WO2011020107A2 - Compositions and methods for detection and treatment of breast cancer - Google Patents

Compositions and methods for detection and treatment of breast cancer Download PDF

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Publication number
WO2011020107A2
WO2011020107A2 PCT/US2010/045645 US2010045645W WO2011020107A2 WO 2011020107 A2 WO2011020107 A2 WO 2011020107A2 US 2010045645 W US2010045645 W US 2010045645W WO 2011020107 A2 WO2011020107 A2 WO 2011020107A2
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cancer
tissue
agent
disclosed
composition
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WO2011020107A3 (en
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Mikell A. Paige
Olga Timofeeva
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Georgetown University
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Georgetown University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
    • A61K47/552—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug one of the codrug's components being an antibiotic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins

Definitions

  • Magnetic resonance imaging provides a solution to the limitations of mammography MRI is virtually uninfluenced by breast density, a problem met in mammography of younger women In conipa ⁇ ng clinical breast examination, mammography, and MRI for detecting invasive breast cancer, the sensitivity was 18%, 33%, and 80%, respectively (Keana et al Magn Reson Med 2005) Howevei, MRI is not without its own limitations The same study showed that specificity with MRI is lower than with mammography, meaning that the ability to distinguish between benign and cancerous images was lower for MRI I ower specificity results in more findings judged as unceitam and requiring a follow-up exam
  • a breast homing peptide that incorporates a fluorescent chromophore, a non-metallic MRI contrast agent, and a therapeutic component can act as a tissue specific theranostic that can detect cancer by dual modality MRI fluorescence imaging, and treat the cancer by specific release of a therapeutic agent in the cancer cell
  • this invention in one aspect, ielates to compositions and methods related to detecting and treating cancer, particularly breast cancer
  • Figure 1 shows two routes to the fluorophore containing ammo acids Methods of attaching the fluorophore to an ammo acid residue that can be employed in SPPS are shown Method 1 is accomplished by tieatmg the 0-succmimide ester of the carboxyl containing fluoropliore with Fmoc-Lys OH This gives selective functionalizatton on the side-chain amine group Fmoc-protected ammo acid (2a) has been synthesized by this method m an overall 51% yield The same method is used to make Fmoc-protected ammo acid (2b), which has an overlapping emission bandw idth with (2a) but has a much higher quantum yield
  • NIR near IR
  • FIG. 16 shows the copper chelate ot bleomycin A5, marketed as Bleocm, attachment to the delivery system
  • bleomycin A5 copper chelate the primary amine is exposed and easily functionalized
  • bleomycin A5-copper chelate is treated with 2-azidoacetic acid, DCC, and HOBt to give the azido-conjugated compound
  • Treatment with 15% aqueous EDTA liberates the bleomycin uonjugdle from the copper (Kriege et al NEJM 2004, 351, 427-437)
  • Figure 6 demonstrates a simple one-step reaction by click chemistry for addition of a theranostic agent to the fully functional diagnostic agent
  • Figure 8 outlines the incorporation of the drug molecule.
  • the diagnostic system is treated with the azido functionalized maytansrnol in the presence of a copper sulfate and sodium ascorbate in a DMSO-waler solvent system to give the completed theranostic agent Synthesis of the complete theranostic agent
  • Figure 9 identifies the structure of disclosed drug entities that can be attached to the diagnostic system as theranostic agents
  • Figure 10 shows the azido functionalized dicarboxyhc acid (13) condensed with the boromc acid under Dean-Stark conditions to give the boromc esters of YK-3 250 and YK 3 237 following the method of Burke and co workers.
  • These azido functionalized drug agents are attached to the theranostic system via click chemistry Boromc ester functionalization for click chemistry attachment
  • Figure 11 shows the functionalization of the dansyl moiety of YK 4 272 and SCG-3- 285 with mono demethylation of the dimethylaimne group followed by conjugation with 2- azidoacetic acid
  • Mono demethylation of the tertiary amine can be accomplished usmg ACE-CI (Olofson et al J Org Chem 1984 49 2081 2082) Conjugation with O-succimmide ester of 2 a7ido-acetic acid gives the requisite azide for attachment to the theranostic system ⁇ ia click chemistry
  • Figure 12 shows Compound MP-201 which is a lead structure optimized to give nitro-containmg qurnazolmone. Dmydroqmnazolinone tubulin inhibitors
  • FIG. 24 Figure 13 shows the expression of ApaseP in cancer cells by nmnunohistostaining usmg an antibody agamst ApaseP ApaseP is richly exprebsed on MCF 7 and MDA MB-231 breast cancer cells. However, ApaseP expression was not detected on PC-3 prostate and A-549 lung cancer cells A) Fluorescence ApaseP antibody shown as light gray areas B) DIC C) DAPI staining of the nuclei shown as light gray areas
  • Figure 14 shows the delivery efficiency of the delivery system and the importance of the Tat sequence
  • the distribution of the homing peptide with or without the Tat cell penetrating sequence was observed in MCF7 breast cancer cells
  • the dansylated breast homing peptide that incorporates the Tat cell penetrating sequence efficiently penetrates MCF 7 breast cancer cells
  • Panel A shows the fluorescence of the peptide (light gray staining)
  • Panel B shows the DIC image of the cells
  • Panel C shows a merge of A and B
  • 26 figure 15 shows the homing peptide with and without Tat on another metastatic breast cancer cell line, MDA MB 231
  • the dansylated PEGA breast homing peptide conjugated to the TdI sequence shows penetration into MDA MB 231 cells
  • the dansylated PEGA breast homing peptide that does not incorporate the Tat sequence gave a considerably w eaker signal under the same treatment and imaging conditions
  • Panel A shows the fluorescence of the peptide (bright color staining)
  • Panel B shows the DIC miage of the cells
  • Panel C shows a merge of A and B
  • FIG. 27 Figure 16 shows the homing peptide with and without Tat on the prostate cancer cell line PC-3 Regardless of the presence or absence of Tat on the dansylated homing peptide no cell penetration was exhibited This shows the cell-type specificity of the homing peptide
  • Figure 19 shows the immunomstostainmg for ApaseP expression in cancer cells
  • Figure 21 shows images from confocal microscopy cell penetration of A-549 cells
  • Figure 22 shows live-imaging in MCF-7 xenograft model
  • Figure 23 shows STAT3 Hel2A 2 peptide growth inhibition assay in breast cancer and non cancerous breast cells
  • compositions and methods A. Compositions and methods
  • compositions and methods allow for the diagnosis and treatment of cancer (breast, lung, liver, prostate or intestinal) such that ⁇ theranostic approach can be taken
  • cancer breast, lung, liver, prostate or intestinal
  • the methods and compositions disclosed herein also provide a means of treating the a subject
  • the disclosed methods and compositions provide a theianostic approach to treating a disorder, such as cancer, by integrating diagnostics and therapeutics to improve the real-time treatment of a subject having for example, breast cancer
  • Thcranostics are useful in clinical diagnosis and management of a variety of diseases and disorders, which include, but are not limited to, e g , cardiovascular disease, cancer, infectious diseases, Alzheimei's Disease and the piediction of drug toxicity or drug resistance
  • compositions valuable for the diagnosis (or confirmation) and treatment of a disease or disorder such as breast cancer
  • the compositions comprise all or some of the following a detection moiety, a contrasting agent, a homing moiety, a cell-penetrating moiety and a therapeutic agent
  • the components of theianostics can be considered toxic
  • the composition is non-toxic
  • detection moiety 43 The disclosed compositions are comprised of a detection moiety and a contrasting agent The detection moiety can be used for identification purposes, Disclosed herein, the detection moiety can be used to identify cancer present in breast tissue
  • the detection moiety for example a fluorescent label, can specifically define a particular location of interest (i e breast cancer cells)
  • the disclosed composition comprises a detection moiety wherein the detection moiety can be a fluorophore
  • the disclosed composition comprises a fluorophore wherein the fluorophore can be dansyl, TAMRA, a cyanine dye, or a cyanme dye encapsulated m a cyolodext ⁇ n
  • composition comprises a contrasting agent wherein the contrasting agent can be an MRI contiastmg agent
  • composition comprises a MRI contrast agent wherein the contrasting agent can be non-metallic
  • trie disclosed composition comprises a non-metallic contrast agent wherein the contrasting agent can be mtroxide radical or derivative thereof
  • compositions can be used for targeting or homing to specific tissues or cell types based on the presence of d homing moiety
  • the homing moiety targets a specific hgand or environment and therefore can be used to transport different molecules or compositions to that location
  • a homing moiety, specific to breast tissue can be linked to a constrastmg agent and thus tiansport the contrasting agent to the targeted breast tissue
  • composition comprises a detection moiety and a contrasting agent and can further comprise a homing peptide
  • the above disclosed composition comprises a breast tissue-specific homing peptide
  • the disclosed breast tissue-specific homing peptide can be CPGPEGAGC
  • a cell-penetrating molecule is a molecule that can comprise a cell-penetratmg moiety
  • a moiety refers to a part of a molecule
  • Examples of cell penetrating molecules are 1 at, penetratm, pVEC, transportan etc
  • a cell penetrating moiety is a part of a molecule or functional group that is responsible for allowing the molecule containing moiety to penetrate the cell ⁇ cell penetrating molecule is capable of penetrating the cell unlike most homing moieties that target a cell-surface molecule but can not enter the cell
  • the presence of a cell-penetrating molecule allows access m to the cell
  • linking a composition to a cell-penetratmg molecule can ensure intracellular delivery of the composition
  • composition can comprise a cell-penetratmg molecule
  • the cell-penetratmg molecule is Tat
  • the disclosed composition comprises a homing peptide that can localize the composition to the target tissue, such as breast tissue, wherein the cell-penetrating molecule transports the composition into the cells of the tissue
  • the therapeutic agent of the present compositions comprises one or more therapeutic agents, such as chemical compounds, macromolecules, proteins, and the like, which are effective in treating diseases and disorders, such as a cancer
  • the therapeutic agent is present in an amount effective in providing a desired therapeutic effect to an individual, such as a human or animal patient, when the composition is administered to the individual
  • therapeutic agents provided in the therapeutic component of the present compositions can be obtained from public sources or can be synthesized using routine chemical procedures known to persons of ordinary skill in the art
  • the therapeutic agent of the present compositions can comprise a variety of therapeutic agents, including chemotherapeutic agents, anti-inflammatory agents anti-prohferative agents, and the like
  • composition comprises a therapeutic agent
  • the therapeutic agent can be an anti cancer agent
  • the anti cancer agent can be bleomycin
  • the anti-cancer agent can be a STATI inhibitor
  • the STAT3 inhibitor can be STAT3-Hel2A-2
  • peptides that target, bind to and/or home to normal tissue, such as normal breast tissue
  • normal tissue such as normal breast tissue
  • the cyclic nonapeptide CPGPEGAGC SEQ ID NO 1
  • the peptide as well as peptide conjugates are known to target aminopeptidase P
  • peptides that target, bind to and/or home to breast tissue, normal and cancerous
  • CGFECVRQCPERC SEQ ID TNO 3
  • breast CDCRGDCFC
  • SEQ ID NO 4 breast
  • prostate CGRRAGGSC
  • SEQ ID NO 5 pancreas
  • SWCEPGWCR SFQ TD NO 6 or CRVASVLPC
  • YSGKWGW SEQ ID NO 8
  • homing peptides that each have specific homing properties
  • Homing peptides are disclosed in US patent applications 11/670318, 11/967509, H '951819, 10/158566, 09/910582, 12/322371, 09/765086, 11/979624 and 11/777382 and are specifically incorporated herein by reference at least for homing peptide sequences and structures
  • the disclosed peptide can have any suitable length
  • the peptide can have a length of up to 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ammo acids
  • the disclosed polypeptides can be, for example, 4 to about 50 amino acids in length
  • the disclosed polypeptides can be, for example, less than about 50, 49, 48, 47, 46, 45, 44 43 42 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 , 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 ammo acids in length
  • the disclosed peptide can have a length of from 4 to about 10 amino acids, from 4 to about 15 ammo acids, from 4 to about 20 ammo acids, from 4 to about 25 ammo acids, from 4 to about 30 amino acids, from 4 to about 35 ammo acids, from 4 to about 40 amino acids, from 4 to about 45 ammo acids, from 4 to about 50 ammo acids
  • the disclosed peptide can have a length of from 5 to about 10 amino acids, from 5 to about 15 amino acids, from 5 to about 20 ammo acids, from 5 to about 25 ammo acids, from 5 to about 30 amino auds, from 5 to about 35 ammo acids, from 5 to about 40 ammo acids, from 5 to about 45 ammo acids, from 5 to about 50 ammo acids
  • the disclosed peptide can have a length of from 6 to about 10 amino acids, from 6 to about 15 ammo acids, from 6 to about 20 amino acids, from 6 to about 25 amino acids, from 6 to about 30 ammo acids
  • the disclosed peptides can be artificial sequences and can be synthesized in vitro and/or recombmantly
  • the disclosed polypeptides can be peptides that are not naturally occurring prolems and can be peptides that have at least two contiguous sequences that aie not contiguous m a naturally occurring protem
  • the disclosed peptides and compositions also can comprise any combination of two, thiee, or moie of the disclosed peptides or ammo acid sequences
  • peptides comprising any one, two, three, or more of the herein disclosed peptides or ammo acid sequences
  • the peptides can be combined in any suitable manner, including, for example, as a single ammo acid chain (that is a fusion of the peptides), via linkers, via branched linkers, and attached individually or together to a structure
  • bifiinctional peptides which contain one or more of the disclosed peptides fused to one or more second peptides having one or more separate functions
  • Such bitunctional peptides can have at least two functions conferred by different portions of the full-length molecule and can, for example, display pro-apoptotic activity in addition to the ability to taiget the tumor lymphatic
  • multivalent peptides that can include at least two of the disclosed peptides each independently containing one or more of the disclosed ammo acid sequences
  • the multivalent peptide can have, foi example, at least three, at least five oi at least ten of such peptides each independently contaimng a disclosed amino acid sequence
  • the multivalent peptide can have two, three, foui five, six, seven, eight, nine, ten, fifteen or twenty identical or non-identical peptides and/or amino acid sequences
  • the multivalent peptide can contain identical peptides and/or amino acid sequences
  • the multivalent peptide can contain contiguous identical or non-identical peptides and/or ammo acid sequences, which are or are not separated by any intervening amino acids
  • antibodies ' is used herein in a broad sense and includes both polyclonal and monoclonal antibodies
  • immunoglobulin molecules also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as described herein
  • the antibodies are tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods
  • the term "antibody” encompasses, but is not limited to, whole immunoglobulin (i e , an intact antibody) of any class
  • Native antibodies are usually heterotetrame ⁇ c glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains
  • each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages vanes between the heavy chains of different immunoglobulin isotypes
  • Each heavy and light chain also has regularly spaced mtracham disulfide b ⁇ dges
  • Each heavy chain has at one end a variable domain (V (H)) followed by a number of constant domains
  • Each light chain has a vai iable domain at one end (V (L)) and a constant domain at its other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain
  • Particular ammo acid residues are believed to form
  • variable ts used herein to desciibe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen
  • CDRs complementarity determining regions
  • hyperv a ⁇ able regions both in the light chain and the heavy chain variable domains
  • the more highly conserved portions of the variable domains arc called the framew ork (FR)
  • the va ⁇ able domains of native heavy and light chains each comprise four FR regions, largely adoptmg a b-sheet configuration, connected by three CDRs, which foim loops connecting, and in some cases forming part of, the b-sheet structure
  • the CDRs in each chain are held together m close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E A et al , 'Sequences of Protein
  • the term ' antibody or fragments thereof encompasses chimeric antibodies and hyb ⁇ d antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as scFv, sFv, F (ab')2, Fab', Fab and the like, including hybrid fragments
  • fragments of the antibodies that retam the ability to bind their specific antigens are provided
  • Such antibodies and fragments can be made by techniques known in the art and can be screened foi specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane Antibodies, A Laboratory Manual Cold Spring Harbor Publications, New York, (1988))
  • antibody or fragments thereof are conjugates of antibody fragments and antigen binding proteins (single chain antibodies) as described, for example, in U S Pat No 4,704,692, the contents of which ai e hereby incorporated by reference
  • the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific ammo acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or unpaired compared to the non-modified antibody or antibody fragment
  • modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio longevity, to alter its secretory characteristics, etc
  • the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide
  • Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment (Zoller, M J Curr Opm Biotechnol 3 348-354, 1992)
  • antibody or ' antibodies
  • antibody can also refer to a human antibody and/or a humanized antibody
  • Many non-human antibodies e g , those derived from mice, rats, or rabbits
  • are naturally antigenic in humans and thus can give rise to undesirable immune responses when administered to humans Therefore, the use of human or humanized antibodies m the disclosed methods serves to lessen the chance that an antibody administered to a human can evoke an undesirable immune response
  • human antibodies ot the disclosed methods and compositions can be prepared using any technique Examples of techniques for human monoclonal antibody production include those desc ⁇ bed by Cole et al (Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p 77, 1985) and by Boerner et al (J Immunol , 147 (1) 86-95, 1991) Human antibodies of the disclosed methods and compositions (and fragments thereof) can also be produced using
  • the human antibodies of the disclosed methods and compositions can also be obtained from transgenic animals
  • transgenic, mutant mice that arc capable of producing a full repertoire of human antibodies, in response to immunization, have been desc ⁇ bed (see, e g , Jakobovits et al ,Proc Natl Acad Sa USA, 90 2551-255 (1993), Jakobo ⁇ its et al , Nature, 362 255 258 (1993), Bruggermann et al , Year m Immunol , 7 33 (1993))
  • J (H)) antibody heavy chain joining region
  • the homozygous deletion of the antibody heavy chain joining region (J (H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge
  • Antibodies having the desired activity are selected using Env CD4-CO- receptor complexes as desc ⁇
  • the antibodies are generated in other species and 'humanized" for administration in humans
  • Humanized forms of non human (e g , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as scFv, sFv, Fv, Fab, Fab', F (ab')2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin
  • Humanized antibodies include human immunoglobulins (recipient antibody) m which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity
  • humanized antibodies may also comprise residues that are found neither m the recipient antibody nor m the imported CDR or framework sequences
  • the humanized antibody can comprise substantially all of at least one, and typically Iwo, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence
  • the humanized antibody optimally also can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human
  • a humanized antibody has one or more ammo acid residues introduced mto it from a source that is non human These non-human amino acid residues are often reterred to as 'import" residues, which are typically taken from an ' import" variable domain Humamzation can be essentially performed following the method of Winter and co-workers (Jones et al , Nature, 321 522-525 (1986), JAiechmann et al , Nature, 332 323 327 (1988), Veihoeyen et al , Science, 239 1534 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody Accordingly, such ' humanized ' antibodies are chimeric antibodies (U S Pat No 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species Tn practice, humanized antibodies are
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences
  • Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art
  • Computer programs are a ⁇ ailable which illustrate and display probable three dimensional conformational structures of selected candidate immunoglobulin sequences Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence i e , the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen
  • 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
  • the CDR residues are directly and most substantially involved in influencing antigen binding (see, WO 94/04679, published 3 March 1994) Also, disclosed
  • monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i e , the individual antibodies within the population are identical except for possible naturally occurring mutations that may he present in a small subset of the antibody molecules
  • the monoclonal antibodies herein specifically include "chimeric" antibodies m which a portion of the heavy and/or light cham is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U S Pat No 4,816,567 and Morrison et al , Proc Natl Acad Sci OSA, 81 6851-6855 (1984))
  • Monoclonal antibodies of the disclosed methods and compositions can be prepared using hyb ⁇ doma methods, such as those described by Kohler and Milstein, Nature, 256 495 (1975) hi a hyb ⁇ doma method, a mouse or other appropnate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that can specifically bind to the immunizing agent Altemativelj, the lymphocytes may be immunized in vitto, e g , using the complexes described herein
  • Transgenic animals e g , mice
  • J (H) antibody heavy chain joining legion
  • chimeric and germ line mutant mice results in complete inhibition of endogenous antibody production
  • Transfer of the human germ-lme immunoglobulin gene array in such germ line mutant mice can result in the production of human antibodies upon antigen challenge (see, e g , Jakobovits et al , Proc Natl Acad Sci USA, 90 2551-255 (1993), Jakobovits et al , Nature, 362 255-258 (1993), Braggemann et al , Year in Immuno , 7 33 (1993))
  • Human antibodies can also be produced in phage display libraries (Hoogenboom et al , J MoI Biol 227 381 (1991), Marks et al
  • peripheral blood lymphocytes ' PBLs '
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non human mammalian sources are desired
  • the lymphocytes are then fused with an immortalized cell hne usmg a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell
  • an immortalized cell hne usmg a suitable fusing agent, such as polyethylene glycol
  • Immortalized cell lmes are usually transformed mammalian cells, including myeloma cells of rodent, bovine, equine, and human ongm
  • rat or mouse myeloma cell lines are employed
  • the hyb ⁇ doma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfased, immortalized cells For example, if the parental cells lack the enzyme hypox
  • the clones may be subcloned by limiting dilution or FACS sorting procedures and grown by standard methods Suitable culture media for this purpose include, for example, Dulbecco's Modified Fagle s Medium and RPMI- 1640 medium Alternatively, the hyb ⁇ doma cells may be grown in vivo as ascites m a mammal
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A Sepharose, protem G, hydroxylapante chromatography, gel electtophoresis, dialysis, or affinity chromatography
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described m U S Pat No 4,816,567 (Cabilly et al )
  • DNA encoding the monoclonal antibodies of the disclosed methods and compositions can be ieadily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of mu ⁇ ne antibodies)
  • Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e g , as described in U S Patent No 5,804,440 to Burton et al and U S Patent No 6,096,441 to Barbas et al
  • polypeptide fragments of the disclosed composition can be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide m an expression system capable of producing the polypeptide fragments thereof, such as an adenovirus or baculovims expiession system Foi example, one can determine the active domain of an antibody from a specific hybndoma that can cause a biological effect associated with the interaction of the antibody with the antigen
  • ammo acids found to not contribute to either the activity or the binding specificity or affinity of the antibody can be deleted without a loss in the respective activity
  • ammo or carboxy-te ⁇ ninal amino acids are sequentially removed from either the native or the modified non-immunoglobulin molecule or the immunoglobulin molecule and the respective activity assayed in one of many available assays
  • a fragment of an antibody comprises a modified antibody wherein at least one ammo acid has been
  • the fragments include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids lesidues, piovided the activity of the fragment is not significantly altered or nnpaued compared to the nonmodified antibody or antibody fragment
  • modifications can pro ⁇ ide for some additional property, such as to remove or add ammo acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc hi any case, the fragment must possess a bioactive pioperty, such as binding activity, iegulation of binding at the binding domam, etc
  • Functional or active regions of the antibody may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide Such methods are readily apparent to a skilled practitioner m the art and can include site-specific mutagenesis of the nucleic acid encoding the antigen (Zoller MJ et al Nucl Acids Res 10 6487-
  • a variety of immunoassay formats may be used to select antibodies that selectively bind with a particular protein, variant, or fragment
  • solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein, protem variant, or fragment thereof See Harlow and Lane Antibodies, A Laboratory Manual Cold Spring Harbor Publications, New York, (1988), for a desc ⁇ ption of immunoassay formats and conditions that could be used to determine selective binding
  • the binding affinity of a monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al , Anal Biochem , 107 2 20 (1980)
  • Antibodies of the disclosed compositions are piefeiably admimsteied to a subject in a pharmaceutically acceptable earner Suitable earners and then- formulations are described in Remington The Science and Practice of Pharmacy (19th ed ) ed A R Gennaro, Mack Publishmg Company, Easton, PA 1995 Typically, an approp ⁇ ate amount of a pharmaceutically- accep table salt is used in the formulation to render the formulation isotonic
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringers solution and dextrose solution
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7 5
  • Further earners include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are m the form of shaped articles, e g , films, liposomes or microparticles It will be apparent to those persons skilled in the art that certain carriers may be more
  • the antibodies can be administered to the subject, patient, or cell by injection (e g , intravenous, intraperitoneal, subcutaneous, intramuscular), or by other methods such as
  • compositions identified by screening with disclosed compositions / combinatorial chemistry 4. Compositions identified by screening with disclosed compositions / combinatorial chemistry
  • compositions can be used as targets for any combinatorial technique to identify molecules or macromolecular molecules that interact with the disclosed compositions in a desired way
  • nucleic acids, peptides, and related molecules disclosed herein can be used as targets for the combinatorial approaches
  • compositions that are identified through combinatorial techniques or screening techniques in which the compositions have the sequences disclosed herein, or portions thereof, are used as the target m a combinato ⁇ al or screening protocol
  • conjugate 104 Also provided herein is a conjugate comprising any one or more of the herein disclosed peptides and one or more moieties
  • the moiety can be a substance that acts upon the target cell(s) or tissue to bring about a desired effect
  • the disclosed conjugate can target, bind to and'or home to breast, lung, liver, pancreas or intestine
  • Tumor lymphatics such as lymphatic vessels in and aiound tumors, and/or lymphdngiogenic vessels
  • the disclosed peptides preferably selectively bind to tumor lymphatics
  • the effect can for example, be the labeling, activating repressing, or killing of the target cell(s) or tissue
  • the moiety can be, for example, a therapeutic moiety or a detectable moiety, a cytotoxic agent, an anti lymphangiogemc agent, a cancer chemotherapeutic agent, a pro- apoptotic polypeptide, a grafted polypeptide, a virus, a cell, or a liposome
  • the moiety can be a small molecule, pharmaceutical drug, toxm, fatty acid, detectable marker, conjugating tag, nanoparticle, or enzyme
  • the moiety of the disclosed conjugate can be an anti cancer agent, such as bleomycin, or pro-apoptotic peptide
  • pro-apopototic peptides are tumor necrosis factor (Curnis et al , Cancer Res 64, 565-71, 2004) and tachyplesin (Chen et al , Cancer res 61, 2434-8, 2001)
  • Many other anti cancer agents and pro-apoptotic peptides and compounds are known and can be used with and m the disclosed composition
  • small molecules and pharmaceutical drugs that can be conjugated to a peptide are known m the art
  • the moiety can be a cytotoxic small molecule or drug that kills the target cell
  • the small molecule or drug can be designed to act on any critical cellular function or pathway
  • the small molecule or drug can inhibit the cell cycle, activate protein degradation, induce apoptosis, modulate kinase activity, or modify cytoskeletal proteins Any known oi newly discovered cytotoxic small molecule or drugs is contemplated for use with the peptides
  • the moiety can be d toxm that kills the taigeted cell
  • toxins include abrin, modcccin, ncin and diphtheria toxm
  • Other known or newly discovered toxins are contemplated for use with the provided conjugates
  • Fatty acids (i e , lipids) that can be conjugated to the provided conjugates include those that allow the efficient incorporation of the peptide into liposomes
  • the fatty acid is a polar lipid
  • the fatty acid can be a phospholipid
  • the provided conjugates can comprise either natural or synthetic phospholipid
  • the phospholipids can be selected from phospholipids containing saturated or unsaturated mono or disubstituted fatty acids and combinations thereof 1 hese phospholipids can be dioleoylphosphatidylcholrne, dioleoylphosphatidylsenne, dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol, dioleoylphosphatidic acid, palrmtoyloleoylphosphatidylcholme,
  • palmitoyloleoylphosphatidylse ⁇ ne palmitoyloleoylphosphatidylethanolamine, palmitoyloleoylphophdtidylglyt-erol, palmitoyloleoylphosphatidic aud,
  • palmitelaidoyloleoylphosphatidylcholrne palmitelaidoyloleoylphosphatidylscnnc, palmitelaidoyloleoylphosphatidylethanolamrne, pahnitelaidoyloleoylphosphatidylglycerol, palmitelaidoyloleoylphosphatidic acid my ⁇ stoleoyloleoylphosphatidylcholme, my ⁇ stoleoyloleoylphosphatidylse ⁇ ne, my ⁇ stoleoyloleoylphosphatidylethanoamine, my ⁇ stoleoyloleoylphosphatidylglyoerol, myristoleoyloleoylphosphatidic acid, dilinolcoylphosphatidylcholinc, dilinolcoylphosphatidylse ⁇ ne,
  • phospholipids may also be the monoacylated de ⁇ vatives of phosphatidylcholine (lysophophatidylidylcholine), phosphatidylse ⁇ ne (lysophosphatidylserrne), phosphatidylethanolamme (lysophosphatidylethanolamine), phophatidylglycerol (lysophosphatidylglycerol) andphosphat
  • Fluorophores are compounds or molecules that luminesce Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength
  • Representative fluorophores include, but are not limited to, 1,5 IAEDANS, 1,8-ANS, 4- Methylumbelhfeione, 5-carboxy-2,7-dichlorofluorescean, 5- Carboxyfluorescem (5 FAM), 5 Carbo ⁇ ynapthofluorescein, 5-Carboxyteteamethylrhodaniine (5- TAMRA), 5 -Hydroxy Tryptamine (5-HAT), 5-ROX (carboxy-X-rhodamine), 6- Carboxyrhodamine 6G, 6-CR 6G, 6- JOE, 7-Amino-4-methylcouma ⁇ n, 7-Aminoactinomycin D (7 AAD), 7 Hydroxy 4 I methylcoumann, 9-Amino-6-chloro-2-methoxyacridine (ACMA), ABQ, Acid
  • Haematoporphyrm Hoechst 33258, Hoechst 33342, Hoechst 34580, HPTS, Hydroxycouma ⁇ n, Hydroxystilbamidme (FluoroGold), Hydroxytryptamine, Indo-1, high calcium, Indo-1 low calcium, lndodicarbocyamne (DiD), Indotncarbocyanine (DiR), Intrawhite Cf, JC-I, JO JO-I, JO-PRO-I, LaserPro, Lauiodan, LDS 751 (DNA), LDS 751 (RNA), Leucophor PAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine, Lissamine Rhodamme B, Calcein/Ethidium homodimer, LOLO-I, LO-PRO-I, , Lucifer Yellow, Lyso Tracker Blue, Lyso Tracker Blue- White, Lyso Tracker Green, Lyso Tracker Red, Ly
  • nanoshell is a nanoparticle having a discrete dielectric or semi-conducting core section surrounded by one or more conducting shell layers
  • U S Patent No 6,530,944 is hereby incorporated by reference herein in its entirety for its teaching of the methods of making and using metal nanoshells
  • Nanoshells can be formed with a core of a dielectric or inert mate ⁇ al such as silicon, coated with a mate ⁇ al such as a highly conductive metal which can be excited using radiation such as near infrared light (approximately 800 to 1300 nm) Upon excitation, the nanoshells emit heat The resulting hyperthermia can kill the surrounding cell(s) or tissue
  • the combined diameter of the shell and core of the nanoshells ranges from the tens to the hundreds of nanometers Near infrared light is advantageous for its ability to penetrate tissue Other types ot radiation can also be used, depending on the selection of the selection of the
  • the moiety can be covalently linked to the disclosed peptide
  • the moiety can be linked to the amino terminal end of the disclosed peptide
  • the moiety can be linked to the carboxy terminal end of the disclosed peptide
  • the moiety can he linked to an amino acid within the disclosed peptide
  • the herein provided conjugates can further compose a linker connecting the moiety and disclosed peptide
  • the disclosed peptide can also be conjugated to a coating molecule such as bovme serum albumin (BS ⁇ ) (see Tkachenko et al , (2003) J Am Chem Soc 125, 4700-4701) that can be used to coat the Nanoshells with the peptide
  • BS ⁇ bovme serum albumin
  • Protein ciosshnkers that can be used to crosslink the moiety to the disclosed peptide are known m the art and are defined based on utility and structure and include DSS (Disuccimmidylsuberate), DSP (Dithiobisfsuccimmidylpropionate)), DTSSP (3,3 -Dithiobis (sulfosuccuiimidylpropionate)), SULFO BSOCOFS (Bisp- ⁇ ulfosuc ⁇ rumdooxycarbonjloxy) ethyl]sulfone), BSOCOES (Bis[2-(succimmdooxycarbonyloxy)ethyl]sulfone) SULFO DST (Disulfosuccimmdyltartrate), DST (Disuccmimdyltartrate), SULFO EGS (Ethylene glycolbis(succinimidylsuccmate)), EGS (Ethylene glycolbis(sulf
  • the moiety of the disclosed conjugate can be a cellular internalization transporter or sequence
  • the cellular internalization sequence can be any internalization sequence known or newly discovered in the art, or conservative variants thereof
  • Non limiting examples of cellular internalization transporters and sequences include Antennapedia sequences, TAT, HIV-Tat, Penetratm, Antp-3A (Antp mutant), Buform II, Transpoi tan, MAP (model amphipathic peptide) K-FGF, KuVO, P ⁇ on, pVEC, Pep-1, SynBl, Pep-7, HN-I, BGSC (Bis Guamdinium-Spermidme- Cholesterol, and BGTC (Bis-Guamdimum-Tren-Cholesterol) (see Table 1)
  • HIV-Tat GRKKRRORPPQ (SEQ ID NO I l)
  • Antp 3A RQIAIWFQNRRMKWAA (SEQ ID NO 13)
  • Transportan GWTLNSAGYLLGKINKALAALAKKIL (SEQ ID NO 16) model amphipathic KLALKLALKALKAALKLA (SEQ ID NO 17) peptide (MAP)
  • the provided polypeptide can further comprise the ammo acid sequence SEQ ID NO 10, SEQ ID NO 11 (Bucci, M et al 2000 Nat Med 6, 1362-1367), SEQ ID NO 12 (Deiossi, D , et al 1994 Biol Chem 269, 10444-10450), SEQ ID NO 13 (Fischer, P M et al 2000 J Pept Res 55, 163 172), SEQ ID NO 14 (Frankel, A D & Pabo, C O 1988 Cell 55,1189 1193, Green, M & Loewenstem, P M 1988 Cell 55, 1179-1188), SEQ ID NO 15 (Park, C B , et al 2000 Proc Natl Acad Sci USA 97, 8245-8250), SEQ ID NO 16 (Pooga, M , et al 1998 FASEB J 12, 67-77), SEQ lD NO 17 (Oehlke, J et al 1998 Bioclum Biophys Acta 1414, 127-H
  • the provided polypeptide can further comprise BGSC (Bis-Guamdmium-Spe ⁇ mdine- Cholesteiol) oi BGTC (Bis-Guanidnuum-Tren Cholesterol) (Vigneron, J P et al 1998 Pioc Natl Acad Sci USA 93, 9682 9686)
  • BGSC Bis-Guamdmium-Spe ⁇ mdine- Cholesteiol
  • BGTC Bis-Guanidnuum-Tren Cholesterol
  • Protein variants and derivatives are well understood by those of skill in the art and in can involve amino acid sequence modifications
  • amino acid sequence modifications typically fall into one or more of three classes substitutional, insertional or deletio ⁇ al variants
  • Insertions include amino and/oi caiboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues Insertions ordinarily can be smaller insertions than those of ammo or carboxyl terminal fusions, for example, on the order of one to four residues
  • Immunogenic fusion protem derivatives such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer imniunogenicity to the target sequence by cross linking in vitro oi by recombinant cell culture tiansformed with DNA encoding the fusion
  • Deletions are characterized by the removal of one or more ammo acid residues from the protem sequence Typically, no more than about from 2 to 6 residues are deleted at any one site within the protem molecule
  • substitutions that are less conservative than those in Table 2, i e , selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone m the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain
  • substitutions which in general are expected to produce the greatest changes in the protein properties can be those in which (a) a hydrophihc residue, e g seryl or threonyl, is substituted for (or by) a hydrophobic residue, e g leucyl, lsoleucyl, phenylalanyl, valyl or alanyl, (b) a cysteine or proline is substituted foi (01 by) any other residue, (c) a residue having an electropositive side chain, e g , lysyl argmyl, or
  • substitutions include combinations such as, for example, GIy, Ala, VaI, lie, Leu, Asp, GIu Asn, GIn, Ser, Thr, Lys, Arg, and Phe, Tyr Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein
  • Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosyladon (Asn-X-Thi/Sei) or O-glycosylation (Ser or Thr)
  • Deletions of cysteine or other labile residues also may be desirable Deletions or substitutions of potential proteolysis sites, e g Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutammyl or histidyl residues
  • Certain post-translational denvatizations are the result of the action of recombinant host cells on the expressed polypeptide Glutammyl and asparaginyl residues are frequently post translationally deamidated to the corresponding glutamyl and asparyl residues Alternatively, these residues are deamidated under mildly acidic conditions
  • Other post- translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-ammo groups of lysine, argmuie, and histidine side chains (T E Creighton, Proteins Structure and Molecular Properties, W H Freeman & Co , San Francisco pp 79-86 [1983]), acetylation of the N terminal amine and, in some instances, amidation of the C-termmal caiboxyl
  • variants of these and other polypeptides herein disclosed which have at least, 65%, 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence
  • homology can be calculated after aligning the two sequences so thai the homology is at its highest level
  • nucleic acids that can encode those protein sequences are also disclosed This would include all degeneiate sequences related to a specific protem sequence, i e all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protem sequences
  • each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protem sequence
  • Ammo acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc ), altered specificity (e g , abroad spectrum of biological activities), reduced antigenicity, and others
  • D-amino acids can be used to generate moie stable peptides, because D ammo acids are not recognized by peptidases and such Systematic substitution of one or more ammo acids of a consensus sequence with a D amino acid of the same type (e g , D-lysme m place of L- Iysine) can be used to generate more stable peptides Cysteine residues can be used to cyclize or attach two or moie peptides together This can be beneficial to constrain peptides into particular conformations (Rizo and Gierasch Ann Rev Biochem 61 387 (1992), incorporated herein by reference)
  • variants of genes and proteins herem disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level
  • a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods
  • a first sequence has 80 peicent homology, as defined herein
  • hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a punier Oi a probe and a gene
  • Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derrv atives in a nucleotide specific manner
  • G interactmg with C or 4 interacting with T are sequence driven mteractions
  • sequence driven interactions occur on the Watson Crick face or Hoogsteen face of the nucleotide
  • the hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules can hybridize
  • selective hybridization conditions can be defined as stringent hybridization conditions
  • stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps
  • the conditions of hybridization to achieve selective hybridization may involve hybridization in high iomc strength solution (6X SSC or 6X SSPE) at a temperature that is about 12 25°C below the Tm (the melting temperature at which half of the molecules dissociate from then * hybridization partners) followed by washing at a combmation of temperature and salt concentration chosen so that the washing temperature is about 5°C to 2O 0 C below the Tm
  • the temperature and salt conditions are readily determined empirically m preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of inteiest and then washed under conditions of different st ⁇ ngencies Hybridization temperatures are typically higher for DNA-RNA and RNA
  • selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 7% 76, 77 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-lrmitmg nucleic acid
  • the non limiting primer is in for example, 10 or 100 or 1000 fold excess This type of assay can be performed at under conditions where both the limiting and non limiting primer are for example, 10 fold or 100 fold or 1000 fold below their kj, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules
  • selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the p ⁇ mer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89
  • composition or method meets any one of these criteria for determining hybridization either collectn ely or singly it is a composition or method that is disclosed herein
  • sequences related to the homing peptide and cell penetrating sequence for example, and other disclosed genes, these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein
  • compositions and methods which can be used to deliver nucleic acids to cells, either m vitro or in vivo
  • these methods and compositions can largely be broken down into two classes viral based delivery systems and non-viral based delivery systems
  • the nucleic acids can be delivered through a number of direct delivery systems suuh db, electroporation hpofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic matenal in cells or earners such as catiomc liposomes
  • Appropriate means for transfection, including viral vectors, chemical transfectants, or physico mechanical methods such as electroporation and direct diffusion of DNA are described by, for example, Wolff, J A , et al , Science, 247, 1465 1468, (1990), and Wolff J A Nature, 352, 815-818, (1991)Such methods
  • compositions can also be administered in vivo in a pharmaceutically acceptable carrier
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable i e , the material may bo administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or intei acting in a deleterious manner with any of the other components of the phaimaceutical composition m which it is contained
  • the earner would naturally be selected to minimize any degradation of the act ⁇ e ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art
  • compositions may be administered orally, parenterally (e g , intravenously), by intramuscular injection, by intrapeiitoneal injection transdermal ⁇ , extra corporeally, topically or the like, although topical intranasal administration or administration by inhalant is typically preferred
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one oi both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosohzation of the nucleic acid or vector The latter may be effective when a large number of animals is Io be treated simultaneously
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism Delivery can also be directly to any area of the respiratory system (e g , lungs) via intubation
  • the exact amount of the compositions required can vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucle
  • 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 of suspension in liquid prior to injection, or as emulsions
  • injection Injectables can be prepared in conventional forms either as> liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained See, e g
  • the materials may be m solution or suspension (for example, incorporated into microparticlcs, liposomes, or cells) These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al , Bioconiugate Chem . 2 447 451, (1991), Bagshawe, K D , Br J Cancer. 60 275-281, (1989), Bagshawe, et al , Br J Cancer. 58 700-703, (1988), Senter, et al , Bioiom iigate Chem , 4 3-9, (1993), Battelli, et ⁇ l , Cancer Immunol Immunother .
  • Vehicles such as 'stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma) receptor mediated targetmg of DNA through cell specific ligands, lymphocyte directed tumor targetmg, and highly specific therapeutic retroviral taigetmg of murine glioma cells m vivo
  • 'stealth and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma) receptor mediated targetmg of DNA through cell specific ligands, lymphocyte directed tumor targetmg, and highly specific therapeutic retroviral taigetmg of murine glioma cells m vivo
  • receptors are involved m pathways of endocytosis, either constitutive or hgand induced These receptors cluster m clath ⁇ n-coated pits, enter the cell via clathrrn-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated protems, clearance of macromolecules, opportunistic entry of viruses and toxms, dissociation and degradation of hgand, and receptor-level regulation Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of hgand, hgand valency, and
  • compositions including antibodies, can be used therapeutically m combination with a pharmaceutically acceptable earner
  • compositions can be administered intramuscularly or subcutaneously Other compounds can be administered according to standard procedures used by those skilled in the art
  • compositions may include carneis, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice
  • compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated Administration may be topically (including ophthalmically, vaginally, rectally lntranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous intraperitoneal or intramuscular injection
  • the disclosed antibodies can be administered intravenously, intraperitoneal ⁇ , intramuscularly, subcutaneously, mtracavity, or transdermally
  • Preparations for parenteral administration include ste ⁇ le aqueous or non aqueous solutions, suspensions, and emulsions
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate
  • Aqueous earners include water, alcoholic/aqueous solutions, emulsions or suspensions, including salme and buffered media
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replemshers (such as those based on Ringer's dextrose), and the like
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents and inert gases and the like
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, supposito ⁇ es, sprays, liquids and powders
  • Comentional pharmaceutical carriers, aqueous, pow der or oily bases, thickeners and the like may be necessary or desirable
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets Thickeners, flavorings diluents, emulsifiers, dispersing aids or binders may be desirable
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic auds such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fuina ⁇ c acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, t ⁇ alkyl and aryl amines and substituted ethanolamines
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic auds such as formic acid, acetic
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired ctfect in which the symptoms disorder are effected I he dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic leactions, and the like Generally, the dosage can vary with the age, condition, sex and extent of the disease m the patient and can be determined by one of skill in the art The dosage can be adjusted by the individual physician m the event of any counte ⁇ ndications Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days
  • homology and identity mean the same thing as similarity
  • lhe word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences
  • Many of the methods for determining homology between two evolutionanly related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionanly related or not
  • variants of genes and proteins herein disclosed typically have at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, SO, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence
  • genes for determining the homology of two proteins or nucleic acids such as genes For
  • the homology can be calculated after aligning the two sequences * so that the homology is at its highest level
  • a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above
  • a first sequence has 80 pei cent homology, as defined heiem, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does, not have 80 percent homology to the second sequence ds calculated by any of the other calculation methods
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Nccdlcman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods
  • a first sequence has 80 percent homology, as defined herein, to a second sequence
  • a method of targeting one or more moieties, to different regions, breast tissue for example, in a subject can involve administering to the subject a conjugate comprising any one or more of the herein disclosed peptides and the one or more moieties
  • the one oi more moieties can be detection moieties, such as those disclosed herein Ihus, the method can further comprise detecting cancer m the subject by detecting the presence of the conjugate in the subject's cells
  • the detection moiety can be a fluorophore, such as TAMRA, DANSYL, a cyanme dye, or a cyanrne dye encapsulated in a cyclodext ⁇ n
  • the one or more moieties can be homing peptides, such as those disclosed herein
  • the homing peptide can be breast tissue-specific, such as CPGPEG ⁇ GC
  • the one or more moieties can be cell-penetrating peptides, such as those disclosed herein
  • the cell-penetrating peptide allows the composition to enter the cell, such as Tat
  • the one or more moieties can be a contrasting agent, such as those disclosed herein
  • the contrasting agent can be an MRI contrasting agent
  • the MRI contrast agent can be non-metallic, such as a mtroxide radical or deroative thereof
  • the one or more moieties can be therapeutic moieties, such as those disclosed herein
  • targeting of the moiety to the cancer cells of the subject can provide a therapeutic effect in the subject
  • the therapeutic moiety can be an anticancer therapeutic such as the chemotherapeutic agent, bleomycin
  • composition disclosed herein can be non-toxic
  • a method of detecting cancer such as a dual modality detection method
  • the method can involve administering a composition composing a detectable moiety and a contrasting agent to the subject, performing MRI on the subject, recording the output from
  • the presence of cancer can be detected by the presence of a kinetic decay m the contrasting agent followed by the emergence of a fluorescent signal
  • the MRI contrast can kmetically decay on the order of a 1 to 8 minute tune interval followed by concomitant emergence of a fluoiescent signal
  • the MRI contrast can continue and the delayed fluorescence response can be realized
  • these agents are not specific to cancer, but specific to breast tissue, administration of the agent to a patient can result jn enhanced MRI signal from the entire breast
  • regions that arc cancerous can have reduced MRI signal compared to the other regions of the breast These regions can also result in a fluorescent signal whereas non-cancerous regions of the bteast can have minimal to no fluorescent signal
  • the disclosed method can detect breast cancer
  • the disclosed method comp ⁇ ses a contrast agent linked to a homing peptide
  • the homing peptide can be breast tissue specific
  • the disclosed method comprises the breast tissue-specific homing peptide CPGPEGAGC
  • the disclosed method comprises a non-metallic contrast agent
  • the non-metallic contrast agent can be a mtroxide radical or de ⁇ vativc thereof
  • the method comprises a detection moiety
  • the detection moiety can be a fluorophore
  • the fluorophore can be DANSYL, TAMRA, a cyanme dye, or a cjanine dye encapsulated in a cyclodextrin
  • the disclosed method comp ⁇ ses a peptide-based dual modality detection system
  • 178 Also provided is a method of treating cancer in a subject comprising administering to the subject a composition comprising a detection moiety, a contrasting agent, a homing peptide, a cell-penetiating molecule and a therapeutic agent
  • the method of treating cancer in a subject further comprises detecting cancer in a subject 180
  • the method of treating cancel m a subject fiirthei comprising detecting cancer in a subject comprises a dual modality detection method of detecting cancer in a subjecting comprising administering a composition to the subject, performing Magnetic Resonance Imaging (MRI) on the subject, performing flourescence imaging on the subject, recording the output from the MRI and the flourescence imaging, and comparing the output of the MRI and the output of the flourescence imaging to a control
  • MRI Magnetic Resonance Imaging
  • the method of treating cancer in a subject further comprising detecting cancer in a subject comprises a composition comprising a breast tissue specific homing peptide wherein the homing peptide can be CPGPEGAGC
  • the method of treating cancer m a subject further comprising detecting cancer in a subject comprises a composition comprising a fluorophore as the detection moiety
  • the fluorophore can be dansyl, TAMRA, a cyanine dye, or a cyanine dye encapsulated in a cyclodext ⁇ n
  • the method of treating cancer in a subject further comprising detecting cancer in a subject comprises a composition comprising a non metallic MRI contrasting agent
  • the non-metallic contrasting agent is a mtroxide iadical oi derivative thereof
  • the method of treating cancer m a subject further composing detecting cancer in a subject composes a composition comprising an anti-cancer theiapeutic agent
  • the anti-cancer therapeutic agent can be bleomycin
  • the method of treating cancer m a subj ect further comprising detecting cancer in a subject comprises a composition comprising Tat as the cell-penetrating molecule
  • the disclosed method of treating cancer in a subject uses a non-toxic composition
  • compositions can be used to treat any disease where uncontrolled cellulai prolifeiation occurs such as cancers
  • a non-lirmtmg list of different types of cancers can be as follows lymphomas (Hodgkrns and non Hodgkms), leukemias, carcinomas, carcinomas of solid tissues, squamous cell carcinomas, adenocarcinomas, saicomas, gliomas, high giade gliomas, blastemas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas,
  • hypoxic tumors myelomas, AIDS-related lymphomas or sarcomas, metastatic cancers, or cancers in general
  • a representative but non-limitmg list of cancers that the disclosed compositions can be used to treat is the following lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkm'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 cancels such as small cell lung cancer and non-small cell lung cancer,
  • composition disclosed herein such as the disclosed peptides and conjugates, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to he treated
  • the compositions may be admimsteied orally, parenterally (e g , intravenous, subcutaneous, intraperitoneal, or intramuscular injection), , by inhalation, extracorporeally, topically (including transdermally, ophthalmically, vaginally, rectally, tntranasally) or the like
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosohzation of the nucleic acid or vector Administration of the compositions by inhalant can be through the nose oi mouth via delivery by a spraying or droplet mechanism Delivery can also be directly to any area of the respiratory system (e g , lungs) via intubation
  • Parenteral administration of the composition is generally characte ⁇ zed by injection Injectables can be prepared m conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions A more recently revised approach for parenteral administration rnvoh es use of a
  • compositions required can vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like Thus, it is not possible to specify an exact amount for every composition However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein Thus, effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art Useful dosage ranges lor the administration of the compositions are those large enough to produce the desired effect The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like Generally, the dosage can vary with the age, condition, sex and extent of the disease m the patient, route of administration, or whether other drugs are included in the regimen, and call be deteimmed by one of skill in the art The dosage can be adjusted by the individual physician in the event of any counter
  • a typical daily dosage of the disclosed peptides used alone might iange from about 1 ⁇ g/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above
  • kits for administering compositions such as those disclosed herein, the kit comprising a composition and a means tor administering the composition to a subject
  • kits also can contain protocols for administering the compositions
  • kits comprising a composition, comprising a detection moiety and contrastmg agent, and articles for delivery to a subject
  • compositions can be used in a variety of ways as research tools Other uses are disclosed, apparent from the disclosure, and/or will be understood by those in the art
  • compositions disclosed heiein and the compositions necessary to perform the disclosed methods can be made using any method known to ttiose of skill in the art for that particular reagent or compound unless otherwise specifically noted
  • the nucleic acids such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be pioduced using enzymatic methods or any other known method
  • Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N Y , 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of MiHigen-Biosearch, Burlington, MA or ABI Model 380B) Synthetic methods useful for making oligonucleotides are also described by Ikuta et al , Ann Rev Biochem 53 323-356 (1984), (phosphot ⁇ ester and phosphite-
  • One method of producing the disclosed proteins is to link tw o or more peptides or polypeptides together by protein chemistry techniques
  • peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fnioc (9-fluorenylmetliyloxycarbonjl) 01 Boc (tert -butyloxycarbonoyl) chemistry (Applied Biosystcms, Inc Foster City, C ⁇ )
  • Fnioc (9-fluorenylmetliyloxycarbonjl) 01 Boc (tert -butyloxycarbonoyl) chemistry Applied Biosystcms, Inc Foster City, C ⁇
  • a peptide or polypeptide corresponding to the disclosed proteins for example, can be synthesized by Standard chemical reactions
  • a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cle
  • enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al , Biochemistry, 30 4151 (1991))
  • 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 the chenioselecttve reaction of an unprotected synthetic peptide— thioester with another unprotected peptide segment containing an ammo-terminal Cys residue to give a thioester-linked mtermediate as the initial covalent product Without a change m the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form
  • unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al Science, 256 221 (1992))
  • This technique has been used to synthesize analogs of protein domains as w e)1 as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al , 1 echmques in Protein Chemistry IV Academic Press, New York, pp 257 267 (1992))
  • a whole body imaging system such as a MRI
  • a module and/or component which for example, a) produces a tissue specific record, which identifies the decay of contrasting agents, identifies one or more detection moieties, creates a set of data, and/or performs a dual modality analysis, such as a dual modality analysis alone or m any combination hi particular
  • the modules and components within the imaging system responsible for determining the presence of cancer can be linked to the modules and/or components responsible for identifying and/or manipulating tissue-specific data sets
  • the presence of cancer can be determined by the moment the decay of the contrasting agent starts and quenching of the fluorophore stops
  • the methods and systems herein can have the data, in any form uploaded by a person operating a device capable of performing the methods disclosed herein
  • the methods can also be associated with the whole body imaging system as desciibed herein, either incorporated into these systems or being on device which is connected to them
  • Disclosed herein is a method of detecting cancer in ⁇ subject wherein the method is a computer implemented method
  • the method further comprises the step of outputting results from the dual modality detection
  • 207 Disclosed herein is a method of analyzing a subject comprising, receiving a tissue-specific record of the subject, wherein the record contains the kinetic decay of the contrasting agent, measuring the amount of decay and the amount of fluorescence, and outputting results from the dual modality detection
  • the method of analyzmg a subject comprises a computer implemented method
  • the method of analyzing a subj cct composes receiving the tissue-specific record wherein the tissue specific record can be from a storage medium
  • the method of analyzing a subject comprises receiving the tissue specific record wherein the tissue-specific record can be from a computer system
  • the method of analyzing a subj ect comprises receiving the tissue-specific record wherem the tissue-specific iecord can be from a whole body imaging system
  • the method of analyzing a subject comprises receiving the tissue specific record wherem the tissue-specific record can be via a computer netwoik
  • a method of analyzing the presence of cancer m a subject comprising, recommending the performance of receiving a tissue-specific record of the subject, wherem the record contains the kinetic decay of the contrasting agent, measuring the amount of decay and the amount of fluorescence, and outputti ⁇ g results from the dual modality detection
  • the disclosed method comprises the steps of receiving an output from any of the disclosed methods of analyzing and recommending treatment by adminstermg a composition comprising a detection moiety, a contrastmg agent, a homing peptide, a cell-penetrating peptide and a therapeutic agent
  • Disclosed herein is one or more computer readable media storing program codes thdt, upon execution by one or more computer systems, causes the computer systems to perform any of the disclosed methods
  • Disclosed herein is a computer program product compiising a computer usable memory adapted to be executed to implement any of the disclosed methods
  • the computer program disclosed above comp ⁇ ses a logic processing module, a configuration file piocessing module, a data organization module, and data display organization module, that are embodied upon a computer readable medium
  • a computer program product comprising a computer usable medium having a computer readable program code embodied therein, said computer readable program code adapted to be executed to implement a method for generating the dual modality detection of any of the previously disclosed methods, said method further composing providing a system, wherein the system comp ⁇ ses distinct software modules, and wherem the distinct
  • softwaie modules comprise a logic processing module, a configuration file piocessmg module, a data organization module, and a data display organization module
  • the computer program product further comprises a computeri7ed system configuied foi performing the method
  • the computer program product further comprises the outputting of the results from the dual modality detection
  • Disclosed heiein is a computer-readable medium having stored thereon instructions that, when executed on a programmed processor perform any of the disclosed methods
  • a dual modality detection system comprising a data store capable of storing tissue specific data, a system processor composing one or more processing elements, the one or more processing elements programmed or adapted to receive tissue-specific data comprising the kinetic decay of the contrasting agent and the presence of fluorescence, store the tissue-specific data in the data store, compare the reduction m the contrast agent to the increase in fluorescence, and output a treatment recommendation based upon the comparison of the decay in contrasting enhancement with the mcreased fluorescence
  • the dual modality detection system receives the tissue-specific data fiom a computer system
  • the dual modality detection system receives the tissue-specific data via a computer network
  • the dual modality detection system further comprises a whole body imaging system
  • the functionality and approaches discussed above, or portions thereof can be embodied m instructions executable by a computer, where such instructions are stored in and/or on one or more computer readable storage media
  • Such media can include primary storage and/or secondary storage integrated with and/or within the computer such as RAM and/oi a magnetic disk, and/or separable from the computer such as on a solid state device or removable magnetic or optical disk
  • the media can use any technology as would be known to those skilled m the art, including, without limitation, ROM, RAM, magnetic, optical, paper, and/or solid state media technology
  • a control can refer to the results from an expe ⁇ ment in whioh the subjects or objects or reagents etc are treated as ui a parallel expe ⁇ ment except for omission of the procedure or agent or variable etc under test and which is used as a standard of comparison in judging experimental effects
  • the control can be used to determine the effects related to the procedure or agent or variable etc For example, if the effect of a test compound on a cell was in question, one could a) simply record the characteristics of the cell in the presence of the compound, b) pel form a and tben also record the effects of adding a control compound with a known activity or lack of activity and then compare effects of the test compound to
  • targeting or “homing” can refer to the preferential movement, binding, and/or accumulation of a compound or composition, such as the disclosed compositions, in or at, for example, target tissue, target cells, and/or target structures as compared to non target tissue, cells and/or structures
  • molecule refers to a hiological or diemicdl entity that exists in the form of a chemical molecule or molecules
  • Many molecules are of the type referred to as organic molecules (compounds containing carbon atoms, among others, connected by covalent bonds), although some molecules do not contain carbon (including simple molecular gases such as molecular oxygen and more complex molecules such as some sulfur- based polymers)
  • the general ternV'molecule' includes numerous descriptive classes or groups of molecules, such as proteins, nucleic acids, carbohydrates, steroids, organic pharmaceuticals, receptors, antibodies, and lipids When appropriate, one or more of these more descriptive terms (many of which, such as "protein,” themselves describe overlapping gioups of compounds) can be used herein because of application of the method to a subgroup of molecules, w lthout detracting from the intent to have such compounds be repiesentative of both the general class "molecules"and the named subclass, such as proteins Unless specifically indicated, the word molecule would include the specific compound
  • the "subject” can include, for example, domesticated animals, such as cats, dogs, etc , livestock (c g , cattle, horses, pigs, sheep, goats, etc ), laboratory animals (e g , mouse, rabbit, rat, guinea pig, etc ) mammals, non-human mammals, primates, non-human p ⁇ mates, rodents, birds, reptiles, amphibians, fish, and any other animal
  • livestock c g , cattle, horses, pigs, sheep, goats, etc
  • laboratory animals e g , mouse, rabbit, rat, guinea pig, etc
  • mammals non-human mammals, primates, non-human p ⁇ mates, rodents, birds, reptiles, amphibians, fish, and any other animal
  • the subject can be a mammal such as a p ⁇ mate or a human
  • the subject can also be a non-human
  • the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder hi addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • a typical system may include a system processor comprising one or more processing elements in communication with a system data store (SDS) comprising one or more storage elements.
  • SDS system data store
  • the system processor may be programmed and/or adapted to perform the functionality described herein.
  • the system may include one or more input devices for receiving input from users and/or software applications.
  • the system may include one or more output devices for presenting output to users and/or software applications.
  • the output devices may include a monitor capable of displaying to a user graphical representation of the described analytic functionality.
  • the described functionality may be supported using a computer including a suitable system processor including one or more processing elements such as a CELERON, PENTIUM, XEON, CORE 2 DUO or CORE 2 QUAD class microprocessor (Intel Corp., Santa Clara, C ⁇ ) or SEMPRON, PHENOM, OPTERON, ATHLON X2 or ATHLON 64 X2 (AMD Corp., Sunnyvale, CA), although other general purpose processors could be used.
  • the functionality as further described below, may be distributed across multiple processing elements.
  • the term processing element may refer to (1) a process running on a particular piece, or across particular pieces, of hardware, (2) a particular piece of hardware, or either (1) or (2) as the context allows.
  • Some implementations can include one or more limited special purpose processors such as a digital signal processor (DSP), application specific integrated circuits (ASIC) or a field programmable gate arrays (FPGA). Further, some implementations can use combinations of general purpose and special purpose processors.
  • DSP digital signal processor
  • ASIC application specific integrated circuits
  • FPGA field programmable gate arrays
  • the envn eminent further includes a system data store (SDS) that could include a variety of primary and secondary storage elements
  • SDS would include registers and RAM as part of the primary storage
  • the primary storage may in some implementations mclude other forms of memory such as cache memory, non-volatile memory (e g , FLASH, ROM, EPROM, etc ), etc
  • the SDS may also include secondary storage including smgle, multiple and/or varied servers and storage elements
  • the SDS may use internal storage devices connected to the system processor
  • a single processing element supports all of the functionality
  • a local hard disk d ⁇ ve may sen e as the secondary storage of the SDS, and a disk operating system executmg on such a single processing element may act as a data server receiving and servicing data requests
  • the different information used in the systems and methods tor respiratory analysis as disclosed herem may be logically or physically segregated within a single device serving as secondary storage for the SDS, multiple related data stores accessible thiough a unified management system, which together serve as the SDS, or multiple independent data stores individually accessible through disparate management systems, which may in some implementations be collectively ⁇ lewed as the SDS
  • the various storage elements that compose the physical architecture of the SDS may be centrally located or distributed across a variety of diverse locations
  • 239 ⁇ computer network or like terms are one or more computers in operable communication with each other
  • Computer implemented or like terms refers to one or more steps being actions being performed by a computei, computer system, or computer network
  • a computer program product or like terms refers to product which can be implemented and used on a computer, such as software
  • a dual modality analysis or like terms is the analysis of two components in a system
  • tissue-specific data or values refers to acqui ⁇ ng this data or values It can be acquired by, for example, collection, such as through a machine, such as an MRI machine and system It can also be acquired by downloading or getting data that has already been collected, and for example, stored in a way m which it can be ret ⁇ eved at a later time
  • Outputting or like terms means an analytical result after processing data by an algorithm
  • tissue-specific record or like terms is any collection of tissue-specific data
  • tissue specific data series or like terms refers to any collection of tissue-specific data
  • the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder Such amelioration only requires a reduction or alteration, not necessarily elimination
  • the term "earner' means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids oi facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose
  • a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects m the subject
  • cell as used heiem also refers to mdividual cells, cell lines, oi cultures de ⁇ ved from such cells
  • a "culture' refers to a composition comp ⁇ smg isolated cells of the same
  • co-culture is used to designate when more than one type of cell arc cultured together in the same dish with either full or partial contact with each other
  • the term ' stable is generally understood in the art as meaning less than a certain amount, usually 10%, loss of the active ingredient undei specified storage conditions for a stated period of time
  • the time required for a composition to be considered stable is relative to the use of each product and is dictated by the commercial practicalities of producing the product, holding it for quality control and inspection, shipping it to a wholesaler or direct to a customer where it is held again in storage before its eventual use Including a safety factor of a few months time, the minimum product life for pharmaceuticals ib usually one year and prefeiably more than 18 months
  • the term 'stable references these market realities and the ability to store and transport the product at readily attainable environmental conditions such as refrigerated conditions, 2°C to 8°C
  • Example 1 Breast tissue-specific cancer theranostics: Therapeutic dual- modality MRI/NIR imaging agents
  • a new smart agent as a thcranostic for the diagnosis and treatment ot breast cancer is desc ⁇ bed
  • the agent incorporates a dual modality magnetic resonance imaging/near infrared imaging component and a releasable therapeutic component
  • This agent can enable research opportunities in tissue selectivity by interactions with cell surface proteins, imaging by MRI and MR of dynamic chemical e ⁇ ents in cells, and methods to deliver cancer drugs directly to the site of interest
  • this system can facilitate in vivo mechanistic studies of these drugs m breast cancer modeling
  • the detection system is a key feature of the pioposed tlieraiiostic, which is designed to dynamically respond to the intracellular chemical environment of cancer cells
  • the detection system is based on three physical properties of paramagnetic rntroxide radicals mtroxide radicals can serve as Tl contrast enhancement agents ior MRI (Keana et al Magn Reson Med 2005, 5 525 536), mtroxide radicals quench fluorescence (Blough et al J Am Chem Soc 1988, 110 1915 1917), and mtroxide radicals are reduced to the dianiagnetic hydroxylamine in cancer cells (Hyodo et al Cancer Res 2006, 66 9921 9928)
  • the reduction of mtroxides to the hydroxylamine constitutes the dynamic chemical event which can be used for detection, because the resulting hydroxylamme is neither MRI active nor able to quench fluorescence Therefore, the detection system reports thefilmnce of cancer in a patient by displaying a
  • Nitroxide paramagnetic spin labels are derived from secondary amines and exist as stable radicals
  • the Tl lelaxivities of mtroxides result in MRI contrast enhancement
  • Murali and co-workers showed a preferential reduction of mtroxides in tumors compared with normal tissues by MRI (Hyodo ct al Cancer Res 2006 66 9921-9928) This property of mtroxides m is key in the disclosed detection system
  • Tile detection system and theranostic agent can be evaluated in animal models Io verify the ability of the conjugated homing peptide to distribute in the breast tissue
  • Mice containing breast cancer xenografts in the mammary glands can then be treated with this agent to verify dual modality detection of the tumor Imaging can be accomplished using a Bruker 7 Tesla MRI instrument and a CRi Maestro In- Vivo Fluorescence Imaging System These instruments allow for noninvasive whole animal imaging of live mice, desflurane is used for anesthesia during imaging of the mice
  • Fluorescence imaging is a simple and cost effective method to determine the parameters outlined above
  • these agents are designed for dual modality imaging and the strategy relies on double confirmation of cancer Therefore, once the half life of the agent is determined by fluorescence imaging, MRI imaging is employed Contrast enhancement is quantified and the kinetics for loss of Tl-weighted contrast enhancement are measured, which is due to reduction of the spin label The loss of MRI contrast correlates with the kinetics for the emerging fluorescence signal
  • a single-agent theranostic that addresses detection, diagnosis, and therapy of breast cancer
  • fluorescence imaguig as a new modality for detecting breast cancer that can be coupled with MRI for dual confirmation of the presence of bi east cancer
  • a ti eatment option is also disclosed that utilizes the same detection system for detecting the cancer, which allows monitoring the distribution and efficacy ol the therapy And, this system does not employ ionizing radiation or toxic metals, which are current problems faced with mammography and MRI imaging agents, respectively
  • a simple peptide platform that can be quickly adapted for the implementation of new and emerging technologies
  • the disclosed methods and compositions invoh e the design of 'intelligent agents that can discriminate between tissue types and are activated by the chemical environment that is exclusive to cancer
  • These agents are also valuable tools for biologists and enable them to visualize biochemical e ⁇ ents by MRI and fluorescence imaging
  • a spm radical is incorporated in the agent, which has the dual role of quenching fluorescence and enhancing Tl-weighted contrast by MRI
  • This agent enables physicists to examine energy transfer within the cell that results m reduction of the radical to the anion
  • the energy required by cancer cells to proliferate is derived from an elaborate system of redox reactions that involves formation of NADH NADH participates in the electron transport chain, which eventually results m the production of ATP, the mam energy earner of the cell
  • the agent can be used to monitor redox chemistry m living systems by MRI imaging of the spm-label Tl contrast agent and concomitantly by fluorescent imaging
  • Tins agent can aid in diagnosing a malignant lesion and concurrently report the effectiveness of the drug during treatment of the lesion
  • the use of the agent can result in fewer invasive surgeries for benign lesions due to false-positive detection by mammography, because the agent contams a dual reporting component that can assess the chemical environment that is particular to cancer A reduced toxicity in treating the patient is expected, because the agent is specific to breast tissue, resulting in a higher effective dose at the breast at an overall reduced systemic dosage
  • the designed agent allows foi accurate detection of early breast tumors in younger at risk patients, who would normally be poor candidates for mammography, because of the increased density of breast tissue and the hazards associated with X-ray radiation
  • the versatility of the disclosed methods and compositions is expected to stimulate further research in adapting our technology to other cancers as well as other diseases that have peculiar chemical characteristics
  • the designed agent is used for breast cancer screening Although screening by MRI is currently not economically feasible, fluorescence-imaging equipment is low cost and can easily become standard And, fluorescence imaging does not involve radiation as does mammography, the current standard for breast cancer screening For this system, a positive detection by fluorescence imaging would lequire secondary confirmation by MRI using the same agent In addition, treatment ot breast cancer patients using this technology can allow immediate assessment of drug delivery to the disease site And, the agent readily allows for imaging of the tumor to evaluate the effectiveness of treatment over time
  • the disclosed methods and compositions are the development of a new smart biomate ⁇ al, and more specifically a combined therapeutic -diagnostic agent coined as a theranostic
  • the advantages of this type of system include the ability to monitor drug distribution to the disease site and rapidly assess the efficacy of drug treatment in individual patients
  • the agent allows for the optional attachment of a variety of drug entities
  • the disclosed methods and compositions specifically address breds>t cancer, i disease that can have a very promising prognosis if detected early
  • breds>t cancer i disease that can have a very promising prognosis if detected early
  • remaining problems in breast cancer include false positive detection by mammography and MRI resulting in unnecessary invasive surgery, false-negative detection especially m youngei women with denser breast tissue, systemic toxicity oi first line therapies for advanced breast cancer, and early assessment of the effectiveness of a breast cancer treatment
  • the disclosed methods and compositions have a dual modality MRI/NIR imaging agent that incorporates a therapeutic component It is designed to give smart agents that can doubly confirm the presence of breast cancer by dual modality magnetic resonance imaging/near infrared imaging (MRI/NTR) and deliver a therapeutic agent specifically to the cancer Currently, a radiologist can examine X-ray and/or MRI images and assess the presence of cancerous lesions The detection of dynamic chemical processes to confirm the presence ot a cancerous chemical environment are also disclosed herein
  • the disclosed agent contains 5 distinct components as follows 1.
  • a spin-radical Tl contrast component gives MRI image enhancement 2 ⁇ near infrared (NIR) chromophore can allow for fluorescence imaging 3.
  • a breast homing peptide sequence results in specific distribution of the agent to breast tissue 4
  • a Tat sequence allows for cell penetiation And in combination with the breast homing sequence, Tat confers tissue/cell type specificity (see preliminary results) 5
  • a therapeutic agent is covalently attached to the system
  • the peptide- based feature of the proposed agent may impart additional specificity for cancer cells Proteases are more active m cancer cells than normal cells, which are expected to confer specificity for release of (he therapeutic agent to cancer cells (Kobhnsl ⁇ et al CIm Chim Acta 2000, 291 113- 135) Additionally, the only by-products of our agent other than the therapeutic moiety are simple ammo acids
  • the disclosed methods and compositions are a multi-faceted approach to breast cancer detection and treatment using a new smart material that combines diagnosis and therapy in a single agent
  • the disclosed methods and compositions include the optional attachment of a variety of drag entities, w hich ultimately leads toward personalized medicine
  • the disclosed agent is sensitive to the chemical environment of cancer cells In conjunction with the fluorescence imaging, a dual-confirmation strategy to increase the specificity of detection is used Additionally, the nature of the disclosed imaging agents can also avoid two other properties associated with MRI imaging agents, namely the toxicity of transition metals normally used in MRI imaging and distribution of the agent to the region of mterest
  • Homing peptides are short sequences derived fiom phage display libraries that have the unique property of homing to specific organs.
  • these zip codes refer to cell-surface interactions and do not reflect the intracellular delivery of these peptides
  • a breast homing peptide with a Tat sequence can be used to deliver the disclosed agents mto cells (Deshayes et al Cell MoI Life Sci 2005, 62 1839-1849) Myrberg and co workers published a related study using a different cell-penetrating peptide, pVEC, for intracellular delivery of breast homing peptides (Myrberg et al Bioconjugate Chem 2008, 19 70-75)
  • the breast homing peptide is a cyclic nonapeptide with the sequence cCPGPEO ⁇ QC
  • the peptide and even peptide conjugates of the homing sequence have been shown to distribute to breast tissue possibly via interaction with a membrane-bound prohne-specific ammopeptidase P (APaseP) (Essler et al PNAS 2002, 99 2252-2257)
  • a membrane-bound prohne-specific ammopeptidase P (APaseP)
  • the homing peptide sequence alone does
  • the disclosed delivery system is based on a 9-amino acid peptide that was discovered by phage display libraries The unique property of this peptide is its ability to 'home' to breast tissue, presumably by interacting with ammopeptidase P (APaseP) protein expressed in breast tissue (Esslei et al PNAS 2002, 992252-2257)
  • Trastuzumab (Herceptin) is a monoclonal antibody that targets the HER2 receptor tyrosine kinase and was approved by the FDA in 1998 for the treatment of breast cancer
  • monoclonal antibodies are designed to recognize an extracellular protein motif and effectively home to its target
  • trastuzumab covalently attached to the cytotoxic tubulin inhibitor niaytansmoid (DMl) entered phase II clinical trials and shows considei able potential as a new therapy foi metastatic breast cancer (Vukelja et al Cancer Res 2009, 69(2 Suppl), Abstract n
  • Tat is a transcription-activating factor derived from HIV-I, and is essential for viral gene expression
  • the 86 ammo acid sequence contains a basic region (amino acids 49 - 58) that is responsible for cell penetration (Deshayes et al Cell MoI Life Sci 2005, 62 1839-1849)
  • This cell-penetrating Tat sequence (ammo acids 49 -57) was incorporated into the homing peptide and induced cell penetration of the peptides
  • the combination of the Tat sequence with the breast homing peptide conferred tissue specificity for breast derived cells
  • tins specificity has been coirelated with the expression of ApaseP, the putative recogmtion protein of the breast homing peptide
  • examination of human tissue arrays revealed that ApaseP expression is especially high in human breast tissue indicating that mouse- de ⁇ ved breast homing sequence can have utility in humans
  • a theranostic agent is defined as a single agent that can be used to diagnose and simultaneously treat a disease
  • the single-agent theranostic has clinical utility and is designed as a fully functional diagnostic system with the option of attachmg a drug molecule
  • the therapeutic agent is attached to the same system used for the diagnosis
  • a one-step [3+2] Huisgen cycloaddition is used This methodology falls under the paradigm of 'click chemistry, which is a synthetic strategy that can accommodate the attachment of a variety of different drags (KoIb et al Angew Chem Int Ed 2001 , 40 2004-2021 )
  • the advantage ot this system is that the physician can monitor the effectiveness of the treatment with the same detection system that was used to diagnose the disease And, the physician can have a number of treatment
  • the disclosed agents require three amino acids as follows the fluorapliore, the spin-label, and the therapeutic agent Each route is very general, and can be adapted to the synthesis of the other disclosed ammo acids
  • SPPS solid-phase peptide synthesis
  • Figure 1 provides an example of how the fluorophore containing amino acids are made
  • Method 1 is accomphshcd by treating the O succmimide ester of the carboxyl containing fluorophore with Fmoc-Lys-OH This gives selective funUionahzation on the side-chain amine group
  • the synthesis of Fmocprotected amino acid (2a) was accomplished by this method in an overall 51 % yield I he same method can be used to make Fmoc-protected ammo acid (2b), which has an overlapping emission bandwidth with (2a) but has a much higher quantum yield
  • the final building block required is the therapeutic moiety This can be attached by dick chemistry under Huisgen [3+2] cycloaddition conditions and allows the drug to be attached to the completed peptide
  • the strategy for this approach is to give a fully functional diagnostic system with optional attachment of the therapeutic agent Pn order to accomplish this, the synthesis of the diagnositic system on solid support includes a propargylglyeine residue Unlike in the case for the NIR fluorophore, which is attached Io the ammo acid by click chemistry before SPPS, the therapeutic moiety is attached to the completed reagent after synthesis, cleavage from the solid support, and purification This gives the option of attaching a variety of therapeutic agents m a simple one-step procedure
  • the only preiequisite for attachment is that the drug agent must be functionahzed with an azide group
  • Maytansme has several advantages 1.
  • the maytansme analog DMl attached to the antibody trastuzumab is currently m phase II clinical tnals 2.
  • Maytdnsme is reported to be a tubulin inhibitor with the same mechanism of action as the clinically used agents vincristine and vinblastine 3.
  • Maytansme is commercially available and can be selectively functionalized at the secondary alcohol And, 4 Maytansme activity requires an N-methyl alanine residue that can readily be functionahzed with an azide
  • conjugation to the diagnostic system involves one step Briefly, the azido-contarnmg drug agent (12) in thedepositednce of the peptide system is treated with a copper (I) source in a DMSO-H 2 O sohent system As mentioned above, this strategy gives a general one step attachment of a variety of drug entities
  • bleomycin as the therapeutic component of the proposed theranostic agent
  • Bleomycin was chosen for several reasons to include the following bleomycin is an established and well-characterized chemotherapeutic drug in the clinic, bleomycin is itself a pentapeptido, a targeted delivery system to the breast can immediately give a new indication for this drug, selective modification to give azido-bleomy ⁇ n can be achieved in a simple one-step reaction, and its molecular mechanism of action suggests that chemical modification can not alter the effectiveness of this drug
  • Bleomycin is administered as a mixture of structurally related compounds marketed under the name Blenoxanc (Hecht, In Cancer Chemotherapeutic Agents 1995) Blenoxane is administered intravenously and is used to treat lymphomas, squamous cell carcinomas, testicular caicmomas, and malignant pleural effusions
  • bleomycin A5 marketed as Bleocm
  • the copper chelate of bleomycin A5, marketed as Bleocm can be used for attachment to the disclosed delivery system
  • the bleomycins are complex glycopeptides and chemical modification can be challenging
  • bleomycins strongly chelate transition metals at their N-terminus domain
  • the primary amine is exposed and easily functionahzed ( Figure 5)
  • bleomycin A5-copper chelate is treated with 2-azidodcetic acid, DCC and HOBt to give the azido-conjugated compound
  • Treatment with 15% aqueous EDTA liberates the bleomycm comugate from the copper (Xu et al Bioorg Med Chem Lett 2005, 15 3996-3999)
  • the functionalized bleomycm is conjugated to the proposed delivery system in one step under click chemistry conditions for the Huisgen [3+2J cycloaddition
  • the cycloaddition requires Cu (I) for the cycloaddition
  • the copper saturated adduct can be investigated for conjugation to the proposed delivery system
  • the antitumor activity of bleomycm is attributed to its ability to induce double strand DNA damage
  • the region of bleomycm that was modified is at the bis-tmazole tether extending from the C-terminus of bleomycm, which has been shown to thread between double- stranded DNA before inducing cleavage
  • Analogs of bleomycm attached to a solid support at the C terminus showed reactivity identical to that of free bleomycm for sequence-selective cleavage of duplex DNA, suggesting that the threading mechanism is not important for the activity of bleomycm (Abraham et al J AmerChem Soc 2001, 123 5167-5175) Therefore, the modified bleomycm retains antitumor activity even when attached to the disclosed system Because bleomycm is required to enter the cell membrane and then the nuclear membrane in order to cleave DNA, this system can efficiently dehvei the bleomycm
  • a further component of the disclosed theranostic agent is the attachment of the drag molecule
  • Click chemistry via a Huisgen 1,3-drpolar cyclodddilion is a convenient method to connect two highly fcnctionahzed groups in a single stop with high yield and purity
  • This design strategies the synthesis of a fully functional dual modality detection system that can be elaborated with a number of therapeutic components by covalently attaching new drug entities using a single-step click reaction
  • the complete detection system is assembled by SPPS using commercially available Fmoc-propargylglycme as the initial residue After cleavage from the bead and HPLC memeification, the agent is fully functional as the proposed detection system
  • Modular addition of the therapeutic agent involves a simple one-step reaction by click chemistry as shown in Figure 6
  • the only requirement is that the drug is labeled with an a7ide functionality
  • the fully functional detection system can be used for initial
  • FIG. 7 outlines the SPPS of the complete diagnostic system the agent is based on a peptide sequence that incorporates the following a cell-penetratmg Tat sequence, tissue- specific PEGA breast homing motif, a fluorescent chromophore, a chemosensitive MRI contrast/fluorescence quencher moiety, and a therapeutic agent
  • the peptide is a 21- mer, which can easily be accommodated on solid phase Eight of the 11 required amino acids are commercially available
  • One disulfide bridge must be installed, which can be accomplished by treatment of the unprotected cysteine side chains with iodine in the presence of air Because our agent only contains two cysteine residues, selectivity is not an issue Cleavage from the solid support and global deprotection is effected by treatment with TFA Note the alkyne of 1he prapargyglycme residue allows foi mild and selective attachment of our azido-functionahzed drag molecule However, even without the attachment of the drug molecule, all
  • the theranostic agents have a pharmacological effect
  • the homing peptide, the homing peptide-Tat conjugate, the homing peptide-Tat maytansinoid conjugate, and the homing peptide-Tat-maytansmoid-NIR fluorophore-MRI mtroxide conjugate, as well as the diagnostic agent comprising other therapeutics (i e bleomycin), can be evaluated on breast cancer cells (MCF-7 and MDA-MB-231) m ⁇ itro by MTT assay to obtain the Gl 50 -values
  • MCF-7 and MDA-MB-2311 breast cancer cells
  • m ⁇ itro by MTT assay to obtain the Gl 50 -values
  • the results provide an in vitro toxicity profile for the different segments of the theranostic agent as well as a reference for in vivo studies
  • Normal endothelial cell lines, HlJVEC and HMVEC can be used as controls to assess
  • gleevec a clinically used targeted cancer therapeutic
  • Gleevec s therapeutic mdex ranged from 57 to 243, as defined by the iatio of EDso in BCR-ABL-ncgative cells (dose limiting side effect) to ED 5 Q m BCR-ABL-positive cells (therapeutic effect)
  • Cytotoxic drugs in general tend to have a very nanow therapeutic index
  • the chemotherapeutic agent 5-FU has an in vitro therapeutic index of only 2 3 when administered by IP injection on tumor bearing mice (ligo et al Biochem Pharmacol 1988, 37 1609-1613)
  • the cytotoxicity of the trastuzumab-DMl conjugate requires cleavage of the maytansinoid group
  • Two possible scenarios for a narrow therapeutic mdex for disclosed agent are as follows 1 high toxicity is met at low concentrations due to rapid cleavage from the peptide, or 2 high concentrations of the agent is required to have an effect, because of slow cleavage hi both cases, the therapeutic index is narrowed
  • both scenarios can be addressed by attenuating the linker region
  • non-peptidic linkers can be explored as is used for the trastuzumab DMl conjugate
  • peptidic or disulfide bond linkages can be explored Disulfide linkages are attractive, because they can be further fine-tuned by increasing ste ⁇ c bulk adjacent to the disulfide linkage
  • disulfide bond cleavage is redox sensitive, which follows the mechanism for reducing the spin-label MPJ contrast moiety of the
  • the synthesized agents can be subjected to wild type mice to determine acute toxicity
  • the method can be performed following the Acute Oral Toxicity- Up and Down Procedure published by the EPA (OECD Guides for testing of chemicals,
  • LD50 is calculated for both the diagnostic system and the theranostic agent that incorporates a drug moiety
  • the dosage for treating the annuals can be determined hi order to achieve maximal therapeutic effect and minimal toxicity, the animals at can be dosed at 30% the LD50 value which is assumed to be the maximally tolerated dose This rough calculation is based on a 6- ammal per experiment study assuming a hneai relationship between dosage and lethality It is expected that 30% of the dosage that kills 50% of the mice is approximately the highest dosage that will not kill any of the mice in a study of 6 animals per expe ⁇ ment
  • mice can be treated with the detection system that does not incorporate the therapeutic component
  • the detection system can be treated with sodium borohydnde prior to administration to the animal, to reduce the nitroxide radical and remove its ability to reduce fluorescence
  • This agent can then be used to verify localization of the homing peptide to the breast tissue by fluorescence imaging
  • the limit of detection for the agent can be defined as the concentration (mg of agent / kg animal weight) that results in a signal-to-noise ratio of 3 1
  • the quantity of agent delivered can be estimated by fluorescence imaging using the Lambert-Beer law
  • the practical limits ⁇ f quantification canbe determined as the lowest concentration to give a relative standard deviation of ⁇ 10%
  • mice can be treated with the fully functional detection system with the mtroxide radical intact
  • the half-life for the free radical by non-specific degradation in a normal mouse can be determined by plotting the relative intensity of the fluorescent signal over a 96-h tune pe ⁇ od
  • the plasma levels of the agent over a 96-h time period can be determined by HPLC Degradation and'or metabolism of the agent m the plasma can be determined by HPLC
  • cancer therapeutics that can be used with the disclosed theranostic system There are a number of potential cancer drags with various mechanisms of action These drugs can be used in place of maytansuioid and bleomycin as the therapeutic component of the theianostic agent
  • the time course for dosing can be determined based on the half-hie of the agent in the circulatory system as determined in the wild- type mice MRI and fluorescence imaging can be used to monitor tumor growth
  • the disclosed agent can be used for diagnosing and treating breast cancer by homing to the breast tissue, identifying the presence of cancel, and impeding tumor growth
  • combretastatm A-4 has an IC50 value of 2 0 ⁇ 0 2 ⁇ M
  • Phase II clinical trials on a related phosphate analog of combietastatin A- 4 suggest that fosbretabulm is safe and canbe advanced to phase in clinical trials for anaplastic thyroid cancer (Mooney et al Thyroid 2009, l ⁇ 233-240)
  • YK-3 237 was considerably potent against a numbei of cancer cell lines with GI50 values in the sub nanomolar range for several breast cancer cell lines This compound can act as a SIRTl activator
  • YK-3-237 is 10 times more potent m activating SIRTl deacetylasc activity
  • the boromc acid group is functionalized
  • the free boromc acid is necessary for activity and therefore requires a handle for conjugation to the peptide Burke and co workers recently reported a sp 3 - hybidized boronate ester that is cleaved under mild aqueous conditions (Burke et al J Am Chem Soc 2007, 129 6716-6717)
  • the strategy involves an internal coordination between a nitrogen atom and the boron atom
  • the azido functionalized dicaiboxylic acid (13) is condensed with the boronic acid under Dean-Stark conditions to give the boronic esters of YK 3 250 and YK 3 237 as shown in Figure 10
  • These azido- functionalized drug agents can be attached to the theranostic system via click chemistry The release oi the drug can occui by kinetic hydrolysis of the agent m
  • 318 YK-4-272 is a fluorescent HDAC mhibitoi developed by Dr Kong This drag inhibits pan HDAC activity with an IC50 value of 125 nM More interestingly, this agent shows some selectivity toward class II isoforms of HDAC, especially HDAC 6 In addition, a crystal
  • MLC myosin light cham
  • SCG-3 285 is a potent inhibitor of cell proliferation for several breast cancer cell lines with GI50 values around 500 nM
  • Biaryl-substitutcd dihydroquinazolmones are a class of potent tubulin inhibitors
  • Compound MP-201 is a lead structure optimized to give the mtro-containing qurnazolmone shown in Figure 9
  • MP-201 induces 100% depolymenzation of tubulin at 15 ⁇ M, inhibits tubulin polymerization with an IC50 value of 0 76 ⁇ 0 01 ⁇ M and displaces [ 3 H]colchicme 33 ⁇ 3 3% at 5 ⁇ M and 60 ⁇ 2 8% at 50 ⁇ M
  • An asymmetric synthesis to the optimized compound has recently been reported (Chinigo et al J Med Chem 2008, 51 4620-4631)
  • the S enantiomer of the optimized mtro-contarning analog inhibits tubulin assembly with an IC50 of 1 1 ⁇ 0 2 ⁇ M, displaces [ 3 H]colchicine 28 ⁇ 1 % at 5
  • ApaseP was determined to be differentially expressed in human tissues A tissue microanay analysis showed that ApaseP was indeed highly expressed in human breast tissue (both normal and cancerous), but not in the lung, esophagus, nor the stomach APaseP is the putative recognition protein for the breast homing peptide These results mdicate that the breast homing peptide canalso distribute to the breast in humans Most notably the low expression levels of ApaseP in the lung suggest that the lungs may be completely bypassed as an accumulation site, and therefore reduce the pulmonary toxicity of bleomycin
  • the breast homing peptide can also be a substrate for APaseP, because it contains potential recognition motifs for cleavage by APaseP APaseP cleaves at N terminal X P Z sequences, and the breast homing peptide contains two X-P-Z sequences, albeit not at the N- terminal
  • the location of these sequences and the cyclic nature of the bieast homing peptide can increase the half life of this peptide (Essler et al PNAS 2002, 99 2252-2257)
  • the X P Z sequence can be placed at different locations m the cyclic system to maximize stability but maintain recognition
  • ApaseP is richly expressed on MCF 7 and MDA-MB-231 breast cancer cells (F lgure 13)
  • ApaseP expression was not detected on PC 3 prostate and A 549 lung cancer cells Binding to ApaseP can be A prerequisite for cell penetiation
  • This agent has cluneal utility for the detection of breast cancer In the event bieast cancer is detected, the disclosed agent can then be used for the snmrltaneous treatment and monitoring of the cancer
  • the proposed theranostic agent is designed to accommodate a variety of drug entities by a simple chemical attachment, which allows for a personal medicine approach to patient treatment
  • a versatile platform is introduced that can be adapted for new and emerging technologies as they aie developed
  • STAT3 is especially critical for breast cancer stem-like cell survival and proliferation (Zhoe et al PNAS 2007)
  • STAT3 regulates cell motility and invasiveness through non-transc ⁇ ptional mechanisms (Gao et al Sci STKE 2006)
  • Inhibition of STAT3 signaling in breast cancer can have enhanced anti-tumor effects, mediated by cell cycle arrest and induction of stem-like cancer tell death, disruption of angiogenesis, and interference with tumor-cell migration and invasion (Turkson bxpert Opin Ther Targets 2004) Therefore, STAT3 is a potential drug target for breast cancer
  • STAT3 inhibition in general has resulted m only mildly potent agents that induce 50% growth inhibition of breast cancer cells in the range of 13 4 to 100 ⁇ M (Song et al PNAS 2005, Coleman et al J Med Chem 2005, Schust et al Anal Biochem 2004, Schust et al Chem Biol 2006, Siddiquee ct al PNAS 2007, Bhasin et al Bioorg Med Chem Lett 2008, Jing et al Cancer Res 2004, Xu et al PIoS ONE 2009)
  • STAT3 N-domain protein-protein interactions has resulted m a more potent STAT3 inhibitor named STAT3 N-domain protein-protein interactions has resulted m a more potent STAT3 inhibitor named STAT
  • Homing peptide technology refers to short peptide sequences derived from phage display libraries that have the unique property of homing to specific organs Ruoslahti and Pasqualmi revealed that different organs have distinct zip codes within the endothelium vascular, and appiop ⁇ ately programmed peptide sequences can be used to homo to these zip codes (Pasqualmi et al Nature 1996) The peptide and even peptide conjugates of the homing sequence have been shown to distribute to breast tissue presumably via interaction with membrane-bound prolme-specific aminopeptidasc P (APaseP) (Essler et al PN ⁇ S 2002) The STAT3 Hel2A 2 retro-inverso peptide can be conjugated to a breast homing peptide for development as a potential breast cancel therapeutic
  • a breast homing peptide conjugated to the STAT3-Hel2A-2 retro-mverso peptide can distribute specifically to the breast for increased effective drug concentration at the breast with an overall reduced systemic concentration
  • 336 Dcsc ⁇ bcd herein is a novel delivery system for a STAT3 inhibitor that targets the N termmal domain of STAT3
  • a modular synthesis that allows for simple attachment of the delivery component to the therapeutic component in a single step can be performed Incorporating a fluoiescent label on the homing peptide sequence will allow for following the distribution of the agent by live imaging in vivo
  • a se ⁇ es of linking groups can be used to synthesize conjugates of the breast homing peptide and the STAT3 Hel2A-2 retro im erso peptide
  • the disclosed agents can be evaluated for cell penetration of MCF-7 and MDA-MB-231 cancer cells, specificity tor breast-dc ⁇ vcd cells, and homing potential
  • FIG. 17 A schematic for synthesis of the disclosed compounds is outlined in Figure 17 As shown, the agent is based on a peptide sequence that incorporates the following a cell penetrating Tat sequence, the tissue-specific PEGA breast homing motif a fluorescent chromophore, and a synthetic handle for attachment of the STAT3-Hel2A-2 peptide
  • the cell-penetrating Tat component is necessary for internalization of an agent into the cytoplasm of the cell.
  • preliminary results indicate that the promiscuity of the Tat sequence is not conferred to the homing peptide when the two units are conjugated together
  • FIG. 342 Figure 18 outlines a series of linkers that are proposed for synthesis and biological evaluation
  • An aliphatic tether and polyethylene glycol tether can determine the distance iequuement between the delivery peptide system and the STAT3 IM2A 2 peptide that can be necessary to retam the homing properties and STAT3 inhibition activity of each entity
  • a poly- lysine tether can be a potential proteolytic site for release of the STAT3-Hel2A-2 peptide in vivo
  • Poly-lysine can be targeted for proteolytic cleavage at tumor sites (Wcisslodcr ct al Nature 1999), and this can provide an increased selectivity in treating the cancer
  • a squarane tether can be a rigid system that can be an uncleavable linker with reduced rotational degrees of freedom in comparison to the aliphatic or PEG tethers
  • the drug can be cleaved from the homing peptide once in the cancerous tissue
  • a selective release strategy can be implemented
  • a disulfide linkage can be used to connect the two umts
  • the disulfide bond can serve as a selective releasing moiety by taking advantage of the hypoxic intracellular redox chemistry that is particular to cancer cells, which would chemically reduce the S-S bond into two fragments (Hyodo et al Cancer Res 2006)
  • the disclosed compounds can be biologically evaluated The disclosed compounds can be evaluated for their ability to induce apoptosis, to specifically associate with STAT3 o ⁇ er STATl, and to decrease STAT3 driven transcription
  • In vivo modeling can be used to determine biodistribution, tox icity, and efficacy in reducing tumor volume
  • the ability of the disclosed agent to cross the cell membrane and its specificity for breast derived cells can be assessed as follows
  • the peptide can be conjugated to a fluorescent reporter group and then exposed to MCF 7 or MDA-MB-231 breast cancer cells.
  • the 9-amino acid Tat cell-penetrating sequence can be used to address the concern of penetrating the cell membrane
  • the Tat sequence is sufficient to allow specific penetration of the homing peptide into the cell
  • the peptide containing the Tat sequence distributes to the cytoplasm of MCF-7 cells the peptide that docs not contain the Tat sequence shows very poor penetration into the cytoplasm of MCF-7 cells
  • a similar phenomenon is observed for MDA-MB 231 cells, where treatment with the homing peptide conjugated to the Tat sequence shows clear penetiation into the cell
  • For the cells treated with the homing peptide not conjugated to the Tat sequence a fluorescent signal is observed, but the homogeneity of the fluorescence signifies non-specific binding and not penetration into the cell
  • Tat-sequence did not confer non-specific penetration into cells
  • A- 549 human lung cancer cells were treated with the dansylated peptide containing the Tat sequence or the dansylated peptide without the Tat sequence
  • the peptide treatment showed no difference from control, indicating that the Tat sequence does not impart non-selective penetration of the peptide into any type of cell
  • the limit of detection for this agent will be defined as the concentiation (mg of agent / kg animal weight) that results in a signal to noise ratio of 3 1
  • the quantity of agent delivered can be estimated by fluorescence imaging based on the intensity of the fluorescent signal
  • the practical limits of quantification will be determined as the lowest concentration to give a relative standard deviation of ⁇ 10%
  • STAT3-Hel2A-2 is an optimized peptide with impressive specificity for breast cancer cells over normal cells and modest potency with GI 50 ⁇ alues in the low micromolar range
  • the cancerous cell lines are more sensitive to STAT3-Hel2A 2 treatment than the non-cancerous breast cell line MCF-IOA
  • This screen can be used for the disclosed analogs to determine an m vitro therapeutic index Loss of cell viability due to caspase- dependent apoptosis can also be determined by analysis of PARP cleavage MCF 7 cells are caspase-3 deficient and therefore apoptosis can be determined by annexin V binding
  • the disclosed analogs can then be evaluated for interaction v, ith STAT3 in living cells by fluorescence resonance energy transfer (FRET) microscopy
  • FRET fluorescence resonance energy transfer
  • the TAMRA fluorophore can act as an acceptor to the donor eGFP tag, which results in FRET (a change in fluorescence wavelength) when the two components are in close proximity Indeed this was observed when HEK293 cells stably transefected with eGFP on the N-te ⁇ runus of STAT3 were treated with TAMRA-labeled STAT3 Hel2A 2
  • the disclosed analogs can contain a TAMRA fluorophore and this assay can be used to determine if the analogs interact with STAT3
  • the luciferase reporter vector can be employed for the acute phase response element (APRE) containing STAT3 DNA-bindmg elements and a remla lucitcrase reporter vector The APRE reporter group has been demonstrated to be activated in MCF-7 cells by leukemia virus.
  • APRE acute phase response element
  • Athymic female Balb/c nude mice can be purchased from the National Cancer Institute (NCI)
  • NCI National Cancer Institute
  • MCF 7 and MDA-MB-231 tumor xenografts presenting different levels of STAT3 activation, are the models of choice, as indicated by favorable cellular data
  • surgical implantation of the breast cancer cells into the breast tissue of the mice can be required MCF-7 cells are estrogen-dependent, and athymic nude mice are estrogen deficient Theiefore, this model can require the implantation of an estrogen-releasing pellet As shown m Figure 22, these models have been generated previously Tumors can be grown to 72-94 mm 3 before the treatment begms
  • the disclosed analogs can be subjected to acute toxicity studies m wild-type mice using the Acute Oral Toxicity- Up and Down Procedure (OECD 2001) This method allows foi the determination of estimated LD 50 values for the compounds with minimal amounts of material
  • the LD 50 can be used to determine the dosage for treating the animals that will give us maximal therapeutic effect and minimal toxicity
  • the animals can be docscd at 30% the LD 50 value This rough calculation is based on a 6-animal experiment, where 30% of the LD 50 is approximately the highest dosage that will not kill any of the mice in the study of 6 animals pel experiment This estimate can be adjusted downward if it appears that the animals are experiencing toxicity, or the dosage can be adjusted upwards if the animals tolerate this dosage well hut the fluoi escent signal m the imager is insufficient Pre ⁇ ious experiences have shown that these mice models can tolerate 30 mg/kg dosage of the fluorescentlj -tagged homing peptide and g ⁇ e a sufficient fluorescence signal when imaged
  • the mice can be divided into treatment groups
  • the test concentrations of the disclosed agents can be obtained by diluting with PBS
  • Tumor-bearmg mice can be injected with either the disclosed agents or vehicle control, once every other day for 4 v, eeks by tail vein injection
  • Tumor volumes and distribution of the agent by fluorescence imaging can be monitored at least twice weekly
  • Tumor and normal tissues can be obtained from treated and untreated annuals, fixed m 10% buffered formalin, blocked m paraffin, sectioned and stained with hematoxylin and eosin for histopathological examination
  • Tumor volumes measured at different time points for each mouse can be correlated for the tumor volumes measured at each time pomt, peicentages with respect to the initial tumor volume (day 0, the first day of dosmg) can be calculated and used as the outcome for further analysis Changes in tumor volumes can be analyzed using linear mixed effect models to compare differences m these changes among groups A pair wise comparison of interest can be made among these subgroups Each comparison can be performed with a two- group univariate repeated measures ANOVA with Greenhouse Geisser correction Six tumor volume measurements from the same mouse can be correlated
  • the baseline tumor volume is estimated to be 83 mm 3 (72-94 mm 3 ) It is assumed that the decreases in tumor volume at the end of the study can be approximately 50 100% for each drag (0 42 mm 3 ) In the pow er calculation tumor growth (percentage) between the subsets can be compared The different dose levels for each treatment can be combined.
  • the STAT3-Hel2A-2 retro-mverso peptide has been evaluated for growth inhibition of breast cancer cells, selectivity for STAT3 over STATl by FRET analysis, and inhibition of the transcriptional activity of STAT3 Disclosed herein is the combination of a novel STAT3 inhibition snategy to a homing peptide for development as a new breast cancel therapeutic

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Abstract

Disclosed herein are compositions and methods relating to detecting breast cancer. Further provided are methods and compositions for treating breast cancer.

Description

COMPOSITIONS AND METHODS FOR DETECTION AND TREATMENT OF BREAST CANCER
I. CROSS-REFERENCE TO RELATED APPLICATIONS
1 This application claims benefit of U S Provisional Application No 61/234,163, filed August 14, 2009, which is hereby incorporated herein by reference in its entirety
IL STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
2 This invention was made with government support under fedeial giant BC076376 awarded by the Department of Defense-Breast Cancer Research Program The Government has certain rights to this invention
III.REFERENCE TO SEQUENCE LISTING
3 The Sequence Listing submitted August 16, 2010 as a text file named "24716_12_9001_2010_08_16__MIC AFD_Sequence_listing_text file tart," created on August 13, 2010, and having a size of 7,691 bytes is hereby incorporated by reference pursuant to 37
C r R § 1 52(e)(5)
IV. Background
4 Breast cancer is the second leading cancer killer of women resulting in 40,000 deaths each year There arc approximately 180,000 new cases of breast cancer diagnosed each year However, follow-up studies on breast cancer patients show a clear correlation between early detection and patient survival It is estimated that 1 m 8 women have a lifetime risk of developing breast cancer The age specific probability of developing breast cancer mcreases considerably after age 40 (Jemal et al CA Cancer J Clin 2008, 58 71096) For younger women, the disease can bo especially devastating, affecting not only women, but also their husbands and their children
5 The data strongly suggest that at its inception breast cancer is a progressive disease, rather than a systemic disease, and early diagnosis and treatment can have the most significant outcome on reducing patient mortality Currently, high-quality mammography is the most effective method presently available for breast cancer screening However, lie uiveats m mammography include the following false positive detection results m up to 85% of non- malignant lesions requiring invasive biopsies and false-negative detection results m up to 20% of cancers being missed, particularly in younger women who have denser breast tissue that obstruct detection of the cancer Furthermore, the technique of mammography involves taking an X-ray of each breast The radiation from X-ray imaging is considered harmM with a greater risk in younger women, and therefore mammography is usually only recommended for women over 40 years of age unless the younger patient has a significant πsk for the cancer (Tabar et al rnt J Gynaecol Obslet 2003)
6 Magnetic resonance imaging (MRI) provides a solution to the limitations of mammography MRI is virtually uninfluenced by breast density, a problem met in mammography of younger women In conipaπng clinical breast examination, mammography, and MRI for detecting invasive breast cancer, the sensitivity was 18%, 33%, and 80%, respectively (Keana et al Magn Reson Med 2005) Howevei, MRI is not without its own limitations The same study showed that specificity with MRI is lower than with mammography, meaning that the ability to distinguish between benign and cancerous images was lower for MRI I ower specificity results in more findings judged as unceitam and requiring a follow-up exam
7 Presented herein is a new detection strategy with the option of simultaneous drug delivery The advantages of this system are as follows 1 Direct imaging of intracellular chemical events can signify the presence of cancer to address the problems with false positive and false-negative detection in mammography and MRI 2 Fluorescence imaging can be employed, which uses harmless non-ionizing radiation as opposed to the X-rays> used in mammography 3 The imaging agents can be designed to work m tandem response to chemical environments in cancer cells, giving double confirmation of the presence of cancer 4 An organic spin-label can be used as the Tl-contrast enhancement agent m place of toxic transition metals normally found in MRI contrast agents 5 The system can be tissue-specific allowing for directed distribution of the agent specifically to the bieast 6 A modular synthetic assembly of the system can allow for the option of attaching a therapeutic moiety to the detection system 7 And, by design the entire system can be peptide-based so that the system is readily degraded in cancer cells, where peptidase activity is the highest, resulting in specific ielease of the therapeutic agent at the target
8 A breast homing peptide that incorporates a fluorescent chromophore, a non-metallic MRI contrast agent, and a therapeutic component can act as a tissue specific theranostic that can detect cancer by dual modality MRI fluorescence imaging, and treat the cancer by specific release of a therapeutic agent in the cancer cell
V. Summary 9 In accordance with the purposes of this invention, as embodied and broadly described herein, this invention, in one aspect, ielates to compositions and methods related to detecting and treating cancer, particularly breast cancer
10 It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed
VI. Brief Description of the Figures
11 Figure 1 shows two routes to the fluorophore containing ammo acids Methods of attaching the fluorophore to an ammo acid residue that can be employed in SPPS are shown Method 1 is accomplished by tieatmg the 0-succmimide ester of the carboxyl containing fluoropliore with Fmoc-Lys OH This gives selective functionalizatton on the side-chain amine group Fmoc-protected ammo acid (2a) has been synthesized by this method m an overall 51% yield The same method is used to make Fmoc-protected ammo acid (2b), which has an overlapping emission bandw idth with (2a) but has a much higher quantum yield
12 A near IR (NIR) dye encapsulated in an α-cyclodextrm molecule The synthesis of the encapsulated NIR dye is shown in equation 2 of Figure 1 following the protocol of Anderson and co-workers (Simon et al Chem Commun 2008, 2897-2899)
13 Figure 2 outlines how commercially available mtroxide (T) is condensed with N- hydroxysuccmimide under DCC couplmg conditions Treatment with Fmoc-Lys-OH in the presence of Hunig's base gives the Fmoc-protected ammo acid building block in 97% yield
14 Figure 3 shows the attachment of the fluorophore and the spin label
15 Figure 4 outlines how commercially available maytansinol (10) is conjugated to azido functionahzed N-methylalanine (11) by DCC coupling to give compound (12). Carboxyhc acid (11) is synthesized in one step from N methylalanme and the hydroxysuccinimide ester of 2 azidoacetic acid
16 Figure 5 shows the copper chelate ot bleomycin A5, marketed as Bleocm, attachment to the delivery system In the case of bleomycin A5 copper chelate, the primary amine is exposed and easily functionalized Following the method of Xu and co-workers, bleomycin A5-copper chelate is treated with 2-azidoacetic acid, DCC, and HOBt to give the azido-conjugated compound Treatment with 15% aqueous EDTA liberates the bleomycin uonjugdle from the copper (Kriege et al NEJM 2004, 351, 427-437) 17 Figure 6 demonstrates a simple one-step reaction by click chemistry for addition of a theranostic agent to the fully functional diagnostic agent
18 Figure 7 outlines the Solid Phase Peptide Synthesis (SPPS) of the complete diagnostic system
19 Figure 8 outlines the incorporation of the drug molecule. The diagnostic system is treated with the azido functionalized maytansrnol in the presence of a copper sulfate and sodium ascorbate in a DMSO-waler solvent system to give the completed theranostic agent Synthesis of the complete theranostic agent
20 Figure 9 identifies the structure of disclosed drug entities that can be attached to the diagnostic system as theranostic agents
21 Figure 10 shows the azido functionalized dicarboxyhc acid (13) condensed with the boromc acid under Dean-Stark conditions to give the boromc esters of YK-3 250 and YK 3 237 following the method of Burke and co workers. These azido functionalized drug agents are attached to the theranostic system via click chemistry Boromc ester functionalization for click chemistry attachment
22 Figure 11 shows the functionalization of the dansyl moiety of YK 4 272 and SCG-3- 285 with mono demethylation of the dimethylaimne group followed by conjugation with 2- azidoacetic acid Mono demethylation of the tertiary amine can be accomplished usmg ACE-CI (Olofson et al J Org Chem 1984 49 2081 2082) Conjugation with O-succimmide ester of 2 a7ido-acetic acid gives the requisite azide for attachment to the theranostic system \ ia click chemistry
23 Figure 12 shows Compound MP-201 which is a lead structure optimized to give nitro-containmg qurnazolmone. Dmydroqmnazolinone tubulin inhibitors
24 Figure 13 shows the expression of ApaseP in cancer cells by nmnunohistostaining usmg an antibody agamst ApaseP ApaseP is richly exprebsed on MCF 7 and MDA MB-231 breast cancer cells. However, ApaseP expression was not detected on PC-3 prostate and A-549 lung cancer cells A) Fluorescence ApaseP antibody shown as light gray areas B) DIC C) DAPI staining of the nuclei shown as light gray areas
25 Figure 14 shows the delivery efficiency of the delivery system and the importance of the Tat sequence The distribution of the homing peptide with or without the Tat cell penetrating sequence was observed in MCF7 breast cancer cells The dansylated breast homing peptide that incorporates the Tat cell penetrating sequence efficiently penetrates MCF 7 breast cancer cells However the dansylated breast homing peptide without a Tat sequence show s poor penetration of MCI1 7 breast cancer cells Panel A shows the fluorescence of the peptide (light gray staining) Panel B shows the DIC image of the cells Panel C shows a merge of A and B
26 figure 15 shows the homing peptide with and without Tat on another metastatic breast cancer cell line, MDA MB 231 The dansylated PEGA breast homing peptide conjugated to the TdI sequence shows penetration into MDA MB 231 cells However the dansylated PEGA breast homing peptide that does not incorporate the Tat sequence gave a considerably w eaker signal under the same treatment and imaging conditions Panel A shows the fluorescence of the peptide (bright color staining) Panel B shows the DIC miage of the cells Panel C shows a merge of A and B
27 Figure 16 shows the homing peptide with and without Tat on the prostate cancer cell line PC-3 Regardless of the presence or absence of Tat on the dansylated homing peptide no cell penetration was exhibited This shows the cell-type specificity of the homing peptide
28 Figure 17 show s the synthesis of the homing peptide component
29 Figure 18 show s the conjugation of the homing peptide and STAT3 Hel2A 2
30 Figure 19 shows the immunomstostainmg for ApaseP expression in cancer cells A) fluorescence ApaseP antibody shown by bright staining B) DIC C) DAPI staining of the nuclei in blue
31 Figure 20 show s images from confocal microscopy cell penetration of MCF7 and MDΛ MB 231 cells Λ) fluorescence of peptide shown b> light gray staining B) DIC C) merge
32 Figure 21 shows images from confocal microscopy cell penetration of A-549 cells A) fluorescence of peptide shown by bright staining B) DIC C) merge
33 Figure 22 shows live-imaging in MCF-7 xenograft model
34 Figure 23 shows STAT3 Hel2A 2 peptide growth inhibition assay in breast cancer and non cancerous breast cells
35 Figure 24 shows FRET selectivity assay
36 Figure 25 shows the inhibition of STAT3 transcriptional activity
VII. Detailed Description
37 The present disclosed embodiment may be understood more readily by reference to the following detailed description of preferred embodiments of the disclosed methods and compositions and the Examples included therein and to the Figures and their previous and following description
38 Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that the disclosed methods and compositions are not limited to specific synthetic methods, specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not mtended to he limiting
A. Compositions and methods
1. Theraiiostics
39 The disclosed compositions and methods allow for the diagnosis and treatment of cancer (breast, lung, liver, prostate or intestinal) such that <ι theranostic approach can be taken Thus, m addition to diagnosing or confirming the presence of cancer, the methods and compositions disclosed herein also provide a means of treating the a subject The disclosed methods and compositions provide a theianostic approach to treating a disorder, such as cancer, by integrating diagnostics and therapeutics to improve the real-time treatment of a subject having for example, breast cancer
40 Thcranostics are useful in clinical diagnosis and management of a variety of diseases and disorders, which include, but are not limited to, e g , cardiovascular disease, cancer, infectious diseases, Alzheimei's Disease and the piediction of drug toxicity or drug resistance
41 Disclosed herein are compositions valuable for the diagnosis (or confirmation) and treatment of a disease or disorder, such as breast cancer The compositions comprise all or some of the following a detection moiety, a contrasting agent, a homing moiety, a cell-penetrating moiety and a therapeutic agent
42 hi many mstances, the components of theianostics can be considered toxic In one embodiment of the disclosed composition, the composition is non-toxic
a) 5 parts
(1) detection moiety 43 The disclosed compositions are comprised of a detection moiety and a contrasting agent The detection moiety can be used for identification purposes, Disclosed herein, the detection moiety can be used to identify cancer present in breast tissue The detection moiety, for example a fluorescent label, can specifically define a particular location of interest (i e breast cancer cells)
44 Any detection moiety known in the art or otherwise contemplated by a person having ordinary skill in the art for use with the presently disclosed and claimed invention is encompassed by the scope of the presently disclosed and claimed invention Particular non- limitmg examples of detection moieties that may be utilized in accordance with the presently disclosed and claimed invention have been described in detail herein above
45 In one embodiment, the disclosed composition comprises a detection moiety wherein the detection moiety can be a fluorophore
46 hi one embodiment, the disclosed composition comprises a fluorophore wherein the fluorophore can be Dansyl, TAMRA, a cyanine dye, or a cyanme dye encapsulated m a cyolodextπn
(2) contrasting agent
47 hi order to achieve effective contrast between magnetic resonance (MR) images of different tissue types, it has long been known to admimstei to the subject contrast agents winch affect relaxation times in the zones in which they are administered or at which the} congregate By shortening the relaxation tunes of the imaging nuiiei (the nuclei whose MR signal is used to generate the image) the strength of the MR signal is changed and image contrast is enhanced
48 The above disclosed composition comprises a contrasting agent wherein the contrasting agent can be an MRI contiastmg agent
49 In one embodiment the composition comprises a MRI contrast agent wherein the contrasting agent can be non-metallic
50 In one embodiment trie disclosed composition comprises a non-metallic contrast agent wherein the contrasting agent can be mtroxide radical or derivative thereof
(3) homing moiety
51 The disclosed compositions can be used for targeting or homing to specific tissues or cell types based on the presence of d homing moiety The homing moiety targets a specific hgand or environment and therefore can be used to transport different molecules or compositions to that location For example, a homing moiety, specific to breast tissue, can be linked to a constrastmg agent and thus tiansport the contrasting agent to the targeted breast tissue
52 The disclosed composition comprises a detection moiety and a contrasting agent and can further comprise a homing peptide
53 In one embodiment, the above disclosed composition comprises a breast tissue- specific homing peptide
54 In one embodiment, the disclosed breast tissue-specific homing peptide can be CPGPEGAGC
(4) cell-penetrating molecule and cell penetrating moiety
55 A cell-penetrating molecule is a molecule that can comprise a cell-penetratmg moiety A moiety refers to a part of a molecule Examples of cell penetrating molecules are 1 at, penetratm, pVEC, transportan etc A cell penetrating moiety is a part of a molecule or functional group that is responsible for allowing the molecule containing moiety to penetrate the cell Λ cell penetrating molecule is capable of penetrating the cell unlike most homing moieties that target a cell-surface molecule but can not enter the cell The presence of a cell-penetrating molecule allows access m to the cell Thus, linking a composition to a cell-penetratmg molecule can ensure intracellular delivery of the composition
56 The disclosed composition can comprise a cell-penetratmg molecule
57 In one embodiment, the cell-penetratmg molecule is Tat
58 In one embodiment, the disclosed composition comprises a homing peptide that can localize the composition to the target tissue, such as breast tissue, wherein the cell-penetrating molecule transports the composition into the cells of the tissue
(5) therapeutic agent.
59 The therapeutic agent of the present compositions comprises one or more therapeutic agents, such as chemical compounds, macromolecules, proteins, and the like, which are effective in treating diseases and disorders, such as a cancer
60 The therapeutic agent is present in an amount effective in providing a desired therapeutic effect to an individual, such as a human or animal patient, when the composition is administered to the individual
61 Therapeutic agents provided in the therapeutic component of the present compositions can be obtained from public sources or can be synthesized using routine chemical procedures known to persons of ordinary skill in the art Thus, the therapeutic agent of the present compositions can comprise a variety of therapeutic agents, including chemotherapeutic agents, anti-inflammatory agents anti-prohferative agents, and the like
62 The disclosed composition comprises a therapeutic agent
63 In one embodiment, the therapeutic agent can be an anti cancer agent
64 In one embodiment, the anti cancer agent can be bleomycin
65 In one embodiment, the anti-cancer agent can be a STATI inhibitor The STAT3 inhibitor can be STAT3-Hel2A-2
2. Homing Peptides
66 Disclosed herem are peptides that target, bind to and/or home to normal tissue, such as normal breast tissue For example, the cyclic nonapeptide CPGPEGAGC (SEQ ID NO 1) specifically homes to breast tissue and the peptide as well as peptide conjugates are known to target aminopeptidase P
67 Disclosed herem are peptides that target, bind to and/or home to breast tissue, normal and cancerous
68 Disclosed herein arc peptides that target lung (CGFECVRQCPERC, SEQ ID TNO 3), breast (CDCRGDCFC, SEQ ID NO 4), prostate (CGRRAGGSC, SEQ ID NO 5), pancreas (SWCEPGWCR, SFQ TD NO 6 or CRVASVLPC, SEQ ID NO 7) or intestine (YSGKWGW, SEQ ID NO 8)
69 Also disclosed herein are homing peptides that each have specific homing properties Homing peptides are disclosed in US patent applications 11/670318, 11/967509, H '951819, 10/158566, 09/910582, 12/322371, 09/765086, 11/979624 and 11/777382 and are specifically incorporated herein by reference at least for homing peptide sequences and structures
70 The disclosed peptide can have any suitable length For example, the peptide can have a length of up to 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ammo acids The disclosed polypeptides can be, for example, 4 to about 50 amino acids in length The disclosed polypeptides can be, for example, less than about 50, 49, 48, 47, 46, 45, 44 43 42 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 , 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 ammo acids in length
71 For example, the disclosed peptide can have a length of from 4 to about 10 amino acids, from 4 to about 15 ammo acids, from 4 to about 20 ammo acids, from 4 to about 25 ammo acids, from 4 to about 30 amino acids, from 4 to about 35 ammo acids, from 4 to about 40 amino acids, from 4 to about 45 ammo acids, from 4 to about 50 ammo acids For example, the disclosed peptide can have a length of from 5 to about 10 amino acids, from 5 to about 15 amino acids, from 5 to about 20 ammo acids, from 5 to about 25 ammo acids, from 5 to about 30 amino auds, from 5 to about 35 ammo acids, from 5 to about 40 ammo acids, from 5 to about 45 ammo acids, from 5 to about 50 ammo acids For example, the disclosed peptide can have a length of from 6 to about 10 amino acids, from 6 to about 15 ammo acids, from 6 to about 20 amino acids, from 6 to about 25 amino acids, from 6 to about 30 ammo acids, from 6 to about 35 amino acids, from 6 to about 40 amino acids, from 6 to about 45 ammo acids, from 6 to about 50 amino acids For example, the disclosed peptide can have d length of from 7 to about 10 ammo acids, from 7 to about 15 amino acids, from 7 to about 20 ammo acids, from 7 to about 25 amino acids, from 7 to about 30 ammo acids, from 7 to about 35 ammo acids, from 7 to about 40 amino acids, from 7 to about 45 amino acids, from 7 to about 50 amino acids For example, the disclosed peptide can have a length of from 8 to about 10 ammo acids, from 8 to about 15 ammo acids, from 8 to about 20 amino acids, from 8 to about 25 amino acids from 8 to about 30 amino acids, from 8 to about 35 amino acids, from 8 to about 40 ammo acids, from 8 to about 45 amino acids, from 8 to about 50 amino acids For example, the disclosed peptide can have a length of from 9 to about 10 amino acids, from 9 to about 15 amino acids, from 9 to about 20 ammo acids, from 9 to about 25 ammo acids, from 9 to about 30 amino acids, from 9 to about 35 amino acids, from 9 to about 40 ammo acids, from 9 to about 45 amino acids, from 9 to about 50 ammo acids
72 The disclosed peptides can be artificial sequences and can be synthesized in vitro and/or recombmantly The disclosed polypeptides can be peptides that are not naturally occurring prolems and can be peptides that have at least two contiguous sequences that aie not contiguous m a naturally occurring protem
73 The disclosed peptides and compositions also can comprise any combination of two, thiee, or moie of the disclosed peptides or ammo acid sequences Thus, disclosed are peptides comprising any one, two, three, or more of the herein disclosed peptides or ammo acid sequences The peptides can be combined in any suitable manner, including, for example, as a single ammo acid chain (that is a fusion of the peptides), via linkers, via branched linkers, and attached individually or together to a structure Also disdos>ed are bifiinctional peptides, which contain one or more of the disclosed peptides fused to one or more second peptides having one or more separate functions Such bitunctional peptides can have at least two functions conferred by different portions of the full-length molecule and can, for example, display pro-apoptotic activity in addition to the ability to taiget the tumor lymphatic
74 Also disclosed are multivalent peptides that can include at least two of the disclosed peptides each independently containing one or more of the disclosed ammo acid sequences The multivalent peptide can have, foi example, at least three, at least five oi at least ten of such peptides each independently contaimng a disclosed amino acid sequence In some aspects, the multivalent peptide can have two, three, foui five, six, seven, eight, nine, ten, fifteen or twenty identical or non-identical peptides and/or amino acid sequences In some aspects, the multivalent peptide can contain identical peptides and/or amino acid sequences In some aspects, the multivalent peptide can contain contiguous identical or non-identical peptides and/or ammo acid sequences, which are or are not separated by any intervening amino acids
3. Antibodies
75 The term "antibodies ' is used herein in a broad sense and includes both polyclonal and monoclonal antibodies In addition to intact immunoglobulin molecules also included in the term "antibodies" are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as described herein The antibodies are tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods
76 As used herein, the term "antibody" encompasses, but is not limited to, whole immunoglobulin (i e , an intact antibody) of any class Native antibodies are usually heterotetrameπc glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages vanes between the heavy chains of different immunoglobulin isotypes Each heavy and light chain also has regularly spaced mtracham disulfide bπdges Each heavy chain has at one end a variable domain (V (H)) followed by a number of constant domains Each light chain has a vai iable domain at one end (V (L)) and a constant domain at its other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain Particular ammo acid residues are believed to form an interface between the light and heavy chain variable domains The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino acid sequences of their constant domains Dependmg on the ammo acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes Theie are five major classes of human immunoglobulins IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (lsotypes), e g IgG-I, IgG-2, IgG-3, and IgG 4, IgA-I and IgA-2 One skilled in the art would recognize the comparable classes for mouse The heavy chain constant domains that correspond to the different classes of immunoglobulms are called alpha, delta, epsilon, gamma, and mu, respectively
77 The term "variable" ts used herein to desciibe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen However, the variability is not usually evenly distributed through the vaπable domains of antibodies It is typically concentrated in three segments called complementarity determining regions (CDRs) or hyperv aπable regions both in the light chain and the heavy chain variable domains The more highly conserved portions of the variable domains arc called the framew ork (FR) The vaπable domains of native heavy and light chains each comprise four FR regions, largely adoptmg a b-sheet configuration, connected by three CDRs, which foim loops connecting, and in some cases forming part of, the b-sheet structure The CDRs in each chain are held together m close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E A et al , 'Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md (1987)) The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular toxicity
78 As used herein, the term ' antibody or fragments thereof encompasses chimeric antibodies and hybπd antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as scFv, sFv, F (ab')2, Fab', Fab and the like, including hybrid fragments Thus, fragments of the antibodies that retam the ability to bind their specific antigens are provided Such antibodies and fragments can be made by techniques known in the art and can be screened foi specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane Antibodies, A Laboratory Manual Cold Spring Harbor Publications, New York, (1988))
79 Also included within the meaning of "antibody or fragments thereof are conjugates of antibody fragments and antigen binding proteins (single chain antibodies) as described, for example, in U S Pat No 4,704,692, the contents of which ai e hereby incorporated by reference
80 The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific ammo acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or unpaired compared to the non-modified antibody or antibody fragment These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio longevity, to alter its secretory characteristics, etc In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment (Zoller, M J Curr Opm Biotechnol 3 348-354, 1992)
81 As used herein, the term "antibody" or ' antibodies" can also refer to a human antibody and/or a humanized antibody Many non-human antibodies (e g , those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans Therefore, the use of human or humanized antibodies m the disclosed methods serves to lessen the chance that an antibody administered to a human can evoke an undesirable immune response
(a) Human antibodies
82 The human antibodies ot the disclosed methods and compositions can be prepared using any technique Examples of techniques for human monoclonal antibody production include those descπbed by Cole et al (Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p 77, 1985) and by Boerner et al (J Immunol , 147 (1) 86-95, 1991) Human antibodies of the disclosed methods and compositions (and fragments thereof) can also be produced using
phage display libraries (Hoogenboom et al , / MoI BwI , 227 381, 1991 , Marks et al , / MoI Biol , 222 581, 1991)
83 The human antibodies of the disclosed methods and compositions can also be obtained from transgenic animals For example, transgenic, mutant mice that arc capable of producing a full repertoire of human antibodies, in response to immunization, have been descπbed (see, e g , Jakobovits et al ,Proc Natl Acad Sa USA, 90 2551-255 (1993), Jakobo\its et al , Nature, 362 255 258 (1993), Bruggermann et al , Year m Immunol , 7 33 (1993)) Specifically, the homozygous deletion of the antibody heavy chain joining region (J (H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge Antibodies having the desired activity are selected using Env CD4-CO- receptor complexes as descπbed herein
(b) Humanized antibodies
84 Optionally, the antibodies are generated in other species and 'humanized" for administration in humans Humanized forms of non human (e g , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as scFv, sFv, Fv, Fab, Fab', F (ab')2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin Humanized antibodies include human immunoglobulins (recipient antibody) m which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity
In some instances, Fv framework residues, of the human immunoglobulin are replaced by corresponding non-human residues Humanized antibodies may also comprise residues that are found neither m the recipient antibody nor m the imported CDR or framework sequences In general, the humanized antibody can comprise substantially all of at least one, and typically Iwo, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence The humanized antibody optimally also can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin (Jones et al , Natuie, 321 522-525 (1986) Riechmann et al , Nature, 332 323- 327 (1988), and Presta, Cιιrr Op Struct Biol , 2 593-596 (1992))
85 Methods for humanizing non-human antibodies are well known in the art Generally, a humanized antibody has one or more ammo acid residues introduced mto it from a source that is non human These non-human amino acid residues are often reterred to as 'import" residues, which are typically taken from an ' import" variable domain Humamzation can be essentially performed following the method of Winter and co-workers (Jones et al , Nature, 321 522-525 (1986), JAiechmann et al , Nature, 332 323 327 (1988), Veihoeyen et al , Science, 239 1534 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody Accordingly, such ' humanized ' antibodies are chimeric antibodies (U S Pat No 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species Tn practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some t R residues are substituted by residues from analogous sites in rodent antibodies
86 The choice of human variable domains, both light and heavy, to be used m making the humanized antibodies is very important in order to reduce antigenicity According to the "best-fit" method the sequence of the variable domain ofa rodent antibody is screened against the entiie library of known human variable domam sequences The human sequence which is closest to that of the rodent is (hen accepted as the human framework (FR) for the humanized antibody (Sims et al , J Immunol , 151 2296 (1993) and Chothia et al , J MoI 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 framewoik may 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))
87 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, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art Computer programs are a\ ailable which illustrate and display probable three dimensional conformational structures of selected candidate immunoglobulin sequences Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence i e , the analysis of residues that influence the ability of the 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) Also, disclosed are chimeric antibodies as well as fully human antibodies, such as monoclonal antibodies
(c) Monoclonal Antibodies
88 The term monoclonal antibody as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i e , the individual antibodies within the population are identical except for possible naturally occurring mutations that may he present in a small subset of the antibody molecules The monoclonal antibodies herein specifically include "chimeric" antibodies m which a portion of the heavy and/or light cham is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U S Pat No 4,816,567 and Morrison et al , Proc Natl Acad Sci OSA, 81 6851-6855 (1984))
89 Monoclonal antibodies of the disclosed methods and compositions can be prepared using hybπdoma methods, such as those described by Kohler and Milstein, Nature, 256 495 (1975) hi a hybπdoma method, a mouse or other appropnate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that can specifically bind to the immunizing agent Altemativelj, the lymphocytes may be immunized in vitto, e g , using the complexes described herein
90 Transgenic animals (e g , mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed For example, it has been descπbed that the homozygous deletion of the antibody heavy chain joining legion (J (H)) gene in chimeric and germ line mutant mice results in complete inhibition of endogenous antibody production Transfer of the human germ-lme immunoglobulin gene array in such germ line mutant mice can result in the production of human antibodies upon antigen challenge (see, e g , Jakobovits et al , Proc Natl Acad Sci USA, 90 2551-255 (1993), Jakobovits et al , Nature, 362 255-258 (1993), Braggemann et al , Year in Immuno , 7 33 (1993)) Human antibodies can also be produced in phage display libraries (Hoogenboom et al , J MoI Biol 227 381 (1991), Marks et al , T MoI Biol , 222 581 (1991)) The techniques of Cote ct al and Bocmcr ct al are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p 77 (1985), Boeiner et al , J Immunol , 147 (1) 86-95 (1991))
91 Generally, either peripheral blood lymphocytes (' PBLs ') are used m methods of producing monoclonal antibodies if cells of human origin are desired, or spleen cells or lymph node cells are used if non human mammalian sources are desired The lymphocytes are then fused with an immortalized cell hne usmg a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, "Monoclonal Antibodies Principles and Pi actice ' Academic Press, (1986) pp 59-103) Immortalized cell lmes are usually transformed mammalian cells, including myeloma cells of rodent, bovine, equine, and human ongm Usually, rat or mouse myeloma cell lines are employed The hybπdoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfased, immortalized cells For example, if the parental cells lack the enzyme hypoxanthme guanine phosphonbosyl transferase (HGPRT or HPRT), the culture medium for the hybπdomas typically can include hypoxanthme, ammoptenn, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producmg cells, and are sensitive to a medium such as HAT medium More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego Calif and the American Type Culture Collection, Rockville, Md Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J Immunol , 133 3001 (1984), Brodeur et al , "Monoclonal Antibody Pioduction Techniques and Applications" Marcel Dekker, Inc , New York, (1987) pp 51-63) The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against tissue-specific antigens, for example Preferably, the binding specificity of monoclonal antibodies produced by the hybπdoma cells is determined by immunopreoipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme linked immunoabsorbent assay (ELISA) Such techniques and assays are known in the art, and are described further in the Examples below or in Harlow and Lane "Antibodies, Λ Laboratory Manual" Cold Spring Harbor Publications, New York, (1988)
92 Aftei the desued hybiidoma cells are identified, the clones may be subcloned by limiting dilution or FACS sorting procedures and grown by standard methods Suitable culture media for this purpose include, for example, Dulbecco's Modified Fagle s Medium and RPMI- 1640 medium Alternatively, the hybπdoma cells may be grown in vivo as ascites m a mammal
93 The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A Sepharose, protem G, hydroxylapante chromatography, gel electtophoresis, dialysis, or affinity chromatography
94 The monoclonal antibodies may also be made by recombinant DNA methods, such as those described m U S Pat No 4,816,567 (Cabilly et al ) DNA encoding the monoclonal antibodies of the disclosed methods and compositions can be ieadily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of muπne antibodies) Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e g , as described in U S Patent No 5,804,440 to Burton et al and U S Patent No 6,096,441 to Barbas et al
95 In vitro methods are also suitable for preparing monovalent antibodies Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art For instance, digestion can be performed using papain Examples of papain digestion are descπbed in WO 94/29348 published Dec 22, 1994 and U S Pat No 4,342,566 Papain digestion of antibodies typically produces two identical antigen binding fragments called Fab fragments, each with a single antigen binding site, and a residual Fc fragment Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen
(d) Antibody fragments
96 Also disclosed are fragments of antibodies which have bioactivity The polypeptide fragments of the disclosed composition can be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide m an expression system capable of producing the polypeptide fragments thereof, such as an adenovirus or baculovims expiession system Foi example, one can determine the active domain of an antibody from a specific hybndoma that can cause a biological effect associated with the interaction of the antibody with the antigen For example, ammo acids found to not contribute to either the activity or the binding specificity or affinity of the antibody can be deleted without a loss in the respective activity For example, in various embodiments, ammo or carboxy-teπninal amino acids are sequentially removed from either the native or the modified non-immunoglobulin molecule or the immunoglobulin molecule and the respective activity assayed in one of many available assays In another example, a fragment of an antibody comprises a modified antibody wherein at least one ammo acid has been substituted for the naturally occurring ammo acid at a specific position, and a portion of either ammo terminal or carboxy terminal amino acids, or even an internal region of the antibody, has been replaced with a polypeptide fragment or other moiety, such as biotin, which can facilitate m the purification of the modified antibody For example, a modified antibody can be fused to a maltose binding piotein, through either peptide chemistry or cloning the lespective nucleic acids encoding the two polypeptide fragments into an expression vector such that the expression of the coding region results m a hybrid polypeptide The hybrid polypeptide can be affinity purified by passmg it over an amylosc affinity column, and the modified antibody receptor can then be separated from the maltose binding region by cleavmg the hybrid polypeptide with the specific protease factor Xa (See, for example, New England Biolabs Product Catalog, 1996, pg 164 ) Similar purification procedures are available for isolating hybrid proteins from eukaryotic cells as well
97 The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids lesidues, piovided the activity of the fragment is not significantly altered or nnpaued compared to the nonmodified antibody or antibody fragment These modifications can pro\ ide for some additional property, such as to remove or add ammo acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc hi any case, the fragment must possess a bioactive pioperty, such as binding activity, iegulation of binding at the binding domam, etc Functional or active regions of the antibody may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide Such methods are readily apparent to a skilled practitioner m the art and can include site-specific mutagenesis of the nucleic acid encoding the antigen (Zoller MJ et al Nucl Acids Res 10 6487-500 (1982)
98 A variety of immunoassay formats may be used to select antibodies that selectively bind with a particular protein, variant, or fragment For example, solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein, protem variant, or fragment thereof See Harlow and Lane Antibodies, A Laboratory Manual Cold Spring Harbor Publications, New York, (1988), for a descπption of immunoassay formats and conditions that could be used to determine selective binding The binding affinity of a monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al , Anal Biochem , 107 220 (1980)
(e) Administration of antibodies
99 Antibodies of the disclosed compositions are piefeiably admimsteied to a subject in a pharmaceutically acceptable earner Suitable earners and then- formulations are described in Remington The Science and Practice of Pharmacy (19th ed ) ed A R Gennaro, Mack Publishmg Company, Easton, PA 1995 Typically, an appropπate amount of a pharmaceutically- accep table salt is used in the formulation to render the formulation isotonic Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringers solution and dextrose solution The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7 5 Further earners include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are m the form of shaped articles, e g , 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 antibody being administered
100 The antibodies can be administered to the subject, patient, or cell by injection (e g , intravenous, intraperitoneal, subcutaneous, intramuscular), or by other methods such as
infusion that ensure its delivery to the bloodstream in an effective form Local or intravenous injection is preferred
101 Effective do sages and schedules for administering the antibodies may be determined empirically, and making such determinations is. wilhm the skill in the art Those skilled in the art will understand that the dosage of antibodies that must be administered will vary depending on, for example, the subject that will receive the antibody, the route of administration, the particular type of antibody used and other drugs being administered Guidance in selecting appropriate doses for antibodies is found in the literature on therapeutic uses of antibodies, e g , Handbook of Monoclonal Antibodies, Ferrone et al , eds , Noges Publications, Park Ridge, N T , (1985) ch 22 and pp 303-357, Smith et al , Antibodies in Human Diagnosis and Therapy, Haber et al , eds , Raven Press, New York (1977) pp 365-389 A typical daily dosage of the antibody used alone might range from about 1 μg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above
4. Compositions identified by screening with disclosed compositions / combinatorial chemistry
(1) Combinatorial chemistry
102 The disclosed compositions can be used as targets for any combinatorial technique to identify molecules or macromolecular molecules that interact with the disclosed compositions in a desired way The nucleic acids, peptides, and related molecules disclosed herein can be used as targets for the combinatorial approaches Also disclosed are the compositions that are identified through combinatorial techniques or screening techniques in which the compositions have the sequences disclosed herein, or portions thereof, are used as the target m a combinatoπal or screening protocol
103 It is understood that when using the disclosed compositions in combinatorial techniques or screening methods, molecules, such as macromolecular molecules, can be identified that have particular desired properties such as inhibition or stimulation or the target molecule's function The molecules identified and isolated when using the disclosed compositions are also disclosed Thus, the products produced using the combinatorial or screemng approaches that involve the disclosed compositions are also considered herein disclosed
S. Conjugate 104 Also provided herein is a conjugate comprising any one or more of the herein disclosed peptides and one or more moieties In general, the moiety can be a substance that acts upon the target cell(s) or tissue to bring about a desired effect In some aspects, the disclosed conjugate can target, bind to and'or home to breast, lung, liver, pancreas or intestine
105 Tumor lymphatics, such as lymphatic vessels in and aiound tumors, and/or lymphdngiogenic vessels The disclosed peptides preferably selectively bind to tumor lymphatics Thus, the effect can for example, be the labeling, activating repressing, or killing of the target cell(s) or tissue
106 The moiety can be, for example, a therapeutic moiety or a detectable moiety, a cytotoxic agent, an anti lymphangiogemc agent, a cancer chemotherapeutic agent, a pro- apoptotic polypeptide, a grafted polypeptide, a virus, a cell, or a liposome Thus, the moiety can be a small molecule, pharmaceutical drug, toxm, fatty acid, detectable marker, conjugating tag, nanoparticle, or enzyme For example, the moiety of the disclosed conjugate can be an anti cancer agent, such as bleomycin, or pro-apoptotic peptide Examples of pro-apopototic peptides are tumor necrosis factor (Curnis et al , Cancer Res 64, 565-71, 2004) and tachyplesin (Chen et al , Cancer res 61, 2434-8, 2001) Many other anti cancer agents and pro-apoptotic peptides and compounds are known and can be used with and m the disclosed compositions, conjugates and methods
107 Examples of small molecules and pharmaceutical drugs that can be conjugated to a peptide are known m the art The moiety can be a cytotoxic small molecule or drug that kills the target cell The small molecule or drug can be designed to act on any critical cellular function or pathway For example, the small molecule or drug can inhibit the cell cycle, activate protein degradation, induce apoptosis, modulate kinase activity, or modify cytoskeletal proteins Any known oi newly discovered cytotoxic small molecule or drugs is contemplated for use with the peptides
108 The moiety can be d toxm that kills the taigeted cell Non-limiting examples of toxins include abrin, modcccin, ncin and diphtheria toxm Other known or newly discovered toxins are contemplated for use with the provided conjugates
109 Fatty acids (i e , lipids) that can be conjugated to the provided conjugates include those that allow the efficient incorporation of the peptide into liposomes Generally, the fatty acid is a polar lipid Thus, the fatty acid can be a phospholipid The provided conjugates can comprise either natural or synthetic phospholipid The phospholipids can be selected from phospholipids containing saturated or unsaturated mono or disubstituted fatty acids and combinations thereof 1 hese phospholipids can be dioleoylphosphatidylcholrne, dioleoylphosphatidylsenne, dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol, dioleoylphosphatidic acid, palrmtoyloleoylphosphatidylcholme,
palmitoyloleoylphosphatidylseπne, palmitoyloleoylphosphatidylethanolamine, palmitoyloleoylphophdtidylglyt-erol, palmitoyloleoylphosphatidic aud,
palmitelaidoyloleoylphosphatidylcholrne, palmitelaidoyloleoylphosphatidylscnnc, palmitelaidoyloleoylphosphatidylethanolamrne, pahnitelaidoyloleoylphosphatidylglycerol, palmitelaidoyloleoylphosphatidic acid myπstoleoyloleoylphosphatidylcholme, myπstoleoyloleoylphosphatidylseπne, myπstoleoyloleoylphosphatidylethanoamine, myπstoleoyloleoylphosphatidylglyoerol, myristoleoyloleoylphosphatidic acid, dilinolcoylphosphatidylcholinc, dilinolcoylphosphatidylseπne,
dilrnoleoylphosphatidylethanolamine, dilmoleoylphosphatidylglycerol, dilrnoleoylphosphatidic acid, palmiticlmoleoylphosphatidylcholme, palmiticlmoleoylphosphatidylseπne, palmiticlmoleoylphosphatidylethanolamme, palmiticlinoleoylphosphatidylglycerol, palmiticlinoleoylphosphatidic acid These phospholipids may also be the monoacylated deπvatives of phosphatidylcholine (lysophophatidylidylcholine), phosphatidylseπne (lysophosphatidylserrne), phosphatidylethanolamme (lysophosphatidylethanolamine), phophatidylglycerol (lysophosphatidylglycerol) andphosphatidic acid (lysophosphatidic acid) The monoacyl chain in these lysophosphatidyl deπvatives may be pahmtoyl, oleoyl, palmitoleoyl lmoleoyl myπstoyl or myπstoleoyl The phospholipids can also be synthetic Synthetic phospholipids are readily available commercially from various sources, such as AVANTI Polar Lipids (Albaster, Ala ), Sigma Chemical Company (St Louis, Mo ) These synthetic compounds may be varied and may have variations in their fatty acid side chains not found in naturally occurring phospholipids The fatty acid can have unsaturated fatty acid side chains with C14, Cl 6, Cl 8 or C20 chains length m either or both the PS or PC Synthetic phospholipids can have dioleoyl (18 I)-PS, palmitoyl (16 0)-oleoyl (18 I)-PS, dimynstoyl (14 O)-PS, dipalimtoleoyl (16 1) PC, dipahmtoyl (16 O)-PC, dioleoyl (18 1) PC, palmitoyl (16 0)-oleoyl (18 I)-PC, and myiistoyl (14 0)-oleoyl (18 I)-PC as constituents Thus as an example, the provided conjugates can comprise palmitoyl 16 0 110 The moiety of the disclosed conjugate can be a detection moiety Detectable moieties/ markers include any substance that can be used to label or stain a target tissue 01 cell(s) Non-lrmitmg examples of detectable markers include radioactive isotopes, enzymes, fluorophores, and quantum dots (Qdot®) For example, the detection moiety can be an enzyme, biotm, metal, or epitope tag Other known or newly discovered detectable markers arc contemplated for use with the provided conjugates
111 Fluorophores are compounds or molecules that luminesce Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength Representative fluorophores include, but are not limited to, 1,5 IAEDANS, 1,8-ANS, 4- Methylumbelhfeione, 5-carboxy-2,7-dichlorofluorescean, 5- Carboxyfluorescem (5 FAM), 5 Carboλynapthofluorescein, 5-Carboxyteteamethylrhodaniine (5- TAMRA), 5 -Hydroxy Tryptamine (5-HAT), 5-ROX (carboxy-X-rhodamine), 6- Carboxyrhodamine 6G, 6-CR 6G, 6- JOE, 7-Amino-4-methylcoumaπn, 7-Aminoactinomycin D (7 AAD), 7 Hydroxy 4 I methylcoumann, 9-Amino-6-chloro-2-methoxyacridine (ACMA), ABQ, Acid Fuchsin, Acπdme Orange, Acπdine Red, Acπdine Yellow, Acnflavui, Acπflavin Fculgcn SITSA, Acquoπn (Photoprotem), AFPs AutoFluorescent Protein (Quantum Biotechnologies) see SgGFP, SgBFP, Alexa Fluor 350™, Alexa Fluor 430™, Alexa Fluor 488™, Alexa Fluor 532™, Alexa Fluor 546™, Alexa Fluor 568™, Alexa Fluor 594™, Alexa Fluor 633™, Alexd Fluor 647™, Alexa Fluor 660™, Alexa Fluor 680™, Alizarm Complexon, Alizarin Red, Allophycocyamn (APC), AMC, AMCA-S, Ammomethylcoumaπn (AMCA), AMCA-X, Aminoactmomycm D, Aminocoumaπn, AmIm Blue, Anlhrocyl slearale, APC-Cy7, APTRA-BTC, APTS, Astrazon Bnlhant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrme, AITO TAG™ CBQCA, ATTO-TAG™ FQ, Auramme, Aurophospmne G, Aurophosphme, BAO 9 (Bisarmnophenylox-ddidzole), BCECF (highpH), BCECF (low pH), Beibeπne Sulphate, Beta Lactamase, BFP blue shifted GFP (Y66H), Blue Fluorescent Protein, BFP/GFP FRET, Bimane, Bisbenzemide, Bisbenzmude (Hoechst), bis- BTC, BlancophorFFG, Blancophor SV, BOBO™ -1, BOBO™-3, Bodipy492/515, Bodipy493/503, Bodipy500/510, Bodipy, 505/515, Bodipy 530/550, Bodipy 542/563, Bodipy 558/568, Bodipy 564/570, Bodipy 576/589, Bodipy 581/591, Bodipy 630/650-X, Bodipy 650/665-X, Bodipy 665/676, Bodipy Fl, Bodipy FL ATP, Bodipy Fl-Ceramide, Bodipy R6G SE, Bodipy TMR, Bodipy TMR-X conjugate, Bodipy TMR-X, SE, Bodipy TR, Bodipy TR ATP, Bodipy TR-X SE, BO PRO™ 1, BO PROIM 3, Brilliant Sulphoflavrn FF, BTC, BTC-5N, Calcein, Calcein Blue, Calcium Crimson - , Calcium Green, Calcium Green-1 Ca2+ Dye, Calcium Green-2 Ca2H , Calcium Green-5N Ca2+, Calcium Green-C 18 Ca2+, Calcium Orange, Calcofluor White, Carboxy-X-rhodamine (5-ROX), Cascade Blue™, Cascade Yellow, Catecholamine, CCF2 (GeneBlazer), CFDA, CFP (Cyan Fluorescent Protein), CFP/YFP FRET, Chlorophyll, Chromomycm A, Chromomycin A, CL-NERF, CMFDA, Coelenterazine, Coelenterazme cp, Coelenterazme f, Coelenterazine fcp, Coelenterazme h, Coclcnteiazme hep, Coelenterdzme ip, Coelenterazme n, Coelenterazme O, Coumaπn Phalloidin, C-phycocyanine, CPM I Methylcoumann, CTC, CTC Formazan, Cy2™, Cy3 1 8, Cy3 5™, Cy3™, Cy5 1 8, Cy5 51M, Cy5™, Cy7™, Cyan GFP, cyclic AMP Fluorosensor (FiCRhR), Dabcyl, Dansyl, Dansyl Amine, Daiisyl Cadavenne, Dansyl Chloride, Dansyl DHPE, Dansyl fluoride, DAPI, Dapoxyl, Dapoxyl 2, Dapoxyl 3'DCFDA, DCFH (Dichlorodihydro£luorescem Diacetate), DDAO, DHR (Dihydorhodamrne 123), Di-4-ANEPPS, Di-S-ANEPPS (non-ratio), DiA (4-Di 16-ASP), Dichlorodihydro fluorescein Diacetate (DCFH), DiD- Lipophilic Tracer, DiD (DiICl 8(5)), DIDS, Dihydorhodamine 123 (DHR), DiI (DilC18(3)), I Dmitrophenol, DiO (DiOC 18(3)), DiR, DiR (DiIC 18(7)), DM NERF (high pH), DNP, Dopamine, DsRed, DTAF, DY-630-NHS, DY-635-NHS, EBFP, ECFP, EGFP, ELF 97, Eosrn, Erythrosm, Erythrosin ITC, Ethidium Bromide, Ethidium homodimer-1 (EthD-1), Euchrysm, EukoLight, Europium (111) chloπde, EYFP, Fast Blue, FDA, Feulgen (Pararosamline), FIF (Formaldehyd Induced Fluorescence), FITC, Flazo Orange, Fluo-3, Fluo-4, Fluorescein (FITC), Fluorescein DiaceUte, Fluoro Emerald, Fluoro Gold (Hydroxystilhamidme), Fluor-Ruby, FluorX, FM 1 43™, FM 4- 46, Fura Red™ (high pH), Fura Red™/Fluo-3, Fura-2, Fura-2/BCECF, Genacryl Bπlliant Red B, Genacryl Brilliant Yellow 10GF, Gendcryl Pink 3G, Genacryl Yellow 5GF, GeneBlazer, (CCF2), GFP (S651 ), GFP red shifted (rsGFP), GFP wild type' non-UV excitation (wtGFP), GFP wild type, UV excitation (wtGFP), GFPuv, Gloxalic Acid, Granular blue,
Haematoporphyrm, Hoechst 33258, Hoechst 33342, Hoechst 34580, HPTS, Hydroxycoumaπn, Hydroxystilbamidme (FluoroGold), Hydroxytryptamine, Indo-1, high calcium, Indo-1 low calcium, lndodicarbocyamne (DiD), Indotncarbocyanine (DiR), Intrawhite Cf, JC-I, JO JO-I, JO-PRO-I, LaserPro, Lauiodan, LDS 751 (DNA), LDS 751 (RNA), Leucophor PAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine, Lissamine Rhodamme B, Calcein/Ethidium homodimer, LOLO-I, LO-PRO-I, , Lucifer Yellow, Lyso Tracker Blue, Lyso Tracker Blue- White, Lyso Tracker Green, Lyso Tracker Red, Lyso Tracker Yellow, LysoSensor Blue, LysoSensor Green, LysoSensor Yellow/Blue, Mag Green, Magddla Red (Phloxin B), MUg-Fura Red, Mag-Fura 2, Mag-Fura-5, Mag-lndo-1, Magnesium Green, Magnesium Orange, Malachite Green, Manna Blue, I Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, Merocyamn, Methoxycoumdnn, Mitotracker Gieen FM, Mitoti acker Oiange, Mitoti acker Red, Mitomycin, Monobromobimane, Monobromobimane (mBBr-GSH), Monochlorobimane, MPS (Methyl Green Pyronme Stilbene), NBD, NBD Amine, Nile Red, Nitrobenzoxedidole, Noiadrenaline, Nuclear Fast Red, i Nuclear Yellow, Nylosan Brilliant lavin E8G, Oregon Green™, Oregon Green™ 488, Oregon Green1"1 500, Oregon Green™ 514, Pacific Blue, Pararosaniline (Feulgen), PBFI, PE-Cy5, PE-Cy7, PerCP, PerCP-Cy5 5, PE-TexasRed (Red 613), Phloxin B (Magdala Red), Phorwite AR, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphme 3R, PhotoResist, Phycoerythπn B [PE], Phycoerythπn R [PE], PKH26 (Sigma), PKH67, PMIA, Pontochrome Blue Black, POPO-1, POPO-3, PO-PRO-1, PO- 1 PRO-3, Pitmulme, Procion Yellow, Propidium lodid (Pl), PyMPO, Pyrene, Pyronme, Pyromnc B, Pyrozal Bπlliant Flavin 7GF, QSY 7, Qumacπne Mustard, Resorafm, RH 414, Rhod 2, Rhoddmine, Rhodamme 110, Rhodamine 123, Rhodamine 5 GLD, Rhodamine 6G, Rhodamine B, Rhodamme B 200, Rhodamine B extra, Rhodamine BB, Rhodamme BG, Rhodamine Green, Rhodamme Phallicidme, Rhodamme Phalloidine, Rhoddmine Red, Rhoddmine WT, Rose Bengal, R-phycocyamne, R-phycoerythπn (PE), rsGFP, S65A, S65C, S65L, S65T, Sapphire GFP, SBFI, Serotonin, Sevron Brilliant Red 2B, Sevron Bπlliant Red 4G, Sevron I Brilliant Red B, Sevron Orange, Se\ ron Yellow L, sgBFP™ (supei glow BFP), sgGFP™ (super glow GFP), SITS (Pπmuline, Stilbene Isothiosulphonic Acid), SNAFL calcem, SNAFL-I, SNAFL-2, SNARF calcem, SNARFl, Sodium Green, SpectrumAqua, SpectrumGreen, SpectrnmOrange, Spectrum Red, SPQ (6-methoxy- N-(3 sulfopropyl) qumohmum), Stilbene Sulphoihodamnie B and C, Sulphorhodamme Extra, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22 SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64 SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Green, SYTOX Orange, Tetracycline, Tetrameftylrhodamme (TRITC), Texas Red™, Texas Red-X™ conjugate, Thiadicarbocyamne (DiSC3), Thiazme Red R, Thiazole Orange, Thio flavin 5, Thio flavin S, Thioflavin TON, Thiolyte, Thiozole Orange, Tmopol CBS (Calcofluor White), TIER, TO-PRO 1, TO PRO 3 TO PRO-5, TOTO 1, TOTO- 3, TπColor (PE-Cy5), TRITC TetramethylRodammelsoThioCyanate, True Blue, Tru Red, Ultrahte, Uramne B, Uvitex SFC, wt GFP, WW 781, X-Rhodamine XRITC, Xylene Orange, Y66F Y66H, Y66W, Yellow GFP YFP, YO PRO-I YO- PRO 3, YOYO-1,YOYO 3 Sybr Green, Thiazolc orange (mtcrchelatmg dyes), semiconductor nanoparticles such as quantum dots, or caged fluorophore (which can be activated with light or other electromagnetic energy source) or a combination thereof
112 The moiety can be a nanoparticle, such as a heat generating nanoshell As used herein, "nanoshell" is a nanoparticle having a discrete dielectric or semi-conducting core section surrounded by one or more conducting shell layers U S Patent No 6,530,944 is hereby incorporated by reference herein in its entirety for its teaching of the methods of making and using metal nanoshells Nanoshells can be formed with a core of a dielectric or inert mateπal such as silicon, coated with a mateπal such as a highly conductive metal which can be excited using radiation such as near infrared light (approximately 800 to 1300 nm) Upon excitation, the nanoshells emit heat The resulting hyperthermia can kill the surrounding cell(s) or tissue The combined diameter of the shell and core of the nanoshells ranges from the tens to the hundreds of nanometers Near infrared light is advantageous for its ability to penetrate tissue Other types ot radiation can also be used, depending on the selection of the nanoparticle coating and targeted cells Examples include x-rays, magnetic fields, electπc fields, and ultrasound The particles can also be used to enhance imaging, especially using infrared diffuse photon imaging methods Targeting molecules can be antibodies or fragments thereof, ligands for specific receptors, or other proteins specifically binding to the surface of the cells to be targeted
113 The moiety can be covalently linked to the disclosed peptide The moiety can be linked to the amino terminal end of the disclosed peptide The moiety can be linked to the carboxy terminal end of the disclosed peptide The moiety can he linked to an amino acid within the disclosed peptide The herein provided conjugates can further compose a linker connecting the moiety and disclosed peptide The disclosed peptide can also be conjugated to a coating molecule such as bovme serum albumin (BSΛ) (see Tkachenko et al , (2003) J Am Chem Soc 125, 4700-4701) that can be used to coat the Nanoshells with the peptide
114 Protein ciosshnkers that can be used to crosslink the moiety to the disclosed peptide are known m the art and are defined based on utility and structure and include DSS (Disuccimmidylsuberate), DSP (Dithiobisfsuccimmidylpropionate)), DTSSP (3,3 -Dithiobis (sulfosuccuiimidylpropionate)), SULFO BSOCOFS (Bisp-^ulfosucαrumdooxycarbonjloxy) ethyl]sulfone), BSOCOES (Bis[2-(succimmdooxycarbonyloxy)ethyl]sulfone) SULFO DST (Disulfosuccimmdyltartrate), DST (Disuccmimdyltartrate), SULFO EGS (Ethylene glycolbis(succinimidylsuccmate)), EGS (Ethylene glycolbis(sulfosuccmmudylsuccinate)) DPDPB (1,2 Di[3' (2' pyridyldithio) propionamido]butane), BSSS (Bis(sulfosuccinimdyl) suberate), SMPB (Succinimdyl-4-(p-maleimidophenyl) butyrate), SULFO SMPB
(Sultosuccinimdyl-4-(p-malcirmdophcnyl) butyrate), MBS (3 Maleimidobenzoyl-N- hydroxysuccimmide ester), SULFO MBS (3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester) STAB (N-Succinιmιdyl(4-iodoacetyl) ammobenzoate), SULFO SIAB (N- Sulfosuccinmiidyl(4 iodoacetyl)ammobenzoate), SMCC (Succiniimdyl-4 (N maleimidomethyl) cyclohexane 1 carboxylate), SULFO SMCC (Sulfosucunimidyl 4 (N-maleimidomethyl) cyclohexane 1 carboxylate), NHS LC SPDP (Succinimidyl-6 [3 (2 pyridyldithio) propionamido) hexanoate), SULFO NHS LC SPDP (Sulfosuccimmidyl-6-[3-(2-pyπdyldithio) propionamido) hexanoate), SPDP (N-Succininidyl-3-(2 pyridyldithio) propionate), NHS BROMOACETATE (N-Hydroxysuccimmidylbromoacetate), NHS IODOACETATE (N- Hydroxysuccinimidjhodoacetate), MPBH (4-(N-Maleimidophenyl) butyric acid hydrazide hydrochloride), MCCII (4-(N-Maleimidomethyl) cyclohexane- 1-carboxyhc acid hydrazide hydrochloride), MBH (m Maleimidobeαzoic acid hydrazidehydrochloπde), SULFO EMCS (N (epsilon Maleimidocaproyloxy) sulfosuccimmide), EMCS (N-fepsilon-Maleimidocaproyloxy) succimmide), PMPl (N (p Maleimidophenyl) isocyanate) KMUH (N (kappa
Maletmidoundecanoic acid) hydrazide) LC SMCC (Succinimidyl 4-(N-malermidomethyl)- cyclohexane-l-carboxy(6-amidocaproate)), SULFO GMBS (N (gamma-Maleimidobutryloxy) sulfosuccimmide ester), SMPH (Succπuimdyl-δ-^eta-maleimidopropionamidohexanoate)), SULFO KMUS (N-(kappa-Maleimidoundecanoyloxy)sulfosuccinimide ester), GMBS (N (gamma Maleimidobutyrloxy) succimmide), DMP (Drmethylpimelimidate hydrochloride), DMS (Dimethylsubenmidate hydrochloride), MHBH(Wood s Reagent) (Methyl p hydroxybenzimidate hydrochloride, 98%) DMA (Dimethyladipimidate hydrochloride)
115 The moiety of the disclosed conjugate can be a cellular internalization transporter or sequence The cellular internalization sequence can be any internalization sequence known or newly discovered in the art, or conservative variants thereof Non limiting examples of cellular internalization transporters and sequences include Antennapedia sequences, TAT, HIV-Tat, Penetratm, Antp-3A (Antp mutant), Buform II, Transpoi tan, MAP (model amphipathic peptide) K-FGF, KuVO, Pπon, pVEC, Pep-1, SynBl, Pep-7, HN-I, BGSC (Bis Guamdinium-Spermidme- Cholesterol, and BGTC (Bis-Guamdimum-Tren-Cholesterol) (see Table 1)
Table 1. Cell Internalization Transporters
Name Sequence SEQ ID NO
Antp RQPKTWFPNRRSPWKK (SEQ ID NO 10)
HIV-Tat GRKKRRORPPQ (SEQ ID NO I l)
Penetiatrn RQIKIWFQNRRMKWKK (SEQ ID NO 12)
Antp 3A RQIAIWFQNRRMKWAA (SEQ ID NO 13)
Tat RKKRRQRRR (SEQ ID NO 14)
Bufoπn π TRSSRAGLQFPVGRVHHLLRK (SEQ ID NO 15)
Transportan GWTLNSAGYLLGKINKALAALAKKIL (SEQ ID NO 16) model amphipathic KLALKLALKALKAALKLA (SEQ ID NO 17) peptide (MAP)
K-FGF AAVALLPΛVLLALLAP (SEQ ID NO 18)
Ku70 VPMLK PMLKE (SEQ ID NO 19)
Pπon MANLGYWLLALFVTMWTDVGLCKKR (SEQ ID NO 20)
PKP
pVEC LLIILRRRIRKQAHAHSK (SEQ ID NO 21)
Pep-1 KETWWETWWTEWbQPKKKRKV (SEQ ID NO 22)
SynBl RGGRLSYSRRRFSTSTGR (SEQ ID NO 23)
Pep 7 SDLWEMMMVSLACQY (SEQ ID NO 24)
HN-I TSPLNIHNGQKL (SFQ TD NO 25)
Figure imgf000030_0001
116 Thus, the provided polypeptide can further comprise the ammo acid sequence SEQ ID NO 10, SEQ ID NO 11 (Bucci, M et al 2000 Nat Med 6, 1362-1367), SEQ ID NO 12 (Deiossi, D , et al 1994 Biol Chem 269, 10444-10450), SEQ ID NO 13 (Fischer, P M et al 2000 J Pept Res 55, 163 172), SEQ ID NO 14 (Frankel, A D & Pabo, C O 1988 Cell 55,1189 1193, Green, M & Loewenstem, P M 1988 Cell 55, 1179-1188), SEQ ID NO 15 (Park, C B , et al 2000 Proc Natl Acad Sci USA 97, 8245-8250), SEQ ID NO 16 (Pooga, M , et al 1998 FASEB J 12, 67-77), SEQ lD NO 17 (Oehlke, J et al 1998 Bioclum Biophys Acta 1414, 127-H9), SEQ ID NO 18 (Lin, Y Z et al 1995 J Biol Chem 270 14255- 14258), SEQ ID NO 19 (Sawada M , et al 2003 Nature Cell Biol 5, 352 357), SEQ ID NO 20 (Lundberg, P et al 2002 Biochem Biophys Res Commun 299, 85-90), SEQ ID NO 21 (Elmquist, A , et al 2001 Exp Cell Res 269, 237-244) SEQ ID NO 22 (Moms, M C et al
2001 Nature Biotechnol 19, 1173-1176), SEQ ID NO 23 (Rousselle, C et al 2000 MoI Pharmacol 57 679-686), SEQ IDNO 24 (Gdo, C et al 2002 Bioorg Med Chem 10, 4057- 4065), or SEQ ID NO 25 (Hong, F D & Clayman G L 2000 Cancer Res 60, 6551-6556) The provided polypeptide can further comprise BGSC (Bis-Guamdmium-Speπmdine- Cholesteiol) oi BGTC (Bis-Guanidnuum-Tren Cholesterol) (Vigneron, J P et al 1998 Pioc Natl Acad Sci USA 93, 9682 9686) The preceding references are hereby incorporated herein by reference in their entirety for the teachings of cellular internalization vectors and sequences Any other internalization sequences now known or later identified can be combined w ith a polypeptide disclosed herein
6. Polypeptides and Peptides
a) Protein variants
117 Protein variants and derivatives are well understood by those of skill in the art and in can involve amino acid sequence modifications For example, amino acid sequence modifications typically fall into one or more of three classes substitutional, insertional or deletioπal variants Insertions include amino and/oi caiboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues Insertions ordinarily can be smaller insertions than those of ammo or carboxyl terminal fusions, for example, on the order of one to four residues Immunogenic fusion protem derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer imniunogenicity to the target sequence by cross linking in vitro oi by recombinant cell culture tiansformed with DNA encoding the fusion Deletions are characterized by the removal of one or more ammo acid residues from the protem sequence Typically, no more than about from 2 to 6 residues are deleted at any one site within the protem molecule These Λ ariants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis Ammo acid substitutions are typically of single lesidues, but can occur at a number of different locations at once, insertions usually can be on the order of about from 1 to 10 ammo acid residues, and deletions can range about from 1 to 30 residues Deletions or insertions preferably are made in adjacent pairs, i e a deletion of 2 residues or insertion of 2 residues Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a final construct The mutationh must not plate the sequence out of reading frame and pieferably can not cieate complementary regions that could produce secondary mRNA structure Substitutional variants are those in which at least one residue has been removed and A different residue inserted in its place Such substitutions generally are made in accordance with the following Table 2 and are referred to as conservative substitutions
TABLE 2. Amino Acid Substitutions Original Residue Exemplary
Figure imgf000032_0001
Substitutions
others are known in the art
Ala Ser
Arg Lys, GIn
Asn GIn His
Asp GIu
Cys Ser
GIn Asn, Lys
GIu Asp
GIy Pro
His Asn,Gln
lie Leu, VaI
Leu He, VaI
Lys Arg GIn
Met Leu, He
Phe Met, Leu, Tyr
Sei Thr
Thr Ser
Trp Tyr
Tyr Trp Phe
VaI He Leu
118 Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 2, i e , selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone m the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain The substitutions which in general are expected to produce the greatest changes in the protein properties can be those in which (a) a hydrophihc residue, e g seryl or threonyl, is substituted for (or by) a hydrophobic residue, e g leucyl, lsoleucyl, phenylalanyl, valyl or alanyl, (b) a cysteine or proline is substituted foi (01 by) any other residue, (c) a residue having an electropositive side chain, e g , lysyl argmyl, or hishdyl, is substituted for (or by) an electronegative residue, e g , glutamyl or aspartyl, or (d) a residue having a bulky side chain, e g phenylalanine, is substituted for (or by) one not having a side chain, e g , glycine, in this case, (e) by increasing the number of sites for sulfation and/or glycosylation
119 For example, the replacement of one amino acid residue with anothei that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another The substitutions include combinations such as, for example, GIy, Ala, VaI, lie, Leu, Asp, GIu Asn, GIn, Ser, Thr, Lys, Arg, and Phe, Tyr Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein
120 Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosyladon (Asn-X-Thi/Sei) or O-glycosylation (Ser or Thr) Deletions of cysteine or other labile residues also may be desirable Deletions or substitutions of potential proteolysis sites, e g Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutammyl or histidyl residues
121 Certain post-translational denvatizations are the result of the action of recombinant host cells on the expressed polypeptide Glutammyl and asparaginyl residues are frequently post translationally deamidated to the corresponding glutamyl and asparyl residues Alternatively, these residues are deamidated under mildly acidic conditions Other post- translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-ammo groups of lysine, argmuie, and histidine side chains (T E Creighton, Proteins Structure and Molecular Properties, W H Freeman & Co , San Francisco pp 79-86 [1983]), acetylation of the N terminal amine and, in some instances, amidation of the C-termmal caiboxyl
122 Specifically disclosed are variants of these and other polypeptides herein disclosed which have at least, 65%, 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence Those of skill in the art readily understand how to determine the homology of two proteins For example, the homology can be calculated after aligning the two sequences so thai the homology is at its highest level
123 Another way of calculating homology can be performed by published algorithms Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman Adv Appl Math 2 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J MoL Biol 48 443 (1970), by the search for similarity method of Pearson and Lipman, Proc Natl Acad Sci U S A 85 2444 (1988), by computerized implementations oi these algorithms (GAP, BESTFIT, FASTA, and TFASTA m the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr , Madison, WI), or by inspection
124 The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed 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 are herein incorporated by reference for at least material related to nucleic acid alignment
125 It is understood that the description oi conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations
126 As this specification discusses vaπous proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed This would include all degeneiate sequences related to a specific protem sequence, i e all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protem sequences Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protem sequence
127 It is understood that there are numerous ammo acid and peptide analogs which can be incorporated mto the disclosed peptides Foi example, there are numerous D ammo acids or ammo acids which have a different Junctional substituent than the ammo acids shown m Table 2 The opposite stereo isomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs These amino auds can ieadily be incorpoiated into polypeptide chains by charging tRNA molecules with the ammo acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog ammo acid mto a peptide chain in a site specific way (Thorson et al , Methods in Molec Biol 77 43-73 (1991), Zollcr, Current Opinion in Biotechnology, 3 348-354 (1992), Ibba, Biotechnology & Genetic Engineering Reviews 13 197-216 (1995), Cahill et al , TIBS, 14(10) 400-403 (1989), Benner, TIB Tech, 12 158-163 (1994), Ibba and Henneckc, Bio/technology, 12 678 682 (1994) all of which are herein incorporated by reference at least for material related to amino acid analogs)
128 Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage For example, linkages for amino acids or ammo acid analogs can include CH2NH-, -CH2S-, -CH2-CH2 -, -CH=CH- (us and trans), -COCH2 -, - CH(OH)CH2-, and -CHH2SO— (These and others can be found in Spatola, A F m Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B Wemstein, eds , Marcel Dekker, New York, p 267 (1983), Spatola, A F , Vega Data (March 1983), VoI 1, Issue 3, Peptide Backbone Modifications (general review), Morley, Trends Pharm Sci (1980) pp 463-468, Hudson, D et al , Int J Pept Prot Res 14 177-185 (1979) (--CH2NH-, CH2CH2-), Spatola et al Life Su 38 1243-1249 (1986) (-CH H2-S), Hann J Chem Soc Porkm Trans 1307-314 (1982) (-CH-CH-, cis and trans), Almqmst et al J Med Chem 23 1392-1398 (1980) (-COCH2-), Jennings- White et al Tetrahedron Lett 23 2533 (1982) (-COCH2-), Szelke et al European Appln, EP 45665 CA (1982) 97 39405 (1982) (- CH(OH)CH2-), Holladay et al Tetrahedron Lett 24 4401-4404 (1983) (-C(OH)CH2-), and Hruby Life Sci 31 189 199 (1982) (-CH2-S-), each of which is incorporated herein by ieference A particularly preferred non-pcptide linkage is -CH2NH- It is understood that peptide analogs can have more than one atom between the bond atoms, such as b alanine, g-aminobutync acid, and the lrke
129 Ammo acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc ), altered specificity (e g , abroad spectrum of biological activities), reduced antigenicity, and others
130 D-amino acids can be used to generate moie stable peptides, because D ammo acids are not recognized by peptidases and such Systematic substitution of one or more ammo acids of a consensus sequence with a D amino acid of the same type (e g , D-lysme m place of L- Iysine) can be used to generate more stable peptides Cysteine residues can be used to cyclize or attach two or moie peptides together This can be beneficial to constrain peptides into particular conformations (Rizo and Gierasch Ann Rev Biochem 61 387 (1992), incorporated herein by reference)
131 There arc a variety of actions, such as determining homology/identity of nucleic acids or proteins, hybridization, expression, delivery, and pharmaceutical formulations which are applicable for the general and specific compositions disclosed
7. Homology/identity
132 It is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein is through defining the variants and derivatives in terms of homology to specific known sequences Specifically disclosed are variants of these and other genes and proteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 93, 94, 95, 96, 97, 98, 99 percent homology to the stated sequence Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level
133 In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herem, is through defining the variants and derivatives in terms ot homology to specific known sequences This identity of particular sequences disclosed herein is also discussed elsewhere herein In general, variants of genes and proteins herem disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level
134 Another way of calculating homology can be performed by published algorithms Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv Appl Math 2 482 (1981) by the homology alignment algorithm of Needleman and Wunsch, J MoL Biol 48 443 (1970), by the search for similarity method of Pearson and Lipman, Proc Natl Acad Sci U S A 85 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr , Madison, WI), or by inspection
135 The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M Science 244 48-52, 1989, Jaeger et al Proc Natl Acad Sa USA 86 7706 7710, 1989, Jaeger et al Methods Fnzymol 183 281 306, 1989 which aie herein incorporated by reference for at least material related to nucleic acid alignment It is understood that any of the methods typically can he used and that in certain instances the results of these various methods may diffei, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein
136 For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above For example a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods As yet anothei example, a first sequence has 80 peicent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages)
8. Hybridization/selective hybridization
137 The term hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a punier Oi a probe and a gene Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derrv atives in a nucleotide specific manner For example, G interactmg with C or 4 interacting with T are sequence driven mteractions Typically sequence driven interactions occur on the Watson Crick face or Hoogsteen face of the nucleotide The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules can hybridize
138 Parameters for selective hybridization between two nucleic acid molecules are well known to those of skill in the art For example, in some embodiments selective hybridization conditions can be defined as stringent hybridization conditions For example, stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps For example, the conditions of hybridization to achieve selective hybridization may involve hybridization in high iomc strength solution (6X SSC or 6X SSPE) at a temperature that is about 12 25°C below the Tm (the melting temperature at which half of the molecules dissociate from then* hybridization partners) followed by washing at a combmation of temperature and salt concentration chosen so that the washing temperature is about 5°C to 2O0C below the Tm The temperature and salt conditions are readily determined empirically m preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of inteiest and then washed under conditions of different stπngencies Hybridization temperatures are typically higher for DNA-RNA and RNA RNA hybridizations The conditions can be used as described above to achieve stringency, or as is known in the art (Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd Ed , Cold Spring Harbor Laboratory, Cold Spiuig Harbor, New York, 1989, Kunkel et al Methods Enzymol 1987 154 367, 1987 which is herein incorporated by reference for material at least related to hybridization of nucleic auds) A preferable stringent hybridization condition for a DNA DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C Stringency of hybridization and washing, if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for Likewise, stringency of hybridization and washing, if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness ot any area wherein high homology is desired, all as known in the art
139 Another way tu define selective hybridization is by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 7% 76, 77 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-lrmitmg nucleic acid Typically, the non limiting primer is in for example, 10 or 100 or 1000 fold excess This type of assay can be performed at under conditions where both the limiting and non limiting primer are for example, 10 fold or 100 fold or 1000 fold below their kj, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their kd
140 Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the pπmer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the pπmer molecules are extended Preferred conditions also mclude those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation
141 Just as with homology, it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient For example if 80% hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein
142 It is understood that those of skill in the art understand that if a composition or method meets any one of these criteria for determining hybridization either collectn ely or singly it is a composition or method that is disclosed herein
a) Sequences
143 There are a variety of sequences related to the homing peptide and cell penetrating sequence, for example, and other disclosed genes, these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein
144 It is understood that the description related to this sequence is applicable to any sequence disclosed herein unless specifically indicated otherwise Those of skill m the art understand how to lesolve sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences
9 Deliver j of the compositions to cells
145 There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either m vitro or in vivo These methods and compositions can largely be broken down into two classes viral based delivery systems and non-viral based delivery systems For example, the nucleic acids can be delivered through a number of direct delivery systems suuh db, electroporation hpofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic matenal in cells or earners such as catiomc liposomes Appropriate means for transfection, including viral vectors, chemical transfectants, or physico mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff, J A , et al , Science, 247, 1465 1468, (1990), and Wolff J A Nature, 352, 815-818, (1991)Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein In certain cases, the methods can he modified to specifically function with large DNA molecules Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the earner
10. Pharmaceutical carriers/Delivery of pharmaceutical products
146 As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier By "pharmaceutically acceptable ' is meant a material that is not biologically or otherwise undesirable i e , the material may bo administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or intei acting in a deleterious manner with any of the other components of the phaimaceutical composition m which it is contained The earner would naturally be selected to minimize any degradation of the actπ e ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art
147 The compositions may be administered orally, parenterally (e g , intravenously), by intramuscular injection, by intrapeiitoneal injection transdermal^, extra corporeally, topically or the like, although topical intranasal administration or administration by inhalant is typically preferred As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one oi both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosohzation of the nucleic acid or vector The latter may be effective when a large number of animals is Io be treated simultaneously Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism Delivery can also be directly to any area of the respiratory system (e g , lungs) via intubation The exact amount of the compositions required can vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like 1 hus, it is not possible to specify an exact amount for e\ery composition However, an appropπate amount can be determined by one of ordinary skill in the art using only routine expeπmentation given the teachings herein
148 Parenteral administration of the composition, if used, is generally characterized by injection Injectables can be prepared in conventional forms either as> liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained See, e g
U S Patent No 3,610,795, which is incorporated by reference herein
149 The materials may be m solution or suspension (for example, incorporated into microparticlcs, liposomes, or cells) These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al , Bioconiugate Chem . 2 447 451, (1991), Bagshawe, K D , Br J Cancer. 60 275-281, (1989), Bagshawe, et al , Br J Cancer. 58 700-703, (1988), Senter, et al , Bioiom iigate Chem , 4 3-9, (1993), Battelli, et <ιl , Cancer Immunol Immunother . 35 421-425, (1992), Pietersz and McKcnzic. Immunolog Reviews. 129 57-80, (1992), and Roffler, et al , Biochem Pharmacol. 42 2062 2065, (1991)) Vehicles such as 'stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma) receptor mediated targetmg of DNA through cell specific ligands, lymphocyte directed tumor targetmg, and highly specific therapeutic retroviral taigetmg of murine glioma cells m vivo The following references arc examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al , Cancer Research. 49 6214- 6220, (1989), and Litzmger and Hαang, Biochimica et Biophvsica Acta. 1 104 179-187, (1992)) In general, receptors are involved m pathways of endocytosis, either constitutive or hgand induced These receptors cluster m clathπn-coated pits, enter the cell via clathrrn-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated protems, clearance of macromolecules, opportunistic entry of viruses and toxms, dissociation and degradation of hgand, and receptor-level regulation Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of hgand, hgand valency, and hgand concentration Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Blown and Greene, DNA and Cell Biology 10 6, 399 409 (1991))
a) Pharmaceutically Acceptable Carriers
150 The compositions, including antibodies, can be used therapeutically m combination with a pharmaceutically acceptable earner
151 Pharmaceutical carriers are known to those skilled in the art These most typically would be standard earners for administration of drugs to humans, including solutions such as sterile water, salme, and buffered solutions at physiological pH The compositions can be administered intramuscularly or subcutaneously Other compounds can be administered according to standard procedures used by those skilled in the art
152 Pharmaceutical compositions may include carneis, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice
Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like
153 The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated Administration may be topically (including ophthalmically, vaginally, rectally lntranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous intraperitoneal or intramuscular injection The disclosed antibodies can be administered intravenously, intraperitoneal^, intramuscularly, subcutaneously, mtracavity, or transdermally
154 Preparations for parenteral administration include steπle 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 Aqueous earners include water, alcoholic/aqueous solutions, emulsions or suspensions, including salme and buffered media Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils Intravenous vehicles include fluid and nutrient replenishers, electrolyte replemshers (such as those based on Ringer's dextrose), and the like Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents and inert gases and the like
155 Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositoπes, sprays, liquids and powders Comentional pharmaceutical carriers, aqueous, pow der or oily bases, thickeners and the like may be necessary or desirable
156 Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets Thickeners, flavorings diluents, emulsifiers, dispersing aids or binders may be desirable
157 Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic auds such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fuinaπc acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tπalkyl and aryl amines and substituted ethanolamines
b) Therapeutic Uses
158 The dosage ranges for the administration of the compositions are those large enough to produce the desired ctfect in which the symptoms disorder are effected I he dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic leactions, and the like Generally, the dosage can vary with the age, condition, sex and extent of the disease m the patient and can be determined by one of skill in the art The dosage can be adjusted by the individual physician m the event of any counteπndications Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days
11. Sequence Similarities
159 It is understood that as discussed herein the use of the terms homology and identity mean the same thing as similarity Thus, for example, if the use of lhe word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences Many of the methods for determining homology between two evolutionanly related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionanly related or not
160 In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences This identity of particular sequences disclosed herein is also discussed elsewhere herem hi general, variants of genes and proteins herein disclosed typically have at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, SO, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes For
example, the homology can be calculated after aligning the two sequences* so that the homology is at its highest level
161 Another way of calculating homology can be performed by published algorithms Optimal alignment of sequences foi comparison can be conducted by the local homology algorithm of Smith and Waterman Λdv Λppl Math 2 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J MoL Biol 48 443 (1970), by the search for similarity method of Pearson and Lipman, Proc Natl Acad Sci U S A 85 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr , Madison, WI), or by inspection
162 The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed 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 are herein incorporated by reference for at least material related to nucleic acid alignment It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein
163 For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above For example, a first sequence has 80 pei cent homology, as defined heiem, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does, not have 80 percent homology to the second sequence ds calculated by any of the other calculation methods As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Nccdlcman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods As yet another example, a first sequence has 80 percent homology, as defined herein to a second sequence if the first sequence is calculated to have 80 peicent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods can often result in different calculated homology percentages) B. Methods
1. Targeting
164 Provided herein is a method of targeting one or more moieties, to different regions, breast tissue for example, in a subject The method can involve administering to the subject a conjugate comprising any one or more of the herein disclosed peptides and the one or more moieties The one oi more moieties can be detection moieties, such as those disclosed herein Ihus, the method can further comprise detecting cancer m the subject by detecting the presence of the conjugate in the subject's cells
165 The detection moiety can be a fluorophore, such as TAMRA, DANSYL, a cyanme dye, or a cyanrne dye encapsulated in a cyclodextπn
166 The one or more moieties can be homing peptides, such as those disclosed herein The homing peptide can be breast tissue-specific, such as CPGPEGΛGC
167 The one or more moieties can be cell-penetrating peptides, such as those disclosed herein The cell-penetrating peptide allows the composition to enter the cell, such as Tat
168 The one or more moieties can be a contrasting agent, such as those disclosed herein The contrasting agent can be an MRI contrasting agent The MRI contrast agent can be non-metallic, such as a mtroxide radical or deroative thereof
169 The one or more moieties can be therapeutic moieties, such as those disclosed herein Thus, wherein the subject has cancer, targeting of the moiety to the cancer cells of the subject can provide a therapeutic effect in the subject The therapeutic moiety can be an anticancer therapeutic such as the chemotherapeutic agent, bleomycin
170 The composition disclosed herein can be non-toxic
2. Detecting
171 Also provided is a method of detecting cancer, such as a dual modality detection method The method can involve administering a composition composing a detectable moiety and a contrasting agent to the subject, performing MRI on the subject, recording the output from
the MRI and the flouiescence imaging and comparing the output of the MRI and the output of the flouresoence imaging to a control
172 In one embodiment, the presence of cancer can be detected by the presence of a kinetic decay m the contrasting agent followed by the emergence of a fluorescent signal For cancer cells the MRI contrast can kmetically decay on the order of a 1 to 8 minute tune interval followed by concomitant emergence of a fluoiescent signal However, for normal cells, the MRI contrast can continue and the delayed fluorescence response can be realized Because these agents are not specific to cancer, but specific to breast tissue, administration of the agent to a patient can result jn enhanced MRI signal from the entire breast However, regions that arc cancerous can have reduced MRI signal compared to the other regions of the breast These regions can also result in a fluorescent signal whereas non-cancerous regions of the bteast can have minimal to no fluorescent signal
173 In one embodiment the disclosed method can detect breast cancer
174 In one embodiment, the disclosed method compπses a contrast agent linked to a homing peptide In one embodiment of the disclosed method, the homing peptide can be breast tissue specific In one embodiment, the disclosed method comprises the breast tissue-specific homing peptide CPGPEGAGC
175 Pn one embodiment, the disclosed method comprises a non-metallic contrast agent In one embodiment, the non-metallic contrast agent can be a mtroxide radical or deπvativc thereof
176 In one embodiment the method comprises a detection moiety In one embodiment, the detection moiety can be a fluorophore hi one embodiment, the fluorophore can be DANSYL, TAMRA, a cyanme dye, or a cjanine dye encapsulated in a cyclodextrin
177 hi one embodiment, the disclosed method compπses a peptide-based dual modality detection system
3. Treating
178 Also provided is a method of treating cancer in a subject comprising administering to the subject a composition comprising a detection moiety, a contrasting agent, a homing peptide, a cell-penetiating molecule and a therapeutic agent
179 In one embodiment, the method of treating cancer in a subject further comprises detecting cancer in a subject 180 In one embodiment, the method of treating cancel m a subject fiirthei comprising detecting cancer in a subject comprises a dual modality detection method of detecting cancer in a subjecting comprising administering a composition to the subject, performing Magnetic Resonance Imaging (MRI) on the subject, performing flourescence imaging on the subject, recording the output from the MRI and the flourescence imaging, and comparing the output of the MRI and the output of the flourescence imaging to a control
181 In one embodiment, the method of treating cancer in a subject further comprising detecting cancer in a subject comprises a composition comprising a breast tissue specific homing peptide wherein the homing peptide can be CPGPEGAGC
182 In one embodiment, the method of treating cancer m a subject further comprising detecting cancer in a subject comprises a composition comprising a fluorophore as the detection moiety In one embodiment of the disclosed method, the fluorophore can be Dansyl, TAMRA, a cyanine dye, or a cyanine dye encapsulated in a cyclodextπn
183 In one embodiment, the method of treating cancer in a subject further comprising detecting cancer in a subject comprises a composition comprising a non metallic MRI contrasting agent In one embodiment, the non-metallic contrasting agent is a mtroxide iadical oi derivative thereof
184 In one embodiment, the method of treating cancer m a subject further composing detecting cancer in a subject composes a composition comprising an anti-cancer theiapeutic agent In one embodiment, the anti-cancer therapeutic agent can be bleomycin
185 ha one embodiment the method of treating cancer m a subj ect further comprising detecting cancer in a subject comprises a composition comprising Tat as the cell-penetrating molecule
186 In one embodiment, the disclosed method of treating cancer in a subject uses a non-toxic composition
187 The disclosed compositions can be used to treat any disease where uncontrolled cellulai prolifeiation occurs such as cancers A non-lirmtmg list of different types of cancers can be as follows lymphomas (Hodgkrns and non Hodgkms), leukemias, carcinomas, carcinomas of solid tissues, squamous cell carcinomas, adenocarcinomas, saicomas, gliomas, high giade gliomas, blastemas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas,
hypoxic tumors, myelomas, AIDS-related lymphomas or sarcomas, metastatic cancers, or cancers in general
188 A representative but non-limitmg list of cancers that the disclosed compositions can be used to treat is the following lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkm'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 cancels such as small cell lung cancer and non-small cell lung cancer,
neuroblastoma/glioblastoma, ovaπan cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers, testicular cancer, colon and rectal cancers, prostatic cancer, or pancreatic cancer
4 Administration
189 A composition disclosed herein, such as the disclosed peptides and conjugates, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to he treated For example, the compositions may be admimsteied orally, parenterally (e g , intravenous, subcutaneous, intraperitoneal, or intramuscular injection), , by inhalation, extracorporeally, topically (including transdermally, ophthalmically, vaginally, rectally, tntranasally) or the like
190 As used herein, ' topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosohzation of the nucleic acid or vector Administration of the compositions by inhalant can be through the nose oi mouth via delivery by a spraying or droplet mechanism Delivery can also be directly to any area of the respiratory system (e g , lungs) via intubation
191 Parenteral administration of the composition, if used, is generally characteπzed by injection Injectables can be prepared m conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions A more recently revised approach for parenteral administration rnvoh es use of a
slow release or sustained release system such that a constant dosage is maintained See, e g , U S Patent No 3,610,795, which is incorporated by reference herein
192 The exact amount of the compositions required can vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like Thus, it is not possible to specify an exact amount for every composition However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein Thus, effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art Useful dosage ranges lor the administration of the compositions are those large enough to produce the desired effect The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like Generally, the dosage can vary with the age, condition, sex and extent of the disease m the patient, route of administration, or whether other drugs are included in the regimen, and call be deteimmed by one of skill in the art The dosage can be adjusted by the individual physician in the event of any counter indications Dosage can vary, and can be admmistered in one or more dose administrations daily, for one or several days Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products
193 For example, a typical daily dosage of the disclosed peptides used alone might iange from about 1 μg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above
194 Follow mg administration of a disclosed composition, the efficacy of a therapeutic moiety can be assessed in various ways well known to the skilled practitioner For instance, one of ordinary skill m the art will understand that a composition disclosed herem is efficacious in treating or inhibiting cancer in a subject by observing that the composition reduces tumor growth or prevents a further increase in lymphangiogenesis
C. Kits
195 The materials described above as well as other materials can be packaged together m any suitable combination as a kit useful for performing or aiding m the perforαidnce of, the disclosed method It is useful if the kit components in a given kit are designed and adapted for use together m the disclosed method For example disclosed are kits for administering compositions, such as those disclosed herein, the kit comprising a composition and a means tor administering the composition to a subject The kits also can contain protocols for administering the compositions
196 Disclosed is a kit comprising a composition, comprising a detection moiety and contrastmg agent, and articles for delivery to a subject
D. Uses
197 The disclosed compositions can be used in a variety of ways as research tools Other uses are disclosed, apparent from the disclosure, and/or will be understood by those in the art
E. Methods of making the compositions
198 The compositions disclosed heiein and the compositions necessary to perform the disclosed methods can be made using any method known to ttiose of skill in the art for that particular reagent or compound unless otherwise specifically noted
1. Nucleic acid synthesis
199 For example, the nucleic acids, such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be pioduced using enzymatic methods or any other known method Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spπng Harbor, N Y , 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of MiHigen-Biosearch, Burlington, MA or ABI Model 380B) Synthetic methods useful for making oligonucleotides are also described by Ikuta et al , Ann Rev Biochem 53 323-356 (1984), (phosphotπester and phosphite-triester methods), and Narang et al , Methods Enzymol , 65 610 620 ( 1980), (phosphotπester method) Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et al , Bioconjug Chem 5 3 7 (1994)
2. Peptide synthesis
200 One method of producing the disclosed proteins is to link tw o or more peptides or polypeptides together by protein chemistry techniques For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fnioc (9-fluorenylmetliyloxycarbonjl) 01 Boc (tert -butyloxycarbonoyl) chemistry (Applied Biosystcms, Inc Foster City, CΛ) One skilled m the art can readily appreciate that a peptide or polypeptide corresponding to the disclosed proteins, for example, can be synthesized by Standard chemical reactions For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the lesm, thereby exposing a terminal group which is functionally blocked on the other fragment By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof (Grant GA (1992) Synthetic Peptides A User Guide W H Freeman and Co , N Y (1992), Bodansky M and Trost B , Ed (1993) Principles of Peptide Synthesis Springer Verlag Inc , NY (which is herein incorporated by reference at least for material related to peptide synthesis) Alternatively, the peptide or polypeptide is independently synthesized in vivo as described herein Once isolated, these independent peptides or polypeptides may be linked to form a peptide or fragment thereof via similar peptide condensation reactions
201 For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (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 the chenioselecttve reaction of an unprotected synthetic peptide— thioester with another unprotected peptide segment containing an ammo-terminal Cys residue to give a thioester-linked mtermediate as the initial covalent product Without a change m the reaction conditions, this intermediate undergoes spontaneous, 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 I et al , Biochemistry, 30 3128 (1991), Rajarathnam K et al , Biochemistτy 33 6623-30 (1994))
202 Alternatively, unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al Science, 256 221 (1992)) This technique has been used to synthesize analogs of protein domains as w e)1 as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al , 1 echmques in Protein Chemistry IV Academic Press, New York, pp 257 267 (1992))
F. Machines, Apparati, and Systems
203 Disclosed herein are machines, apparati, and systems, which are designed to perform the various methods disclosed herein It is understood that these can be multipurpose machines having modules and/or components dedicated to the performance of the disclosed methods For example, a whole body imaging system, such as a MRI, can be modified as descπbed herein so that it contains a module and/or component which for example, a) produces a tissue specific record, which identifies the decay of contrasting agents, identifies one or more detection moieties, creates a set of data, and/or performs a dual modality analysis, such as a dual modality analysis alone or m any combination hi particular, the modules and components within the imaging system responsible for determining the presence of cancer, can be linked to the modules and/or components responsible for identifying and/or manipulating tissue-specific data sets In certain embodiments the presence of cancer can be determined by the moment the decay of the contrasting agent starts and quenching of the fluorophore stops
204 Thus, the methods and systems herein can have the data, in any form uploaded by a person operating a device capable of performing the methods disclosed herein The methods can also be associated with the whole body imaging system as desciibed herein, either incorporated into these systems or being on device which is connected to them
205 Disclosed herein is a method of detecting cancer in ά subject wherein the method is a computer implemented method
206 In one embodiment, the method further comprises the step of outputting results from the dual modality detection
207 Disclosed herein is a method of analyzing a subject comprising, receiving a tissue- specific record of the subject, wherein the record contains the kinetic decay of the contrasting agent, measuring the amount of decay and the amount of fluorescence, and outputting results from the dual modality detection
208 In one embodiment the method of analyzmg a subject comprises a computer implemented method
209 In one embodiment, the method of analyzing a subj cct composes receiving the tissue-specific record wherein the tissue specific record can be from a storage medium
210 In one embodiment, the method of analyzing a subject comprises receiving the tissue specific record wherein the tissue-specific record can be from a computer system
211 In one embodiment the method of analyzing a subj ect comprises receiving the tissue-specific record wherem the tissue-specific iecord can be from a whole body imaging system
212 hi one embodiment, the method of analyzing a subject comprises receiving the tissue specific record wherem the tissue-specific record can be via a computer netwoik
213 Disclosed herein is a method of analyzing the presence of cancer m a subject comprising, recommending the performance of receiving a tissue-specific record of the subject, wherem the record contains the kinetic decay of the contrasting agent, measuring the amount of decay and the amount of fluorescence, and outputtiπg results from the dual modality detection
214 In one embodiment, the disclosed method comprises the steps of receiving an output from any of the disclosed methods of analyzing and recommending treatment by adminstermg a composition comprising a detection moiety, a contrastmg agent, a homing peptide, a cell-penetrating peptide and a therapeutic agent
215 Disclosed herein is one or more computer readable media storing program codes thdt, upon execution by one or more computer systems, causes the computer systems to perform any of the disclosed methods
216 Disclosed herein is a computer program product compiising a computer usable memory adapted to be executed to implement any of the disclosed methods
217 hi one embodiment, the computer program disclosed above, compπses a logic processing module, a configuration file piocessing module, a data organization module, and data display organization module, that are embodied upon a computer readable medium
218 Disclosed herein is a computer program product, comprising a computer usable medium having a computer readable program code embodied therein, said computer readable program code adapted to be executed to implement a method for generating the dual modality detection of any of the previously disclosed methods, said method further composing providing a system, wherein the system compπses distinct software modules, and wherem the distinct
softwaie modules comprise a logic processing module, a configuration file piocessmg module, a data organization module, and a data display organization module
219 In one embodiment, the computer program product further comprises a computeri7ed system configuied foi performing the method
220 In one embodiment, the computer program product further comprises the outputting of the results from the dual modality detection
221 Disclosed heiein is a computer-readable medium having stored thereon instructions that, when executed on a programmed processor perform any of the disclosed methods
222 Disclosed herein is a dual modality detection system, the system comprising a data store capable of storing tissue specific data, a system processor composing one or more processing elements, the one or more processing elements programmed or adapted to receive tissue-specific data comprising the kinetic decay of the contrasting agent and the presence of fluorescence, store the tissue-specific data in the data store, compare the reduction m the contrast agent to the increase in fluorescence, and output a treatment recommendation based upon the comparison of the decay in contrasting enhancement with the mcreased fluorescence
223 In one embodiment, the dual modality detection system receives the tissue-specific data fiom a computer system
224 In one embodiment, the dual modality detection system receives the tissue-specific data via a computer network
225 In one embodiment, the dual modality detection system further comprises a whole body imaging system
1. Systems, machines, and computer readable medium
226 In addition, or instead, the functionality and approaches discussed above, or portions thereof, can be embodied m instructions executable by a computer, where such instructions are stored in and/or on one or more computer readable storage media Such media can include primary storage and/or secondary storage integrated with and/or within the computer such as RAM and/oi a magnetic disk, and/or separable from the computer such as on a solid state device or removable magnetic or optical disk The media can use any technology as would be known to those skilled m the art, including, without limitation, ROM, RAM, magnetic, optical, paper, and/or solid state media technology
227 G Definitions
1. "a", "an", and "the"
228 As used m the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise Thus, tor example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like
2. Optional or optionally
229 The subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not
3. control or control levels or control cells
230 The standard by which a change is measured, for example, the controls are not subjected to the experiment, but are instead subjected to a defined set of parameters, or the controls are based on pre- or post treatment levels They can either be run in parallel with or before or after a test run, or they can be a pre-determined standard For example, a control can refer to the results from an expeπment in whioh the subjects or objects or reagents etc are treated as ui a parallel expeπment except for omission of the procedure or agent or variable etc under test and which is used as a standard of comparison in judging experimental effects Thus, the control can be used to determine the effects related to the procedure or agent or variable etc For example, if the effect of a test compound on a cell was in question, one could a) simply record the characteristics of the cell in the presence of the compound, b) pel form a and tben also record the effects of adding a control compound with a known activity or lack of activity and then compare effects of the test compound to the control compound In certain circumstances once a conti ol is performed the control can be used as a standard, m which the control expeπment does not have to be performed again and m other circumstances the control expeπement should be run in parallel each time a comparison can be made
4. targeting or homing
231 The preferential movement, binding and/or accumulation of a targeted compound or composition, such as the disclosed compositions, at a site or a location as compared to a non- targeted compound or composition For example, in the context of in vivo administration to a subject, "targeting" or "homing" can refer to the preferential movement, binding, and/or accumulation of a compound or composition, such as the disclosed compositions, in or at, for example, target tissue, target cells, and/or target structures as compared to non target tissue, cells and/or structures
5. Molecule
232 As used herein, the terms "molecule" or like terms refers to a hiological or diemicdl entity that exists in the form of a chemical molecule or molecules Many molecules are of the type referred to as organic molecules (compounds containing carbon atoms, among others, connected by covalent bonds), although some molecules do not contain carbon (including simple molecular gases such as molecular oxygen and more complex molecules such as some sulfur- based polymers) The general ternV'molecule' includes numerous descriptive classes or groups of molecules, such as proteins, nucleic acids, carbohydrates, steroids, organic pharmaceuticals, receptors, antibodies, and lipids When appropriate, one or more of these more descriptive terms (many of which, such as "protein," themselves describe overlapping gioups of compounds) can be used herein because of application of the method to a subgroup of molecules, w lthout detracting from the intent to have such compounds be repiesentative of both the general class "molecules"and the named subclass, such as proteins Unless specifically indicated, the word molecule would include the specific compound and salts thereof, such as pharmaceutically acceptable salts
6. subject
233 As used throughout, by a "subject" is meant an individual Thus, the "subject" can include, for example, domesticated animals, such as cats, dogs, etc , livestock (c g , cattle, horses, pigs, sheep, goats, etc ), laboratory animals (e g , mouse, rabbit, rat, guinea pig, etc ) mammals, non-human mammals, primates, non-human pπmates, rodents, birds, reptiles, amphibians, fish, and any other animal The subject can be a mammal such as a pπmate or a human The subject can also be a non-human
7. treating, treat or treatment
234 The medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder hi addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
8. computer readable media, computer program product, processors.
Computer usable memory, computer systems
235. In some embodiments, instructions stored on one or more computer readable media that, when executed by a system processor, cause the system processor to perform the methods described above, and in greater detail below. Further, some embodiments may include systems implementing such methods in hardware and/or software, A typical system may include a system processor comprising one or more processing elements in communication with a system data store (SDS) comprising one or more storage elements. The system processor may be programmed and/or adapted to perform the functionality described herein. The system may include one or more input devices for receiving input from users and/or software applications. The system may include one or more output devices for presenting output to users and/or software applications. In some embodiments, the output devices may include a monitor capable of displaying to a user graphical representation of the described analytic functionality.
236. The described functionality may be supported using a computer including a suitable system processor including one or more processing elements such as a CELERON, PENTIUM, XEON, CORE 2 DUO or CORE 2 QUAD class microprocessor (Intel Corp., Santa Clara, CΛ) or SEMPRON, PHENOM, OPTERON, ATHLON X2 or ATHLON 64 X2 (AMD Corp., Sunnyvale, CA), although other general purpose processors could be used. In some embodiments, the functionality, as further described below, may be distributed across multiple processing elements. The term processing element may refer to (1) a process running on a particular piece, or across particular pieces, of hardware, (2) a particular piece of hardware, or either (1) or (2) as the context allows. Some implementations can include one or more limited special purpose processors such as a digital signal processor (DSP), application specific integrated circuits (ASIC) or a field programmable gate arrays (FPGA). Further, some implementations can use combinations of general purpose and special purpose processors. 237 The envn eminent further includes a system data store (SDS) that could include a variety of primary and secondary storage elements In one preferred implementation the SDS would include registers and RAM as part of the primary storage The primary storage may in some implementations mclude other forms of memory such as cache memory, non-volatile memory (e g , FLASH, ROM, EPROM, etc ), etc The SDS may also include secondary storage including smgle, multiple and/or varied servers and storage elements For example, the SDS may use internal storage devices connected to the system processor In implementations where a single processing element supports all of the functionality a local hard disk dπve may sen e as the secondary storage of the SDS, and a disk operating system executmg on such a single processing element may act as a data server receiving and servicing data requests
238 It will be understood by those skilled in the ait that the different information used in the systems and methods tor respiratory analysis as disclosed herem may be logically or physically segregated within a single device serving as secondary storage for the SDS, multiple related data stores accessible thiough a unified management system, which together serve as the SDS, or multiple independent data stores individually accessible through disparate management systems, which may in some implementations be collectively \ lewed as the SDS The various storage elements that compose the physical architecture of the SDS may be centrally located or distributed across a variety of diverse locations
9. Computer network
239 Λ computer network or like terms are one or more computers in operable communication with each other
10. Computer implemented
240 Computer implemented or like terms refers to one or more steps being actions being performed by a computei, computer system, or computer network
11. Computer program product
241 A computer program product or like terms refers to product which can be implemented and used on a computer, such as software
12. Dual modality analysis
242 A dual modality analysis or like terms is the analysis of two components in a system
13. Obtaining 243 Obtaining as used in the context of data or values, such as tissue-specific data or values refers to acquiπng this data or values It can be acquired by, for example, collection, such as through a machine, such as an MRI machine and system It can also be acquired by downloading or getting data that has already been collected, and for example, stored in a way m which it can be retπeved at a later time
14. Outputting results
244 37 Outputting or like terms means an analytical result after processing data by an algorithm
15. Tissue-specific recoi d
245 Λ tissue-specific record or like terms is any collection of tissue-specific data
16. Tissue-specific data
246 A tissue specific data series or like terms refers to any collection of tissue-specific data
17. therapeutically effective
247 The amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder Such amelioration only requires a reduction or alteration, not necessarily elimination The term "earner' means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids oi facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects m the subject
18. comprise, comprising, comprises
248 Throughout the description and claims of this specification, the woid "comprise" and variations of the word, such as "composing" and 'comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers or steps
19. Cell
249 The term "cell" as used heiem also refers to mdividual cells, cell lines, oi cultures deπved from such cells A "culture' refers to a composition compπsmg isolated cells of the same
or a diffeient type The term co-culture is used to designate when more than one type of cell arc cultured together in the same dish with either full or partial contact with each other
20 Stable
250 When used with respect to pharmaceutical compositions the term ' stable is generally understood in the art as meaning less than a certain amount, usually 10%, loss of the active ingredient undei specified storage conditions for a stated period of time The time required for a composition to be considered stable is relative to the use of each product and is dictated by the commercial practicalities of producing the product, holding it for quality control and inspection, shipping it to a wholesaler or direct to a customer where it is held again in storage before its eventual use Including a safety factor of a few months time, the minimum product life for pharmaceuticals ib usually one year and prefeiably more than 18 months As used herein, the term 'stable" references these market realities and the ability to store and transport the product at readily attainable environmental conditions such as refrigerated conditions, 2°C to 8°C
21 Components
251 It is understood that -wherever the word cancer appears without breast as a modifier, it is understood that breast cancer is also disclosed
252 Disclosed arc the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein These and other materials are disclosed heiein, and it is understood that when combinations, subsets, interactions, groups, etc of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A D is disclosed, then even it each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C- D, C-E, and C-F are considered disclosed Likewise any subset or combination of these is also disclosed Thus, for example, the sub group of Λ-E, B-F, and C-E would be considered disclosed This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be
pel formed with any specific embodiment 01 combination of embodiments of the disclosed methods
22 References
253 Throughout this application, various publications are referenced The disclosures of these publications in their entireties are hereby incorporated by reference mto this application m order to more fully describe lhe state of the art to which this pertains The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon
H. Examples
1. Example 1 Breast tissue-specific cancer theranostics: Therapeutic dual- modality MRI/NIR imaging agents
254 A new smart agent as a thcranostic for the diagnosis and treatment ot breast cancer is descπbed The agent incorporates a dual modality magnetic resonance imaging/near infrared imaging component and a releasable therapeutic component This agent can enable research opportunities in tissue selectivity by interactions with cell surface proteins, imaging by MRI and MR of dynamic chemical e\ ents in cells, and methods to deliver cancer drugs directly to the site of interest Because of the dual modality imaging component and tissue specificity, this system can facilitate in vivo mechanistic studies of these drugs m breast cancer modeling
255 The detection system is a key feature of the pioposed tlieraiiostic, which is designed to dynamically respond to the intracellular chemical environment of cancer cells The detection system is based on three physical properties of paramagnetic rntroxide radicals mtroxide radicals can serve as Tl contrast enhancement agents ior MRI (Keana et al Magn Reson Med 2005, 5 525 536), mtroxide radicals quench fluorescence (Blough et al J Am Chem Soc 1988, 110 1915 1917), and mtroxide radicals are reduced to the dianiagnetic hydroxylamine in cancer cells (Hyodo et al Cancer Res 2006, 66 9921 9928) The reduction of mtroxides to the hydroxylamine constitutes the dynamic chemical event which can be used for detection, because the resulting hydroxylamme is neither MRI active nor able to quench fluorescence Therefore, the detection system reports the piesence of cancer in a patient by displaying a kinetic decay in MRI contrast enhancement followed by the emergence of a fluorescent signal
256 Nitroxide paramagnetic spin labels are derived from secondary amines and exist as stable radicals The Tl lelaxivities of mtroxides result in MRI contrast enhancement Murali and co-workers showed a preferential reduction of mtroxides in tumors compared with normal tissues by MRI (Hyodo ct al Cancer Res 2006 66 9921-9928) This property of mtroxides m is key in the disclosed detection system
257 Tile detection system and theranostic agent can be evaluated in animal models Io verify the ability of the conjugated homing peptide to distribute in the breast tissue Mice containing breast cancer xenografts in the mammary glands can then be treated with this agent to verify dual modality detection of the tumor Imaging can be accomplished using a Bruker 7 Tesla MRI instrument and a CRi Maestro In- Vivo Fluorescence Imaging System These instruments allow for noninvasive whole animal imaging of live mice, desflurane is used for anesthesia during imaging of the mice
258 Fluorescence imaging is a simple and cost effective method to determine the parameters outlined above However, these agents are designed for dual modality imaging and the strategy relies on double confirmation of cancer Therefore, once the half life of the agent is determined by fluorescence imaging, MRI imaging is employed Contrast enhancement is quantified and the kinetics for loss of Tl-weighted contrast enhancement are measured, which is due to reduction of the spin label The loss of MRI contrast correlates with the kinetics for the emerging fluorescence signal
259 MRI inherently gives high resolution at the expense of sensitivity Although MRI allows accurate monitoring of tumor growth independent to the le\el of contrast enhancement, dual confirmation of the presence ot a cancerous chemical environment is best Gadolinium, a very common transition metal used for MRI contrast enhancement, has 7 unpaired electrons in its 4f orbitals giving it a very large magnetic moment The effect of this element on spin-lattice (7\) relaxation rates m tissues results in enhanced T\ weighted images (Weinmann et al Am I Roentgenol 1984, 142 619-624) However, the disclosed agent contains an organic mtroxide radical that contains a single unpaired electron
260 Disclosed herein is a single-agent theranostic that addresses detection, diagnosis, and therapy of breast cancer Also disclosed is the use of fluorescence imaguig as a new modality for detecting breast cancer that can be coupled with MRI for dual confirmation of the presence of bi east cancer A ti eatment option is also disclosed that utilizes the same detection system for detecting the cancer, which allows monitoring the distribution and efficacy ol the therapy And, this system does not employ ionizing radiation or toxic metals, which are current problems faced with mammography and MRI imaging agents, respectively Also disclosed is a simple peptide platform that can be quickly adapted for the implementation of new and emerging technologies
261 The disclosed methods and compositions invoh e the design of 'intelligent agents that can discriminate between tissue types and are activated by the chemical environment that is exclusive to cancer These agents are also valuable tools for biologists and enable them to visualize biochemical e\ ents by MRI and fluorescence imaging In particular the dynamics of cell penetration can be directly observed with these agents A spm radical is incorporated in the agent, which has the dual role of quenching fluorescence and enhancing Tl-weighted contrast by MRI This agent enables physicists to examine energy transfer within the cell that results m reduction of the radical to the anion For example, the energy required by cancer cells to proliferate is derived from an elaborate system of redox reactions that involves formation of NADH NADH participates in the electron transport chain, which eventually results m the production of ATP, the mam energy earner of the cell The agent can be used to monitor redox chemistry m living systems by MRI imaging of the spm-label Tl contrast agent and concomitantly by fluorescent imaging
262 The disclosed methods and compositions, have broad impact on health Currently, strategies to increase success in diagnosis and treatment m the clinic are receiving considerable attention Tins agent can aid in diagnosing a malignant lesion and concurrently report the effectiveness of the drug during treatment of the lesion The use of the agent can result in fewer invasive surgeries for benign lesions due to false-positive detection by mammography, because the agent contams a dual reporting component that can assess the chemical environment that is particular to cancer A reduced toxicity in treating the patient is expected, because the agent is specific to breast tissue, resulting in a higher effective dose at the breast at an overall reduced systemic dosage And, the designed agent allows foi accurate detection of early breast tumors in younger at risk patients, who would normally be poor candidates for mammography, because of the increased density of breast tissue and the hazards associated with X-ray radiation Overall, the versatility of the disclosed methods and compositions is expected to stimulate further research in adapting our technology to other cancers as well as other diseases that have peculiar chemical characteristics
263 The designed agent is used for breast cancer screening Although screening by MRI is currently not economically feasible, fluorescence-imaging equipment is low cost and can easily become standard And, fluorescence imaging does not involve radiation as does mammography, the current standard for breast cancer screening For this system, a positive detection by fluorescence imaging would lequire secondary confirmation by MRI using the same agent In addition, treatment ot breast cancer patients using this technology can allow immediate assessment of drug delivery to the disease site And, the agent readily allows for imaging of the tumor to evaluate the effectiveness of treatment over time
264 The disclosed methods and compositions are the development of a new smart biomateπal, and more specifically a combined therapeutic -diagnostic agent coined as a theranostic The advantages of this type of system include the ability to monitor drug distribution to the disease site and rapidly assess the efficacy of drug treatment in individual patients The agent allows for the optional attachment of a variety of drug entities These advantages constitute a personalized medicine approach for treating disease
265 The disclosed methods and compositions specifically address breds>t cancer, i disease that can have a very promising prognosis if detected early However remaining problems in breast cancer include false positive detection by mammography and MRI resulting in unnecessary invasive surgery, false-negative detection especially m youngei women with denser breast tissue, systemic toxicity oi first line therapies for advanced breast cancer, and early assessment of the effectiveness of a breast cancer treatment
266 The disclosed methods and compositions have a dual modality MRI/NIR imaging agent that incorporates a therapeutic component It is designed to give smart agents that can doubly confirm the presence of breast cancer by dual modality magnetic resonance imaging/near infrared imaging (MRI/NTR) and deliver a therapeutic agent specifically to the cancer Currently, a radiologist can examine X-ray and/or MRI images and assess the presence of cancerous lesions The detection of dynamic chemical processes to confirm the presence ot a cancerous chemical environment are also disclosed herein
267 The disclosed agent contains 5 distinct components as follows 1. A spin-radical Tl contrast component gives MRI image enhancement 2 Λ near infrared (NIR) chromophore can allow for fluorescence imaging 3. A breast homing peptide sequence results in specific distribution of the agent to breast tissue 4 A Tat sequence allows for cell penetiation And in combination with the breast homing sequence, Tat confers tissue/cell type specificity (see preliminary results) 5 A therapeutic agent is covalently attached to the system The peptide- based feature of the proposed agent may impart additional specificity for cancer cells Proteases are more active m cancer cells than normal cells, which are expected to confer specificity for release of (he therapeutic agent to cancer cells (Kobhnslα et al CIm Chim Acta 2000, 291 113- 135) Additionally, the only by-products of our agent other than the therapeutic moiety are simple ammo acids
268 The key to the dynamic detection component of this system is the nature of the covalent grouping of the MRI and NlR contrast agents Briefly, the spin label of the MRI contrast component quenches the fluorescent signal from the diromophore (Green et al J Am Chem Soc 1990, 112 7337 7346) However, the stability of the spm-labcl is sensitive to the redox environment of the cancer cell and undergoes chemical reduction, which can be measured by MRI (Hyodo et al Cancer Res 2006, 66 9921 992R) Theiefore, as the MRI contrast is lost by chemical reduction of the spin-label, a concomitant fluorescent signal is formed The redox chemistry of cancer cells is more reducing than m normal cells Therefore, a kinetic loss of contrast in the MRI with concomitant contrast enhancement in fluorescence m cells is exhibit a cancerous chemical environment
269 The disclosed methods and compositions are a multi-faceted approach to breast cancer detection and treatment using a new smart material that combines diagnosis and therapy in a single agent Within the theranostics paradigm, the disclosed methods and compositions include the optional attachment of a variety of drag entities, w hich ultimately leads toward personalized medicine
270 Currently, existing challenges and barrieis to the field of theranostics include the design and syntheses of multi faceted agents The disclosed peptide based template can he used to incorporate various imaging agents and therapeutics In addition, this system is amenable to solid-phase peptide synthesis which can easily be automated Because of the simplicity of this system, a number of new theranostics are possible Our initial entry can consist of a redox- sensitive MRVNIR reporter group and a synthetic handle for general attachment of various therapeutic agents
271 Breast cancer detection has advanced considerably And, follow-up studies on breast cancer patients show a clear correlation between early detection and patient survival The data strongly suggest that at its inception breast cancer is a progressive disease, rather than a systemic disease, and early diagnosis and treatment can have the most significant outcome on i educing patient mortality Currently, high-quality mammography is the most effective method presently available for breast cancer screening However, the caveats in mammography include the follow mg false-positive detection results m up to 85% of non-malignant lesions requiring invasive biopsies, and talso-negative detection results in up to 20% of cancers being missed, particularly in younger women who have denser breast tissue that obstruct detection of the cancer Furthermore, the technique of mammography involves taking an X-ray of each breast The radiation from X-ray imaging is considered harmful with a greater risk in younger women, and therefore mammography is usually only recommended for women over 40 years of age unless the younger patient has a significant risk for the cancer (Tabar et al Int J Gynaecol Obstet 2003, 82 319 326) The disclosed methods and compositions do not employ ionizing radiation such as X-ray, and the pioblems with false-positive and ialse-negative detection by monitoring the chemical environment of the cell to determine if a lesion is cancerous is alleviated with our dual modality system
272 The disclosed agent is sensitive to the chemical environment of cancer cells In conjunction with the fluorescence imaging, a dual-confirmation strategy to increase the specificity of detection is used Additionally, the nature of the disclosed imaging agents can also avoid two other properties associated with MRI imaging agents, namely the toxicity of transition metals normally used in MRI imaging and distribution of the agent to the region of mterest
273 Tissue-specificity is addressed with the homing peptide backbone that makes up the bulk the proposed agent Homing peptides are short sequences derived fiom phage display libraries that have the unique property of homing to specific organs The discovery of homing peptide technology by Ruoslahti and Pasquah re\ ealed that different organs have distinct zip codes within the endothelium vascular, and appropiiately programmed peptide sequences can be used to home to these zip codes (Pasqualmi et al Nature 1996, 380 Z364 366) However, these zip codes refer to cell-surface interactions and do not reflect the intracellular delivery of these peptides
274 A breast homing peptide with a Tat sequence, derived from HTV that allows for cell penetration, can be used to deliver the disclosed agents mto cells (Deshayes et al Cell MoI Life Sci 2005, 62 1839-1849) Myrberg and co workers published a related study using a different cell-penetrating peptide, pVEC, for intracellular delivery of breast homing peptides (Myrberg et al Bioconjugate Chem 2008, 19 70-75) The breast homing peptide is a cyclic nonapeptide with the sequence cCPGPEOΛQC The peptide and even peptide conjugates of the homing sequence have been shown to distribute to breast tissue possibly via interaction with a membrane-bound prohne-specific ammopeptidase P (APaseP) (Essler et al PNAS 2002, 99 2252-2257) In agreement with Myrberg s work, the homing peptide sequence alone does not penetrate the cell surface of breast cancer cells
275 The disclosed delivery system is based on a 9-amino acid peptide that was discovered by phage display libraries The unique property of this peptide is its ability to 'home' to breast tissue, presumably by interacting with ammopeptidase P (APaseP) protein expressed in breast tissue (Esslei et al PNAS 2002, 992252-2257) Trastuzumab (Herceptin) is a monoclonal antibody that targets the HER2 receptor tyrosine kinase and was approved by the FDA in 1998 for the treatment of breast cancer Analogous to the system herein, monoclonal antibodies are designed to recognize an extracellular protein motif and effectively home to its target Recently, trastuzumab covalently attached to the cytotoxic tubulin inhibitor niaytansmoid (DMl) entered phase II clinical trials and shows considei able potential as a new therapy foi metastatic breast cancer (Vukelja et al Cancer Res 2009, 69(2 Suppl), Abstract nr33) However, despite the incredible success of trastuzumab and the potential of trasluzumdb DMl, there are several drawbacks to using monoclonal antibodies for therapy such as stability, manufacturability, and cost Disclosed is a 21-amino acid peptide-based agent
276 Monoclonal antibodies are an inci edible strategy foi cancer tieatment However, antibodies must be refrigerated, have a short shelf life, and require fermentation technology for manufacture These special requirements all contribute to a major barrier to this strategy, high cost A Canadian study showed that m 2005, trastuzumab treatment cost was between $28,350 and $49,915 per patient, adding a significant cost burden to their health care system (Druker et al Current Oncology 2008, 15 136-142) In the US, trastuzumab costs $2928 89 foi 440 mg, which translates to a yearly regiment costing approximately $50,000 for a 70 Kg woman (Fleming T, ed Redbook 2005 Ed Montvale, NJ Thomson PDR, 2005) One of the major challenges associated with monoclonal antibody therapy is manufacture and storage ot the agent Classical chemical synthesis of drugs average less than $5 per gram However, antibody pioduction incuis costs between $100 and $1000 per gram (Molowa et al 2001) Peptides can be manufactured as low as $1 per gram per amino acid residue, which calculates to approximately $21 per gram for our proposed agent 277 The manufacture of antibodies i nvolves a fermentation process using live cells, which require cell culture medium and medium supplements The complexity of the fermentation process introduces several routes for contamination Modifications to the antibody cannot be achieved readily However, unexpected post-transcπptional modifications including various glycosylation events can occur due to slight changes to the fermentation environment The one advantage of antibodies is the reduced toxicity associated with the agent The disclosed compositions methods, and systems takes advantage of using a peptide-based material, which has low toxicity Unlike antibodies, the relatively small size of the agent is amenable to chemical synthesis, and can be iepioducibly manufactured Chemical synthesis also allows for controlled modifications and easy adjustments to the structure And, unlike with antibodies, incorporation of various imaging and therapeutic moieties to this system can readily be
Figure imgf000069_0001
by standard solid-phase peptide synthesis (SPPS) protocols
278 Tat is a transcription-activating factor derived from HIV-I, and is essential for viral gene expression The 86 ammo acid sequence contains a basic region (amino acids 49 - 58) that is responsible for cell penetration (Deshayes et al Cell MoI Life Sci 2005, 62 1839-1849) This cell-penetrating Tat sequence (ammo acids 49 -57) was incorporated into the homing peptide and induced cell penetration of the peptides Further, the combination of the Tat sequence with the breast homing peptide conferred tissue specificity for breast derived cells In addition, tins specificity has been coirelated with the expression of ApaseP, the putative recogmtion protein of the breast homing peptide And, examination of human tissue arrays revealed that ApaseP expression is especially high in human breast tissue indicating that mouse- deπved breast homing sequence can have utility in humans
279 Disclosed herein, a theranostic agent is defined as a single agent that can be used to diagnose and simultaneously treat a disease However, the question arises whether it is reasonable to mclude a therapeutic component to a diagnostic agent, and in effect unnecessarily medicate patients that are diagnosed as cancer free The single-agent theranostic has clinical utility and is designed as a fully functional diagnostic system with the option of attachmg a drug molecule Once the disease is diagnosed, the therapeutic agent is attached to the same system used for the diagnosis To attach the drug, a one-step [3+2] Huisgen cycloaddition is used This methodology falls under the paradigm of 'click chemistry, which is a synthetic strategy that can accommodate the attachment of a variety of different drags (KoIb et al Angew Chem Int Ed 2001 , 40 2004-2021 ) The advantage ot this system is that the physician can monitor the effectiveness of the treatment with the same detection system that was used to diagnose the disease And, the physician can have a number of treatment options tailored to the patient, because different drugs can be attached to the system using click chemistry
280 There is a need of a personalized medicme approach to patient care by creating a theianostic agent that can simultaneously detect cancer, doubly confirm the presence of a cancerous chemical environment, and treat the tumor with an interchangeable therapeutic component This approach is exceptionally innovative, because the latest techniques and strategies for both cancer detection and treatment have been combined into a simple single agent that is amenable to process-scale manufacture This strategy is unconventional, because the ability to monitor and manipulate intricate processes of biology, chemistry, and physics were incorporating while maintaining a practicality that can allow significant progress within the allotted two-year time frame This strategy is very logical and codifies the strategies that arc essential to the theranostics concept
281 The disclosed agents require three amino acids as follows the fluorapliore, the spin-label, and the therapeutic agent Each route is very general, and can be adapted to the synthesis of the other disclosed ammo acids
282 Modern solid-phase peptide synthesis (SPPS) is a convenient method to assemble peptides of increasing lengths The best methods available utilize Fmoc-protected ammo acids that are attached to a solid support m a step- wise fashion Peptides containing 20 or more ammo acids are ioutinely synthesized by this technique The disclosed peptide agent is a 21 mer made up ot 8 commercially available ammo acids and the 3 disclosed amino acids
283 Figure 1 provides an example of how the fluorophore containing amino acids are made Method 1 is accomphshcd by treating the O succmimide ester of the carboxyl containing fluorophore with Fmoc-Lys-OH This gives selective funUionahzation on the side-chain amine group The synthesis of Fmocprotected amino acid (2a) was accomplished by this method in an overall 51 % yield I he same method can be used to make Fmoc-protected ammo acid (2b), which has an overlapping emission bandwidth with (2a) but has a much higher quantum yield
284 Three problems that arise with fluorescence imaging are depth of light penetration, stability of available fluorophores under physiological conditions, and the solubility of fluorophores that have the appropriate photophysical properties (Frangiom et al Curr Opm Chem Biol 2003 7 626 634) For depth of light penetration, the two fhiorophores above are suitahlc for mouse modeling However, for increased tissue penetration, a near IR (NIR) fluorophore such as a cyanine dye with an emission wavelength above 700 nm is required (Jaffer et al J Am Med Assoc 2005, 293 855 862) Unfortunately, cyanme dyes tend to be unstable in vivo, not soluble, and considerably toxic The increased toxicity with NIR dyes is partly attributed to their hpophihcity Tndocyanrne green is an FDA-approvcd NIR dye that addresses this toxicity by attachmg several hydrophilic sulfate groups to the dye However, mdocyantne green is still susceptible to photobleaching and radical formation by redox mechanisms To address this problem, the disclosed agent uses an NIR dye encapsulated m an α-cyclodextrin molecule The synthesis of the encapsulated NIR dye is shown in equation 2 of Figure 1 following the protocol of Anderson and co-workers (Simon et al Chem Commun 2008, 2897- 2899) This encapsulation procedure gives rotaxane (5a) a 34% yield In order to attach the dye to the agent, monofunctionalization of the dye is required Moπo-azido a cyclodextπn is commerudlly available and is expected to give the monofunctionalized rotaxane (5b) The click chemistry protocol for [3+2] cycloaddition with Fmoc- protected propargylglycine gives the requisite ammo acid for SPPS attachment to the proposed peptide
285 The spm-labeled ammo acid is the next building block needed for the synthesis of the disclosed detection system This synthetic reaction is outlined m Figure 2
286 Attachment of a fluorophore, specifically mtrobenzoxadiazole, to the spin label is shown m Figure 3 Condensation of 4-chloro-7-mtrobenzofUr azane with 6-atmnocaproic acid (35% yield) is followed by activation of the carboxyhc acid by formation of the
Nhydroxysuccimmide ester (62% yield) Coupling with the amine side chain of Fmoc-Lys-OH gives the appropriately fimctionalized building block for incorporation in the proposed agent by SPPS In an analogous fashion, the spinlabeled ammo acid building block is assembled by initial activation ot commercially available 2,2,5,5, - tetramethyl-3 pyrrolm-l-oxyl-3-carboxylic acid by formation of the N hydroxysuccmimide ester (61 %) followed by coupling to the amme side chain of Fmoc-Lys- OH
287 The final building block required is the therapeutic moiety This can be attached by dick chemistry under Huisgen [3+2] cycloaddition conditions and allows the drug to be attached to the completed peptide The strategy for this approach is to give a fully functional diagnostic system with optional attachment of the therapeutic agent Pn order to accomplish this, the synthesis of the diagnositic system on solid support includes a propargylglyeine residue Unlike in the case for the NIR fluorophore, which is attached Io the ammo acid by click chemistry before SPPS, the therapeutic moiety is attached to the completed reagent after synthesis, cleavage from the solid support, and purification This gives the option of attaching a variety of therapeutic agents m a simple one-step procedure The only preiequisite for attachment is that the drug agent must be functionahzed with an azide group
288 Maytansme is a natural product originally isolated from the bark of the African shrub Maytenus ovatus by Kupchan and co-workers (Kupchan ct al J Am Chem Soc 1972,
94 1354-1356 ) Maytansme has several advantages 1. The maytansme analog DMl attached to the antibody trastuzumab is currently m phase II clinical tnals 2. Maytdnsme is reported to be a tubulin inhibitor with the same mechanism of action as the clinically used agents vincristine and vinblastine 3. Maytansme is commercially available and can be selectively functionalized at the secondary alcohol And, 4 Maytansme activity requires an N-methyl alanine residue that can readily be functionahzed with an azide
289 As shown in Figure 4, conjugation to the diagnostic system involves one step Briefly, the azido-contarnmg drug agent (12) in the piesence of the peptide system is treated with a copper (I) source in a DMSO-H2O sohent system As mentioned above, this strategy gives a general one step attachment of a variety of drug entities
290 Also disclosed herein is the use of bleomycin as the therapeutic component of the proposed theranostic agent Bleomycin was chosen for several reasons to include the following bleomycin is an established and well-characterized chemotherapeutic drug in the clinic, bleomycin is itself a pentapeptido, a targeted delivery system to the breast can immediately give a new indication for this drug, selective modification to give azido-bleomyαn can be achieved in a simple one-step reaction, and its molecular mechanism of action suggests that chemical modification can not alter the effectiveness of this drug
291 Bleomycin is administered as a mixture of structurally related compounds marketed under the name Blenoxanc (Hecht, In Cancer Chemotherapeutic Agents 1995) Blenoxane is administered intravenously and is used to treat lymphomas, squamous cell carcinomas, testicular caicmomas, and malignant pleural effusions
(http //www fda gov/CDER/foi/label/2003/50443slr035_blenoxane_lbl pdf) The most severe side effect of bleomycin treatment is late-onset pulmonary fibrosis, which usually presents several months after therapy has been completed ( Abid et al Cuπ Opm Oncol 2001, 13 242- 248) Conjugation of bleomycin to the disclosed system can result in lower pulmonary toxicity, because the agent distributes specifically to the breast and does not accumulate in the lung In addition, oveiall systemic toxicity from bleomycin treatment is predicted, because of an increased effective concentration of the drug at reduced dosages
292 The copper chelate of bleomycin A5, marketed as Bleocm, can be used for attachment to the disclosed delivery system In general, the bleomycins are complex glycopeptides and chemical modification can be challenging However, bleomycins strongly chelate transition metals at their N-terminus domain In the case of bleomycin A5 coppei chelate, the primary amine is exposed and easily functionahzed (Figure 5) Following the method of Xu and co-workers, bleomycin A5-copper chelate is treated with 2-azidodcetic acid, DCC and HOBt to give the azido-conjugated compound Treatment with 15% aqueous EDTA liberates the bleomycm comugate from the copper (Xu et al Bioorg Med Chem Lett 2005, 15 3996-3999)
293 The functionalized bleomycm is conjugated to the proposed delivery system in one step under click chemistry conditions for the Huisgen [3+2J cycloaddition The cycloaddition requires Cu (I) for the cycloaddition In the event the cycloaddition reaction is not facile due to chelatation of the Cu (I) cation with bleomyin (Oppenheimer ett al J Biol Chem 1981, 256 1514 1517), the copper saturated adduct can be investigated for conjugation to the proposed delivery system
294 The antitumor activity of bleomycm is attributed to its ability to induce double strand DNA damage The region of bleomycm that was modified is at the bis-tmazole tether extending from the C-terminus of bleomycm, which has been shown to thread between double- stranded DNA before inducing cleavage Analogs of bleomycm attached to a solid support at the C terminus showed reactivity identical to that of free bleomycm for sequence-selective cleavage of duplex DNA, suggesting that the threading mechanism is not important for the activity of bleomycm (Abraham et al J AmerChem Soc 2001, 123 5167-5175) Therefore, the modified bleomycm retains antitumor activity even when attached to the disclosed system Because bleomycm is required to enter the cell membrane and then the nuclear membrane in order to cleave DNA, this system can efficiently dehvei the bleomycm into the cell and proved a more efficacious effect on cancer cells
295 A further component of the disclosed theranostic agent is the attachment of the drag molecule This comprises a one step reaction to connect the drug molecule to the detection system Click chemistry via a Huisgen 1,3-drpolar cyclodddilion is a convenient method to connect two highly fcnctionahzed groups in a single stop with high yield and purity This design strategies the synthesis of a fully functional dual modality detection system that can be elaborated with a number of therapeutic components by covalently attaching new drug entities using a single-step click reaction The complete detection system is assembled by SPPS using commercially available Fmoc-propargylglycme as the initial residue After cleavage from the bead and HPLC puiification, the agent is fully functional as the proposed detection system Modular addition of the therapeutic agent involves a simple one-step reaction by click chemistry as shown in Figure 6 The only requirement is that the drug is labeled with an a7ide functionality The fully functional detection system can be used for initial patient diagnosis, and then an appropπate therapeutic agent is attached to the same detection system for treatment of the patient
296 Figure 7 outlines the SPPS of the complete diagnostic system the agent is based on a peptide sequence that incorporates the following a cell-penetratmg Tat sequence, tissue- specific PEGA breast homing motif, a fluorescent chromophore, a chemosensitive MRI contrast/fluorescence quencher moiety, and a therapeutic agent As shown, the peptide is a 21- mer, which can easily be accommodated on solid phase Eight of the 11 required amino acids are commercially available One disulfide bridge must be installed, which can be accomplished by treatment of the unprotected cysteine side chains with iodine in the presence of air Because our agent only contains two cysteine residues, selectivity is not an issue Cleavage from the solid support and global deprotection is effected by treatment with TFA Note the alkyne of 1he prapargyglycme residue allows foi mild and selective attachment of our azido-functionahzed drag molecule However, even without the attachment of the drug molecule, all elements of the diagnostic system is in place and is expected to be fully functional for the detection of cancer lesions
297 By convention, peptide sequences are written from N to C termini However, as shown in Figure 7, current SPPS methods require synthesis of the peptides from C to N Synthesis of the proposed agents can commence as follows A rink resm is used to give the non charged terminal amide at the C-terminus The cyclic PEGA homing motif is formed by oxidative disulfide bond formation of the unprotected cysteine side chain residues with iodine Λnd finally, the completed compound is cleaved from the resin with TFA, which results m global deprotection of the amino acid side-chain protecting groups The PFGA homing moiety with a C-termmal dansyl fluorescent tag, the PEGA homing moiety with a C terminal I AMRA fluorescent tag, and the PEGA homing moiety with both a C-termmal dansyl fluorescent tag and an N-terminal Tat sequence have been made
298 Incorporation of the drug molecule is accomplished as shown in Figure 8. The diagnostic system is treated with the azido-mnctionalized rnayUnsmol in the presence of a copper sulfate and sodium ascorbate in a DMSO-water solvent system to gnc the completed theranostic agent
299 The theranostic agents have a pharmacological effect The homing peptide, the homing peptide-Tat conjugate, the homing peptide-Tat maytansinoid conjugate, and the homing peptide-Tat-maytansmoid-NIR fluorophore-MRI mtroxide conjugate, as well as the diagnostic agent comprising other therapeutics (i e bleomycin), can be evaluated on breast cancer cells (MCF-7 and MDA-MB-231) m \itro by MTT assay to obtain the Gl50-values The results provide an in vitro toxicity profile for the different segments of the theranostic agent as well as a reference for in vivo studies Normal endothelial cell lines, HlJVEC and HMVEC, can be used as controls to assess potential systemic toxicity Compaπng the GI50 values for HUVEC or HMVFC cells with breast cancel cells provides a therapeutic index The therapeutic index is defined as the toxic dose divided by the effective dose for 50% of the population (TD50 ED-;o) Cancer drugs tend to have a relatively low therapeutic index The maytansinoid family of compounds is 100 to 1000-fold more cytotoxic than vmcπstine and vinblastine toward cancer cell lines in vitro (Widdiso et al J Med Chem 2006, 49 4392-4408), and has a 10% lethal dose of 040 mg/kg m mice (Issell et al Cancer Treat Rev 1978, 5 199-207) The therapeutic index for maytansine is not reported However, several phase I and 11 clinical trials failed to demonstrate therapeutic benefits at tolerable doses (Widdiso et al T Med Chem 2006, 49 4392- 4408) Bleomycin is reported to have an in vitro therapeutic index of 8 6 when compaπng the IC90 value for the non-leukemic permanent muπne hematopoietic progenitor (PMHP) cells as the 'normal cells' and the IC90 value for breast tumor cells (Tueni et al Cancer Res 1989, 49 1099-
1102) In comparison, gleevec, a clinically used targeted cancer therapeutic, is very well tolerated in patients Gleevec s therapeutic mdex ranged from 57 to 243, as defined by the iatio of EDso in BCR-ABL-ncgative cells (dose limiting side effect) to ED5Q m BCR-ABL-positive cells (therapeutic effect) (Topaly et al Br J Cancer 2002, 86 M87-1493) Cytotoxic drugs in general tend to have a very nanow therapeutic index For example, the chemotherapeutic agent 5-FU has an in vitro therapeutic index of only 2 3 when administered by IP injection on tumor bearing mice (ligo et al Biochem Pharmacol 1988, 37 1609-1613) Ideally, an m vitro therapeutic index in the range of that shown for gleevec greater than 50, and an in vivo therapeutic index of greater than 20, a 10-fold improvement over currently used cytotoxic agents, ib achieved
300 The toxicity of breast homing peptides has briefly been addressed Myrberg and co-workers concluded that the toxicity of a breast homing peptide-pVec-chlorambucil conjugate was due piedominately to the cytotoxic chlorambucil moiety The experiment also verified the homing properties of this system in vivo (Myrberg et al Bioconjugate Chem 2008, 19 70-75) However, the disclosed molecule compi ises a different cell-penetrating peptide sequence, Tat rn place of pVec, and a different cytotoxic moiety
301 According to Chan and co-workers, the cytotoxicity of the trastuzumab-DMl conjugate requires cleavage of the maytansinoid group Two possible scenarios for a narrow therapeutic mdex for disclosed agent are as follows 1 high toxicity is met at low concentrations due to rapid cleavage from the peptide, or 2 high concentrations of the agent is required to have an effect, because of slow cleavage hi both cases, the therapeutic index is narrowed However, both scenarios can be addressed by attenuating the linker region To strengthen the conjugation, non-peptidic linkers can be explored as is used for the trastuzumab DMl conjugate For more labile connections, peptidic or disulfide bond linkages can be explored Disulfide linkages are attractive, because they can be further fine-tuned by increasing steπc bulk adjacent to the disulfide linkage And, disulfide bond cleavage is redox sensitive, which follows the mechanism for reducing the spin-label MPJ contrast moiety of the disclosed system
302 The synthesized agents can be subjected to wild type mice to determine acute toxicity The method can be performed following the Acute Oral Toxicity- Up and Down Procedure published by the EPA (OECD Guides for testing of chemicals,
http //www epa gov/oppfeadl/harmomzation/) An estimated LD50 can be calculated from this piocedure, which allows for determination of an approximate dosage to give the animals An
LD50 is calculated for both the diagnostic system and the theranostic agent that incorporates a drug moiety
303 Animal studies can be performed to study human hi east tumor models m mice The breast tumors can be placed m the mammary fat pad of the mice Female nude athymic mice can be used Because of reduced estrogen levels m these mice, estrogen supplementation can be used for MCF-7 breast cancel xenogiaft models
304 Based on the estimated LD50 value from the Acute Oral Toxicity Up and Down Procedure, the dosage for treating the annuals can be determined hi order to achieve maximal therapeutic effect and minimal toxicity, the animals at can be dosed at 30% the LD50 value which is assumed to be the maximally tolerated dose This rough calculation is based on a 6- ammal per experiment study assuming a hneai relationship between dosage and lethality It is expected that 30% of the dosage that kills 50% of the mice is approximately the highest dosage that will not kill any of the mice in a study of 6 animals per expeπment
305 Normal wild-type mice can be treated with the detection system that does not incorporate the therapeutic component The detection system can be treated with sodium borohydnde prior to administration to the animal, to reduce the nitroxide radical and remove its ability to reduce fluorescence This agent can then be used to verify localization of the homing peptide to the breast tissue by fluorescence imaging The limit of detection for the agent can be defined as the concentration (mg of agent / kg animal weight) that results in a signal-to-noise ratio of 3 1 The quantity of agent delivered can be estimated by fluorescence imaging using the Lambert-Beer law The practical limits αf quantification canbe determined as the lowest concentration to give a relative standard deviation of < 10%
306 Secondly, the mice can be treated with the fully functional detection system with the mtroxide radical intact The half-life for the free radical by non-specific degradation in a normal mouse can be determined by plotting the relative intensity of the fluorescent signal over a 96-h tune peπod
307 Finally, the plasma levels of the agent over a 96-h time period can be determined by HPLC Degradation and'or metabolism of the agent m the plasma can be determined by HPLC
308 The mimmal amount of agent necessary for detection can be determined In combination with the calculated LD50 values, the concentration range that can be used for treating the animals foi optimal results is determined A time-course experiment determines the half life of the free radical and the peptide system m normal mice
309 Imaging of mtroxide radicals, by MRI and characterizing ledox activity by measuring the intensity of Tl-weighted images as a function of time in mouse xenograft models is reported (Matsumoto et al Clin Cancer Res 2006, 12 2455-2462) The SPPS methodology can he used to modify the disclosed agents in order to improve their half-life, if necessary SPPS allows rapid synthesis of the disclosed agents, and therefore modifications to the system can be made fairly quickly To increase the signal, multiple residues of the nitroxide-containing amino acid can be conjugated to the peptide to attenuate the signal To increase the half life, spacing the nitroxide-containing residues with pegylated lysmes can protect the radical hi a related imaging strategy that utilizes protease activation of fluorescent probes, Weissleder and co-workers report selective protcasc-mediated degradation of pegylated lysine-contaimng peptides at tumor sites in mouse models (Weissleder et al Nat Biotechnol 1999, 17 375-378) Disclosed herein, umeihng the mtroxide in tumors allows for chemical reduction of the mtroxide in a second step by the redox environment of the cancer
310 Disclosed are cancer therapeutics that can be used with the disclosed theranostic system There are a number of potential cancer drags with various mechanisms of action These drugs can be used in place of maytansuioid and bleomycin as the therapeutic component of the theianostic agent
311 Optimization of the detection system and theranostic agent can be performed m xenograft models utilizing the conditions foi imaging determined with the wild-type mice Mice at various stages of tumor growth can be subjected to tail-vein injection of the diagnostic agent that does not incorporate the therapeutic moiety MRI and fluorescence imaging can be used to ascertain the minimal size tumor that can be detected
312 Mice bearing breast tumor xenografts (tumor -volume=100 mm3) can be subjected to tail-vein injection of the theranos>tic agent at 30% the LD50 The time course for dosing can be determined based on the half-hie of the agent in the circulatory system as determined in the wild- type mice MRI and fluorescence imaging can be used to monitor tumor growth
313 The disclosed agent can be used for diagnosing and treating breast cancer by homing to the breast tissue, identifying the presence of cancel, and impeding tumor growth
314 Disclosed are cancel therapeutics that can be mcorpoiated to the ttieranostic agent m place of the maytansmoid moiety Some compounds that can be used are tubulin inhibitors (YK 3 250 and MP-2Ol), a SIRTl activator (YK-3 237), an HDAC inhibitor (YK-4-272), and an MLCP inhibitor (SCG-3-285) (Figure 9)
315 YK-3-250 is a boronic acid bioisostere of combretastatom A 4 developed by Dr YaIi Kong (Kong et al Ohem Biol 2005, 12 1007-1014) This compound has a GI50 value of less than 10 nM across a panel of breast cancer cell lines, and is a very potent inhibitor of tubulin polymerization with an IC50=l 5 ± 0 2 μM In comparison, combretastatm A-4 has an IC50 value of 2 0 ± 0 2 μM Phase II clinical trials on a related phosphate analog of combietastatin A- 4, fosbretabulm, suggest that fosbretabulm is safe and canbe advanced to phase in clinical trials for anaplastic thyroid cancer (Mooney et al Thyroid 2009, lθ 233-240)
316 Dr Kong developed a related boromc acid chalconc analog, YK-3-237, that surprisingly showed little inhibition of tubulin polymerization (IC50=31 0 ± 3 4 μM) However, YK-3 237 was considerably potent against a numbei of cancer cell lines with GI50 values in the sub nanomolar range for several breast cancer cell lines This compound can act as a SIRTl activator In comparison with resveratrol, a well-known SIRTl activator, YK-3-237 is 10 times more potent m activating SIRTl deacetylasc activity
317 hi order to attach YK-3-250 or YK-3-237 to the theranostic system, the boromc acid group is functionalized The free boromc acid is necessary for activity and therefore requires a handle for conjugation to the peptide Burke and co workers recently reported a sp3- hybidized boronate ester that is cleaved under mild aqueous conditions (Burke et al J Am Chem Soc 2007, 129 6716-6717) The strategy involves an internal coordination between a nitrogen atom and the boron atom Following the method of Burke and co workers, the azido functionalized dicaiboxylic acid (13) is condensed with the boronic acid under Dean-Stark conditions to give the boronic esters of YK 3 250 and YK 3 237 as shown in Figure 10 These azido- functionalized drug agents can be attached to the theranostic system via click chemistry The release oi the drug can occui by kinetic hydrolysis of the agent m the cell to give the free boromc acid
318 YK-4-272 is a fluorescent HDAC mhibitoi developed by Dr Kong This drag inhibits pan HDAC activity with an IC50 value of 125 nM More interestingly, this agent shows some selectivity toward class II isoforms of HDAC, especially HDAC 6 In addition, a crystal
7g structure of the HDAC inhibitor SAHA bound to HDAC 8 reveals that the aliphatic chain reaches into a cavity of the protein and the hydroxamic acid moiety binds to a zinc atom (Vanπim et al PNAS 2004,101 15064-15069) Importantly, the opposite end of the molecule corresponding to the dansyl group m YK 4 272, is rn the solvent exposed region of the protein Therefore the drug can be attached to the lheranostic system via the dimethylamme on the dansyl group Functionalization of the dansyl group is descπbed below
319 A myosin light chain phosphatase (MLCP) inhibitor, SCG 3 285, that also incorporates a dansyl moiety has been developed 1 μM of SCG-3-285 causes considerable increase in phosphorylation of myosin light cham (MLC) in PC-3 cells Cell cycle analysis shows that treatment with 1 μM of SCG 3 285 causes G2/M arrest in PC 3 cells In addition, the NCI 60 cell line screen revealed that SCG-3 285 is a potent inhibitor of cell proliferation for several breast cancer cell lines with GI50 values around 500 nM
320 Functionalization of the dansyl moiety of YK-4 272 and SCG-3-285 can commence with mono-demethylation of the drmethylamine group follow ed by conjugation with 2-azidoacetic acid Mono-demethylation of the tertiary amine can be accomplished using ACE CI as reported (Figure 11) (Olofson et al J Org Chem 1984, 49 2081 2082) Conjugation with O succinimide ester of 2-azido-acetic acid gives the requisite azide for attachment to the theranostic system via click chemistry
321 Biaryl-substitutcd dihydroquinazolmones are a class of potent tubulin inhibitors Compound MP-201 is a lead structure optimized to give the mtro-containing qurnazolmone shown in Figure 9 MP-201 induces 100% depolymenzation of tubulin at 15 μM, inhibits tubulin polymerization with an IC50 value of 0 76 ± 0 01 μM and displaces [3H]colchicme 33 ± 3 3% at 5 μM and 60 ± 2 8% at 50 μM An asymmetric synthesis to the optimized compound has recently been reported (Chinigo et al J Med Chem 2008, 51 4620-4631) The S enantiomer of the optimized mtro-contarning analog inhibits tubulin assembly with an IC50 of 1 1 ± 0 2 μM, displaces [3H]colchicine 28 ± 1 % at 5 μM and 66 ± 1 % at 50 μM and inhibits proliferation of MDA-MB-435 cancel cells w ith a GI50 of 0 10 nM Two regions have been identified that arc amenable to functionalization on the 6 position of the dΛydroqumazolmone ring and on the ortho-position of the terminal phenyl ring Modifications at these positions aie straightforward MP-201 has been modified by replacing the methyl group on the terminal phenyl group with - [18F]CH2 group for evaluation as a PET agent There are at least two possible methods of connecting this potential drug to the disclosed theranostic system First, synthesis of the agent with an OH substituent on the terminal phenyl group can allow for condensation with 2- dzidoacetic acid to install the lequisite azido group for attacbment to the theranositic system via click chemistry Secondly, considering the success with boromc acid bioisosteres, a boronic acid moiety can be incorporated in place of the nitro group This agent can be evaluated for its ability to mlubit tubulin assembly, displace [3H]colchicine, and inhibit breast cancer cell proliferation The same strategy shown aboΛ e for attachment of the drug to the theranostic system can be used for the boronic acid (Figure 12)
322 The incorporation of different cancer therapeutics to the disclosed theianostic system can enable further development of these agents Successful implementation of these agents can lead to furthei funding and eventual drug development
a) Results
323 The agents disclosed above can be evaluated for two aspects, tissue specificity and pharmacological effectiveness
324 In order to demonstrate tissue specificity, ApaseP was determined to be differentially expressed in human tissues A tissue microanay analysis showed that ApaseP was indeed highly expressed in human breast tissue (both normal and cancerous), but not in the lung, esophagus, nor the stomach APaseP is the putative recognition protein for the breast homing peptide These results mdicate that the breast homing peptide canalso distribute to the breast in humans Most notably the low expression levels of ApaseP in the lung suggest that the lungs may be completely bypassed as an accumulation site, and therefore reduce the pulmonary toxicity of bleomycin
325 The breast homing peptide can also be a substrate for APaseP, because it contains potential recognition motifs for cleavage by APaseP APaseP cleaves at N terminal X P Z sequences, and the breast homing peptide contains two X-P-Z sequences, albeit not at the N- terminal The location of these sequences and the cyclic nature of the bieast homing peptide can increase the half life of this peptide (Essler et al PNAS 2002, 99 2252-2257) The X P Z sequence can be placed at different locations m the cyclic system to maximize stability but maintain recognition
326 Expression of ApaseP in cancer cells was determined by immunohisto staining using an antibody against ApaseP The results show clearly that ApaseP is richly expressed on MCF 7 and MDA-MB-231 breast cancer cells (F lgure 13) However, ApaseP expression was not detected on PC 3 prostate and A 549 lung cancer cells Binding to ApaseP can be A prerequisite for cell penetiation
327 lhe delivery efficiency of the proposed delivery system and the importance of the Tat sequence were assessed The distribution of the homing peptide with or without the Tat cell- penetrating sequence was observed m MCF7 breast cancer cells As shown in Figure 14, the dansylated breast homing peptide that incorporates the Tat cell penetrating sequence efficiently penetrates MCF-7 bredbt cancer cells However, the dansylated breast homing peptide v, lthout a Tat sequence shows poor penetration of MCF 7 breast cancer cells
328 Another breast cancer cell line, MDA-MB-231 was also examined As> shown in Figure 1 i, the dansylated PFGA breast homing peptide conjugated to the Tat sequence shows penetration mto MDA MB 231 metastatic breast cancer cells However, the dansylated PEGA breast homing peptide that does not incorporate the Tat sequence gave a consideiably weakei signal under the same treatment and imaging conditions The homogeneity of the signal that is observed can be due to non-specific binding and no penetration mto the cell The breast hommg peptide did not penetrate ApaseP negative PC-3 prostate cancer cells (Figure 16) or A-549 lung cancer cells, regardless of the presence of Tat The experiments were repeated with the hommg peptide containing a TAMRA fluorophore but no Tat sequence This agent allowed evaluation at a different excitement and emission channel The data indicate that incorporation of Tat is required for penetration of the disclosed agent mto breast cancer cells An apparent correlation with ApaseP expression and cell penetration is noted These results are further confirmed with a breast hommg peptide that contains a TΛMRA fluorophore and Tat
b) Conclusion
329 The synthesis and evaluation of a new theranostic agent is disclosed The disclosed methods and compositions are designed to detect cancer and give double confirmation by dual modality MRI/fluorescence imaging of a dynamic chemical event that occurs in cancer cells Secondly, an optional therapeutic agent can be covalently attached to the agent by a simple one-step click chemistry reaction Finally the platfoim in which this agent is synthesi7ed is an
18-amino acid peptide that homes to breast tissue and can penetrate ApaseP positive breast cancer cells
330 This agent has cluneal utility for the detection of breast cancer In the event bieast cancer is detected, the disclosed agent can then be used for the snmrltaneous treatment and monitoring of the cancer The proposed theranostic agent is designed to accommodate a variety of drug entities by a simple chemical attachment, which allows for a personal medicine approach to patient treatment And, finally a versatile platform is introduced that can be adapted for new and emerging technologies as they aie developed
2. Example 2 rTissuc-spccific STAT3 Inhibition for Breast Cancer Therapy
a) Background
331 Early detection and treatment has resulted in a dramatic reduction in breast cancer mortality Despite this success, breast cancer continues to be the second leading cancer killer of women resulting in 40,000 deaths each >ear The oncogenic transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) is activated in more than 60% of malignant breast tumors (Gaicia et al , Cell Growth Differ 1997, Bowman et al , Oncogene
2000, Dechow et al PNAS 2004) Activation of STAT3 correlates with a poorer prognosis, presumably due to concomitant elevation of activated Src and Survivm expression (Diaz et al CIm Cancer Res 2006)
332 Constitutive STAT3 activity promotes breast cancer aggressiveness through suppression of pro-apoptotic genes (Battle et al Curr MoI Med 2002, Nm et al Cancer Res
2001, Zhang et al PNAS 2005) and activation of genes that promote proliferation, survival, invasion, and angiogenesis (Wei et al Oncogene 2003, Ling eta al Cancer Res 2005, Song et al PNAS 2005) STAT3 is especially critical for breast cancer stem-like cell survival and proliferation (Zhoe et al PNAS 2007) In addition, STAT3 regulates cell motility and invasiveness through non-transcπptional mechanisms (Gao et al Sci STKE 2006) Inhibition of STAT3 signaling in breast cancer can have enhanced anti-tumor effects, mediated by cell cycle arrest and induction of stem-like cancer tell death, disruption of angiogenesis, and interference with tumor-cell migration and invasion (Turkson bxpert Opin Ther Targets 2004) Therefore, STAT3 is a potential drug target for breast cancer
333 Current strategies in targeting STAT3 include inhibiting tyrosine phosphorylation of STAT3, SH2-domam dependent dimeπzation of STAT3 translocation of STAT3 to the nucleus, or binding of STAT3 to DNA (Desnvieres et al J Mammary Gland Biol Neoplasia 2006) However, STAT3 inhibition in general has resulted m only mildly potent agents that induce 50% growth inhibition of breast cancer cells in the range of 13 4 to 100 μM (Song et al PNAS 2005, Coleman et al J Med Chem 2005, Schust et al Anal Biochem 2004, Schust et al Chem Biol 2006, Siddiquee ct al PNAS 2007, Bhasin et al Bioorg Med Chem Lett 2008, Jing et al Cancer Res 2004, Xu et al PIoS ONE 2009) A novel strategy targeting STAT3 N-domain protein-protein interactions has resulted m a more potent STAT3 inhibitor named STAT3- Hel2A 2 S IAl 3-Hel2A-2 is a retro inverso peptide with GI50 values in the low micromolar range (<10 μM) against breast cancer cells (Timofeeva et al ACS Chem Biol 2007) Despite the increased potency of STAT3-Hel2A-2, Yue and Turkson have indicated that a major impediment to clinical development for STAT3 inhibitors is that the modest potency that can be achieved with drugs that disrupt protein-protein interactions does not provide a sufficiently wide therapeutic mdex to achieve clinical efficacy (Yue et al Expert Opm Investig Drugs 2009) Herein, a new strategy is presented to increase the therapeutic index of STAT3-Hel2A-2 retro- mveiso peptide by specifically increasing its concentration at breast tissues with an overall reduced systemic concentration using a breast tissue-specific molecular homing device
334 Homing peptide technology refers to short peptide sequences derived from phage display libraries that have the unique property of homing to specific organs Ruoslahti and Pasqualmi revealed that different organs have distinct zip codes within the endothelium vascular, and appiopπately programmed peptide sequences can be used to homo to these zip codes (Pasqualmi et al Nature 1996) The peptide and even peptide conjugates of the homing sequence have been shown to distribute to breast tissue presumably via interaction with membrane-bound prolme-specific aminopeptidasc P (APaseP) (Essler et al PNΛS 2002) The STAT3 Hel2A 2 retro-inverso peptide can be conjugated to a breast homing peptide for development as a potential breast cancel therapeutic
335 A breast homing peptide conjugated to the STAT3-Hel2A-2 retro-mverso peptide can distribute specifically to the breast for increased effective drug concentration at the breast with an overall reduced systemic concentration
b) Materials and Methods
336 Dcscπbcd herein is a novel delivery system for a STAT3 inhibitor that targets the N termmal domain of STAT3 A modular synthesis that allows for simple attachment of the delivery component to the therapeutic component in a single step can be performed Incorporating a fluoiescent label on the homing peptide sequence will allow for following the distribution of the agent by live imaging in vivo
337 The disclosed methods can be divided into two parts as follow s 1 Chemistry (linker optimization) the design, chemical synthesis, and characterization of the homing properties of the proposed agents 2 Biology (m vitro and in vivo) a m vitro characterization of the agents against STAT3 and b. in vivo evaluation in tumor models
(1) Chemistry
338 Disclosed herein is the design and synthesis of a seπes of breast homing peptide - STAT3 Hel2A 2 fluorescent label conjugates for biological evaluation A seπes of linking groups can be used to synthesize conjugates of the breast homing peptide and the STAT3 Hel2A-2 retro im erso peptide The disclosed agents can be evaluated for cell penetration of MCF-7 and MDA-MB-231 cancer cells, specificity tor breast-dcπvcd cells, and homing potential
(a) Design and chemical synthesis of the agent
339 A schematic for synthesis of the disclosed compounds is outlined in Figure 17 As shown, the agent is based on a peptide sequence that incorporates the following a cell penetrating Tat sequence, the tissue-specific PEGA breast homing motif a fluorescent chromophore, and a synthetic handle for attachment of the STAT3-Hel2A-2 peptide
340 The cell-penetrating Tat component is necessary for internalization of an agent into the cytoplasm of the cell As detailed below, preliminary results indicate that the promiscuity of the Tat sequence is not conferred to the homing peptide when the two units are conjugated together
341 In order to attach the STAT3-Hel2A-2 retro-inverso peptide to the breast homing peptide, a 'click' chemistry approach can be used Click chemistry is a new paradigm in organic synthesis that allows for efficient coupling of complex entities in high j ield The Huisgen [3+2] cycloaddition is the most common application of the click chemistry concept and can be utilized lor the disclosed compounds The linker region can be optimized by synthesizing a scπcs of connecting groups and evaluating each compound for its ability to selectively penetrate the cell membrane of breast cancer cells and home to the breast in mouse models
342 Figure 18 outlines a series of linkers that are proposed for synthesis and biological evaluation An aliphatic tether and polyethylene glycol tether can determine the distance iequuement between the delivery peptide system and the STAT3 IM2A 2 peptide that can be necessary to retam the homing properties and STAT3 inhibition activity of each entity A poly- lysine tether can be a potential proteolytic site for release of the STAT3-Hel2A-2 peptide in vivo Poly-lysine can be targeted for proteolytic cleavage at tumor sites (Wcisslodcr ct al Nature 1999), and this can provide an increased selectivity in treating the cancer A squarane tether can be a rigid system that can be an uncleavable linker with reduced rotational degrees of freedom in comparison to the aliphatic or PEG tethers These methods can help prevent premature proteolytic cleavage of the STAT3 inhibitor from the homing peptide before distributing to the breast and help retam the function of each group by sufficicnctly separating the flexible aliphatic (or PEG) tether and the homing and STAT3 inhibitory moieties
343 In one embodiment, the drug can be cleaved from the homing peptide once in the cancerous tissue A selective release strategy can be implemented To that end, a disulfide linkage can be used to connect the two umts The disulfide bond can serve as a selective releasing moiety by taking advantage of the hypoxic intracellular redox chemistry that is particular to cancer cells, which would chemically reduce the S-S bond into two fragments (Hyodo et al Cancer Res 2006)
(b) Biological evaluation (homing properties)
344 The disclosed compounds can be biologically evaluated The disclosed compounds can be evaluated for their ability to induce apoptosis, to specifically associate with STAT3 o\ er STATl, and to decrease STAT3 driven transcription In vivo modeling can be used to determine biodistribution, tox icity, and efficacy in reducing tumor volume
345 The homing property of the PEGA sequence for breast tissue is presumed to be due to affinity for aminopeptidase P (ApaseP) MCF 7 and MDA-MB-231 human breast cancer cells indeed show considerable ApaseP expression as shown by lmmunohistochemistry, whereas PC 3 human prostate cancer cells and A-549 human lung cancer cells show minimal expression of ApaseP (Figure 19) Therefore, the disclosed analogs, which contain the same amino acid sequence for association with ApaseP can also have affinity for breast-derived cells
346 The ability of the disclosed agent to cross the cell membrane and its specificity for breast derived cells can be assessed as follows The peptide can be conjugated to a fluorescent reporter group and then exposed to MCF 7 or MDA-MB-231 breast cancer cells The
distribution of the peptide can be observed by multi-photon confocal microscopy Preliminary data is outlined below The disclosed compounds can be evaluated using tbe disclosed methods
347 Designing compounds that can penetrate the cell membrane is a great concern when designing peptide based drugs The 9-amino acid Tat cell-penetrating sequence can be used to address the concern of penetrating the cell membrane As shown in Figure 20, the Tat sequence is sufficient to allow specific penetration of the homing peptide into the cell Using a dansylated substrate for visualization, the peptide containing the Tat sequence distributes to the cytoplasm of MCF-7 cells
Figure imgf000087_0001
the peptide that docs not contain the Tat sequence shows very poor penetration into the cytoplasm of MCF-7 cells A similar phenomenon is observed for MDA-MB 231 cells, where treatment with the homing peptide conjugated to the Tat sequence shows clear penetiation into the cell For the cells treated with the homing peptide not conjugated to the Tat sequence a fluorescent signal is observed, but the homogeneity of the fluorescence signifies non-specific binding and not penetration into the cell These results indicate that the Tat sequence is sufficient to provide penetration across the cell membrane of breast cancer cells The disclosed compounds can be subjected to this screen to ensuie that the disclosed agents are able to cross the cell membrane for drag delivery
348 A concern with using Tat for cell penetration is potential non-selective penetration, thereby overcoming the homing pioperties of the peptide It was determined that the Tat-sequence did not confer non-specific penetration into cells As shown in Figure 21, A- 549 human lung cancer cells were treated with the dansylated peptide containing the Tat sequence or the dansylated peptide without the Tat sequence In both cases, the peptide treatment showed no difference from control, indicating that the Tat sequence does not impart non-selective penetration of the peptide into any type of cell This is in agreement with immunohistostammg experiments that showed low expression of ApaseP in A-549 cells New compounds can be subjected to this screen to ensure cell-type specificity
349 Specific distribution to the breast tissue can be evaluated in vivo The fluorescent properties of the dansyl reporter group are not sufficient for live imaging in mouse models Therefore, a TAMRA reporter group was used, which has an absorption λmax at 544 run and an emission X013x at 572 nm the breast homing peptide containing the Tat sequence and a TAMRA fluorophore was found to be distributed to the breast of nude mice beaπng MCF 7 breast tumors surgically implanted in the breast tissue (Figure 22) This model requires the implantation of an estrogen-releasing pellet, because of the low estrogen levels in female athyπuc nude mice The disclosed compounds can be e\ aluated in this model to determine the homing potential of these compounds
350 For in vivo evaluation of the homing properties of the drug, the limit of detection for this agent will be defined as the concentiation (mg of agent / kg animal weight) that results in a signal to noise ratio of 3 1 The quantity of agent delivered can be estimated by fluorescence imaging based on the intensity of the fluorescent signal The practical limits of quantification will be determined as the lowest concentration to give a relative standard deviation of < 10%
(2) Biologj
(a) Biological evaluation (STAT3 inhibition)
351 STAT3-Hel2A-2 is an optimized peptide with impressive specificity for breast cancer cells over normal cells and modest potency with GI50 \ alues in the low micromolar range As shown in Figure 23, the cancerous cell lines are more sensitive to STAT3-Hel2A 2 treatment than the non-cancerous breast cell line MCF-IOA This screen can be used for the disclosed analogs to determine an m vitro therapeutic index Loss of cell viability due to caspase- dependent apoptosis can also be determined by analysis of PARP cleavage MCF 7 cells are caspase-3 deficient and therefore apoptosis can be determined by annexin V binding
352 The disclosed analogs can then be evaluated for interaction v, ith STAT3 in living cells by fluorescence resonance energy transfer (FRET) microscopy The TAMRA fluorophore can act as an acceptor to the donor eGFP tag, which results in FRET (a change in fluorescence wavelength) when the two components are in close proximity Indeed this was observed when HEK293 cells stably transefected with eGFP on the N-teπrunus of STAT3 were treated with TAMRA-labeled STAT3 Hel2A 2 The disclosed analogs can contain a TAMRA fluorophore and this assay can be used to determine if the analogs interact with STAT3
353 In order to determine specificity for STAT3 over STATl, FRET efficiency can be compared in HEK293 cells expressing eGFP STATl, eGFP STAT3, or eGFP As shown in Figure 24, the TAMRA labeled STAT3 Hel2A 2 peptide showed highest FRET efficiency in cells expressing eGFP STAT3 and ieduced FRET efficiency in cells that express eGFP STATl or just eGFP This assay indicates that the STAT3-Hel2A-2 retro-mverso peptide is specific for STAT3 This assay can be used to confirm whether this specificity is conferred to the disclosed analogs 354 To evaluate the effect of the disclosed agents on STAT3-dnven transcription, the luciferase reporter vector can be employed for the acute phase response element (APRE) containing STAT3 DNA-bindmg elements and a remla lucitcrase reporter vector The APRE reporter group has been demonstrated to be activated in MCF-7 cells by leukemia inhibitory factor (LIF) As shown m Figure 25, pretreatnient with STAT3 Hel2A 2 for 1 hour inhibited basal and LlT-strmulated expression when compared to control (treatment with DMSO) The luciferase activity was measured and normalized against remla luciferase activity This system can be used to evaluate the ability of the disclosed agents to decrease STAT3-dnven transcription
(b) Biological evaluation (xenograft modeling)
355 Athymic female Balb/c nude mice can be purchased from the National Cancer Institute (NCI) The MCF 7 and MDA-MB-231 tumor xenografts, presenting different levels of STAT3 activation, are the models of choice, as indicated by favorable cellular data In one embodiment, surgical implantation of the breast cancer cells into the breast tissue of the mice can be required MCF-7 cells are estrogen-dependent, and athymic nude mice are estrogen deficient Theiefore, this model can require the implantation of an estrogen-releasing pellet As shown m Figure 22, these models have been generated previously Tumors can be grown to 72-94 mm3 before the treatment begms
(l) Bwttistributwn
356 Biodistnbution experiments can be performed to determine how selectively the drugs are delivered into tumor cells versus normal cells and organs Balb/c nude mice bearing MCF-7 breast tumors can be administered intravenously (TV) by tail-vein injection Animals can then be evaluated by fluorescence imaging
(ιι) Toxicity screen
357 The disclosed analogs can be subjected to acute toxicity studies m wild-type mice using the Acute Oral Toxicity- Up and Down Procedure (OECD 2001) This method allows foi the determination of estimated LD50 values for the compounds with minimal amounts of material The LD50 can be used to determine the dosage for treating the animals that will give us maximal therapeutic effect and minimal toxicity The animals can be docscd at 30% the LD50 value This rough calculation is based on a 6-animal experiment, where 30% of the LD50 is approximately the highest dosage that will not kill any of the mice in the study of 6 animals pel experiment This estimate can be adjusted downward if it appears that the animals are experiencing toxicity, or the dosage can be adjusted upwards if the animals tolerate this dosage well hut the fluoi escent signal m the imager is insufficient Pre\ ious experiences have shown that these mice models can tolerate 30 mg/kg dosage of the fluorescentlj -tagged homing peptide and gπ e a sufficient fluorescence signal when imaged The Acute Oral Toxcity- Up and Down Procedure can allow an estimate of the best dosage for the homing peptide conjugated to the STAT3 Hel2A 2 retro mverso peptide
(in) Efficacy screen
358 Tumors can be measured with an external caliper to a significance of 0 1 mm, and volumes can be calculated (V=HxLxW) When palpable tumors grow (72-94 mm3), the mice can be divided into treatment groups The test concentrations of the disclosed agents can be obtained by diluting with PBS Tumor-bearmg mice can be injected with either the disclosed agents or vehicle control, once every other day for 4 v, eeks by tail vein injection Tumor volumes and distribution of the agent by fluorescence imaging can be monitored at least twice weekly Tumor and normal tissues can be obtained from treated and untreated annuals, fixed m 10% buffered formalin, blocked m paraffin, sectioned and stained with hematoxylin and eosin for histopathological examination
(ιv) Statistical Analysis
359 Tumor volumes measured at different time points for each mouse can be correlated For the tumor volumes measured at each time pomt, peicentages with respect to the initial tumor volume (day 0, the first day of dosmg) can be calculated and used as the outcome for further analysis Changes in tumor volumes can be analyzed using linear mixed effect models to compare differences m these changes among groups A pair wise comparison of interest can be made among these subgroups Each comparison can be performed with a two- group univariate repeated measures ANOVA with Greenhouse Geisser correction Six tumor volume measurements from the same mouse can be correlated
(v) Power Calculation
360 The baseline tumor volume is estimated to be 83 mm3 (72-94 mm3) It is assumed that the decreases in tumor volume at the end of the study can be approximately 50 100% for each drag (0 42 mm3) In the pow er calculation tumor growth (percentage) between the subsets can be compared The different dose levels for each treatment can be combined The
data from subsets can be analyzed using a univariate analysis of variance with the Greenhouse- Gcisscr correction The power analyses can be performed using the nQuery softw are
c) Summary
361 Disclosed herein is a new breast cancel therapeutic, wherein a breast homing peptide is conjugated to a novel S I AT 3 inhibitor STAT3 inhibitors have shown promise as potential therapeutic agents for breast cancer The STAT3-Hel2A-2 retro mverso peptide targets the N-domain protem-protein interaction of STAT3 However, the modest potency of STAT3 inhibitors in general has limited their translation to the clime To address this limitation, the disclosed compounds and methods provide a new stiategy to increase the effective concentration of the therapeutic agent at an overall decreased systemic concentration
362 ApaseP expression, the putative receptor protein for the breast homing peptide, is elevated ni breast cancel cells in comparison to lung or prostate cancer cells It has also been shown that the Tat cell-penetratmg peptide sequence was necessary for cell penetration of the breast homing peptide And, then it was shown that the breast homing peptide conferred cell- type specificity to the Tat sequence, such that the breast homing peptide conjugated to the Tat sequence did not penetrate A-549 lung cancer cells And, finally, live imaging of mice bearing MCF-7 breast cancer xenografts implanted in the breast tissue of the mouse with an estrogen- releasing pellet showed that the homing peptide effectively homed to the breast tissue
363 The STAT3-Hel2A-2 retro-mverso peptide has been evaluated for growth inhibition of breast cancer cells, selectivity for STAT3 over STATl by FRET analysis, and inhibition of the transcriptional activity of STAT3 Disclosed herein is the combination of a novel STAT3 inhibition snategy to a homing peptide for development as a new breast cancel therapeutic
I. SEQUENCES
SEQ ID NO 1 cCPGPEGAGC (breast tesue-speufic homing peptide)
SEQ ID NO 2 EKKRRQRRR (Tdt)
SEQ ID NO 3 CGFECVRQCPERC (lung homing peptide)
SEQ ID NO 4 CDCRGDCFC (breast homing peptide)
SEQ ID NO 5 CGRRAGGSC (prostate homing peptide) SEQ ID NO 6 SWCEPGWCR (pancreas, homing peptide)
SEQ ID NO 7 CRVASVLPC (pancreas homing peptide)
SEQ ID NO 8 YSGKWGW (intestine homing peptide)
SFQ ID NO 9 LDTRYLEQI HKI Y (STAT3-Hel2A-2 peptide)
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Claims

VIII. Claims We claim:
1. A composition comprising a detection moiety and a contrasting agent.
2. The composition of claim 1 , further comprising a homing peptide.
3. The composition of claim 2, further comprising a cell-penetrating molecule.
4. The composition of claim 3, further comprising a therapeutic agent.
5. The composition of claim 1 , wherein the contrasting agent is an MRI contrasting agent.
6. The composition of claim 1 , wherein the MRJ contrast agent is non-metallic.
7. The composition of claim 6, wherein the non-metallic contrast agent is a nitroxide radical or derivative thereof.
8. The composition of claim 2, wherein the homing peptide is breast tissue specific.
9. The composition of claim 8, wherein the breast tissue specific homing peptide is CPGPEGAGC.
10. The composition of claim 1, wherein the detection moiety is a fluorophore.
11. The composition of claim 10, wherein the fluorophore is Dansyl, TAMRA, a cyanine dye, or a cyanine dye encapsulated in a cyclodextrin.
12. The composition of claim 4, wherein the therapeutic agent is an anti-cancer agent.
13. The composition of claim 12, wherein the anti-cancer agent is bleomycin.
14. The composition of claim 12, wherein the anti-cancer agent is a STAT3 inhibitor.
15. The composition of claim 14, wherein the STAT3 inhibitor is STAT3-Hcl2A-2.
16. The composition of claim 3, wherein the cell-penetrating molecule is Tat.
17. The composition of claim 1, wherein the composition is non-toxic.
18. A dual modality detection method of detecting cancer in a subjecting comprising: a) administering a composition of claim 1 to the subject, b) performing Magnetic Resonance
Imaging (MRI) on the subject; c) performing flourescence imaging on the subject, c) recording the output from the MRI and the flourescence imaging, comparing the output of the MRI and the output of the flourescence imaging to a control.
19. The method of claiml8, further comprising determining the presence of cancer by the presence of a kinetic decay in MRI contrast enhancement followed by the emergence of a fluorescent signal.
20. The method of claim 19, wherein the kinetic decay in MRI contrast occurs on the order of a 1 to 8 minute time interval and is followed by concommitant emergence of a fluorescent signal in cancer cells.
21. The method of claiml 8, wherein the cancer is breast cancer.
22. The method of claim 18, wherein the contrast agent is linked to a homing peptide
23. The method of claim22, wherein the homing peptide is specific to breast tissue, lung tissue, prostate tissue, pancreatic tissue or intestinal tissue
24. The method of claim23, wherein the breast tissue specific homing peptide is CPGPEGAGC.
25. The method of claim 22, wherein the contrast agent is non-metallic.
26. The method of claim 25, wherein the non-metallic contrast agent is a nitroxide radical or derivative thereof.
27. The method of claim 18, wherein the dual modality detection system is peptide based.
28. The method of claim 18, wherein the fluorophore is Dansyl, TAMRA, a cyanine dye, or a cyanine dye encapsulated in a cyclodextrin.
29. A method of treating cancer in a subject comprising administering the composition of claim 4 to the subject.
30. The method of claim 29, further comprising detecting cancer in the subject.
31. The method of claim 30, wherein detecting cancer comprises the method of claim 18.
32. The method of claim 29, wherein the homing peptide is specific to breast tissue, lung tissue, prostate tissue, pancreatic tissue or intestinal tissue.
33. The method of claim 32, wherein the breast tissue specific homing peptide is CPGPEGAGC.
34. The method of claim 29, wherein the composition comprises a fluorophore as the detection moiety.
35. The method of claim 33, wherein the fluorophore is Dansyl, TAMRA, a cyanine dye, or a cyanine dye encapsulated in a cyclodextrin.
36. The method of claim 29, wherein the composition comprises a non-metallic MRI contrasting agent.
37. The method of claim 36, wherein the non-metallic contrasting agent is a nitroxide radical or derivative thereof.
38. The method of claim 29, wherein the composition comprises an anti-cancer therapeutic agent.
39. The method of claim 38, wherein the anti-cancer agent is bleomycin.
40. The method of claim 38, wherein the anti-cancer agent is a STAT3 inhibitor.
41. The method of claim 40, wherein the STAT3 inhibitor is STAT3-Hel2A-2.
42. The method of claim 29, wherein the composition comprises Tat as the cell- penetrating molecule.
43. The method of claim 29, wherein the composition is non-toxic.
44. A kit comprising the composition of claim 1 and articles for delivery to a subject.
45. The method of claim 18, wherein the method is a computer implemented method.
46. The method of claim 45, further comprising the step of outputting results from the dual modality detection.
47. A method of analyzing a subject comprising; receiving a tissue-specific record of the subject, wherein the record contains the kinetic decay of the contrasting agent; measuring the amount of decay and the amount of fluorescence, and outputting results from the dual modality detection.
48. The method of claim 47, wherein the method is a computer implemented method.
49. The method of claim 47, wherein receiving the tissue-specific record comprises receiving the tissue-specific record from a storage medium.
50. The method of claim 47, wherein receiving the tissue-specific record comprises receiving the record from a computer system.
51. The method of claim 47, wherein receiving the tissue-specific record comprises receiving the record from a whole body imaging system.
52. The method of claim 47, wherein receiving the tissue-specific record comprises receiving the tissue-specific record via a computer network.
53. A method of analyzing the presence of cancer in a subj ect comprising, recommending the performance of methods in claim 47 to be performed.
54. A method comprising the steps of receiving an output from any of claims 41 and recommending treatment with the method of claim 29.
55. One or more computer readable media storing program code that, upon execution by one or more computer systems, causes the computer systems to perform the method of claims 18, 19, oi- 47.
56. A computer program product comprising a computer usable memory adapted to be executed to implement the method of claims 18, 19, or 47.
57. The computer program of claim 56, comprising a logic processing module, a configuration file processing module, a data organization module, and data display organization module, that are embodied upon a computer readable medium.
58. A computer program product, comprising a computer usable medium having a computer readable program code embodied therein, said computer readable program code adapted to be executed to implement a method for generating the dual modality detection of claims 18, 30, and 47, said method further comprising: providing a system, wherein the system comprises distinct software modules, and wherein the distinct software modules comprise a logic processing module, a configuration file processing module, a data organization module, and a data display organization module.
59. The method of claim 58, further comprising a computerized system configured for performing the method.
60. The method of claim 58, further comprising the outputting of the results from the dual modality detection.
61. A computer-readable medium having stored thereon instructions that, when executed on a programmed processor perform the methods of claim 18, 19, or 47.
62. A dual modality detection system, the system comprising: a data store capable of storing tissue-specific data; a system processor comprising one or more processing elements, the one or more processing elements programmed or adapted to: receive tissue-specific data comprising the kinetic decay of the contrasting agent and the presence of fluorescence; store the tissue-specific data in the data store; compare the reduction in the contrast agent to the increase in fluorescence; and output a treatment recommendation based upon the comparison of the decay in contrasting enhancement with the increased fluorescence.
63. The system of claim 62, wherein the system receives the tissue-specific data from a computer system.
64. The system of claim 62, wherein the system receives the tissue-specific data via a computer network.
65. The system of claim 62, further comprising a whole body imaging system.
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