WO2010135431A2 - Compositions, dispositifs, et procédés relatifs à l'antigène membranaire spécifique de la prostate (psma) - Google Patents

Compositions, dispositifs, et procédés relatifs à l'antigène membranaire spécifique de la prostate (psma) Download PDF

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WO2010135431A2
WO2010135431A2 PCT/US2010/035410 US2010035410W WO2010135431A2 WO 2010135431 A2 WO2010135431 A2 WO 2010135431A2 US 2010035410 W US2010035410 W US 2010035410W WO 2010135431 A2 WO2010135431 A2 WO 2010135431A2
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seq
psma
peptide
sequence
amino acid
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PCT/US2010/035410
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WO2010135431A3 (fr
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Gregory A. Weiss
Jessica Arter
Juan E. Diaz
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The Regents Of The University Of California
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Priority to US13/321,011 priority Critical patent/US8986655B2/en
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Publication of WO2010135431A3 publication Critical patent/WO2010135431A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6445—Kallikreins (3.4.21.34; 3.4.21.35)

Definitions

  • the field of the invention is high-affinity prostate-specific membrane antigen (PSMA) ligand motifs and ligands, compositions comprising same, and methods therefor.
  • PSMA prostate-specific membrane antigen
  • PSMA has received considerable attention since its discovery (Horoszewicz et al. (1987) Anticancer Res. 7:927-35), mostly due to its specifically elevated expression by malignant prostate cells (Israeli et al. (1994) Cancer Res. 54: 1807-11; Wright et al. (1996) Urology 48:326-34). Moreover, normal prostate cells predominantly express the cytosolic form while malignant cells express the full-length membrane bound form (Su et al. (1995) Cancer Res. 55: 1441-3). PSMA expression was also reported, albeit at lower levels, in the brain, kidney, salivary gland and duodenum, (e.g. Renneberg et al. (1999) Urol. Res.
  • PSMA is a 750-amino acid type II transmembrane protein with carboxypeptidase activity (specificity: N-acetyl-asp-glu). Remarkably, the majority of the protein is present in the extracellular space and so forms an attractive target for diagnostic and therapeutic agents.
  • monoclonal antibodies can be raised against PSMA and modified for use in diagnostic imaging (Sodee et al. (1998) Prostate 37: 140-8; Lopes et al. (1990) Cancer Res. 50:6423-6429; Elgamal et al. (1998) Prostate 37(4):261-9; Lamb and Faulds (1998) Drugs Aging 12(4):293-304).
  • PSMA antibodies can also be used in tumor vascular imaging and anti- angiogenesis therapy.
  • monoclonal antibodies are often limited to due various factors. Most significantly, antibodies are relatively large molecules and often problematic in terms of large-scale production and isolation. Moreover, and especially where antibodies are used over an extended period, many antibodies will ultimately elicit an immune response. Still further, even though most antibodies have fairly strong affinity towards their antigen, the high molecular weights of antibodies requires relatively large dosages for tagging or otherwise labeling cells with antibodies.
  • antibody fragments or single chain variable chain fragments can be employed. While such approaches advantageously reduce the molecular weight and in at least some instances overcome limited production volume, numerous problems nevertheless remain.
  • purification of scFvs is typically performed from a recombinant source, which introduces a new set of potentially antigenic components.
  • antibody fragments e.g., Fab or F(ab')2
  • protease activity is often carried over to the preparation of antibody fragments, which is highly undesirable.
  • non-antibody ligands can be used as described in U.S. Pat. No. 6,933,114 where high-affinity RNA aptamers were produced in a SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process to so generate binders to PSMA.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • the inventors reported various high-affinity nuclease resistant RNA sequences that inhibited (at low nM Ki) the peptidase activity of PSMA. While such molecules overcome at least some of the difficulties associated with antibodies, other problems tend to arise.
  • RNA aptamers are often relatively long RNA molecules (e.g., 65-70 bases, and even longer), conformational changes may reduce utility of such molecules.
  • RNA molecules are typically bound or otherwise associated with various serum proteins and so rendered ineffective for binding to PSMA, particularly where nucleic acid analogs are used (e.g., phosphorothioate ) to stabilize the molecule against degradation.
  • nucleic acid analogs e.g., phosphorothioate
  • PSMA ligands are known in the art, there is still a need to provide improved PSMA ligand motifs and/or ligands, compositions, and methods using such improved PSMA ligand motifs and/or ligands.
  • PSMA ligand motifs and/or ligands which are most typically peptide ligands and/or peptidomimetic ligands.
  • PSMA ligand motifs and/or ligands comprise a core motif with typically 13 amino acid residues, of which several positions may be altered, preferably using homologous amino acid exchange.
  • the PSMA ligand motifs and/or ligands contemplated herein exhibit significantly higher affinity to the prostate-specific membrane antigen than heretofore known PSMA ligands and are therefore particularly suitable in the manufacture of high- specificity ligand motifs and/or ligands for in vivo use.
  • contemplated PSMA ligand motifs and/or ligands will preferentially bind to the monomeric form or the dimeric form, and may thus act as a neutral ligand, as an agonist, or as an antagonist of the PSMA catalytic function. Consequently, it should be appreciated that the PSMA ligand motifs and/or ligands presented herein may be employed as diagnostic or therapeutic agents, and/or as a research tool (e.g., in SAR studies).
  • the invention provides peptide PSMA binding moieties having a sequence that preferentially binds a monomeric PSMA relative to dimeric PSMA. In a related aspect, the invention provides peptide binding moieties having a sequence that preferentially binds a dimeric PSMA relative to monomeric PSMA.
  • PSMA ligand motifs and/or ligands comprise a recombinant, an isolated, or a synthetic peptide having the sequence
  • Xi to X 5 are independently a natural amino acid or a non-natural amino acid.
  • Such ligand motifs and/or ligands may be further modified and may further include a protecting group, a linker, or other functional or structural moiety.
  • the ligand motifs and/or ligands according to the inventive subject matter are particularly useful as diagnostic agents and/or therapeutic agents.
  • Xi is an amino acid with a linear side chain
  • X 2 and X 4 are independently an amino acid with a charged side chain
  • X 3 is an amino acid with an aromatic moiety the side chain
  • X5 is an amino acid with a non-polar and a hydrophobic side chain
  • Xi is an amino acid with a linear and uncharged side chain
  • X 2 and X 4 are independently an amino acid with a polar and charged side chain
  • X3 is an amino acid with an aromatic and uncharged moiety in the side chain
  • X5 is an amino acid with a non-polar and hydrophobic side chain.
  • Xi is serine or leucine
  • X 2 and X 4 are independently aspartic acid or glutamic acid
  • X3 is phenylalanine or tyrosine
  • X5 is leucine or tryptophan.
  • suitable first PSMA binding moieties include
  • PSMA binding moieties include CALCEFLG [SEQ ID NO: I].
  • a diagnostic reagent for detection and/or quantification of PSMA in a sample will include a first PSMA binding moiety and a solid carrier or detectable moiety, wherein (a) the first PSMA binding moiety is coupled to the solid carrier, or (b) wherein the first PSMA binding moiety is coupled to or part of the detectable moiety.
  • the first PSMA binding moiety is a recombinant or synthetic peptide having a sequence X ⁇ CVEVX S QNSCX 4 X S [SEQ ID NO: 2], wherein Xi to X 5 are independently a natural amino acid or a non-natural amino acid.
  • the diagnostic reagent may further comprise a second PSMA binding moiety, and in certain embodiments the second PSMA binding moieties include CALCEFLG [SEQ ID NO: 1], LDCVEVFQNSCDW [SEQ ID NO: 3], SECVEVFQNSCDW [SEQ ID NO: 4], SDCVEVFQNSCDW [SEQ ID NO: 5], LECVEVYQNSCEW [SEQ ID NO: 6], SECVEVFQNSCEL [SEQ ID NO: 7], and SDCVEVYQNSCDW [SEQ ID NO: 8].
  • the first PSMA binding moiety may have a sequence that preferentially binds a monomeric PSMA
  • the second PSMA binding moiety may have a sequence that preferentially binds a dimeric PSMA.
  • one or more peptides of the present invention may be modified by covalent or non-covalent coupling to one or more additional species.
  • additional species include, but are not limited to, polypeptides, lipids, nucleic acids, cross- linkers, solid phases, contrast agents, toxins, detectable labels, etc.
  • Such coupling may be direct (e.g, a peptide of the invention may be coupled via a peptide bond to an additional polypeptide sequence) or indirect (e.g., a peptide of the invention may be coupled via a linkage chemistry to a solid phase).
  • one or more peptides of the present invention may be coupled to one or more additional species by electrostatic and/or hydrophobic interactions.
  • a covalent bond may be formed between a peptide of the present invention and one or more additional species at the amino terminus, carboxyl terminus, and/or a side chain of the peptide.
  • a plurality of peptides according to the claimed invention may be coupled to one another and/or to one or more additional species to provide a multivalent binding reagent comprising a plurality of PSMA binding ligands.
  • Two or more binding interactions taking place within the same molecular complex have particularly large effects on the dissociation kinetics (ko ff ), as all interactions must be broken simultaneously before dissociation can take place.
  • the gain in apparent affinity through the avidity effect is substantial for a complex with bivalent interaction but may be still increased by increasing the number of interactions.
  • one or more peptides of the present invention may comprise an additional peptide sequence, such as an affinity marker, detection marker, and/or protease cleavage site.
  • affinity/detection markers include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), poly-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and poly-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • Other suitable tag sequences will be apparent to those of skill in the art.
  • proteolytic enzymes e.g., trypsin, subtilisin, thermolysin, chymotrypsin, papain, etc.
  • protease cleavage sites are known to those of skill in the art.
  • one or more proteolytic enzymes for use in the present methods are selected from the group consisting of factor Xa, enterokinase, thrombin, tobacco etch virus protease, and human rhino virus 3 C protease.
  • an introduced protease cleavage site is selected from the group consisting of IEGR [SEQ ID NO.9], DDDDK [SEQ ID NO.10], LVPRGS [SEQ ID NO.l l], ENLYFQG [SEQ ID NO.12], and LEVLFQGP [SEQ ID N0.13].
  • the peptides of the present invention may be chemically synthesized or expressed using recombinant DNA methodology.
  • one or more peptides of the present invention are expressed as part of a fusion protein with an additional polypeptide sequence.
  • Such recombinant proteins may comprise sequences selected from the group consisting of a viral envelope protein, a viral coat protein, a bacterial outer membrane protein, and a eukaryotic membrane protein.
  • reagents often employ functional groups that couple to amino acid side chains of peptides.
  • Moieties that can be targeted using a cross-linker include primary and ⁇ - amines, sulfhydryls, carbonyls, hydroxyls, and carboxylic acids.
  • many reactive groups can be coupled nonselectively using a cross-linker such as photoreactive phenyl azides.
  • the peptides of the present invention are used in a variety of diagnostic compositions and methods as binding ligands for PSMA.
  • the PSMA binding ligands of the present invention may be used in various sandwich, competitive, or non-competitive assay methods, to generate a signal that is related to the presence or amount of PSMA in cells, tissues, body fluids, etc.
  • Suitable assay formats also include chromatographic, mass spectrographic, protein "blotting,” and cell sorting methods.
  • one or more peptides of the present invention are used as targeting moieties for contrast imaging agents.
  • ultrasound imaging contrast agents that exhibit an affinity for specific tissue types, and especially, disease sites, can be prepared using agents such as liposomes that reflect diagnostic ultrasound and, when conjugated to one or more peptides of the present invention, target themselves to cells and tissues expressing PSMA.
  • the procedure for preparation of ultrasound-reflective liposomes generally involves dispersal (e.g., by sonication) of one or more lipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cholesterol (CH) into a buffer, lyophilization, and reconstitution into a pharmaceutically acceptable excipient.
  • lipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cholesterol (CH)
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PG phosphatidylglycerol
  • CH cholesterol
  • Peptide(s) of the present invention may be directly or indirectly coupled to such lipid components prior to or following dispersal, or may be inserted into the liposome by conjugation to an appropriate hydrophobic moiety.
  • Other "microbubble" ultrasound contrast agents
  • MRI contrast agents can be prepared using Gd-perfluorocarbon nanoparticles conjugated to one or more peptides of the present invention.
  • Other MRI contrast agents for example those based on superparamagnetic iron oxide, are known in the art and may be conjugated in a similar fashion.
  • a method of detecting or quantifying PSMA in a sample will include a step of contacting the sample with contemplated diagnostic reagents under conditions to allow binding of the first PSMA binding moiety to a PSMA in the sample. In another step, binding of the diagnostic reagent to the PSMA is detected using the detectable moiety and/or a labeling agent.
  • one or more peptides of the present invention are used as targeting moieties for delivering molecules to cells and tissues which express PSMA.
  • peptides of the present invention may be directly or indirectly conjugated to a therapeutic agent for delivery to such cells or tissues.
  • a therapeutic agent for delivery to such cells or tissues.
  • Such an approach is analogous to gemtuzumab (MYLOTARG®; Wyeth)), which consists of a humanized mAb targeted to a cell surface marker (CD33) linked to the cytotoxic antibiotic ozogamicin (N-acetyl- ⁇ calicheamicin).
  • Suitable therapeutic agents include radioactive isotopes, antifolates, vinca alkaloids, taxanes, cytokines, nucleic acids such as antisense RNA, ribozymes, short interfering RNA (siRNA), apoptosis-inducing ligands such as TRAIL, and anthracyclines.
  • Such therapeutic agents may be provided encapsulated in particles such as liposomes, which are in turn conjugated to one or more PSMA ligands of the present invention. This list is not meant to be limiting.
  • peptide linkers that are stable in serum but which can be readily degraded in intracellular compartments (e.g., disulphide linkers) may be superior to noncleavable linkers.
  • a PSMA binding moiety coupled to an enzyme in a method akin to ADEPT therapy with antibody targeting.
  • the binding moiety preferentially localizes the enzyme molecule at the target site.
  • the enzyme is designed to rapidly convert a prodrug (inactive drug) to its active (cytotoxic) form.
  • a high dose of prodrug can be administered, where activation would preferentially occur at the target site rather than systemically.
  • the sample is a tissue section or a body fluid
  • the step of contacting is performed by adding the diagnostic reagent onto the tissue section or by injecting the diagnostic reagent into the body fluid.
  • the step of detecting may include ultrasound imaging, radiographic imaging, and/or magnetic resonance imaging, and may also include a step of using an anti-PSMA antibody as the labeling agent.
  • the solid carrier may be configured as an addressable array, a virus electrode, a surface plasmon resonance chip, or as a colored bead.
  • Figures IA- 1C are graphs illustrating exemplary binding results for different PSMA ligand motif presenting phages isolated from phage panning.
  • Figure 2 is a schematic illustration of PSMA binding by two distinct ligand motifs.
  • Figure 3 is a graph illustrating exemplary binding results for enhanced PSMA ligand binding using two distinct ligand motifs.
  • Figure 4 is another graph illustrating exemplary binding results for enhanced PSMA ligand binding using two distinct ligand motifs.
  • Figure 5 is a graph illustrating the differential binding modes of phage-displayed PSMA ligand motifs.
  • ligand motifs and/or ligands contemplated herein are employed in diagnostic compositions and methods where the ligand motifs and/or ligands are labeled or otherwise modified to allow production of a specific signal.
  • the ligand motifs and/or ligands are used in therapeutic compositions and methods where the ligand motifs and/or ligands are modified to interfere with the biological function of PSMA expressed on a cell.
  • PSMA ligand motifs and/or ligands presented herein will have a peptide or peptidomimetic portion that binds to the monomeric form of PSMA and/or the dimeric form of PSMA.
  • This portion will typically have a linear length of between 6 and 20, and more preferably between 7 and 15, and most preferably between 8 and 13 amino acids, and have an apparent affinity to PSMA of equal or less than 10 nM (K D ), more preferably equal or less than 1 nM (K D ), and most preferably equal or less than 0.1 nM (K D ).
  • amino acid refers to a molecule containing both amine and carboxyl functional groups, preferably (but not necessarily) bound to the same carbon atom.
  • especially preferred amino acids are alpha-amino acids (preferably having L- configuration) with the general formula H2N-C(H)(R)-COOH, where R is an organic substituent.
  • suitable amino acids include isoleucine, alanine, leucine, asparagine, lysine, aspartate, methionine, cysteine, phenylalanine, glutamate, threonine, glutamine, tryptophan, glycine, valine, proline, serine, tyrosine, arginine, and histidine.
  • contemplated ligand motifs will have the amino acid sequence of
  • Xi is preferably an amino acid with linear side chain, more preferably an amino acid with linear and uncharged side chain, and most preferably serine or leucine
  • X 2 is preferably an amino acid with a charged side chain, more preferably an amino acid with a polar and charged side chain, and most preferably aspartic acid or glutamic acid
  • X 3 is preferably an amino acid with an aromatic moiety in the side chain, more preferably an amino acid with an aromatic moiety in the side chain that is uncharged, and most preferably phenylalanine or tyrosine
  • X 4 is defined as X 2
  • X 5 is preferably an amino acid with non-polar and hydrophobic side chain, and most preferably leucine or tryptophan.
  • ligand motifs include LDCVEVFQNSCDW [SEQ ID NO: 3], SECVEVFQNSCDW [SEQ ID NO: 4], SDCVEVFQNSCDW [SEQ ID NO: 5], LECVEVYQNSCEW [SEQ ID NO: 6], SECVEVFQNSCEL [SEQ ID NO: 7], and SDCVEVYQNSCDW [SEQ ID NO: 8].
  • LDCVEVFQNSCDW [SEQ ID NO: 3]
  • SECVEVFQNSCEL SEQ ID NO: 7
  • SDCVEVYQNSCDW SEQ ID NO: 8
  • the ligand motifs will comprise naturally occurring amino acids
  • various modifications are also deemed appropriate and include, inter alia, backbone modifications to so form a peptidomimetic moiety as well as side-chain modifications.
  • suitable modifications include use of non-naturally occurring amino acids, D-amino acids, and non-peptide spacers (e.g., alkyl, alkoxy, saccharide, etc.) within the backbone of the peptide or peptidomimetic.
  • non-naturally occurring amino acids e.g., D-amino acids, and non-peptide spacers (e.g., alkyl, alkoxy, saccharide, etc.) within the backbone of the peptide or peptidomimetic.
  • the ligands and ligand compositions of the present invention may be chemically synthesized or expressed using recombinant DNA methodology.
  • one or more ligand motifs and/or ligands may be expressed as part of a fusion protein with an additional polypeptide sequence on the N-terminus and/or the C-terminus.
  • additional polypeptide sequences may be naturally occurring sequences (or portions thereof) with immunogenic function, structural function, and/or catalytic function.
  • a ligand or ligand composition may include one or more ligand motifs.
  • one or more ligand motifs may be modified by covalent or non-covalent coupling to one or more additional species.
  • additional species include, but are not limited to, polypeptides, lipids, nucleic acids, cross-linkers, solid phases, contrast agents, toxins, detectable labels, etc.
  • Such coupling may be direct (e.g, one or more ligand motifs may be coupled via a peptide bond to an additional polypeptide sequence) or indirect (e.g., a one or more ligand motifs may be coupled via a linkage chemistry to a solid phase).
  • non-covalent coupling one or more ligand motifs may be coupled to one or more additional species by electrostatic and/or hydrophobic interactions.
  • covalent coupling a covalent bond may be formed between a ligand motif and one or more additional species at the amino terminus, carboxyl terminus, and/or a side chain of the ligand motif.
  • one or more ligand motifs may comprise an additional peptide sequence, such as an affinity marker, detection marker, and/or protease cleavage site.
  • Suitable affinity/detection markers include glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), poly-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and poly-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • Other suitable tag sequences will be apparent to those of skill in the art.
  • proteolytic enzymes e.g., trypsin, subtilisin, thermolysin, chymotrypsin, papain, etc.
  • protease cleavage sites are known to those of skill in the art.
  • one or more proteolytic enzymes for use in the present methods are selected from the group consisting of factor Xa, enterokinase, thrombin, tobacco etch virus protease, and human rhinovirus 3 C protease.
  • ligand motifs may be used to so provide a desirable effect.
  • a first ligand motif may be used to preferentially bind to the monomer while a second ligand motif (which may or may not be covalently bound to the first ligand) will preferentially bind to the dimer.
  • contemplated PSMA ligands may comprise one, two, or even more ligand motifs, which may be separated by an optional spacer that may or may not be a peptide.
  • first and a second ligand motifs may be in a form in which the ligand motifs are directly covalently coupled to each other, or in which first and second ligand motifs are coupled to each other via a common carrier or other molecule.
  • the ligand motif will include (in addition to an optionally second ligand motif as described above) the heretofore known PSMA peptide ligand having the amino acid sequence of CALCEFLG [SEQ ID NO: I].
  • a plurality of ligand motifs according to the inventive subject matter may be coupled to one another and/or to one or more additional species to provide a multivalent binding reagent comprising a plurality of PSMA binding ligands.
  • Two or more binding interactions taking place within the same molecular complex may have particularly large effects on the dissociation kinetics (ko ff ), as all interactions must be broken simultaneously before dissociation can take place.
  • the gain in apparent affinity through the avidity effect is substantial for a complex with bivalent interaction but may be still increased by increasing the number of interactions.
  • the compounds contemplated herein may comprise in addition to the ligand motif(s) additional moieties that may impart a desirable functionality.
  • suitable additional moieties may include one or more spacers between two ligand motifs to optimize flexibility and/or solubility and so afford increased affinity and/or bioavailability.
  • the spacer between two or more ligand motifs may include a cleavage site specific for PSMA to so generate two or more ligands at the site of catalytic action.
  • additional moieties may be added to the C- and/or N-terminal portion (or intervening portion between two ligand motifs) to allow for detection of the modified compound.
  • moieties include radiolabels (e.g., fluorine, carbon, technetium or indium radiolabel), affinity moieties (e.g., via biotinylation, etc.), enzymatic moieties (e.g., beta galactosidase), fluorescent or luminescent moieties (e.g., green fluorescent protein), MRI imaging reagents (e.g., gadolinium chelates), and so forth.
  • radiolabels e.g., fluorine, carbon, technetium or indium radiolabel
  • affinity moieties e.g., via biotinylation, etc.
  • enzymatic moieties e.g., beta galactosidase
  • fluorescent or luminescent moieties e.g., green fluorescent protein
  • MRI imaging reagents e.g., gadolinium chelates
  • modifications may also be included into the amino acid portion of the motif (e.g., PET/SPECT detectable
  • contemplated moieties also include solid phases, and it is especially preferred that the solid phase is part of a wall of a fluid container (e.g., wall of a micro well, reaction tube, capillary, microfluidic device, etc.).
  • the solid phase may also be a bead or other insoluble material, preferably with a characterized physical parameter.
  • suitable solid phases will include magnetic beads, colored beads, but also filter membranes or affinity membranes.
  • contemplated ligands or ligand motifs may be coupled to a solid phase in an addressable format using coupling methodologies well known in the art.
  • ligand motifs contemplated be may further modified with one or more additional moieties to so impart a desirable function.
  • all ligand motifs and/or ligands may be present in form of a prodrug, a metabolite, and/or a salt with a pharmaceutically acceptable acid or base.
  • prodrug refers to a modification of ligand motifs and/or ligands, wherein the modified compound exhibits less pharmacological activity (as compared to the modified compound) and wherein the modified compound is converted within the body (e.g., in a target cell or target organ) back into the unmodified form through enzymatic or non- enzymatic reactions.
  • conversion of ligand motifs and/or ligands into prodrugs may be useful where the active drug is too toxic for safe systemic administration, or where the contemplated compound is poorly absorbed by the digestive tract or other compartment or cell, or where the body breaks down the ligand motifs and/or ligands before reaching its target.
  • the ligands or ligand motifs according to the inventive subject matter can be modified in numerous manners, and especially preferred modifications include those that improve one or more pharmacokinetic and/or pharmacodynamic parameter.
  • one or more substituents may be added or replaced to achieve a higher AUC in serum.
  • hydrophilic groups may be added.
  • Exemplary suitable protocols for conversion of contemplated ligands or ligand motifs into the corresponding prodrug form can be found in "Prodrugs (Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs)" by Kenneth B.
  • the ligand motifs presented herein may be modified to form a diagnostic reagent, a therapeutic reagent, and/or a reagent suitable for research and development as it relates to PSMA and modulation of PSMA activity.
  • contemplated pharmaceutical compositions comprise a therapeutically effective amount of contemplated ligand motifs and/or ligands (or pharmaceutically acceptable salt, hydrate, or prodrug thereof), and a pharmaceutically acceptable carrier.
  • contemplated compositions are formulated for diagnosis, staging, and/or treatment of prostate cancer and tumor neovasularization.
  • contemplated ligand motifs and/or ligands are included in a composition that is formulated with one or more non-toxic pharmaceutically acceptable carriers.
  • Suitable pharmaceutical compositions are preferably formulated for oral administration in solid or liquid form, or for parenteral injection.
  • pharmaceutical compositions according to the inventive subject matter may be administered to humans and other animals using various routes, including orally, rectally, parenterally, intraperitoneally, vaginally, or topically.
  • suitable pharmaceutical compositions for injection preferably comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, emulsions, or suspensions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, oils, and injectable organic esters
  • Contemplated compositions may also contain various inactive ingredients, including preservatives, wetting agents, emulsifying agents, and/or dispersing agents. Sterility may be ensured by inclusion of antibacterial and/or antifungal agents (e.g., paraben, phenol sorbic acid, chlorobutanol, etc.). Where appropriate, osmotically active agents may be included (e.g., sugars, sodium chloride, etc.).
  • antibacterial and/or antifungal agents e.g., paraben, phenol sorbic acid, chlorobutanol, etc.
  • osmotically active agents may be included (e.g., sugars, sodium chloride, etc.).
  • contemplated compositions may be formulated into solid dosage forms for oral administration, and may therefore be capsules, tablets, pills, powders, and granules.
  • contemplated ligand motifs and/or ligands are mixed with at least one of a pharmaceutically acceptable excipient or carrier (e.g., sodium citrate or dicalcium phosphate), a filler or extender (e.g., starch, lactose, sucrose, glucose, mannitol, or silicic acid), a binder (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, etc.), a humectant (e.g., glycerol), a disintegrating agent (e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, or sodium carbonate), a solution retarding agent (e.g., par
  • Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art.
  • Contemplated compositions may further be formulated to release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
  • Contemplated ligand motifs and/or ligands may also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • liquid dosage forms may contain inert diluents commonly used in the art (e.g., water, or other solvent, solubilizing agents), emulsifiers (e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide), oils (and in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions may also include
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Ligand motifs and/or ligands according to the inventive subject matter can also be administered in form of liposomes, which may be unilamellar, oligolamellar, or polylamellar. Contemplated compositions in liposome form may further contain stabilizers, preservatives, excipients, etc.
  • Preferred lipids for liposome formation include phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.
  • Methods to form liposomes are known in the art (e.g., Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N. Y. (1976), p. 33 et seq.)
  • contemplated ligand motifs and/or ligands in pharmaceutical and/or diagnostic compositions may be varied so as to obtain an amount of contemplated ligand motifs and/or ligands that is effective to achieve the desired diagnostic or therapeutic response for a particular patient, composition, and mode of administration.
  • the selected dosage level will depend upon various factors, including the avidity and activity of the particular ligand motifs and/or ligands, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated.
  • ligand motifs and/or ligands are started at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • dosage levels of about 0.01 mg to about 500 mg, more preferably of about 0.5 mg to about 50 mg of contemplated ligand motifs and/or ligands per kilogram of body weight per day are administered orally to a mammalian patient.
  • the effective daily dose may be divided into multiple doses for purposes of administration, e.g., two to four separate doses per day.
  • contemplated pharmaceutical compositions may also include additional pharmaceutically active compounds, and contemplated additional active compounds include anti-neoplastic and anti-angiogenic agents as further discussed below.
  • one or more ligand motifs are used as targeting moieties for delivering molecules to cells and tissues which express PSMA.
  • one or more ligand motifs may be directly or indirectly conjugated to a therapeutic agent for delivery to such cells or tissues.
  • a therapeutic agent for delivery to such cells or tissues.
  • Such an approach is analogous to gemtuzumab (MYLOTARG®; Wyeth)), which consists of a humanized mAb targeted to a cell surface marker (CD33) linked to the cytotoxic antibiotic ozogamicin (N- acetyl- ⁇ calicheamicin).
  • Suitable therapeutic agents include radioactive isotopes, antifolates, vinca alkaloids, taxanes, cytokines, nucleic acids such as antisense RNA, ribozymes, short interfering RNA (siRNA), apoptosis-inducing ligands such as TRAIL, and anthracyclines.
  • Such therapeutic agents may be provided encapsulated in particles such as liposomes, which are in turn conjugated to one or more PSMA ligands of the present invention.
  • peptide linkers that are stable in serum but which can be readily degraded in intracellular compartments (e.g., disulfide linkers) may be superior to non-cleavable linkers.
  • one or more ligand motifs are used as targeting moieties for directed enzyme prodrug therapy approach (analogous to ADEPT), which specifically aims at causing bystander effects by targeting enzymes to a targeted cell and delivering a prodrug that is locally converted to a chemotherapeutic by the targeted enzyme.
  • contrast agent As used herein, "contrast agent”, “contrast medium” and “imaging agent” relate to any composition administered in vivo to obtain images of an area of interest of a body.
  • the images may be obtained using any imaging technique known in the art.
  • use of such agent provides an enhanced image of the body structures within the area of interest as compared to an image obtained without use of any such agent.
  • one or more ligand motifs are used as targeting moieties for contrast imaging agents.
  • ultrasound imaging contrast agents that exhibit an affinity for specific tissue types, and especially, disease sites, can be prepared using agents such as liposomes that reflect diagnostic ultrasound and, when conjugated to one or more ligand motifs and/or ligands, target themselves to cells and tissues expressing PSMA.
  • the procedure for preparation of ultrasound-reflective liposomes generally involves dispersal (e.g., by sonication) of one or more lipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cholesterol (CH) into a buffer, lyophilization, and reconstitution into a pharmaceutically acceptable excipient.
  • lipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cholesterol (CH)
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PG phosphatidylglycerol
  • CH cholesterol
  • Contemplated ligands may be directly or indirectly coupled to such lipid components prior to or following dispersal, or may be inserted into the liposome by conjugation to an appropriate hydrophobic moiety.
  • Other "microbubble" ultrasound contrast agents are known in
  • MRI contrast agents can be prepared using Gd-perfluorocarbon nanoparticles conjugated to one or more ligand motifs and/or ligands.
  • Other MRI contrast agents for example those based on superparamagnetic iron oxide, are known in the art and may be conjugated in a similar fashion.
  • methods for delivering such targeted contrast agents comprise administering a pharmaceutical formulation comprising one or more such contrast agents to a subject in need thereof.
  • Formulations may be administered by various enteral and parenteral routes, including intravenous, oral, intraperitoneal, subcutaneous, rectal and intrauterine routes of delivery.
  • Such methods may further comprise imaging the subject using an imaging method appropriate to the contrast agent to determine any site(s) of accumulated contrast agent in the subject.
  • Ultrasound is an imaging process that relies on the reflection of sound waves within the body to produce an image thereof.
  • High frequency sound (ultrasonic) waves which are above the range of sound audible to humans, are directed at the region of interest within the body.
  • the waves are reflected back wherever there is a change in the physical parameters of the structures within the body, e.g., a change in density between two adjacent organs.
  • the ultrasound equipment receives the reflected sound waves and transmits them into an image based on the differing levels of intensity of the reflected waves.
  • contrast agent enhances the differences in intensities of the reflected waves.
  • intravenous encapsulated microbubble contrast agents have become an established clinical tool for enhancing medical diagnostic ultrasound and Doppler sensitivity.
  • contrast agents function to enhance the appearance of the blood pool and to define its architecture and integrity.
  • Other contrast agents provide targeted, organ-specific imaging based upon the biodistribution and pharmacokinetics of the circulating contrast agent.
  • Albunex from Molecular Biosystems, San Diego, CA
  • the first commercially available ultrasound contrast agent is a suspension of air- filled albumin microspheres produced by sonication of a heated solution of 5% human albumin.
  • microbubble response relative to a driving acoustic pressure can be divided into three categories: (1) linear scattering, (2) nonlinear scattering, and (3) cavitati on/destruction.
  • Microbubbles produce linear scattering with low acoustic driving pressures and produce non linear scattering with moderate acoustic driving pressures.
  • microbubbles exhibit pressure peaks at the compressional phases of the source thereby providing both harmonic and subharmonic energy greater than the surrounding medium.
  • microbubbles cavitate or destruct as a result of fragmentation and deflation and thus create an associated acoustic emission signal.
  • the absolute values for low, moderate and high acoustic driving pressures are not well defined and depend upon not only the acoustic parameters of the ultrasonic source but also the constituent physical properties of the microbubbles themselves, as well as the fluid surrounding them.
  • the duration of reflectivity was prolonged drastically to a half-lifetime of 866 minutes by incorporating cholesterol into the MLV, although, significantly, the echogenicity was decreased by such incorporation. Although there have been a number of important steps at lengthening the effective imaging half-life of injectable ultrasonic contrast agents using liposomes, there has been an overall reduction in the echogenicity of these agents.
  • MR magnetic resonance
  • This modality relies on detecting the emission of electromagnetic radiation by certain atomic nuclei in the body upon application of pulsed radio frequency signals in the presence of a magnetic field. The resulting magnetic echoes produced when the signal is terminated ultimately are translated into an image.
  • contrast agents are commonly used intravenously to change the local magnetic field in tissue. Generally, abnormal tissue will respond differently in the presence of the contrast agent as compared to normal tissue and will give off a different magnetic echo. Thus, when the magnetic echoes are translated into an image, an image of the tissue abnormalities is provided.
  • Gd2O3 gadolinium oxide
  • MAM albumin microspheres
  • MAM Magnetic Oral MR Contrast Agent
  • HSA human serum albumin
  • magnetite to create albumin microspheres using a modified water-in-oil emulsion polymerization technique.
  • Nonlinear behavior of MAM with increased applied external magnetic field over 0.3-0.9T was observed.
  • the influence of magnetite on T2 relaxation is believed to be due to local field inhomogeneities generated by the large magnetic moment of Fe3O4, which causes dephasing of proton spins and an acceleration of T2 relaxation with negligible To effects.
  • MAM has limited usefulness in conventional MR imaging. Additionally, based on the lower density of iron oxide relative to other heavy metals, iron oxide, and thus MAM, has a very limited utility for other imaging modalities, such as computed tomography.
  • Computed tomography also called computerized axial tomography, is an imaging modality that utilizes a toroidal, or donut-shaped x-ray camera to provide a cross sectional image of the body area of interest.
  • CT computed tomography
  • the contrast agent localizes in a particular body compartment and differentially opacities normal or abnormal tissue.
  • the contrast agent causes the tissue to inhibit passage of x-rays to produce a shadow of positive contrast in the resulting image.
  • Iodine- based contrast agents are considered to be the industry standard with CT.
  • Gd-DTPA contrast agents have been used for certain limited applications in conventional angiography and CT imaging. (Bloem and Wondergem, "Gd- DTPA as a Contrast Agent in CT," Radiology, 171:578-79 (1989)). Perfluorocarbon emulsions have also been evaluated for contrast image enhancement. Perflubron (perfluorooctyl bromide, "PFOB”) emulsified with egg yolk lecithin has been tested for use in US (due to its high density), MR (fluorine nuclei imaging or as a signal void for hydrogen nuclei imaging) and CT imaging (due to its bromine atom).
  • PFOB perfluorooctyl bromide
  • contrast agents may be unable to reach sufficient concentrations in a site of interest (e.g., the site of a tumor) to either produce a strong signal for imaging or to carry optimal amounts of therapeutic agents into the diseased tissues.
  • a site of interest e.g., the site of a tumor
  • One approach to overcome this problem is to develop tumor- targeted agents that are highly sensitive imaging probes and/or are capable of conjugating large amounts of therapeutic agents.
  • Antibody -based targeted contrast media have been found to maintain both the properties of the antibody and the contrast particles.
  • one or more ligand motifs may be used as targeting moieties for binding detectable labels to PSMA.
  • Suitable detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).
  • a detectable reaction product e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.
  • a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, malto
  • Assay devices and methods known in the art can utilize specific binding agents such as the ligand motifs and/or ligands in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of a biomarker of interest, in this case PSMA.
  • Suitable assay formats also include chromatographic, mass spectrographic, and protein "blotting" methods.
  • certain methods and devices, such as biosensors and optical assays may be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S.
  • Patents 5,631,171; and 5,955,377 each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims.
  • robotic instrumentation including but not limited to Beckman ACCESS®, Abbott AXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems are among the immunoassay analyzers that are capable of performing immunoassays.
  • any suitable immunoassay-type format may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like.
  • Contemplated ligand motifs and/or ligands may be immobilized onto a variety of solid supports for use in assays.
  • Solid phases that may be used to immobilize specific binding members include those developed and/or used as solid phases in solid phase binding assays. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple-well plates.
  • An assay strip could be prepared by coating the ligand motifs and/or ligands in an array on solid support.
  • ligand motifs and/or ligands may also be bound to specific zones of assay devices either by conjugating directly to an assay device surface, or by indirect binding.
  • ligand motifs and/or ligands may be immobilized on particles or other solid supports, and that solid support immobilized to the device surface. Consequently, targeted detectable labeled molecules may be used in various sandwich, competitive, or non- competitive assay methods, to generate a signal that is related to the presence or amount of PSMA in cells, tissues, body fluids, etc.
  • Suitable assay formats also include chromatographic, mass spectrographic, protein "blotting", and cell sorting methods.
  • Biological assays require methods for detection, and one of the most common methods for quantitation of results is to conjugate a detectable label to a protein or nucleic acid that has affinity for one of the components in the biological system being studied.
  • Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).
  • a detectable reaction product e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.
  • Detectably labeled PSMA ligand motifs and/or ligands may also find use in flow cytometry.
  • Flow cytometry and the related fluorescence-activated cell sorting provide a method for detecting and sorting a target cell from a heterogeneous mixture of biological cells based upon the specific light scattering and fluorescent characteristics of each cell. It provides fast, objective and quantitative recording of fluorescent signals from individual cells, and can also provide physical separation of cells of particular interest.
  • a beam of light (usually laser light) is directed onto a hydro-dynamically focused stream of fluid.
  • Detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC), as well as one or more fluorescent detectors.
  • FSC Forward Scatter
  • SSC Segment Scatter
  • Each suspended particle from 0.2 to 150 micrometers passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle (e.g., by means of a specific ligand) may be excited, and a combination of scattered and fluorescent light may be picked up by the detectors.
  • fluorescent chemicals found in the particle or attached to the particle e.g., by means of a specific ligand
  • a combination of scattered and fluorescent light may be picked up by the detectors.
  • Coupling of the ligand motifs and/or ligands can be achieved by non-covalent interactions, for example, electrostatic interactions, hydrophobic interactions, Van der Waals interactions, and hydrogen bond interactions. In the alternative, coupling may be by means of covalent bonds.
  • Chemical cross- linkers also useful for preparing such peptide conjugates. Cross-linking agents often employ functional groups that couple to amino acid side chains of peptides.
  • reagents may be classified on the basis of the following: (1) functional groups and chemical specificity; (2) length and composition of the cross-bridge; (3) whether the cross-linking groups are similar (homobifunctional) or different (heterobifunctional); (4) whether the groups react chemically or photochemically; (5) whether the reagent is cleavable; and (6) whether the reagent can be radiolabeled or tagged with another label.
  • Reactive groups that can be targeted using a cross-linker include primary amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids.
  • many reactive groups can be coupled nonselectively using a cross-linker such as photoreactive phenyl azides.
  • Cross-linking reagents contain at least two reactive groups, and are divided generally into homofunctional cross-linkers (containing identical reactive groups) and heterofunctional cross-linkers (containing non-identical reactive groups). While for convenience the following discussion refers to homobifunctional and heterobifunctional cross-linkers (where "bifunctional” refers to the presence of two functional groups), cross-linking reagents having more than two functional groups are well known to the artisan and are within the scope of the invention described herein.
  • Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non- specifically are available from many commercial sources.
  • Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are thiol reactive groups.
  • Maleimides, alkyl and aryl halides, and alpha-haloacyls react with sulfhydryls to form thiol ether bonds, while pyridyl disulfides react with sulfhydryls to produce mixed disulfides.
  • the pyridyl disulfide product is cleavable.
  • Imidoesters are also very useful for protein-protein cross-links.
  • cross-linkers can penetrate cell membranes and cross-link proteins within the membrane to study membrane composition, structure and protein-protein and protein-lipid interactions. Imidoesters are also useful for oligomer formation. For example, cross-linking proteins to form oligomers may reveal if a bivalent, dimeric or trimeric form of the protein is responsible for activity.
  • a nonselective homobifunctional cross-linker is useful for conjugating functional groups, such as hydroxyls for which specific cross-linkers are not available.
  • An example of a nonselective homobifunctional cross-linker is BASED (Product #21564 Pierce Co.).
  • This cross-linker has a long spacer arm and 2 aromatic rings which makes it very hydrophobic with a limited solubility in aqueous systems.
  • This cross-linker also has a large diffusion capacity and may be useful for permeation of biological membranes before conjugation initiates.
  • Heterobifunctional cross-linkers possess two or more different reactive groups that allow for sequential conjugations with specific groups of proteins, minimizing undesirable polymerization or self-conjugation.
  • Heterobifunctional reagents are also used when modification of amines is problematic. Amines may sometimes be found at the active sites of macromolecules, and the modification of these may lead to the loss of activity. Other moieties such as sulfhydryls, carboxyls, phenols and carbohydrates may be more appropriate targets.
  • a two-step strategy allows for the coupling of a protein that can tolerate the modification of its amines to a protein with other accessible groups.
  • a variety of heterobifunctional cross-linkers, each combining different attributes for successful conjugation are commercially available. Cross-linkers that are amine-reactive at one end and sulfhydryl-reactive at the other end are quite common.
  • heterobifunctional reagents If using heterobifunctional reagents, the most labile group is typically reacted first to ensure effective cross-linking and avoid unwanted polymerization.
  • a selection of heterobifunctional reagents that contain at least one photoaffinity group are also commercially available. This selection includes iodinatable and cleavable reagents that react nonspecifically at the azido group and with amines, sulfhydryls, carbohydrates and carbonyls.
  • cross-linker-to-target molar ratios Many factors must be considered to determine optimum cross-linker-to-target molar ratios. Depending on the application, the degree of conjugation is an important factor. For example, when preparing immunogen conjugates, a high degree of conjugation is normally desired to increase the immunogenicity of the antigen. However, when conjugating to an antibody or an enzyme, a low-to-moderate degree of conjugation may be optimal to ensure that the biological activity of the protein is retained. It is also important to consider the number of reactive groups on the surface of the protein. If there are numerous target groups, a lower cross-linker-to-protein ratio can be used. For a limited number of potential targets, a higher cross-linker-to-protein ratio may be required. This translates into more cross-linker per gram for a small molecular weight protein.
  • Conformational changes of proteins associated with a particular interaction may also be analyzed by performing cross-linking studies before and after the interaction. A comparison is made by using different arm-length cross-linkers and analyzing the success of conjugation.
  • the use of cross-linkers with different reactive groups and/or spacer arms may be desirable when the conformation of the protein changes such that hindered amino acids become available for cross-linking.
  • Cross-linkers are available with varying lengths of spacer arms or bridges connecting the reactive ends.
  • the most apparent attribute of the bridge is its ability to deal with steric considerations of the moieties to be linked. Because steric effects dictate the distance between potential reaction sites for cross-linking, different lengths of bridges may be considered for the interaction. Shorter spacer arms are often used in intramolecular cross-linking studies, while intermolecular cross-linking is favored with a cross-linker containing a longer spacer arm.
  • ligand motifs is used on a solid carrier as a sensor or affinity marker to so allow measurement (qualitative or quantitative) of PSMA.
  • suitable devices will include those in which one or more of the ligand motifs is immobilized on a surface to so form an electrode as described in WO2007/104058, which is incorporated by reference herein.
  • the presence of PSMA isolated or on a cell
  • kinetic or SAR studies can be undertaken that help identify binding requirements of a ligand to PSMA or that help determine the interaction of a non-ligand motif compound with PSMA (e.g., using a competitive assay).
  • Further suitable devices will especially include those in which one or more of contemplated ligand motifs are immobilized on a solid carrier in an addressable fashion (e.g., in an addressable x-y grid, or in association with a colored bead). Examples
  • PSMA-binding peptides were selected from phage-displayed peptide libraries with both linear and disulfide-constrained peptide binding motifs.
  • An eight residue long peptide with the amino acid sequence CALCEFLG [SEQ ID NO: 1] was previously identified as a PSMA binder from an 8-mer (X8) library.
  • Phage-displayed ELISAs, quartz crystal microbalance (QCM) studies, and electrochemical impedance spectroscopy (EIS) measurements demonstrated strong binding by CALCEFLG [SEQ ID NO: 1] (also referred to as peptide 1 in the Figures) to PSMA as reported previously.
  • CALCEFLG In addition to CALCEFLG [SEQ ID NO: 1], the inventors also examined a longer 13-mer peptide, selected from a disulfide-constrained library with the amino acid sequence LDCVEVFQNSCDW [SEQ ID NO: 3] (also referred to as peptide 2 in the Figures).
  • Phage-displayed ELISAs demonstrate the varying strengths of [SEQ ID NO: 1] and [SEQ ID NO: 3], in binding to the monomeric and dimeric isoforms of PSMA as can be taken from Figure 5.
  • the graph illustrates phage-displayed peptides binding to 1 ⁇ g/mL PSMA dimer and monomer in an ELISA.
  • Peptide 1 [SEQ ID NO: 1] binds with a higher affinity to PSMA dimer than peptide 2 [SEQ ID NO: 3].
  • Peptide 2 [SEQ ID NO: 3]does not bind PSMA monomer with appreciable affinity.
  • peptide 1 [SEQ ID NO: 1] With efficient binding to both the monomeric and homodimeric PSMA, peptide 1 [SEQ ID NO: 1] is thought to recognize a site on the individual monomer subunit that remains accessible even when the protein dimerizes. However, the apparent affinity of peptide 1 [SEQ ID NO: 1] for dimeric PSMA exceeds affinity for the monomeric isoform. As suggested by this higher apparent affinity for dimeric PSMA, the dimer provides twice the binding sites for peptide 1 [SEQ ID NO: 1] to bind. However, the longer peptide 2 [SEQ ID NO: 3], only binds to dimeric PSMA, and fails to bind the monomer with significant affinity. Based on the two distinct binding modes of the two peptides, the inventors contemplate that two ligand motifs could work in concert, resulting in higher sensitivity and a lower limit of PSMA detection.
  • Affinity maturation can increase the binding strength of a peptide for a target molecule.
  • One effective affinity maturation method applies homologous amino acid substitutions in every position of the peptide sequence, termed homolog shotgun scanning.
  • the technique probes the effects of closely homologous side chain substitutions (e.g., Tyr mutated to a 1 : 1 mixture of Tyr and Phe) in every position, and allows for selection of peptides with improved binding to its target.
  • the peptide sequence was used as a template for a homolog shotgun scanning library synthesized by site-directed mutagenesis.
  • the phage-based ELISA demonstrates that peptides 3 and 4 [SEQ ID NOs: 4 and 5] possess a higher affinity towards the target.
  • the negative control phage which lack a displayed peptide, show no significant binding to PSMA, as expected.
  • a phage-based ELISA with 20 nM phage demonstrates that peptide 3 [SEQ ID NO: 4] possesses -1000 times higher affinity for PSMA compared to the parent peptide 2 [SEQ ID NO: 3], and -100 times higher affinity than that of peptide 1 [SEQ ID NO: 2] based upon comparison of phage concentration versus HRP activity of different peptide-displaying phage as can be taken from Figures IB and 1C.
  • results for phage- based ELISAs are shown comparing binding affinities of peptides to PSMA (1 ⁇ g/mL).
  • Figure IA illustrated peptides selected from homolog shotgun scanning library.
  • Figure IB illustrates relative affinity of selected peptide 3 [SEQ ID NO: 4] versus the previous highest affinity peptide 1 [SEQ ID NO: 2].
  • Figure 1C illustrates a bar graph showing that 20 nM phage displaying peptide 1 [SEQ ID NO: 2] has binding affinity similar to that of 0.2 nM phage displaying peptide 3 [SEQ ID NO: 4], and 20 nM phage displaying parent peptide 2 [SEQ ID NO: 3] has binding affinity similar to 0.02 nM phage displaying peptide 3 [SEQ ID NO: 4].
  • peptide 3 [SEQ ID NO: 4] demonstrates no detectable cross-reactivity for binding transferrin receptor (TfR), a homolog of PSMA (54% sequence identical). It should be noted that the two substitutions incorporated in the sequence of peptide 3 [SEQ ID NO: 4] were LlS and D2E. The serine substitution removes a hydrophobic leucine residue, which provides the peptide with a more polar side chain, which can accept and donate hydrogen bonds (H-bonds).
  • the hydroxyl group likely improves the surface complementarity at the binding interface with PSMA by creating new H-bonding contacts with the protein backbone, or by participating in an extended H-bonding network directly or indirectly through water to other side chains at the binding site.
  • the selection of GIu in place of Asp merely extends the side chain length by one carbon unit. This substitution maintains contribution of the carboxylate functionality, and the length extension of 1.5 A could allow for new H-bonding or salt-bridging opportunities.
  • the core sequence of the selected peptides is critical for the molecular recognition of PSMA, and this core remained conserved in the tightest binding peptides.
  • a second PSMA ligand motif was coupled to a first ligand motif. Specifically, one of the ligand motifs was presented via phage display while the other was attached to the surface of the phage. Of course, it should be appreciated that numerous alternative manners of coupling are also deemed suitable.
  • the negatively charged phage coat of Ml 3 was used to link artificial receptors for multivalent target binding. Three solvent- exposed, negatively charged residues confer an overall net negative charge to the phage coat.
  • a genetically synthesized fusion peptide was constructed from a 14-mer oligopeptide that was made from lysine and arginine residues (total of 8 lysine and 6 arginine, KRl 4), which was linked to the PSMA-binding peptide 1 [SEQ ID NO.2] and termed 1-KR14.
  • the variation of lysine and arginine residues facilitated amplification of the gene sequence by PCR, assisted in subcloning, and helped avoid potential translational problems during ribosomal protein synthesis.
  • an oligolysine (K14) peptide and the PSMA-binding peptide 1 [SEQ ID NO.2] were chemically synthesized in parallel via solid phase synthesis. Then, an azide or alkyne functionality was attached to the N-terminus of each peptide and the two peptides were coupled via a [3+2] cycloaddition reaction, yielding the fused 1-K14 peptide.
  • the 1-K14 peptide wraps the surface of the phage for artificial display of peptide 1 [SEQ ID NO.2].
  • the 1-KR14 peptide was expressed as a C-terminal fusion to maltose-binding protein (MBP).
  • MBP maltose-binding protein
  • TEV Tobacco etch virus
  • Both peptide 1 [SEQ ID NO.2] and the K14 peptide were synthesized separately via solid-phase peptide synthesis. After additional coupling of 4-pentynoic acid to K14, and A- azido-butanoic acid to peptide 1 [SEQ ID NO.2], the two peptides were coupled via [3+2] cycloaddition reaction to provide the desired fusion product 1-K14 (termed peptide 10 in the Figures). After HPLC purification, as well as characterization by MALDI-TOF analysis, the fusion product was isolated in pure form.
  • a phage-based ELISA was performed using purified peptide 10 to determine whether the peptide enhances the binding to immobilized PSMA.
  • the results obtained demonstrated that phage displaying peptide 3 [SEQ ID NO.4] coated with 1-K14, binds with a 16 times higher affinity than the uncoated phage as can be taken from Figure 3.
  • the graph illustrates results for the phage displaying peptide 3 [SEQ ID NO.4], wrapped with peptide 10 binding 1 ⁇ g/mL PSMA dimer.

Abstract

La présente invention concerne des ligands à PSMA, des compositions, et des procédés selon lesquels le ligand est un peptide ayant la séquence X1X2CVEVX3QNSCX4X5 où X1-X5 sont indépendamment un acide aminé naturel ou non naturel ou un peptide ayant la séquence CALCEFLG [SEQ ID n° : 1]. Les aspects particulièrement préférés comprennent des réactifs diagnostiques pour la détection et/ou la quantification de PSMA dans un échantillon, des réactifs thérapeutiques, et des réactifs d'imagerie diagnostiques.
PCT/US2010/035410 2009-05-19 2010-05-19 Compositions, dispositifs, et procédés relatifs à l'antigène membranaire spécifique de la prostate (psma) WO2010135431A2 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103048461A (zh) * 2012-12-18 2013-04-17 苏州浩欧博生物医药有限公司 一种癌抗原ca15-3的纳米磁微粒化学发光测定试剂盒及其制备方法和检测方法
CN103048473A (zh) * 2012-12-18 2013-04-17 苏州浩欧博生物医药有限公司 一种促卵泡生成激素的纳米磁微粒化学发光测定试剂盒及其制备方法和检测方法
WO2018134691A2 (fr) 2017-01-20 2018-07-26 Juno Therapeutics Gmbh Conjugués de surface cellulaire et compositions cellulaires et méthodes associées
WO2018187791A1 (fr) 2017-04-07 2018-10-11 Juno Therapeutics, Inc Cellules génétiquement modifiées exprimant un antigène membranaire spécifique de la prostate (psma) ou une forme modifiée de celui-ci et procédés associés
WO2020097531A1 (fr) * 2018-11-08 2020-05-14 Indi Molecular, Inc. Agents de capture théranostiques, compositions et procédés d'utilisation et de fabrication
CN111182927A (zh) * 2017-12-11 2020-05-19 慕尼黑工业大学 用于成像和腔内放射治疗的psma配体
US11719705B2 (en) 2017-06-15 2023-08-08 Indi Molecular, Inc. IL-17F and IL-17A-specific capture agents, compositions, and methods of using and making
US11723944B2 (en) 2015-03-16 2023-08-15 Indi Molecular, Inc. Botulinum neurotoxin-specific capture agents, compositions, and methods of using and making
US11733246B2 (en) 2020-11-03 2023-08-22 Indi Molecular, Inc. Compositions, imaging, and therapeutic methods targeting folate receptor 1 (FOLR1)
US11884707B2 (en) 2016-09-29 2024-01-30 Regeneron Pharmaceuticals, Inc. Compositions for detection, inhibition and imaging of indoleamine 2, 3-dioxygenase 1 (IDO1) and methods of making and using same

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070020278A1 (en) * 2004-01-09 2007-01-25 Millennium Pharmaceuticals, Inc.*Ewc* Diagnosing and treating cancer
US20080081768A1 (en) * 2006-02-20 2008-04-03 Watt Paul M Methods of constructing and screening libraries of peptide structures
US20090092965A1 (en) * 2006-03-09 2009-04-09 The Regents Of The University Of California Method and Apparatus for Target Detection Using Electrode-Bound Viruses

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070020278A1 (en) * 2004-01-09 2007-01-25 Millennium Pharmaceuticals, Inc.*Ewc* Diagnosing and treating cancer
US20080081768A1 (en) * 2006-02-20 2008-04-03 Watt Paul M Methods of constructing and screening libraries of peptide structures
US20090092965A1 (en) * 2006-03-09 2009-04-09 The Regents Of The University Of California Method and Apparatus for Target Detection Using Electrode-Bound Viruses

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANDERSSON ET AL: 'Pseudogenes, junk DNA, and the dynamics of Rickettsia genomes' MOL. BIOL. EVOL. vol. 18, 2001, pages 829 - 839 *
DATABASE UNIPROT [Online] 03 April 2007 EMBL Database accession no. A3JDN2 *
KANEKO ET AL: 'Complete genomic structure of the bloom-forming toxic cyanobacterium Microcystis aeruginosa NIES-843' DNA RES. vol. 14, 2007, pages 247 - 256 *
MATTHIJNSSENS ET AL: 'Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains' J. VIROL. vol. 82, 2008, pages 3204 - 3219 *
MYERS ET AL: 'Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens' GENOME RES. vol. 16, 2006, pages 1031 - 1040 *

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CN103048461A (zh) * 2012-12-18 2013-04-17 苏州浩欧博生物医药有限公司 一种癌抗原ca15-3的纳米磁微粒化学发光测定试剂盒及其制备方法和检测方法
US11723944B2 (en) 2015-03-16 2023-08-15 Indi Molecular, Inc. Botulinum neurotoxin-specific capture agents, compositions, and methods of using and making
US11884707B2 (en) 2016-09-29 2024-01-30 Regeneron Pharmaceuticals, Inc. Compositions for detection, inhibition and imaging of indoleamine 2, 3-dioxygenase 1 (IDO1) and methods of making and using same
US11517627B2 (en) 2017-01-20 2022-12-06 Juno Therapeutics Gmbh Cell surface conjugates and related cell compositions and methods
WO2018134691A2 (fr) 2017-01-20 2018-07-26 Juno Therapeutics Gmbh Conjugués de surface cellulaire et compositions cellulaires et méthodes associées
WO2018187791A1 (fr) 2017-04-07 2018-10-11 Juno Therapeutics, Inc Cellules génétiquement modifiées exprimant un antigène membranaire spécifique de la prostate (psma) ou une forme modifiée de celui-ci et procédés associés
US11719705B2 (en) 2017-06-15 2023-08-08 Indi Molecular, Inc. IL-17F and IL-17A-specific capture agents, compositions, and methods of using and making
CN111182927B (zh) * 2017-12-11 2023-07-21 慕尼黑工业大学 用于成像和腔内放射治疗的psma配体
CN111182927A (zh) * 2017-12-11 2020-05-19 慕尼黑工业大学 用于成像和腔内放射治疗的psma配体
US11638764B2 (en) 2018-11-08 2023-05-02 Indi Molecular, Inc. Theranostic capture agents, compositions, and methods of using and making
WO2020097531A1 (fr) * 2018-11-08 2020-05-14 Indi Molecular, Inc. Agents de capture théranostiques, compositions et procédés d'utilisation et de fabrication
US11733246B2 (en) 2020-11-03 2023-08-22 Indi Molecular, Inc. Compositions, imaging, and therapeutic methods targeting folate receptor 1 (FOLR1)

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