods Enzymol 267: 336-67; Osborne, M. E. 1997. “Aptamers as therapeutic and diagnostic reagents: problems and prospects”, Curr Opin Chem Biol 1 (1): 5-9; Famook, M. and G. Mayr (1999). ”Curr To Microbiol Immunol 243: 123-36; Hesselberth, J., MP Robertson et al. (2000), “In vitro selection of nucleic acids for diagnostic applications [1] ProC, 5”. 25). The methods of the invention are directed against a target that is unique to, or overexpressed in, around, or on a desired cell (as compared to normal or non-target cells). Aptamers with unique or improved binding properties can be utilized. As used herein, “aptamer” refers to a nucleic acid that binds to a target molecule through interactions or conformations other than the nucleic acid annealing / hybridization described herein. Methods for making and modifying aptamers, and methods for assaying aptamer binding to a target molecule can be assayed or screened by any mechanism known to those of skill in the art (eg, US Pat. No. 6,111,095). 5,861,501, 5,840,867, 5,792,613, 5,780,610, 5,780,449, 5,756,291, See 5,631,146 and 5,582,981; and WO 92/14843, WO 91/19813, and WO 92/05285, each of which is a reference. As incorporated herein).

  Aptamers are single-stranded or double-stranded DNA or single-stranded RNA molecules that recognize and bind to a desired target molecule by its shape. See, for example, WO 92/14843, WO 91/19813, and WO 92/05285. US Pat. No. 5,270,163 issued to Gold et al., Tuerk et al. (1990) Science 249: 505-510, Szostak et al. (1990) Nature 346: 818-822 and Joyce (1989) Gene 82:83. The SELEX procedure described in -87 can be used for selection of target specific RNA or DNA aptamers. In the SELEX approach, oligonucleotides are constructed, and a random sequence region of n-mers, preferably nucleotides, is sandwiched between two polymerase chain reaction (PCR) primers thereby forming a “landomer pool” of oligonucleotides. ing. The construct is then contacted with the target molecule under conditions that facilitate binding of the oligonucleotide to the target molecule. These oligonucleotides that bind to the target molecule are (a) separated from those oligonucleotides that do not bind to the target molecule using conventional methods such as filtration, centrifugation, chromatography, etc .; (b) from the target molecule (C) amplified using conventional PCR techniques to form a ligand-rich pool of oligonucleotides. Additional rounds of binding, separation, dissociation and amplification are performed until an aptamer with the desired binding affinity, specificity or both is obtained. The identified final aptamer sequence can then be prepared chemically or by in vitro transcription.

The length of the random sequence region can range from 20 to 150 residues or more and can be even longer if multiple random oligonucleotides are merged into one pool by ligation or other methods. . (Bartel, DP and JW Szostak (1993). “Isolation of new ribozymes from a large pool of random sequences [see comments].” Science 261 (5127): 41. The individual number of random sequence populations is typically at least 10 ¹³ and can easily exceed 10 ¹⁵ . For most pools, this means that there are all possible 25-mers or more and a proportionally small number of more than 25 motifs. Due to the redundancy of biological sequences, the sequence diversity of most random sequence pools is probably comparable to that of the Earth's biosphere.

  Aptamers have been selected for the surprising range of targets that range from ions to supramolecular structures such as small organics, peptides, proteins, viruses and tissues. (Famulok, M. and G. Mayer (1999). "Aptamers as tools in molecular biology and immunology", Curr Top Microbiol Immunol 243: 123-36; Xu, W. A. D. 96.t. -Peptide aptamers recognize amino acid sequence and bind a protein epitope ", Proc Natl Acad Sci U SA 93 (15): e. S, e., P. the prion p rotein PrP ", J Virol 71 (11): 8790-7; Convery, MA, S. Rowsell et al. (1998)." Crystal structure of an RNA aptamer-protein complex at 2.8 Aresion ". Struct Biol 5 (2): 133-9; Homann, M. and H. U. Goringer (1999) “Combinatorial selection of high affinity RNA ligands to live Africans 9” ). In particular, aptamers are a variety of proteins, including many nucleic acid binding proteins such as T4 DNA polymerase (Tuerk, C. and L. Gold (1990). “Systemic evolution of ligands by exponential biomolecules: RNA ligands to bacterial DNA”. , Science 249 (4968): 505-10.) And HTV-1 Rev, (Giver, L., D. Bartel et al. (1993). “Selective optimization of the Rev-binding element of HIV-1”, Nucleic. Res 21 (23): 5509-16) It is selected for a plurality of non-nucleic acid binding protein. In general, anti-protein aptamers appear to recognize the basic portion of the protein surface. For example, the many viral protein arginine-rich motifs (ARMs) are aptamers (Ellington, AD, F. Leclerc et al. (1996). “An RNA groove [news]”, Nat Struct Biol 3 (12): 981-4), phosphate binding pockets of both kinases (Conrad, R., LM Keranen et al. (1994), “Isozyme-specific inhibition of protein C by RNA aptamers”, J Biol Chem 269 (51): 32051-4.) And phosphatases, (Bell, SD, JM Denu et al., (1998). "RNA molecules that that bin. to and inhibit the active site of a tyrosine phosphatase ", J Biol Chem 273 (23): 14309-14) and cytokines such as heparin binding sites and basic fibroblast growth factors on many surface proteins (Jellinek, D. et al. , C. K. Lynott et al., (1993) “High-affinity RNA ligands to basic fibroblast growth factor receptor binding in J. L. Proc Natl Acad Sci U. S. Green et al., (1995) “Potent 2′-amino-”. '-Deoxypyrimidine RNA inhibitors, of basic fibroblast growth factor ", Biochemistry 34 (36): 11363-72) and vascular endothelial growth factor (Jellinek, D., L. S. Green, et al., 94). by high-affinity RNA ligands to basal endothelial growth factor, Biochemistry 33 (34): 10450-6; Green, L. et al. S. , D. Jellinek et al. (1995). Recognized by “Nuclease-resistant nucleic acid ligands to vascular permeability factor / basic endogenous growth factor”, Chem Biol 2 (10): 683-95).

  Aptamers are pockets or cusps on the protein surface, such as antibody binding sites (Tsai, DE, DJ Kenan et al. (1992). “In vitro selection of an RNA immunologically cross-reactive with.” a peptide ”, Proc Natl Acad Sci USA 89 (19): 8864-8) or the active site of the enzyme (Tuerk, C, S. MacDougalse cit ren cit num cit enti num cit num cit num cit num cit num cit num cit num cit num cit num cit num cit num cit num cit num cit num num num num , Proc Natl Acad Sci USA 89 (1 ): 6988-92) appears to have affinity for. Almost all proteins have either surface pockets or basic patches (in fact, even proteins with a negative pI such as T4 DNA polymerase typically contain sites that can induce aptamers). Most aptamers, ie target complexes, have dissociation constants in the nanomolar range. In addition, aptamers recognize their targets with high specificity and can typically discriminate between protein targets that are highly homologous or differ by a few amino acids. (Conrad, R., LM Keranen et al. (1994). “Isozyme-specific inhibition of protein kinase C by RNA aptamers”, J Biol Chem 269 (51): 32051-4; Gold et al., (1995) “Let's get specific; the relation betspecificity and affinity”, Chem Biol 2 (10): 633-8; Hirao, L, M. Spingola et al., 1998. it. of specificity: an experimental analysis with RNA aptamers to S2 coat protein variants ", Mol Divers 4 (2): 75~89).

  The term “antibody” as used herein includes, but is not limited to, any class of complete immunoglobulin (ie, complete antibody). Natural antibodies are usually heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to the heavy chain by one covalent bond, a disulfide bond, but the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced internal chain disulfide bridges. Each heavy chain has several constant regions followed by a variable region (V (H)) at one end. Each light chain has a variable region at one end (V (L)) and a constant region at the other end, the constant region of the light chain is aligned with the first constant region of the heavy chain, and the light chain variable The region is aligned with the variable region of the heavy chain. Certain amino acid residues are thought to form the interface between the light and heavy chain variable regions. The light chains of antibodies from all vertebrate species can be assigned to one of two distinct types called kappa (κ) and lambda (λ) based on the amino acid sequence of their constant regions. Depending on the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of human immunoglobulins, namely IgA, IgD, IgE, IgG and IgM, some of which are further subclasses (isotypes) such as IgG-1, IgG-2, IgG-3 and IgG. -4; can be divided into IgA-1 and IgA-2. Those skilled in the art will recognize classes that can be compared to mice. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called α, delta, epsilon, gamma, and mu, respectively.

  The term “variable” is used herein to describe a particular portion of a variable region that differs in sequence between antibodies, and the binding and specificity of each particular antibody for that particular antigen. Used for. Usually, however, variability is not uniformly distributed throughout the variable region of the antibody. Typically, variability is concentrated in three parts called complementarity determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable regions. The more highly conserved portions of variable regions are called the framework (FR). Each of the natural heavy and light chain variable regions contains four FR regions, forming a loop connecting parts of the β-sheet structure, and optionally connected by three CDRs that form part of the β-sheet structure It has a β sheet structure. The CDRs of each chain are held together in close proximity to the CDRs of the other chains by the FR region and contribute to the formation of the antigen binding site of the antibody (Kabat EA et al., “Sequences of Proteins of Immunological. "Interest", National Institutes of Health, Bethesda, Md. (1987)). The constant region is not directly involved in binding the antibody to the antigen, but exhibits various effector functions such as antibody involvement in antibody-dependent cytotoxicity.

  As used herein, the term “antibody or fragment thereof” refers to chimeric and hybrid antibodies having double or multiple antigens or epitope specificity, and F (ab ′) 2, Fab ′, Fab, including hybrid fragments. And so on. Thus, a fragment of an antibody is provided that retains the ability to bind a specific antigen to that fragment. For example, a fragment of an antibody that retains integrin binding activity is included within the meaning of the term “antibody or fragment thereof”. Specifically, an antibody or fragment thereof that retains αVbeta3 binding activity is included within the meaning of the term “antibody or fragment thereof”. Such antibodies and fragments can be made by techniques known in the art, and in terms of specificity and activity, according to the methods set forth in the Examples and the general methods for producing antibodies and screening antibodies for specificity and activity. Can be screened (see Harlow and Lane, Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).

  In addition, the meaning of “antibody or a fragment thereof” includes, for example, a complex of an antibody fragment and an antigen-binding protein (single chain antibody) described in US Pat. No. 4,704,692, the contents of which are incorporated by reference. Incorporated herein.

  If desired, antibodies are made in other species and “humanized” for administration to humans. Humanized forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ′, F (ab ′) that contain minimal sequence derived from non-human immunoglobulin. 2) or other antigen-binding subsequence of the antibody). A humanized antibody is a residue derived from the CDR of a non-human species such as a mouse, rat or rabbit whose donor complementarity determining region (CDR) has the desired specificity, affinity and ability. Human immunoglobulins (recipient antibodies) that are replaced by (antibodies). In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has at least one, typically two, variable regions in which all or substantially all of the FR regions corresponding to the CDR regions of a non-human immunoglobulin are those regions of a human immunoglobulin consensus sequence. Substantially all of Also, humanized antibodies optimally comprise at least a portion of an immunoglobulin constant region (Fc), and typically comprise human immunoglobulin constant regions (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)).

  Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source other than human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable region. Humanization is basically performed by the method of Winter et al. (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534- 1536 (1988)) can be carried out by substituting rodent CDRs or CDR sequences for the corresponding sequences of human antibodies. Accordingly, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567), with a portion substantially smaller than a fully human variable region corresponding to a non-human species. It is replaced by a sequence. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. .

  The selection of both the light and heavy chain human variable regions used to make the humanized antibody is very important to reduce antigenicity. In accordance with the “best-fit” method, the variable region sequences of rodent antibodies are screened against an entire library of known human variable region sequences. The human sequence closest to the rodent sequence is then accepted as the human framework (FR) of the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993) and Chothia et al., J. Mol. Biol. 196: 901 (1987)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol., 151). : 2623 (1993)).

  It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies are prepared by analyzing parent sequences and various conceptual humanized products using a three-dimensional model of parental and humanized sequences according to the disclosed method. Is done. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display the predicted three-dimensional conformation structure of selected candidate immunoglobulin sequences. A close examination of these representations allows analysis of the possible role of residues in the function of a candidate immunoglobulin sequence, ie, analysis of residues that affect the ability of a candidate immunoglobulin to bind its antigen. . In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen (s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding (see WO 94/04679 published 3 March 1994).

  Transgenic animals (eg, mice) that can produce a complete repertoire of human antibodies after immunization in the absence of endogenous immunoglobulin production can be used. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (J (H)) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene sequences to such germline mutant mice results in the production of human antibodies following antigen challenge (eg, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90 : 2551-255 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); see Bruggemann et al., Year in Immuno., 7:33 (1993)). Human antibodies can also be produced in phage display libraries (Hoogenboom et al., J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991)). . The techniques of Cote et al. And Boerner et al. Can also be used for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147. (L): 86-95 (1991)).

  The term “monoclonal antibody” as used herein refers to an antibody that is obtained from a substantially homogeneous population of antibodies. That is, each antibody comprising the population is identical except for possible naturally occurring mutations that may be present in small amounts. As used herein, a monoclonal antibody specifically refers to a portion of a heavy chain and / or light chain that is identical or homologous to a corresponding sequence of an antibody derived from a specific species, or a specific Including “chimeric” antibodies belonging to the class or subclass of an antibody, while the remaining part (s) of this chain are identical or homologous to or different from the corresponding sequence of an antibody from another species As long as the antibody class or subclass, and antibody fragments exhibit the desired activity, they belong to such antibody fragments (US Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

Monoclonal antibodies can be obtained from Kohler and Milstein, Nature, 256: 495 (1975) or Harlow and Lane. Antibodies, A Laboratory Manual. It can be prepared using the hybridoma method as described in Cold Spring Harbor Publications, New York, (1988). In hybridoma methods, a mouse or other suitable host animal is typically immunized with an immunizing agent to induce lymphocytes that produce or can produce antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. For example, the immunizing agent is human epithelial cell mucin (Muc-1; a 20 amino acid core repeat for the Muc-1 glycoprotein present on breast and pancreatic cancer cells), Ha-ras oncogene product, p53, carcinoembryonic antigen (CEA), raf oncogene product, gp100 / pme117, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, BAGE, GAGE, tyrosinase, gp75, Melan-A / Mart-1, gp100, HER2 / Neu, EBV-LMP1 & 2, HPV-F4, 6, 7, prostate specific antigen (PSA), HPV-16, MUM, α-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, ras oncogene product, HPV E7, Wilm tumor antigen-1, telomerase, vitronectin Scepter _{(α v β 3), CA125} (MUC16), CD4, CD20, CD30 (Siglec-2), CD30, TNFRSF1), CD33 (Siglec-3), CD52 (CAMPATH-1), CD56 (NCAM), CD66e ( CEA), CD80 (B7-1), CD140b (PDGFRβ), CD152 (CTLA4), CD227 (PEM, MUC1, mucin-1), EGFR (HER1, ErbB1), EpCam, GD3 ganglioside, HER2 (HER2 / neu, ErbB2) ), PSMA, Siaryl Lewis, VEGF, and melanoma ganglioside. Traditionally, the production of monoclonal antibodies has depended on the availability of purified proteins or peptides used as immunogens. More recently, DNA-based immunity has been shown to be a promising method for inducing a strong immune response to produce monoclonal antibodies. In this method, DNA-based immunity can be used, and a DNA encoding a part of an integrin expressed as a fusion protein with human IgG1 can be obtained by methods well known in the art (for example, Kilpatrick KE et al., Gene gun released DNA). -based immunizations mediate rapid production of murine monoclonal antibodies to the Flt-3 receptor.Hybridoma.1998 Dec; 17 (6): 569~76; Kilpatrick KE, et al., High-affinity monoclonal antibodies to PED / PEA-15 generated using 5 microg of DNA Hyb ridoma.2000 Aug; 19 (4); 297-302, all of which are hereby incorporated by reference for methods of antibody production) and as described in the Examples.

  Another approach to immunize with either purified protein or DNA is to use antigens expressed in baculovirus. Advantages of this system include ease of production, high level expression, and post-translational modifications that are very similar to those found in mammalian systems. Use of this system includes the expression region of an integrin antibody as a fusion protein. The antigen is produced by inserting a gene fragment in frame between the signal sequence of the integrin antibody nucleotide sequence and the mature protein region. This provides a display of foreign proteins on the surface of the virus particle. This method allows immunization with the complete virus and eliminates the need for purification of the target antigen.

  In general, when methods of producing monoclonal antibodies require cells of human origin, peripheral blood lymphocytes ("PBL") are used, or when non-human mammalian sources are required, Spleen cells or lymph node cells are used. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol to produce hybridoma cells (Goding, “Monoclonal Antibodies: Principles and Practices”, Academic Press, (1986) 59- 103). Immortal cell lines are transformed mammalian cells, usually including myeloma cells of rodent, bovine, horse, and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells can be cultured in a suitable medium that can contain one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parent cell lacks the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine (“HAT medium”), This substance prevents the growth of HGPRT-deficient cells. Immortalized cell lines are cell lines that fuse efficiently, retain stable high level expression of antibodies in selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Other immortalized cell lines are described in, for example, the Salk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Rockville, Md. Mouse myeloma strains available from Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., “Monoclonal Production Technologies and Applications Application”. “Marcel Dekker, Inc., New York, (1987) 51-63). The medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies made against the integrin. The binding specificity of monoclonal antibodies produced by hybridoma cells can be measured by in vitro binding experiments such as immunoprecipitation or radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art and are further described below in the Examples or in Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Publications, New York, (1988).

  After identification of the desired hybridoma cells, the clone can be subcloned by limiting dilution or FACS sorting and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as mammalian ascites.

  Monoclonal antibodies secreted by these subclones can be isolated from the medium or ascites by conventional immunoglobulin purification methods such as protein A-sepharose, protein G, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. It can be separated or purified from the liquid.

  Monoclonal antibodies can also be generated by recombinant DNA methods such as those described in US Pat. No. 4,816,567. DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional methods (eg, using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the murine antibody). it can. Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector and the expression vector is then transferred to the host cell, eg monkey COS cells, Chinese hamster ovary (CHO) cells, to obtain monoclonal antibody synthesis in a recombinant host cell. , Plasmacytoma cells, or myeloma cells that do not produce immunoglobulin protein are transfected. This DNA can also be used, for example, by replacing human heavy and light chain constant region coding sequences in place of homologous mouse sequences (US Pat. No. 4,816,567), or in immunoglobulin coding sequences. Modification can be made by covalent attachment of all or part of the coding sequence of an immunoglobulin polypeptide. If desired, such non-immunoglobulin polypeptides can be used in place of the constant region of an antibody or in place of the variable region of one antigen binding site of an antibody to have integrin and other antigens with specificity for different antigens. A chimeric bivalent antibody that constitutes one antigen-binding portion having specificity for the binding portion is prepared.

  In vitro methods are also suitable for preparing monovalent antibodies. Digestion of the antibodies that produce these fragments, particularly the digestion of Fab fragments, can be accomplished using conventional techniques well known in the art. For example, digestion can be performed using papain. Examples of papain digestion can be found in WO 94/29348, published December 22, 1994, US Pat. No. 4,342,566, and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, ( 1988). Antibody papain digestion produces two identical antigen-binding fragments, typically called Fab fragments, each with one antigen-binding site and a remaining Fc fragment. Pepsin treatment yields a single fragment called an F (ab ') 2 fragment that has two antigen-binding sites and can still cross-link antigen.

  The Fab fragment produced by antibody digestion also contains the constant region of the light chain and the first constant region of the heavy chain. Fab 'fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain region containing one or more cysteines from the antibody hinge region. The F (ab ') 2 fragment is a divalent fragment consisting of two Fab' fragments joined by a disulfide bridge at the hinge region. Fab'-SH is the name herein for Fab 'in which the constant region cysteine residue (s) have a free thiol group. Antibody fragments were originally produced as a pair of Fab 'fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

  Also provided is an isolated immunological specific paratope or fragment of the antibody. Specific immunogenic epitopes of the antibody can be separated from the intact antibody by chemical or mechanical disruption of the molecule. The purified fragment thus obtained was tested to determine its immunogenicity and specificity by the methods disclosed herein. The immunoreactive paratope of the antibody is synthesized directly if desired. An immunoreactive fragment is defined as an amino acid sequence of at least about 2 to 5 consecutive amino acids derived from the antibody amino acid sequence.

  One way of producing the proteins that make up an antibody is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, a peptide or polypeptide uses currently available laboratory equipment and uses either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl) chemistry. Can be synthesized chemically. (Applied Biosystems, Inc., Foster City, CA). Those skilled in the art will readily understand that peptides or polypeptides corresponding to antibodies can be synthesized, for example, by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthetic resin, whereas other fragments of the antibody can be synthesized and then cleaved from the resin, thereby functional on the other fragment The end group that is blocked is exposed. By means of a peptide condensation reaction, these two fragments can be covalently linked via a peptide bond at the respective carboxyl terminus and amino terminus to form an antibody or antibody fragment. (Grant GA (1992) Synthetic Peptides: A User Guide. WH Freeman and Co., NY (1992); Bodsky M and Trost B. Ed., 1993) Principles of Peptide. ., NY). Alternatively, as described above, the peptide or polypeptide is independently synthesized in vivo. Once separated, these independent peptides or polypeptides can be combined to form an antibody or antibody fragment via a similar peptide condensation reaction.

  For example, enzymatic ligation of cloned or synthetic peptide segments can combine relatively short peptide fragments to produce larger peptide fragments, polypeptides or all protein regions (Abrahmsen L et al. , Biochemistry, 30: 4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction (Dawson et al., Synthesis of Proteins by Native Chemical Ligation. Science, 266: 776-779 (1994)). The first step is a functional group of an unprotected synthetic peptide-α-thioester and another unprotected peptide segment having a Cys residue at the amino terminus, which provides a thioester bond intermediate as the first covalent product Selective reaction. Without changing reaction conditions, this intermediate undergoes a spontaneous and rapid intramolecular reaction to form a native peptide bond at the ligation site. The application of this natural chemical ligation method to the complete synthesis of protein molecules is exemplified by the preparation of human interleukin 8 (IL-8) (Baggiolini M et al. (1992) FEBS Lett. 307: 97-101). Clark-Lewis I et al., J. Biol. Chem., 269: 16075 (1994); Clark-Lewis I et al., Biochemistry, 30: 3128 (1991); Rajarathnam K et al., Biochemistry 33: 6623-30 (1994) .

  Alternatively, if the bond formed between peptide segments by chemical ligation is a non-natural (non-peptidic) bond, the unprotected peptide segment is chemically linked (Schnolzer, M et al., Science, 256 : 221 (1992)). This technology has been used to synthesize analogs of protein domains as well as large quantities of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry IV. Academic Press, New York, 257-267. Page (1992)).

  Also disclosed are antibody fragments having biological activity. The polypeptide fragment may be a recombinant protein obtained by cloning a nucleic acid encoding the polypeptide into an expression system capable of producing these polypeptide fragments, such as an adenovirus or baculovirus expression system. Good. For example, the active region of a specific hybridoma-derived antibody that can produce a biological effect associated with the interaction of the integrin with the antibody can be determined. For example, amino acids that are found not to be involved in either antibody activity or binding specificity or affinity can be deleted without losing their respective activities. For example, in various embodiments, the amino-terminal or carboxy-terminal amino acid is sequentially deleted from a natural or modified non-immunoglobulin molecule or immunoglobulin molecule and its respective activity is one of many available assays. Assayed. In other examples, antibody fragments include modified antibodies, wherein at least one amino acid is substituted for a naturally occurring amino acid at a specific position, and the amino-terminal amino acid or a portion of the carboxy-terminal amino acid. Or even the internal region of the antibody is replaced with a polypeptide fragment or another moiety, for example with biotin which can facilitate the purification of the modified antibody. For example, fusing a modified antibody with a maltose binding protein by cloning each nucleic acid encoding two polypeptide fragments into an expression vector by peptide chemistry or such that expression of the coding region results in a hybrid polypeptide. it can. The hybrid polypeptide can be affinity purified by passing it through an amylose affinity column and then cleaving the hybrid polypeptide with the specific protease factor Xa (factor Xa) to remove the modified antibody receptor from the maltose binding region. Can be separated. (See, eg, New England Biolabs Product Catalog, 1996, pg. 164). Similar purification methods are available for separating hybrid proteins from eukaryotic cells as well.

  A fragment, whether attached to another sequence or not, can be a specific region or a specific amino acid residue if the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. Includes group insertions, deletions, substitutions, or other selected modifications. These modifications provide several additional properties, such as deleting or adding disulfide-bondable amino acids to increase their bio-longevity, changing their secretion properties, etc. can do. In any case, the fragment must have bioactive properties such as binding activity, modulation of binding at the binding region, and the like. The functional or active region of an antibody can be identified by testing expression and expressed polypeptide following mutagenesis of a particular region of the protein. Such methods are readily understood by those skilled in the art and can include site-directed mutagenesis of the nucleic acid encoding the antigen. (Zoller MJ et al., Nucl. Acids Res. 10: 6487-500 (1982).

  A variety of immunoassay formats can be used to select antibodies that selectively bind to a particular protein, variant, or fragment. For example, solid phase ELISA immunoassays are commonly used to select antibodies that are selectively immunoreactive with certain proteins, protein variants, or fragments thereof. For a description of immunoassay formats and conditions that can be used to measure selective binding, see Harlow and Lane. Antibodies, A Laboratory Manual. See Cold Spring Harbor Publications, New York, (1988). The binding affinity of monoclonal antibodies is described, for example, in Munson et al., Anal. Biochem. 107: 220 (1980), by Scatchard analysis.

Methods of Making the Compositions The compositions disclosed herein and the compositions necessary to practice the disclosed methods are well known to those skilled in the art for that particular reagent or compound, unless otherwise specified. It can be created using any method.

Nucleic acid synthesis For example, nucleic acids can be made using standard chemical synthesis methods, or can be produced using enzymatic methods or any other known method. Such methods can be derived from nucleotide fragment separations following standard enzymatic digestion (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 198). See chapters 5 and 6), eg purely by the cyanoethyl phosphoramidite method using a Milligen or Beckman System 1 Plus DNA synthesizer (eg Milligen-Biosearch, Burlington, MA Model 8700 automatic synthesizer or ABI Model 380B). It can be in the range of synthesis methods. Synthetic methods useful for making oligonucleotides are also described in Ikuta et al., Ann, Rev. Biochem. 53: 323-356 (1984), (phosphotriester and phosphorite-triester methods), (phosphate triester and phosphite triester method), and Narang et al., Methods Enzymol, 65: 610-620 (1980), (phos) In the photoreester method). Protein nucleic acid molecules are described in Nielsen et al., Bioconjug. Chem. 5: 3-7 (1994).

Peptide synthesis A particular advantage of the useful peptides of the present invention is that they are readily synthesized by solid phase methods and can be combined in various ways to achieve the required results in detail. The advantage of using the solid phase method is that this product can be synthesized directly with an amidated or blocked C-terminus that favors the formation of the procytotoxins of the present invention.

  One way of producing the disclosed proteins, eg (melittin) SEQ ID NO: 6, is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, a peptide or polypeptide uses currently available laboratory equipment and uses either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl) chemistry. Can be synthesized chemically. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art will readily understand that peptides or polypeptides corresponding to the disclosed proteins can be synthesized, for example, by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthetic resin, whereas other fragments of the peptide or protein can be synthesized and then cleaved from the resin, thereby producing other fragments. Exposes functionally blocked end groups above. By means of a peptide condensation reaction, these two fragments can be covalently linked via a peptide bond at the respective carboxyl terminus and amino terminus to form an antibody or antibody fragment. (Grant GA (1992) Synthetic Peptides: A User Guide. WH Freeman and Co., NY (1992); Bodsky M and Trost B. Ed., 1993) Principles of Sept. , NY (these are incorporated herein by reference for at least substances related to peptide synthesis) As described herein, the peptide or polypeptide is synthesized independently in vivo. Once separated, these independent peptides or polypeptides can be combined to form a peptide or peptide fragment via a similar peptide condensation reaction.

  For example, enzymatic ligation of cloned or synthetic peptide segments can combine relatively short peptide fragments to produce larger peptide fragments, polypeptides or all protein regions (Abrahmsen L et al. , Biochemistry, 30: 4151 (1991)). Alternatively, natural chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction (Dawson et al., Synthesis of Proteins by Native Chemical Ligation. Science, 266: 776-779 (1994)). The first step is functional group-selective between an unprotected synthetic peptide-thioester and another unprotected peptide segment with a Cys residue at the amino terminus, which provides a thioester bond intermediate as the first covalent product It is a reaction. Without changing reaction conditions, this intermediate undergoes a spontaneous and rapid intramolecular reaction to form a native peptide bond at the ligation site (Baggiolini M et al. (1992) FEBS Lett. 307: 97-101; Clark-Lewis I et al., J. Biol. Chem., 269: 16075 (1994); Clark-Lewis et al., Biochemistry, 30: 3128 (1991); Rajarathnam K et al., Biochemistry 33: 6623-30 (1994)).

  Alternatively, if the bond formed between peptide segments by chemical ligation is a non-natural (non-peptidic) bond, the unprotected peptide segment is chemically linked (Schnolzer, M et al., Science, 256 : 221 (1992)). This technology has been used to synthesize analogs of protein domains as well as large quantities of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry IV. Academic Press, New York, 257-267. Page (1992)).

  Another method of producing the disclosed proteins, such as (melittin) SEQ ID NO: 6, is to use recombinant DNA methods such as those described in US Pat. No. 4,816,567. The DNA encoding the protein can be easily separated and sequenced using conventional methods. Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector and the expression vector is then transferred to the host cell, eg, monkey COS cells, Chinese hamster ovary (CHO) cells, traits, to obtain protein synthesis in a recombinant host cell. A cell tumor cell, or one that does not produce protein, is transfected into a myeloma cell.

Pharmaceutical Compositions Also provided are pharmaceutical compositions comprising the generally disclosed procytotoxins and pharmaceutically suitable excipients to achieve this object of the present invention.

  Another aspect of the present invention is a pharmaceutical composition comprising one or more procytotoxins of the present invention and a pharmaceutically suitable carrier or excipient.

  While the composition of the present invention can be administered to a patient alone, the composition can also be administered in vivo in a pharmaceutically acceptable carrier. “Pharmaceutically acceptable” means a non-biological or non-hazardous substance, ie, a pharmaceutical composition that does not produce or contain any undesirable biological effects This means that the substance can be administered to a subject with a nucleic acid or vector without interacting in a deleterious manner with any of the other components. The carrier is of course selected to minimize any degradation of the active ingredient and to minimize any harmful side effects in the subject, as is well known to those skilled in the art.

  When used, parenteral administration of the composition is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution in suspension in liquid prior to injection, or as emulsions. A very recently revised approach for parenteral administration involves the use of slow or delayed release systems to maintain a constant dose. See, for example, US Pat. No. 3,610,795. This is incorporated herein by reference.

  This material may be a solution, suspension (eg, microparticles, liposomes, or cells in which they are incorporated). These can target specific targets as described above as well as below.

  This material may be a solution, suspension (eg, microparticles, liposomes, or entrapped in cells). They can target specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target proteins specific to tumor tissue (Senter et al., Bioconjugate Chem. 2: 447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60: 275-281, (1989); Bagshawe et al., Br. J. Cancer, 58: 700-703, (1988); Senter et al., Bioconjugate Chem., 4: 3-9, (1993). Battelli et al., Cancer Immunol Immunother., 35: 421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129: 57-80, (1992); and Roffler et al., Bioch; m.Pharmacol, 42: 2062~2065, (1991)). Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs that target colon cancer), receptor-mediated DNA targeting via cell-specific ligands, lymphocyte-induced tumor targeting As well as highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technique to target protein specific to tumor tissue (Hughes et al., Cancer Research, 49: 6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104: 179-187, (1992)). In general, receptors are involved in constitutive or ligand-induced endocytic pathways. Clusters of these receptors in clathrin-coated pits enter cells via clathrin-coated vesicles, pass through acidified endosomes where receptors are sorted, and then recirculate to the cell surface Or stored intracellularly or degraded in lysosomes. Internalization pathways function in a variety of functions such as nutrient absorption, removal of activated proteins, elimination of opportunistic invasion of macromolecules, viruses and toxins, ligand dissociation and degradation, and regulation of receptor concentrations. Many receptors follow one or more intracellular pathways depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-dependent endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10: 6, 399-409 (1991)).

  The composition can be administered orally, parenterally (eg, intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, by topical intranasal administration or by inhalation. Or can be administered topically.

Pharmaceutically Acceptable Carriers The compositions described herein can be used therapeutically in combination with a pharmaceutically acceptable carrier.

  Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th edition), A.M. R. In Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of this pharmaceutically acceptable carrier include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of this solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Still other carriers include delayed release preparations, such as solid hydrophobic polymer semipermeable matrices containing antibodies, which are in the form of, for example, molded films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

  Pharmaceutical carriers are well known to those skilled in the art. These are most typically standard carriers for drug administration to humans, including solutions such as sterile water, saline, and physiological pH buffers. The composition can be administered intramuscularly or subcutaneously. Other compounds are administered according to standard methods practiced by those skilled in the art.

  Pharmaceutical compositions can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. The pharmaceutical composition may contain one or more active ingredients such as antibacterial agents, anti-inflammatory agents, anesthetics and the like.

  The pharmaceutical composition can be administered in a number of ways depending on whether local or systemic treatment is desired and on the site to be treated. Administration is topical (including ophthalmic, vaginal, rectal, nasal), oral, by inhalation, or parenteral, eg, intravenous, subcutaneous, intraperitoneal or intramuscular It may be by internal injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

  Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Water soluble carriers include water, alcoholic / water soluble solutions, emulsions or suspensions containing saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (eg, those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases.

  Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, water, powder or oily bases, thickeners and the like may be necessary and desirable.

  Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous bases, capsules, sachets, pills, powders, granules or tablets. Thickeners, seasonings, diluents, emulsifiers, dispersion aids or binders can also be used.

  Some compositions include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, By reaction with pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono, di, trialkyl and In some cases, it may be administered as a pharmaceutically acceptable acid addition salt or base addition salt formed by reaction with an arylamine and a substituted ethanolamine.

  Since the procytotoxins of the present invention are amphoteric, they can be utilized as free bases, as acid addition salts or as metal salts. The salts must of course be pharmaceutically acceptable and these will include metal salts, particularly the appropriate potassium or sodium salts which are alkali and alkaline earth metal salts. A variety of pharmaceutically acceptable acid addition salts are available. These include acid addition salts prepared from both organic and inorganic acids, for example, pharmaceutically acceptable acid addition salts may be mineral acids. Typical acids that may be mentioned as examples include citric acid, succinic acid, lactic acid, hydrochloric acid and hydrobromic acid. Such products are readily prepared by methods well known to those skilled in the art.

  Furthermore, the compositions of the present invention can also be modified to have longer clearance rates, thus increasing bioavailability by protecting the composition from immune responses and other elimination mechanisms provided by the subject. It can also be increased. In fact, WO 9/2802 shows that PEGylated compounds reduce immunogenicity and antigenicity and disclose that they circulate in the bloodstream considerably longer than unconjugated proteins. For example, PEG (polyethylene glycol) polymer chains can be converted to prolactin variants / prolactin receptors by methods well known in the art, such as the PEGylation method described in Wang et al., Advanced Drug Delivery Reviews; 54 (2002) 547-570. It can bind to an antagonist, which is incorporated herein by reference.

  However, other agents that can increase the elimination half-life of the therapeutic compositions of the present invention are known to those of skill in the art and are contemplated herein.

  In addition, the compositions described herein can be modified with hydroxyethyl starch (HES). HES is a naturally occurring derivative of amylopectin and is degraded by α-amylase in the body. Methods for making HES-protein conjugates are known in the art. See, for example, EP 1398322, DE 2616086 and DE 2646854, which are incorporated herein by reference.

  In all such compositions, the cytolytic peptide is usually the major physiologically active constituent molecule. However, the peptides of the invention can be formulated with another pharmacological agent for combination therapy. When used for cancer treatment, for example, they can be formulated with compatible conventional chemotherapeutic agents.

Therapeutic Uses Effective doses and dosing schedules for the compositions can be determined empirically, and making such determinations is within the skill in the art. The dosage range for administration of this composition is a dosage range that is sufficiently large to produce the desired effect upon which the symptoms of the disease are affected. This dose should not be so high as to cause adverse side effects such as unwanted cross-reactions, anaphylactic reactions, and the like. In general, dosage will vary depending on the patient's age, symptoms, sex and degree of disease, route of administration, or whether other drugs are included in the therapy, and can be determined by one skilled in the art. In case of any contraindications, the dose can be adjusted by the individual physician. The dose can vary and can be administered in one or more doses daily for a day or days. Guidance can be found in the literature regarding appropriate doses for a given class of pharmaceutical products. For example, guidance on selecting appropriate doses of antibodies can be found in the literature on therapeutic use of antibodies, such as Handbook of Monoclonal Antibodies, Ferrone et al., Ed., Noges Publications, Park Ridge, NJ. (1985) Chapter 22 and pages 303-357; Smith et al., Antibodies in Human Diagnostics and Therapy, Haber et al., Ed., Raven Press, New York (1977) 365-389. A typical daily dose of antibody used alone may be from about 1 μg / kg body weight to 100 mg / kg body weight or even more per day depending on the factors described above.

  The effectiveness of this therapeutic composition for treating, inhibiting or preventing tumor formation or tumor growth following administration of the disclosed composition can be assessed by various methods known to the skilled practitioner. For example, one of ordinary skill in the art will find that the compositions disclosed herein in a subject by observing that the composition reduces tumor formation or tumor growth or prevents further increases in tumor formation or tumor growth. It will be appreciated that it is effective in treating or inhibiting tumor formation or tumor growth. Also, the effectiveness of administration of the disclosed composition can be determined by measuring the concentration of tumor-specific antigen present in the blood. Methods for measuring the concentration of tumor specific antigen are well known in the art.

  The compositions that inhibit tumor formation or tumor growth disclosed herein can be administered prophylactically to patients or subjects at risk of tumor formation or tumor growth.

  Other molecules that interact with the tumor and inhibit tumor formation or tumor growth, but do not have specific pharmaceutical functions, but for example to track changes in the chromosomes of cells or for the delivery of diagnostic tools Other molecules that can be used for delivery can be delivered in a manner similar to that described for pharmaceuticals.

  The disclosed compositions and methods can also be used, for example, as a tool for isolating and testing novel candidate drugs for various cancers.

  The disclosed compositions and methods can also be used to study cell death. The disclosed compositions and methods can also be used to study the cellular expression of MMPs in cells.

  The disclosed compositions and methods can also be used to study the effects of different cytotoxins on different cell types, and to study the ability of different enzymes to cleave different proteolytic cleavage sites. It can also be used.

Method for selectively destroying target cells Also disclosed is a method for selectively destroying target cells. This disclosed method typically involves contacting a target cell with the procytotoxin disclosed herein. For example, the procytotoxin can have a cytotoxic peptide attached to LAP via a peptide bond, which is sensitive to cleavage by a target-specific protease. The target cell may be a microcirculation system surrounding the cancer cell and a cell contained in the cancer cell.

  The procytotoxins of the present invention are typically converted to and activated by a target cell-specific activity that is typically converted to a cytotoxic peptide specifically for the target cell. In other words, the target cell has a specific mechanism for converting procytotoxin to cytotoxin. The activated cytotoxic peptide acts on the target cell and selectively destroys the target cell. For example, the activity associated with the cell can be a protease.

  Accordingly, a method is described for selectively destroying a target cell comprising contacting the target cell with a procytotoxin in which a proteolytic cleavage site is provided between the latency-related peptide and the cytotoxic peptide. For example, the target cell can be a cancer cell, but any cell that contains a cell-specific protease can be targeted.

  Target cells can have proteases that selectively cleave proteolytic cleavage sites bound by LAP. Thus, if the procytotoxin contains an MMP cleavage site, target cells with MMP can cleave the proteolytic cleavage site and “activate” the cytolytic peptide. In addition, procytotoxins can include target molecules. The target sequence can itself act as a LAP and can direct procytotoxins to the neovasculature and cytoskeletal elements surrounding the target cell. For example, the procytotoxin can comprise a target molecule selected from the group consisting of an RGD target sequence and an anti-fibronectin ED-B antibody.

  In one embodiment, the procytotoxin comprises a latency-related peptide and a cytotoxic peptide, a proteolytic cleavage site is provided between the latency-related peptide and the cytotoxic peptide, the cytotoxic peptide is a cytolytic peptide, and the protein The degradable cleavage site is an MMP cleavage site and LAP contains a precursor peptide of TGFβ-1, 2, 3, 4 or 5. For example, a procytotoxin can include a latency-related peptide and a cytotoxic peptide, and the cytotoxic peptide in which the proteolytic cleavage site is located between the latency-related peptide and the cytotoxic peptide is melittin and proteolytic The cleavage site is an MMP2 cleavage site and LAP is a precursor peptide of TGFβ-1.

  In another example, a procytotoxin can include a latency-related peptide and a cytotoxic peptide, a proteolytic cleavage site is provided between the latency-related peptide and the cytotoxic peptide, the cytotoxic peptide is an amoebopore, The degradable cleavage site is the MMP2 cleavage site, and LAP is a precursor peptide of TGFβ-1. In either instance, off-target cells that do not contain MMP are unable to activate protoxins and are therefore not affected by protoxins.

  Therapeutic treatment of cancer using the cytolytic peptide-based procytotoxin of the present invention is particularly advantageous because it is known that the cytolytic peptide is absorbed into the cell membrane of the target cell. Thus, even after the target cells are lysed, the cytolytic peptide is prevented from acting on adjacent off-target cells and causing undesired destruction. See, for example, Leippe et al. (1991) Proc. Natl. Acad. Sci. 88: 7659-7663. Furthermore, it should be noted that the agents of the present invention are generally small peptides with a fairly short half-life, at least if no special stabilization steps (above) are performed. Thus, even if a small amount of activated cytolytic peptide leaks out of the surface of a cancer cell, it should be fairly short lived, causing little, if any, destruction of off-target cells.

Methods for treating cancer in a patient Further disclosed are methods of treating cancer in a patient. In general, the method includes administering to the patient a therapeutically effective amount of a procytotoxin. In certain embodiments, the procytotoxin can be based on amoebopore, melittin or a cytolytic peptide derived therefrom.

  Also disclosed is a method of treating a cancer patient comprising administering a therapeutically effective amount of a pharmaceutical composition described herein. For example, the dose of procytotoxin may be about 1 μg / kg body weight to 100 mg / kg body weight or more per day. For example, the dose can be 5 mg / kg to 10 mg / kg. One or more procytotoxins can be administered and combinations of procytotoxins can also be administered. One skilled in the art can readily determine the optimal delivery route, dose, and therapy for a given mammalian host. It will be appreciated that the actual dosage used will vary depending on the particular composition formulated, the particular compound used, the mode of administration and the particular site being treated, the host and the disease. Many factors are considered which modify the action of the drug, including age, weight, sex, diet, time of administration, route of administration, excretion rate, patient symptoms, combination drug, response sensitivity and severity of the disease.

  The disclosed compositions can be used to treat any solid tumor. A representative and non-limiting list of cancers in which the disclosed compositions can be used for therapy is as follows. Lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, kidney cancer, lung cancer, For example, small and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, mouth, throat, larynx and lung squamous cell carcinoma, Colon cancer, cervical cancer, cervical cancer, breast cancer, and epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colon cancer, hematopoietic cancer, testicular cancer, colorectal cancer, prostate cancer, or Pancreatic cancer.

  The compounds disclosed herein can also be used to treat precancerous conditions such as cervical and anal dysplasia, other dysplasia, hyperplasia, hyperplasia, atypical hyperplasia, and neoplasia. . For example, the disclosed procytotoxin can be used to target any angiogenic tissue that expresses MMP2.

  A method of treating a cancer patient comprises administering to the patient a therapeutically effective amount of a procytotoxin comprising a latency-related peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency-related peptide and the cytotoxic peptide Can be included. For example, as described above, the cytotoxic peptide can be a pore-forming cytolytic peptide. In certain embodiments, the cytolytic peptide can be melittin, a melittin analog, or a melittin derivative.

Methods for providing latency to cytotoxins Also disclosed is a method for providing latency to cytotoxins comprising covalently binding cytotoxins to latency related peptides and proteolytic cleavage sites, said latency related peptides and proteins Degradable cleavage sites provide latency to the cytotoxin.

  In general, the procytotoxin is a cytotoxic peptide that is rendered nontoxic by sterically inhibiting charge neutralization and / or formation of conformations that render the peptide toxic. Specifically, the procytotoxin comprises a cytotoxic peptide attached to LAP via a peptide bond, which is sensitive to cleavage by a tumor specific protease. The LAP of the present invention prevents cytotoxic peptides from forming an active conformation. For example, when procytotoxin contacts target cells, tumor-specific proteases cleave the peptide bond between LAP and cytotoxin, thereby allowing the cytotoxic peptide to form a pore conformation and disrupt the cell membrane To.

  A method for providing latency to a cytotoxin can include covalently coupling the cytotoxin to a latency-related peptide and a proteolytic cleavage site, wherein the latency-related peptide and the proteolytic cleavage site are the cytotoxin. To provide latency. As described above, the cytotoxin can be a pore-forming cytolytic peptide. In certain embodiments, the cytolytic peptide can be melittin, a melittin analog, or a melittin derivative. The proteolytic cleavage site can be an MMP cleavage site and the LAP can comprise a precursor peptide of TGFβ-1, 2, 3, 4 or 5.

  A method for providing latency to a cytotoxin can also include covalently coupling the cytotoxin to a latency-related peptide and a proteolytic cleavage site, wherein the cytotoxin forms a conformation that renders the peptide toxic. Thus making the cytotoxic peptide non-toxic.

  A method for providing latency to a cytotoxin can also include covalently coupling the cytotoxin to a latency-related peptide and a proteolytic cleavage site, wherein the latency-related peptide is surrounded by a protective shell around the cytotoxic peptide. (“Caging”), thereby shielding the cytotoxic peptide and preventing or preventing its interaction with other molecules on the cell surface or molecules important for its activity. This can be accomplished by allowing the LAP to form a dimer around the cytotoxic peptide, thereby forming a protective shell or cage around the cytotoxic peptide.

  A method for providing latency to a cytotoxin can also include covalently binding the cytotoxin to a latency-related peptide and a proteolytic cleavage site, wherein the latency-related peptide is formed by the alpha helix structure of the cytotoxin. It is sterically blocked from being done. Furthermore, cytotoxic peptides can be modified to contain negatively charged amino acids, thereby preventing the formation of toxic pore conformation.

Kits Disclosed herein are kits that provide reagents that can be used to practice the methods disclosed herein. The kit may be any reagent or combination of reagents described herein, or any reagent or combination that will be understood or required to practice the disclosed method. Combinations of reagents can be included. For example, the kit can include the procytotoxin described in the particular embodiment of the method and other reagents required for the intended use of the pharmaceutical excipient and procytotoxin.
〔Example〕

  The following examples provide those skilled in the art with a complete disclosure and description of how the compounds, compositions, products, devices, and / or methods described in the claims herein are made and evaluated. And is intended only to illustrate the invention and is not intended to limit the disclosure. For numerical values (eg, quantities, temperatures, etc.), efforts are made to ensure accuracy, but some errors and deviations should be taken into account. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Celsius or ambient temperature, and pressure is at or near atmospheric.

Cloning of melittin-based procytotoxins Generation of double-stranded MMP2 / melittin templates Two complementary oligos, EcoRI / MMP2 / melittin sense strand (SEQ ID NO: 17) and EcoRI / MMP2 / melittin antisense strand (sequence) No. 18) was synthesized using standard methods. The two oligos were mixed at an equal molar ratio and then heated in a 1 liter beaker with boiling water for 5 minutes. This mixture was cooled to room temperature on a laboratory bench to obtain a double-stranded MMP2 / melittin template.

Amplification and Subcloning of Double-Stranded MMP2 / Melittin Template PCR was performed using double-stranded MMP2 / melitin template with primer P1 (SEQ ID NO: 19) and primer P2 (SEQ ID NO: 20) having a phosphorylated 5 ′ end. Amplified with PCR was performed under the following conditions. That is, 94 cycles of 94 ° C. for 5 minutes, then 94 ° C. for 30 seconds, 62.5 ° C. for 30 seconds and 72 ° C. for 30 seconds were performed for 28 cycles, then 72 ° C. for 10 minutes. The PCR product was then purified using the QIAGEN kit (catalog number: 28104).

  After purification of the PCR product, the purified product was digested with EcoRI. Further, the PcR2.1 vector was digested with EcoRI and EcoRV. The EcoRI digested PCR product was then incubated with the EcoRI / EcoRV digested PcR2.1 vector so that the digested PCR product could be inserted into the digested PcR2.1 vector. This resulting plasmid was named pCRMMP2 / melittin.

Amplification of LAP fragment The LAP cDNA fragment was amplified using PCR with plasmid phTGFB-2 (ATCC # 59954) as template. A LAP sense primer containing a HindIII site (SEQ ID NO: 21) and a LAP antisense primer containing an EcoRI site (SEQ ID NO: 22) were used in the PCR reaction.

  PCR was performed under the following conditions. That is, 94 cycles at 94 ° C. for 5 minutes, followed by 25 cycles of 94 ° C. for 30 seconds, 75 ° C. for 30 seconds and 72 ° C. for 30 seconds, then 72 ° C. for 10 minutes. The PCR product was then purified using the QIAGEN kit (catalog number: 28104) and digested with HindIII and EcoRI. In parallel, the pCRMMP2 / melittin plasmid was also digested with HindIII and EcoRI.

  The HindIII / EcoRI digested PCR product was then incubated with the HindIII / EcoRI digested pCRMMP2 / melittin plasmid so that the digested PCR product could be inserted into the digested pCRMMP2 / melittin plasmid. This resulting plasmid was named pCRLAP / mmp2 / melittin.

Preparation of mammalian expression vector The mouse DHFR cDNA fragment was excised from plasmid pSV2-DHFR (ATCC # 37146) using BstX1 and NotI. The pIRES2-EGFP plasmid was also digested with BstX1 and NotI (Clontech).

  The BstX1 / NotI digested DHFR cDNA fragment and the BstX1 / NotI digested pIRES2-EGFP plasmid were then purified. The BstX1 / NotI-digested DHFR cDNA fragment and BstX1 / NotI-digested pIRES2-EGFP plasmid are then inserted into the digested pIRES2-EGFP plasmid under appropriate conditions that allow insertion of the digested DHFR cDNA fragment. Incubated together. This plasmid was named pIRES / DHFR.

  The LAP / mmp2 / melittin fragment was excised from pCRLAP / mmp2 / melittin using HindIII and XhoI and inserted into the pIRES / DHFR plasmid cut with SmaI. The resulting mammalian expression vector was named pLAP / mmp2 / melittin / IRES / DHFR.

CHO cell expression CHO / DHFR- (ATCC # 9096) cells were transfected with the pLAP / mmp2 / melittin / IRES / DHFR plasmid, DMEM + 1.5 g / L sodium bicarbonate, 0.1 mM hypoxanthine, 0.016 mM thymidine, Selection by G418 in medium containing 0.002 mM methotrexate, 10% FBS, 1 × PSN, and 1000 μg / ml G418.

  Single colonies were picked from selection subjects and grown for expression.

  To induce expression, the selected cells were replaced with serum-free medium supplemented with 500 nM methotrexate and incubated overnight. The medium was collected for further purification.

Cloning of melittin-based procytotoxin production of LAP-MMP2-melittin fusion protein The LAP-MMP2-melittin construct was cloned into the pIRES-DHFR vector. This plasmid DNA was then transfected into CHO cells as described in Example 1. 48 hours after transfection in medium containing DMEM + 1.5 g / L sodium bicarbonate, 0.1 mM hypoxanthine, 0.016 mM thymidine, 0.002 mM methotrexate, 10% FBS, 1 × PSN, and 1000 ug / ml G418. Cells were selected by G418. Cells were continuously cultured in G418 medium containing 10% FBS. After G418 selection is complete, the transfected CHO cells are acclimated to medium containing only 1% FBS.

  Medium MTX concentration was increased from 50 nM MTX to 500 nM MTX to induce transfected CHO cells to produce LAP-MMP2-melittin fusion protein. At each MTX concentration, cell lysates were obtained and analyzed by Western analysis using SDS PAGE followed by LAP antiserum (FIG. 1). Western analysis revealed a protein with the expected molecular weight of the LAP fusion protein. This protein was most enhanced at MTX concentrations of 250 nm and 500 nm. A very dark band was also seen at 50 nM MTX concentration, but there was also a mixture of other bands.

  Lysates were obtained from untransfected CHO cells exposed to 500 nM MTX to show that this protein observed in transfected CHO cells was simply an artifact. FIG. 2 shows that this protein is absent in CHO cells that have not been transfected with the LAP MMP2 melittin fusion protein. It can therefore be concluded that the LAP fusion protein is present in lysates obtained from cells transfected with the fusion protein.

  The next step is to see if the LAP fusion protein is secreted into the medium. This was examined by collecting media from cells transfected in the presence of 500 nM MTX. Prior to media collection, cells were exposed to each of the following MTX concentrations: 50 nM, 100 nM, 250 nM, and 500 nM for 48 hours. The medium was then harvested from cells exposed to 500 nM MTX, cell debris was pelleted, and the supernatant was stored at -20 degrees Celsius. In order to concentrate the amount of fusion protein in the medium, the medium was lyophilized. The lyophilized medium was then resuspended in water to a volume approximately 10 times less than the original volume. The LAP MMP2 melittin fusion protein was partially purified from the concentrated medium by hLAP affinity chromatography using a batch method. The sample was analyzed by Western blot using native PAGE followed by hLAP antiserum (FIG. 3). This sample was compared to the protein obtained from the transfected cell lysate. The same band was observed for both of these samples on Western.

  LAP MMP2 melittin was purified from the culture medium by another method. Samples were lyophilized as described above and a Waters HPLC size exclusion column was used for purification. FIG. 4 shows the partially purified protein.

Target cell killing in vitro The in vitro function of the LAP MMP2 melittin fusion protein can also be evaluated. For example, DU-145 prostate cancer cells secreting MMP2 can be seeded on day 0 in 48-well plates. On day 1 these cells can be treated with this fusion protein for 3 hours and then cell lysis is measured by LDH (lactate dehydrogenase) assay. This reaction can be visualized by an LDH assay.

  In addition or alternatively, purified LAP MMP2 melittin fusion protein can be incubated overnight at 37 degrees Celsius in the presence or absence of active MMP2. The next day, treated and untreated samples can be incubated with CHO cells. Cells can be treated in 1 hour and then an LDH assay can be performed to determine whether the cells have lysed. Treatment of the fusion protein with active MMP2 overnight can cleave this protein at the MMP2 cleavage site and release free melittin. When MMP2-treated fusion protein is incubated with CHO cells, the cells show increased cell lysis compared to cells incubated with untreated fusion protein.

Target cell killing in vivo The function of the LAP MMP2 melittin fusion protein can also be demonstrated in vivo. For example, C57 female mice can be inoculated subcutaneously with about 800,000 B16 cells. Seven days after tumor inoculation, mice can be injected intramuscularly with 50 μg of LAP MMP2 melittin plasmid DNA. The mice can then be injected twice more per week. Tumor size decreases and mouse lifespan increases.

  Various publications are referenced throughout this application. The disclosures of these publications are incorporated herein by reference in their entirety in order to more fully describe the state of the art in the technical field of the present invention.

It is a figure which shows the detection of hLAP-MMP2-melittin in the transfected CHO cell. Western blot using 30 μg of LAP CHO (transfected with hLAP-MMP2-melittin) and CHO (untransfected) lysates (searching for μg / μl throughout) followed by SDS PAGE followed by hLAP antiserum Was analyzed. 50 ng hLAP was used as a positive control. It is a figure which shows the induction by the methotrexate of LAP-MMP2-melittin production in the transfected CHO cell. CHO cells were transfected with LAP-MMP2-melittin plasmid DNA in the presence of normal transfection medium. After transfection, the cells were cultured in a medium containing 10% FBS and 50 nM MTX. The amount of FBS was gradually reduced to 1% with the medium. The amount of MTX in the medium was then increased to induce the cells to produce protein. Lysates were obtained from cells at various concentrations of MTX and analyzed by Western blot using SDS PAGE followed by hLAP antiserum. For each MTX concentration, approximately 30 μg of lysate was analyzed and 50 ng hLAP (Western blot positive control) was analyzed. FIG. 5 shows analysis of lysates and supernatants from CHO cells transfected with hLAP-MMP2-melittin under non-denaturing conditions. The hLAP fusion protein (hLAP-MMP2-melittin) present in the medium was concentrated before analysis by affinity chromatography. 30 μg of fusion protein lysate was analyzed and 50 ng hLAP was used as a positive control. All samples were analyzed by Western blot using Native PAGE followed by hLAP antiserum. FIG. 5 shows analysis of partially purified hLAP-MMP2-melittin fusion protein. Purified hLAP-MMP2-melittin fusion protein was analyzed by Western blot using SDS PAGE followed by hLAP antiserum. The fusion protein was partially purified by size exclusion chromatography on a Waters HPLC system.

Claims (62)

  A procytotoxin comprising a latency-related peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency-related peptide and the cytotoxic peptide.   The procytotoxin according to claim 1, wherein the cytotoxic peptide is a cytolytic peptide.   The cytolytic peptides are Ae I, sea anemone cytolysin, erolysin, amatoxin, amoeba dispar derived from entamoeba dispar, amoeba pore homolog, brevinin-2E, brevinin-2E, Barbatridine, Enterococcus faecalis cytolysin, Delta hemolysin, Diphtheria toxin, Vibrio cholerae Ertol cytolysin, Equinatoxin, Aeromonas hydrophilo Lentin (esculentin), granuridine (granlysin), vibrio pa Vibrio parahaemolyticus hemolysin, Streptococcus intermedius intermedirisin, lentiviral cytolytic peptide, actinobacillus actinomicin b , Cell membrane-related lymphotoxin, methenkephalin, neokyotorphin, neokyotolphine fragment 1, neokyotolphine fragment 2, neokyotolphine fragment 3, neokyotolphine fragment 4, NK-rigid , Paradaxin, perforin, perfringolysin O of clostridial perfringens, thetatoxin, faroricin, farotoxin, and streptolysin, an analog of pore-forming cell-lytic peptide, and pore-forming cell-lytic peptide The procytotoxin according to claim 2, selected from the group consisting of:   The procytotoxin according to claim 3, wherein the cytolytic peptide is amoebapore.   5. The procytotoxin according to claim 4, comprising SEQ ID NO: 10 or SEQ ID NO: 12.   The procytotoxin according to claim 3, wherein the cytolytic peptide is melittin.   The procytotoxin according to claim 6, comprising SEQ ID NO: 2 or SEQ ID NO: 4.   The procytotoxin according to claim 1, wherein the latency-related peptide comprises a precursor peptide of TGFβ-1, 2, 3, 4 or 5.   The procytotoxin according to claim 1, wherein the proteolytic cleavage site is a matrix metalloprotease (MMP) cleavage site.   The proteolytic cleavage site according to claim 9, wherein the proteolytic cleavage site is an MMP2 cleavage site.   A pharmaceutical composition comprising the procytotoxin according to claim 1 and a pharmaceutically acceptable excipient.   A method of treating cancer in a patient, comprising: a procytotoxin comprising a latency-related peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency-related peptide and the cytotoxic peptide. Administering a therapeutically effective amount.   The method according to claim 12, wherein the cytotoxic peptide is a cytolytic peptide.   The cytolytic peptide is Ae I, sea anemone cytolysin, erolysin, amatoxin, amoeba pore derived from enthamoeba disper, amoeba pore homolog, blebinin-1E, blebinin-2E, barbatridin, enterococcus faecalis cytolysin , Delta hemolysin, diphtheria toxin, Vibrio cholerae Eltor cell lysin, equinatoxin, Aeromonas hydrophila enterotoxin, esculentin, granuridine, Vibrio parahaemolyticus hemolysin, Streptococcus intermedius inter Medilysine, Lentiviral cytolytic peptide, Actinobacillus actinomycetemcomitans leukotoxin, magainin, melittin, cell membrane related lymphotoxin Metoenkephalin, Neo-Kyotolphine, Neo-Kyotolphine fragment 1, Neo-Kyotolphine fragment 2, Neo-Kyotolphine fragment 3, Neo-Kyotolphine fragment 4, NK-lysine, paradaxin, perforin, Clostridium perfringens perfringolysine O, 14. The method of claim 13, wherein the method is selected from the group consisting of theta toxin, faroricin, farotoxin, and streptricin, an analog of a pore-forming cell lytic peptide, and a derivative of a pore-forming cell lytic peptide.   The method according to claim 14, wherein the cytolytic peptide is amoebapore.   15. The method of claim 14, wherein the procytotoxin comprises SEQ ID NO: 10 or SEQ ID NO: 12.   The method according to claim 14, wherein the cytolytic peptide is melittin.   15. The method of claim 14, wherein the procytotoxin comprises SEQ ID NO: 2 or SEQ ID NO: 4.   The method of claim 12, wherein the latency-related peptide comprises the precursor peptide of TGFβ-1, 2, 3, 4 or 5.   13. The method of claim 12, wherein the proteolytic cleavage site is a matrix metalloprotease (MMP) cleavage site.   21. The method of claim 20, wherein the proteolytic cleavage site is an MMP2 cleavage site.   Cancer is lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancer For example, small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of mouth, throat, larynx and lung , Colon cancer, cervical cancer, cervical cancer, breast cancer, as well as epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colon cancer, hematopoietic cancer, testicular cancer, colorectal cancer, prostate cancer, Or the method of claim 12 selected from the group consisting of pancreatic cancer.   A method for providing latency to a cytotoxin, comprising covalently coupling the cytotoxin to a latency-related peptide and a proteolytic cleavage site, wherein the latency-related peptide and the proteolytic cleavage site are latent to the cytotoxin. How to provide sex.   24. The method of claim 23, wherein the cytotoxic peptide is a cytolytic peptide.   The cell lytic peptide is Ae I, sea anemone cytolysin, erolysin, amatoxin, amoeba pore derived from enthamoeba disper, amoeba pore homolog, brevinin-1E, blebinin-2E, barbatridine, enterococcus faecalis cytolysin , Delta hemolysin, diphtheria toxin, Vibrio cholerae Eltor cell lysin, equinatoxin, Aeromonas hydrophila enterotoxin, esculentin, granuridine, Vibrio parahaemolyticus hemolysin, Streptococcus intermedius inter Medilysine, Lentiviral cytolytic peptide, Actinobacillus actinomycetemcomitans leukotoxin, magainin, melittin, cell membrane related lymphotoxin Metoenkephalin, Neo-Kyotolphine, Neo-Kyotolphine fragment 1, Neo-Kyotolphine fragment 2, Neo-Kyotolphine fragment 3, Neo-Kyotolphine fragment 4, NK-lysine, paradaxin, perforin, Clostridium perfringens perfringolysine O, 25. The method of claim 24, wherein the method is selected from the group consisting of theta toxin, faroricin, farotoxin, and streptricin, a pore-forming cell lytic peptide analog, and a pore-forming cell lytic peptide derivative.   26. The method of claim 25, wherein the cytolytic peptide is amoebapore.   26. The method of claim 25, wherein the procytotoxin comprises the sequence of SEQ ID NO: 10 or SEQ ID NO: 12.   26. The method of claim 25, wherein the cytolytic peptide is melittin.   26. The method of claim 25, wherein the procytotoxin comprises a sequence consisting of SEQ ID NO: 2 or SEQ ID NO: 4.   24. The method of claim 23, wherein the latency-related peptide comprises the precursor peptide of TGFβ-1, 2, 3, 4 or 5.   24. The method of claim 23, wherein the proteolytic cleavage site is a matrix metalloprotease (MMP) cleavage site.   A method for selectively destroying a target cell that is a cancer cell, the target cell comprising a latency-related peptide and a cytotoxic peptide, wherein a proteolytic cleavage site is provided between the latency-related peptide and the cytotoxic peptide Contacting the procytotoxin, wherein the latency-related peptide acts to prevent the procytotoxin from forming a cytolytically active conformation.   33. The method of claim 32, wherein the cytotoxic peptide is a cytolytic peptide.   The cytolytic peptide is Ae I, sea anemone cytolysin, erolysin, amatoxin, amoeba pore derived from enthamoeba disper, amoeba pore homolog, blebinin-1E, blebinin-2E, barbatridin, enterococcus faecalis cytolysin , Delta hemolysin, diphtheria toxin, Vibrio cholerae Eltor cell lysin, equinatoxin, Aeromonas hydrophila enterotoxin, esculentin, granuridine, Vibrio parahaemolyticus hemolysin, Streptococcus intermedius inter Medilysine, Lentiviral cytolytic peptide, Actinobacillus actinomycetemcomitans leukotoxin, magainin, melittin, cell membrane related lymphotoxin Metoenkephalin, Neo-Kyotolphine, Neo-Kyotolphine fragment 1, Neo-Kyotolphine fragment 2, Neo-Kyotolphine fragment 3, Neo-Kyotolphine fragment 4, NK-lysine, paradaxin, perforin, Clostridium perfringens perfringolysine O, 34. The method of claim 33, wherein the method is selected from the group consisting of theta toxin, faroricin, farotoxin, and streptolysin, a pore forming cell lytic peptide analog, and a pore forming cell lytic peptide derivative.   35. The method of claim 34, wherein the cytolytic peptide is amoebapore.   35. The method of claim 34, comprising SEQ ID NO: 10 or SEQ ID NO: 12.   35. The method of claim 34, wherein the cytolytic peptide is melittin.   35. The method of claim 34 comprising SEQ ID NO: 2 or SEQ ID NO: 4.   33. The method of claim 32, wherein the latency-related peptide comprises the precursor peptide of TGFβ-1, 2, 3, 4 or 5.   35. The method of claim 32, wherein the proteolytic cleavage site is a matrix metalloprotease (MMP) cleavage site.   41. The method of claim 40, wherein the proteolytic cleavage site is an MMP2 cleavage site.   A method for selectively destroying a target cell contained in a microcirculation system surrounding a cancer cell, the target cell comprising a latency-related peptide and a cytotoxic peptide, wherein the proteolytic cleavage site is the latency-related peptide and the cytotoxic peptide Wherein the latency-related peptide acts to prevent the procytotoxin from forming a cytolytically active conformation.   43. The method of claim 42, wherein the cytotoxic peptide is a cytolytic peptide.   The cell lytic peptide is Ae I, sea anemone cytolysin, erolysin, amatoxin, amoeba pore derived from enthamoeba disper, amoeba pore homolog, brevinin-1E, blebinin-2E, barbatridine, enterococcus faecalis cytolysin , Delta hemolysin, diphtheria toxin, Vibrio cholerae Eltor cell lysin, equinatoxin, Aeromonas hydrophila enterotoxin, esculentin, granuridine, Vibrio parahaemolyticus hemolysin, Streptococcus intermedius inter Medilysine, Lentiviral cytolytic peptide, Actinobacillus actinomycetemcomitans leukotoxin, magainin, melittin, cell membrane related lymphotoxin Metoenkephalin, Neo-Kyotolphine, Neo-Kyotolphine fragment 1, Neo-Kyotolphine fragment 2, Neo-Kyotolphine fragment 3, Neo-Kyotolphine fragment 4, NK-lysine, paradaxin, perforin, Clostridium perfringens perfringolysine O, 44. The method of claim 43, wherein the method is selected from the group consisting of theta toxin, faroricin, farotoxin, and streptricin, a pore-forming cell lytic peptide analog, and a pore-forming cell lytic peptide derivative.   45. The method of claim 44, wherein the cytolytic peptide is amoebapore.   45. The method of claim 44, comprising SEQ ID NO: 10 or SEQ ID NO: 12.   45. The method of claim 44, wherein the cytolytic peptide is melittin.   45. The method of claim 44, comprising SEQ ID NO: 2 or SEQ ID NO: 4.   43. The method of claim 42, wherein the latency related peptide comprises the precursor peptide of TGFβ-1, 2, 3, 4 or 5.   43. The method of claim 42, wherein the proteolytic cleavage site is a matrix metalloprotease (MMP) cleavage site.   51. The method of claim 50, wherein the proteolytic cleavage site is an MMP2 cleavage site.   A method for selectively destroying a target cell, which is a cancer cell that produces MMP2, wherein the target cell includes a latency-related peptide and a cytotoxic peptide, and a proteolytic cleavage site is located between the latency-related peptide and the cytotoxic peptide. Wherein the latency-related peptide acts to prevent the procytotoxin from forming a cytolytically active conformation.   53. The method according to claim 52, wherein the cancer cells producing MMP2 are tumor microvascular endothelial cells producing MMP2.   53. The method of claim 52, wherein the cytotoxic peptide is a cytolytic peptide.   The cell lytic peptide is Ae I, sea anemone cytolysin, erolysin, amatoxin, amoeba pore derived from enthamoeba disper, amoeba pore homolog, brevinin-1E, blebinin-2E, barbatridine, enterococcus faecalis cytolysin , Delta hemolysin, diphtheria toxin, Vibrio cholerae Eltor cell lysin, equinatoxin, Aeromonas hydrophila enterotoxin, esculentin, granuridine, Vibrio parahaemolyticus hemolysin, Streptococcus intermedius inter Medilysine, Lentiviral cytolytic peptide, Actinobacillus actinomycetemcomitans leukotoxin, magainin, melittin, cell membrane related lymphotoxin Metoenkephalin, Neo-Kyotolphine, Neo-Kyotolphine fragment 1, Neo-Kyotolphine fragment 2, Neo-Kyotolphine fragment 3, Neo-Kyotolphine fragment 4, NK-lysine, paradaxin, perforin, Clostridium perfringens perfringolysine O, 55. The method of claim 54, wherein the method is selected from the group consisting of theta toxin, faroricin, farotoxin, and streptolysin, a pore-forming cell lytic peptide analog, and a pore-forming cell lytic peptide derivative.   56. The method of claim 55, wherein the cytolytic peptide is amoebapore.   56. The method of claim 55, comprising SEQ ID NO: 10 or SEQ ID NO: 12.   56. The method of claim 55, wherein the cytolytic peptide is melittin.   56. The method of claim 55, comprising SEQ ID NO: 2 or SEQ ID NO: 4.   53. The method of claim 52, wherein the latency related peptide comprises the precursor peptide of TGFβ-1, 2, 3, 4 or 5.   53. The method of claim 52, wherein the proteolytic cleavage site is a matrix metalloprotease (MMP) cleavage site.   62. The method of claim 61, wherein the proteolytic cleavage site is an MMP2 cleavage site.

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